Current Role at Stanford

Senior Research Associate, Ophthalmology & Physics
Lab manager, surgeon, micro-surgeon, microscopist
Surgical instrument designer, methods designer
Pre-Major Advisor (18 years), Watson Court Campus Council,
Departmental Property Administrator (DPA)- Ophthalmology,
Emergency Coordinator - Ophthalmology,
Stanford CERT, BAT, Building Response Team (Grant, Byers, Physics/AstroPhysics BRT),
University Safety Partners, certified iSpace coordinator - Ophthalmology,
Instructor - Stanford Clinical Anatomy Summer Program - Eye Dissection
Stanford Bay Area Ophthalmology Course - Lab Coordinator

Honors & Awards

  • 5 patents, Stanford OTL (2009)


  • Retinal Chip to restore vision, Stanford University (11/21/1999 - Present)


    Stanford, CA


Service, Volunteer and Community Work

  • Cupertino Amateur Radio Emergency Service (11/21/2005)


    Cupertino, CA

  • Cupertino CERT (11/25/2009)


    Cupertino, CA

Personal Interests

Rower, Crew, Masters
Biology & Ecology


Professional Interests

Cell and Molecular Biology
Scanning and transmission electron microscopy
Fluorescence microscopy
in-situ hybridization
small animal microsurgery
nanofabrication & tissue engineering
biology and pathology of peroxynitrite
development of new processes and methods
design and development of microsurgical instruments

Professional Affiliations and Activities

  • CPPS, NPMA (2009 - Present)


All Publications

  • Development of Animal Models of Local Retinal Degeneration. Investigative ophthalmology & visual science Lorach, H., Kung, J., Beier, C., Mandel, Y., Dalal, R., Huie, P., Wang, J., Lee, S., Sher, A., Jones, B. W., Palanker, D. 2015; 56 (8): 4644-4652


    Development of nongenetic animal models of local retinal degeneration is essential for studies of retinal pathologies, such as chronic retinal detachment or age-related macular degeneration. We present two different methods to induce a highly localized retinal degeneration with precise onset time, that can be applied to a broad range of species in laboratory use.A 30-μm thin polymer sheet was implanted subretinally in wild-type (WT) rats. The effects of chronic retinal separation from the RPE were studied using histology and immunohistochemistry. Another approach is applicable to species with avascular retina, such as rabbits, where the photoreceptors and RPE were thermally ablated over large areas, using a high power scanning laser.Photoreceptors above the subretinal implant in rats degenerated over time, with 80% of the outer nuclear layer disappearing within a month, and the rest by 3 months. Similar loss was obtained by selective photocoagulation with a scanning laser. Cells in the inner nuclear layer and ganglion cell layer were preserved in both cases. However, there were signs of rewiring and decrease in the size of the bipolar cell terminals in the damaged areas.Both methods induce highly reproducible degeneration of photoreceptors over a defined area, with complete preservation of the inner retinal neurons during the 3-month follow-up. They provide a reliable platform for studies of local retinal degeneration and development of therapeutic strategies in a wide variety of species.

    View details for DOI 10.1167/iovs.14-16011

    View details for PubMedID 26207299

  • Performance of photovoltaic arrays in-vivo and characteristics of prosthetic vision in animals with retinal degeneration VISION RESEARCH Lorach, H., Goetz, G., Mandel, Y., Lei, X., Kamins, T. I., Mathieson, K., Huie, P., Dalal, R., Harris, J. S., Palanker, D. 2015; 111: 142-148


    Loss of photoreceptors during retinal degeneration leads to blindness, but information can be reintroduced into the visual system using electrical stimulation of the remaining retinal neurons. Subretinal photovoltaic arrays convert pulsed illumination into pulsed electric current to stimulate the inner retinal neurons. Since required irradiance exceeds the natural luminance levels, an invisible near-infrared (915 nm) light is used to avoid photophobic effects. We characterized the thresholds and dynamic range of cortical responses to prosthetic stimulation with arrays of various pixel sizes and with different number of photodiodes. Stimulation thresholds for devices with 140 μm pixels were approximately half those of 70 μm pixels, and with both pixel sizes, thresholds were lower with 2 diodes than with 3 diodes per pixel. In all cases these thresholds were more than two orders of magnitude below the ocular safety limit. At high stimulation frequencies (>20 Hz), the cortical response exhibited flicker fusion. Over one order of magnitude of dynamic range could be achieved by varying either pulse duration or irradiance. However, contrast sensitivity was very limited. Cortical responses could be detected even with only a few illuminated pixels. Finally, we demonstrate that recording of the corneal electric potential in response to patterned illumination of the subretinal arrays allows monitoring the current produced by each pixel, and thereby assessing the changes in the implant performance over time.

    View details for DOI 10.1016/j.visres.2014.09.007

    View details for Web of Science ID 000355961200004

    View details for PubMedID 25255990

  • Photovoltaic restoration of sight with high visual acuity NATURE MEDICINE Lorach, H., Goetz, G., Smith, R., Lei, X., Mandel, Y., Kamins, T., Mathieson, K., Huie, P., Harris, J., Sher, A., Palanker, D. 2015; 21 (5): 476-U254

    View details for DOI 10.1038/nm.3851

    View details for Web of Science ID 000354068800014

  • Photovoltaic restoration of sight with high visual acuity. Nature medicine Lorach, H., Goetz, G., Smith, R., Lei, X., Mandel, Y., Kamins, T., Mathieson, K., Huie, P., Harris, J., Sher, A., Palanker, D. 2015; 21 (5): 476-482


    Patients with retinal degeneration lose sight due to the gradual demise of photoreceptors. Electrical stimulation of surviving retinal neurons provides an alternative route for the delivery of visual information. We demonstrate that subretinal implants with 70-μm-wide photovoltaic pixels provide highly localized stimulation of retinal neurons in rats. The electrical receptive fields recorded in retinal ganglion cells were similar in size to the natural visual receptive fields. Similarly to normal vision, the retinal response to prosthetic stimulation exhibited flicker fusion at high frequencies, adaptation to static images and nonlinear spatial summation. In rats with retinal degeneration, these photovoltaic arrays elicited retinal responses with a spatial resolution of 64 ± 11 μm, corresponding to half of the normal visual acuity in healthy rats. The ease of implantation of these wireless and modular arrays, combined with their high resolution, opens the door to the functional restoration of sight in patients blinded by retinal degeneration.

    View details for DOI 10.1038/nm.3851

    View details for PubMedID 25915832

  • Role of molecular photodissociation in ultrafast laser surgery OPTICAL INTERACTIONS WITH TISSUE AND CELLS XXVI Wang, J., Schuele, G., Huie, P., Palanker, D. V. 2015; 9321

    View details for DOI 10.1117/12.2077004

    View details for Web of Science ID 000353616800005

  • SUBVISIBLE RETINAL LASER THERAPY Titration Algorithm and Tissue Response RETINA-THE JOURNAL OF RETINAL AND VITREOUS DISEASES Lavinsky, D., Sramek, C., Wang, J., Huie, P., Dalal, R., Mandel, Y., Palanker, D. 2014; 34 (1): 87-97


    Laser therapy for diabetic macular edema and other retinal diseases has been used within a wide range of laser settings: from intense burns to nondamaging exposures. However, there has been no algorithm for laser dosimetry that could determine laser parameters yielding a predictable extent of tissue damage. This multimodal imaging and structural correlation study aimed to verify and calibrate a computational model-based titration algorithm for predictable laser dosimetry ranging from nondamaging to intense coagulative tissue effects.Endpoint Management, an algorithm based on a computational model of retinal photothermal damage, was used to set laser parameters for various levels of tissue effect. The algorithm adjusts both power and pulse duration to vary the expected level of thermal damage at different percentages of a reference titration energy dose. Experimental verification was conducted in Dutch Belted rabbits using a PASCAL Streamline 577 laser system. Titration was performed by adjusting laser power to produce a barely visible lesion at 20 ms pulse duration, which is defined as the nominal (100%) energy level. Tissue effects were then determined for energy levels of 170, 120, 100, 75, 50, and 30% of the nominal energy at 1 hour and 3, 7, 30, and 60 days after treatment. In vivo imaging included fundus autofluorescence, fluorescein angiography, and spectral-domain optical coherence tomography. Morphologic changes in tissue were analyzed using light microscopy, as well as scanning and transmission electron microscopy.One hundred and seventy percent and 120% levels corresponded to moderate and light burns, respectively, with damage to retinal pigment epithelium, photoreceptors, and at highest settings, to the inner retina. 50% to 75% lesions were typically subvisible ophthalmoscopically but detectable with fluorescein angiography and optical coherence tomography. Histology in these lesions demonstrated some selective damage to retinal pigment epithelium and photoreceptors. The 30% to 50% lesions were invisible with in vivo multimodal imaging, and damage was limited primarily to retinal pigment epithelium, visible best with scanning electron microscopy. Over time, photoreceptors shifted into the coagulated zone, reestablishing normal retinal anatomy in lesions ≤100%, as seen in optical coherence tomography and light microscopy. Transmission electron microscopy at 2 months demonstrated restoration of synapses between shifted-in photoreceptors and bipolar cells in these lesions. Retinal pigment epithelium monolayer restored its continuity after 1 week in all lesions. No damage could be seen <30% level.A retinal laser dosimetry protocol based on the Endpoint Management algorithm provides reproducible changes in retinal morphology in animals with various levels of pigmentation. This algorithm opens doors to clinical trials of well-defined subvisible and nondestructive regimes of retinal therapy, especially important for treatment of macular disorders.

    View details for Web of Science ID 000336958700015

  • Non-damaging Laser Therapy of the Macula: Titration Algorithm and Tissue Response OPHTHALMIC TECHNOLOGIES XXIV Palanker, D., Lavinsky, D., Dalal, R., Huie, P. 2014; 8930

    View details for DOI 10.1117/12.2036870

    View details for Web of Science ID 000334350000021

  • Vasoconstriction by Electrical Stimulation: New Approach to Control of Non-Compressible Hemorrhage SCIENTIFIC REPORTS Mandel, Y., Manivanh, R., Dalal, R., Huie, P., Wang, J., Brinton, M., Palanker, D. 2013; 3

    View details for DOI 10.1038/srep02111

    View details for Web of Science ID 000321287200001

  • Cortical responses elicited by photovoltaic subretinal prostheses exhibit similarities to visually evoked potentials NATURE COMMUNICATIONS Mandel, Y., Goetz, G., Lavinsky, D., Huie, P., Mathieson, K., Wang, L., Kamins, T., Galambos, L., Manivanh, R., Harris, J., Palanker, D. 2013; 4

    View details for DOI 10.1038/ncomms2980

    View details for Web of Science ID 000323624600003

  • Restoration of retinal morphology and residual scarring after photocoagulation ACTA OPHTHALMOLOGICA Lavinsky, D., Cardillo, J. A., Mandel, Y., Huie, P., Melo, L. A., Farah, M. E., Belfort, R., Palanker, D. 2013; 91 (4): E315-E323


    Purpose:  To study healing of retinal laser lesions in patients undergoing PRP using SD-OCT. Methods:  Moderate, light and barely visible retinal burns were produced in patients with proliferative diabetic retinopathy scheduled for PRP using 100-, 20- and 10-ms pulses of 532-nm laser, with retinal spot sizes of 100, 200 and 400 μm. Lesions were measured with OCT at 1 hr, 1 week, 1, 2, 4, 6, 9 and 12 months. OCT imaging was correlated with histology in a separate study in rabbits. Results:  Lesions produced by the standard 100-ms exposures exhibited steady scarring, with the damage zone stabilized after 2 months. For 400- and 200-μm spots and 100-ms pulses, the residual scar area at 12 months was approximately 50% of the initial lesion size for moderate, light and barely visible burns. In contrast, lesions produced by shorter exposures demonstrated enhanced restoration of the photoreceptor layer, especially in smaller burns. With 20-ms pulses, the damage zone decreased to 32%, 24% and 20% for moderate, light and barely visible burns of 400 μm, respectively, and down to 12% for barely visible burns of 200 μm. In the 100-μm spots, the residual scar area of the moderate 100-ms burns was 41% of the initial lesion, while barely visible 10-ms burns contracted to 6% of the initial size. Histological observations in rabbits were useful for proper interpretation of the damage zone boundaries in OCT. Conclusions:  Traditional photocoagulation parameters (400 μm, 100 ms and moderate burn) result in a stable scar similar in size to the beam diameter. Restoration of the damaged photoreceptor layer in lighter lesions produced by shorter pulses should allow reducing the common side-effects of photocoagulation such as scotomata and scarring.

    View details for DOI 10.1111/aos.12045

    View details for Web of Science ID 000334387500015

    View details for PubMedID 23557390

  • Restoration of Retinal Structure and Function after Selective Photocoagulation JOURNAL OF NEUROSCIENCE Sher, A., Jones, B. W., Huie, P., Paulus, Y. M., Lavinsky, D., Leung, L. S., Nomoto, H., Beier, C., Marc, R. E., Palanker, D. 2013; 33 (16): 6800-6808


    CNS neurons change their connectivity to accommodate a changing environment, form memories, or respond to injury. Plasticity in the adult mammalian retina after injury or disease was thought to be limited to restructuring resulting in abnormal retinal anatomy and function. Here we report that neurons in the mammalian retina change their connectivity and restore normal retinal anatomy and function after injury. Patches of photoreceptors in the rabbit retina were destroyed by selective laser photocoagulation, leaving retinal inner neurons (bipolar, amacrine, horizontal, ganglion cells) intact. Photoreceptors located outside of the damaged zone migrated to make new functional connections with deafferented bipolar cells located inside the lesion. The new connections restored ON and OFF responses in deafferented ganglion cells. This finding extends the previously perceived limits of restorative plasticity in the adult retina and allows for new approaches to retinal laser therapy free of current detrimental side effects such as scotomata and scarring.

    View details for DOI 10.1523/JNEUROSCI.1044-12.2013

    View details for Web of Science ID 000317723000012

    View details for PubMedID 23595739

  • Modulation of Transgene Expression in Retinal Gene Therapy by Selective Laser Treatment INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Lavinsky, D., Chalberg, T. W., Mandel, Y., Huie, P., Dalal, R., Marmor, M., Palanker, D. 2013; 54 (3): 1873-1880


    To develop a method for modulation of transgene expression in retinal pigment epithelium (RPE) using scanning laser that spares neurosensory retina.Fifteen pigmented rabbits received subretinal injection of recombinant adeno-associated virus (rAAV-2) encoding green fluorescent protein (GFP). GFP expression was measured using confocal scanning laser ophthalmoscopy (cSLO) fluorescence imaging and immunohistochemistry. To reduce the total expression in RPE by half, 50% of the transfected RPE cells were selectively destroyed by microsecond exposures to scanning laser with 50% pattern density. The selectivity of RPE destruction and its migration and proliferation were monitored using fluorescein angiography, spectral-domain optical coherence tomography (SD-OCT), and light, transmission, and scanning electron microscopy. 5-Bromo-2'-dioxyuridine (BrdU) assay was performed to evaluate proliferation of RPE cells.RPE cells were selectively destroyed by the line scanning laser with 15 μs exposures, without damage to the photoreceptors or Bruch's membrane. RPE cells started migrating after the first day, and in 1 week there was complete restoration of RPE monolayer. Selective laser treatment decreased the GFP fluorescence by 54% as compared to control areas; this was further decreased by an additional 48% following a second treatment 1 month later. BrdU assay demonstrated proliferation in approximately half of the RPE cells in treatment areas.Microsecond exposures produced by scanning laser destroyed RPE cells selectively, without damage to neural retina. Continuity of RPE layer is restored within days by migration and proliferation, but transgene not integrated into the nucleus is not replicated. Therefore, gene expression can be modulated in a precise manner by controlling the laser pattern density and further adjusted using repeated applications.

    View details for DOI 10.1167/iovs.12-10933

    View details for Web of Science ID 000316942400040

  • Vasoconstriction by electrical stimulation: new approach to control of non-compressible hemorrhage. Scientific reports Mandel, Y., Manivanh, R., Dalal, R., Huie, P., Wang, J., Brinton, M., Palanker, D. 2013; 3: 2111-?


    Non-compressible hemorrhage is the most common preventable cause of death on battlefield and in civilian traumatic injuries. We report the use of microsecond pulses of electric current to induce rapid constriction in femoral and mesenteric arteries and veins in rats. Electrically-induced vasoconstriction could be induced in seconds while blood vessels dilated back to their original size within minutes after stimulation. At higher settings, a blood clotting formed, leading to complete and permanent occlusion of the vessels. The latter regime dramatically decreased the bleeding rate in the injured femoral and mesenteric arteries, with a complete hemorrhage arrest achieved within seconds. The average blood loss from the treated femoral artery during the first minute after injury was about 7 times less than that of a non-treated control. This new treatment modality offers a promising approach to non-damaging control of bleeding during surgery, and to efficient hemorrhage arrest in trauma patients.

    View details for DOI 10.1038/srep02111

    View details for PubMedID 23828130

  • In-vivo Performance of Photovoltaic Subretinal Prosthesis OPHTHALMIC TECHNOLOGIES XXIII Mandel, Y., Goetz, G., Lavinsky, D., Huie, P., Mathieson, K., Wang, L., Kamins, T., Manivanh, R., Harris, J., Palanker, D. 2013; 8567

    View details for DOI 10.1117/12.2001750

    View details for Web of Science ID 000325430800005

  • Optical Modulation of Transgene Expression in Retinal Pigment Epithelium OPHTHALMIC TECHNOLOGIES XXIII Palanker, D., Lavinsky, D., Chalberg, T., Mandel, Y., Huie, P., Dalal, R., Marmor, M. 2013; 8567

    View details for DOI 10.1117/12.2001923

    View details for Web of Science ID 000325430800006

  • Hemorrhage Control by Microsecond Electrical Pulses TERAHERTZ AND ULTRASHORT ELECTROMAGNETIC PULSES FOR BIOMEDICAL APPLICATIONS Mandel, Y., Manivanh, R., Dalal, R., Huie, P., Wang, J., Brinton, M., Palanker, D. 2013; 8585

    View details for DOI 10.1117/12.2002303

    View details for Web of Science ID 000325500100010

  • Cortical responses elicited by photovoltaic subretinal prostheses exhibit similarities to visually evoked potentials. Nature communications Mandel, Y., Goetz, G., Lavinsky, D., Huie, P., Mathieson, K., Wang, L., Kamins, T., Galambos, L., Manivanh, R., Harris, J., Palanker, D. 2013; 4: 1980-?


    We have previously developed a wireless photovoltaic retinal prosthesis, in which camera-captured images are projected onto the retina using pulsed near-IR light. Each pixel in the subretinal implant directly converts pulsed light into local electric current to stimulate the nearby inner retinal neurons. Here we report that implants having pixel sizes of 280, 140 and 70 μm implanted in the subretinal space in rats with normal and degenerate retina elicit robust cortical responses upon stimulation with pulsed near-IR light. Implant-induced eVEP has shorter latency than visible light-induced VEP, its amplitude increases with peak irradiance and pulse duration, and decreases with frequency in the range of 2-20 Hz, similar to the visible light response. Modular design of the arrays allows scalability to a large number of pixels, and combined with the ease of implantation, offers a promising approach to restoration of sight in patients blinded by retinal degenerative diseases.

    View details for DOI 10.1038/ncomms2980

    View details for PubMedID 23778557

  • Photovoltaic retinal prosthesis: implant fabrication and performance JOURNAL OF NEURAL ENGINEERING Wang, L., Mathieson, K., Kamins, T. I., Loudin, J. D., Galambos, L., Goetz, G., Sher, A., Mandel, Y., Huie, P., Lavinsky, D., Harris, J. S., Palanker, D. V. 2012; 9 (4)


    The objective of this work is to develop and test a photovoltaic retinal prosthesis for restoring sight to patients blinded by degenerative retinal diseases. A silicon photodiode array for subretinal stimulation has been fabricated by a silicon-integrated-circuit/MEMS process. Each pixel in the two-dimensional array contains three series-connected photodiodes, which photovoltaically convert pulsed near-infrared light into bi-phasic current to stimulate nearby retinal neurons without wired power connections. The device thickness is chosen to be 30 µm to absorb a significant portion of light while still being thin enough for subretinal implantation. Active and return electrodes confine current near each pixel and are sputter coated with iridium oxide to enhance charge injection levels and provide a stable neural interface. Pixels are separated by 5 µm wide trenches to electrically isolate them and to allow nutrient diffusion through the device. Three sizes of pixels (280, 140 and 70 µm) with active electrodes of 80, 40 and 20 µm diameter were fabricated. The turn-on voltages of the one-diode, two-series-connected diode and three-series-connected diode structures are approximately 0.6, 1.2 and 1.8 V, respectively. The measured photo-responsivity per diode at 880 nm wavelength is ?0.36 A W(-1), at zero voltage bias and scales with the exposed silicon area. For all three pixel sizes, the reverse-bias dark current is sufficiently low (<100 pA) for our application. Pixels of all three sizes reliably elicit retinal responses at safe near-infrared light irradiances, with good acceptance of the photodiode array in the subretinal space. The fabricated device delivers efficient retinal stimulation at safe near-infrared light irradiances without any wired power connections, which greatly simplifies the implantation procedure. Presence of the return electrodes in each pixel helps to localize the current, and thereby improves resolution.

    View details for DOI 10.1088/1741-2560/9/4/046014

    View details for Web of Science ID 000306759600027

    View details for PubMedID 22791690

  • Photovoltaic Retinal Prosthesis with High Pixel Density. Nature photonics Mathieson, K., Loudin, J., Goetz, G., Huie, P., Wang, L., Kamins, T. I., Galambos, L., Smith, R., Harris, J. S., Sher, A., Palanker, D. 2012; 6 (6): 391-397


    Retinal degenerative diseases lead to blindness due to loss of the "image capturing" photoreceptors, while neurons in the "image processing" inner retinal layers are relatively well preserved. Electronic retinal prostheses seek to restore sight by electrically stimulating surviving neurons. Most implants are powered through inductive coils, requiring complex surgical methods to implant the coil-decoder-cable-array systems, which deliver energy to stimulating electrodes via intraocular cables. We present a photovoltaic subretinal prosthesis, in which silicon photodiodes in each pixel receive power and data directly through pulsed near-infrared illumination and electrically stimulate neurons. Stimulation was produced in normal and degenerate rat retinas, with pulse durations from 0.5 to 4 ms, and threshold peak irradiances from 0.2 to 10 mW/mm(2), two orders of magnitude below the ocular safety limit. Neural responses were elicited by illuminating a single 70 ?m bipolar pixel, demonstrating the possibility of a fully-integrated photovoltaic retinal prosthesis with high pixel density.

    View details for PubMedID 23049619

  • Toward the development of an artificial cornea: Improved stability of interpenetrating polymer networks JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS Hartmann, L., Watanabe, K., Zheng, L. L., Kim, C., Beck, S. E., Huie, P., Noolandi, J., Cochran, J. R., Ta, C. N., Frank, C. W. 2011; 98B (1): 8-17


    A novel interpenetrating network (IPN) based on poly(ethylene glycol) (PEG) and poly(acrylic acid) was developed and its use as an artificial cornea was evaluated in vivo. The in vivo results of a first set of corneal inlays based on PEG-diacrylate precursor showed inflammation of the treated eyes and haze in the corneas. The insufficient biocompatibility could be correlated to poor long-term stability of the implant caused by hydrolytic degradation over time. Adapting the hydrogel chemistry by replacing hydrolysable acrylate functionalities with stable acrylamide functionalities was shown to increase the long-term stability of the resulting IPNs under hydrolytic conditions. This new set of hydrogel implants now shows increased biocompatibility in vivo. Rabbits with corneal inlay implants are healthy and have clear cornea and non-inflamed eyes for up to 6 months after implantation.

    View details for DOI 10.1002/jbm.b.31806

    View details for Web of Science ID 000291598900002

  • Photovoltaic Retinal Prosthesis OPHTHALMIC TECHNOLOGIES XXI Loudin, J., Mathieson, K., Kamins, T., Wang, L., Galambos, L., Huie, P., Sher, A., Harris, J., Palanker, D. 2011; 7885

    View details for DOI 10.1117/12.876560

    View details for Web of Science ID 000297590500028

  • Anterior capsulotomy with a pulsed-electron avalanche knife JOURNAL OF CATARACT AND REFRACTIVE SURGERY Palanker, D., Nomoto, H., Huie, P., Vankov, A., Chang, D. F. 2010; 36 (1): 127-132


    To evaluate a new pulsed-electron avalanche knife design for creating a continuous curvilinear capsulotomy (CCC) and compare the CCC with a mechanical capsulorhexis.Department of Ophthalmology, Stanford University, Stanford, California, USA.In this study, CCCs were created in freshly enucleated bovine eyes and in rabbit eyes in vivo. The cutting velocity was adjusted by controlling the burst repetition rate, voltage amplitude, and burst duration. Tissue samples were fixed and processed for histology and scanning electron microscopy (SEM) immediately after surgery.The study included 50 bovine eyes and 10 rabbit eyes. By adjusting the electrosurgical waveforms, gas-bubble formation was minimized to permit good surgical visualization. The optimum voltage level was determined to be +/-410 V with a burst duration of 20 mus. Burst repetition rate, continuously adjustable from 20 to 200 Hz with footpedal control, allowed the surgeon to vary linear cutting velocity up to 2.0 mm/s. Histology and SEM showed that the pulsed-electron avalanche knife produced sharp-edged capsule cutting without radial nicks or tears.The probe of the pulsed-electron avalanche knife duplicated the surgical feel of a 25-gauge cystotome and created a histologically smooth capsule cut. It may improve precision and reproducibility of creating a CCC, as well as improve its proper sizing and centration, especially in the face of surgical risk factors, such as weak zonules or poor visibility. FINANCIAL DISCLOSURES: Drs. Palanker and Vankov hold patents to the pulsed electron avalanche knife technology, which are licensed to PEAK Surgical by Stanford University. Drs. Palanker and Chang are consultants to PEAK Surgical. Dr. Vankov is an employee of PEAK Surgical. Neither of the other authors has a financial or proprietary interest in any material or method mentioned.

    View details for DOI 10.1016/j.jcrs.2009.07.046

    View details for Web of Science ID 000277410700021

    View details for PubMedID 20117716

  • Dynamics of retinal photocoagulation and rupture JOURNAL OF BIOMEDICAL OPTICS Sramek, C., Paulus, Y., Nomoto, H., Huie, P., Brown, J., Palanker, D. 2009; 14 (3)


    In laser retinal photocoagulation, short (<20 ms) pulses have been found to reduce thermal damage to the inner retina, decrease treatment time, and minimize pain. However, the safe therapeutic window (defined as the ratio of power for producing a rupture to that of mild coagulation) decreases with shorter exposures. To quantify the extent of retinal heating and maximize the therapeutic window, a computational model of millisecond retinal photocoagulation and rupture was developed. Optical attenuation of 532-nm laser light in ocular tissues was measured, including retinal pigment epithelial (RPE) pigmentation and cell-size variability. Threshold powers for vaporization and RPE damage were measured with pulse durations ranging from 1 to 200 ms. A finite element model of retinal heating inferred that vaporization (rupture) takes place at 180-190 degrees C. RPE damage was accurately described by the Arrhenius model with activation energy of 340 kJ/mol. Computed photocoagulation lesion width increased logarithmically with pulse duration, in agreement with histological findings. The model will allow for the optimization of beam parameters to increase the width of the therapeutic window for short exposures.

    View details for DOI 10.1117/1.3130282

    View details for Web of Science ID 000268536300016

    View details for PubMedID 19566300

  • Effect of shape and coating of a subretinal prosthesis on its integration with the retina EXPERIMENTAL EYE RESEARCH Butterwick, A., Huie, P., Jones, B. W., Marc, R. E., Marmor, M., Palanker, D. 2009; 88 (1): 22-29


    Retinal stimulation with high spatial resolution requires close proximity of electrodes to target cells. This study examines the effects of material coatings and 3-dimensional geometries of subretinal prostheses on their integration with the retina. A trans-scleral implantation technique was developed to place microfabricated structures in the subretinal space of RCS rats. The effect of three coatings (silicon oxide, iridium oxide and parylene) and three geometries (flat, pillars and chambers) on the retinal integration was compared using passive implants. Retinal morphology was evaluated histologically 6 weeks after implantation. For 3-dimensional implants the retinal cell phenotype was also evaluated using Computational Molecular Phenotyping. Flat implants coated with parylene and iridium oxide were generally well tolerated in the subretinal space, inducing only a mild gliotic response. However, silicon-oxide coatings induced the formation of a significant fibrotic seal around the implants. Glial proliferation was observed at the base of the pillar electrode arrays and inside the chambers. The non-traumatic penetration of pillar tips into the retina provided uniform and stable proximity to the inner nuclear layer. Retinal cells migrated into chambers with apertures larger than 10 mum. Both pillars and chambers achieved better proximity to the inner retinal cells than flat implants. However, isolation of retinal cells inside the chamber arrays is likely to affect their long-term viability. Pillars demonstrated minimal alteration of the inner retinal architecture, and thus appear to be the most promising approach for maintaining close proximity between the retinal prosthetic electrodes and target neurons.

    View details for DOI 10.1016/j.exer.2008.09.018

    View details for Web of Science ID 000262395800004

    View details for PubMedID 18955050

  • Computational model of retinal photocoagulation and rupture OPHTHALMIC TECHNOLOGIES XIX Sramek, C., Paulus, Y. M., Nomoto, H., Huie, P., Palanker, D. 2009; 7163

    View details for DOI 10.1117/12.808556

    View details for Web of Science ID 000284821000019

  • High resolution optoelectronic retinal prosthesis OPHTHALMIC TECHNOLOGIES XIX Loudin, J., Dinyari, R., Huie, P., Butterwick, A., Peumans, P., Palanker, D. 2009; 7163

    View details for DOI 10.1117/12.807668

    View details for Web of Science ID 000284821000029

  • Finite element model of thermal processes in retinal photocoagulation OPTICAL INTERACTIONS WITH TISSUE AND CELLS XX Sramek, C., Paulus, Y. M., Nomoto, H., Huie, P., Palanker, D. 2009; 7175

    View details for DOI 10.1117/12.828888

    View details for Web of Science ID 000285714500035

  • A Curvable Silicon Retinal Implant 2009 IEEE INTERNATIONAL ELECTRON DEVICES MEETING Dinyari, R., Loudin, J. D., Huie, P., Palanker, D., Peumans, P. 2009: 551-554
  • Electrosurgery with cellular precision IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Palanker, D. V., Vankov, A., Huie, P. 2008; 55 (2): 838-841


    Electrosurgery, one of the most-often used surgical tools, is a robust but somewhat crude technology that has changed surprisingly little since its invention almost a century ago. Continuous radiofrequency is still used for tissue cutting, with thermal damage extending to hundreds of micrometers. In contrast, lasers developed 70 years later, have been constantly perfected, and the laser-tissue interactions explored in great detail, which has allowed tissue ablation with cellular precision in many laser applications. We discuss mechanisms of tissue damage by electric field, and demonstrate that electrosurgery with properly optimized waveforms and microelectrodes can rival many advanced lasers. Pulsed electric waveforms with burst durations ranging from 10 to 100 micros applied via insulated planar electrodes with 12 microm wide exposed edges produced plasma-mediated dissection of tissues with the collateral damage zone ranging from 2 to 10 microm. Length of the electrodes can vary from micrometers to centimeters and all types of soft tissues-from membranes to cartilage and skin could be dissected in liquid medium and in a dry field. This technology may allow for major improvements in outcomes of the current surgical procedures and development of much more refined surgical techniques.

    View details for DOI 10.1109/TBME.2007.914539

    View details for Web of Science ID 000252622500006

    View details for PubMedID 18270030

  • Pulsed electrical stimulation for control of vasculature: Temporary vasoconstriction and permanent thrombosis BIOELECTROMAGNETICS Palanker, D., Vankov, A., Freyvert, Y., Huie, P. 2008; 29 (2): 100-107


    A variety of medical procedures is aimed to selectively compromise or destroy vascular function. Such procedures include cancer therapies, treatments of cutaneous vascular disorders, and temporary hemostasis during surgery. Currently, technologies such as lasers, cryosurgery and radio frequency coagulation, produce significant collateral damage due to the thermal nature of these interactions and corresponding heat exchange with surrounding tissues. We describe a non-thermal method of inducing temporary vasoconstriction and permanent thrombosis using short pulse (microseconds) electrical stimulation. The current density required for vasoconstriction increases with decreasing pulse duration approximately as t(-0.25). The threshold of electroporation has a steeper dependence on pulse duration-exceeding t(-0.5). At pulse durations shorter than 5 micros, damage threshold exceeds the vasoconstriction threshold, thus allowing for temporary hemostasis without direct damage to surrounding tissue. With a pulse repetition rate of 0.1 Hz, vasoconstriction is achieved approximately 1 min after the beginning of treatment in both arteries and veins. Thrombosis occurs at higher electric fields, and its threshold increases with vessel diameter. Histology demonstrated a lack of tissue damage during vasoconstriction, but vascular endothelium was damaged during thrombosis. The temperature increase does not exceed 0.1 degrees C during these treatments.

    View details for DOI 10.1002/bem.20368

    View details for Web of Science ID 000253250000003

    View details for PubMedID 17918191

  • Effect of pulse duration on size and character of the lesion in retinal photocoagulation ARCHIVES OF OPHTHALMOLOGY Jain, A., Blumenkranz, M. S., Paulus, Y., Wiltberger, M. W., Andersen, D. E., Huie, P., Palanker, D. 2008; 126 (1): 78-85


    To systematically evaluate the effects of laser beam size, power, and pulse duration of 1 to 100 milliseconds on the characteristics of ophthalmoscopically visible retinal coagulation lesions.A 532-nm Nd:YAG laser was used to irradiate 36 retinas in Dutch Belt rabbits with retinal beam sizes of 66, 132, and 330 mum. Lesions were clinically graded 1 minute after placement, their size measured by digital imaging, and their depth assessed histologically at different time points.Retinal lesion size increased linearly with laser power and logarithmically with pulse duration. The width of the therapeutic window, defined by the ratio of the threshold power for producing a rupture to that of a mild coagulation, decreased with decreasing pulse durations. For 132- and 330-mum retinal beam sizes, the therapeutic window declined from 3.9 to 3.0 and 5.4 to 3.7, respectively, as pulse duration decreased from 100 to 20 ms. At pulse durations of 1 millisecond, the therapeutic window decreased to unity, at which point rupture and a mild lesion were equally likely to occur.At shorter pulse durations, the width and axial extent of the retinal lesions are smaller and less dependent on variations in laser power than at longer durations. The width of the therapeutic window, a measure of relative safety, increases with the beam size.Pulse durations of approximately 20 milliseconds represent an optimal compromise between the favorable impact of speed, higher spatial localization, and reduced collateral damage on one hand, and sufficient width of the therapeutic window (> 3) on the other.

    View details for Web of Science ID 000252312800011

    View details for PubMedID 18195222

  • Tissue damage by pulsed electrical stimulation IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Butterwick, A., Vankov, A., Huie, P., Freyvert, Y., Palanker, D. 2007; 54 (12): 2261-2267


    Repeated pulsed electrical stimulation is used in a multitude of neural interfaces; damage resulting from such stimulation was studied as a function of pulse duration, electrode size, and number of pulses using a fluorescent assay on chick chorioallontoic membrane (CAM) in vivo and chick retina in vitro. Data from the chick model were verified by repeating some measurements on porcine retina in-vitro. The electrode size varied from 100 microm to 1 mm, pulse duration from 6 micros to 6 ms, and the number of pulses from 1 to 7500. The threshold current density for damage was independent of electrode size for diameters greater than 300 microm, and scaled as 1/r2 for electrodes smaller than 200 microm. Damage threshold decreased with the number of pulses, dropping by a factor of 14 on the CAM and 7 on the retina as the number of pulses increased from 1 to 50, and remained constant for a higher numbers of pulses. The damage threshold current density on large electrodes scaled with pulse duration as approximately 1/t0.5, characteristic of electroporation. The threshold current density for repeated exposure on the retina varied between 0.061 A/cm2 at 6 ms to 1.3 A/cm2 at 6 micros. The highest ratio of the damage threshold to the stimulation threshold in retinal ganglion cells occurred at pulse durations near chronaxie-around 1.3 ms.

    View details for DOI 10.1109/TBME.2007.908310

    View details for Web of Science ID 000251226200016

    View details for PubMedID 18075042

  • Perspective: Tissue engineering for RPE transplantation in AMD SPEKTRUM DER AUGENHEILKUNDE Stanzel, B. V., Englander, M., Strick, D. J., Sanislo, S. S., Huie, P., Blumenkranz, M. S., Binder, S., Marmor, M. F. 2007; 21 (4): 212-217
  • Optoelectronic retinal prosthesis: system design and performance JOURNAL OF NEURAL ENGINEERING Loudin, J. D., Simanovskii, D. M., VijayRaghavan, K., Sramek, C. K., Butterwick, A. F., Huie, P., McLean, G. Y., Palanker, D. V. 2007; 4 (1): S72-S84


    The design of high-resolution retinal prostheses presents many unique engineering and biological challenges. Ever smaller electrodes must inject enough charge to stimulate nerve cells, within electrochemically safe voltage limits. Stimulation sites should be placed within an electrode diameter from the target cells to prevent 'blurring' and minimize current. Signals must be delivered wirelessly from an external source to a large number of electrodes, and visual information should, ideally, maintain its natural link to eye movements. Finally, a good system must have a wide range of stimulation currents, external control of image processing and the option of either anodic-first or cathodic-first pulses. This paper discusses these challenges and presents solutions to them for a system based on a photodiode array implant. Video frames are processed and imaged onto the retinal implant by a head-mounted near-to-eye projection system operating at near-infrared wavelengths. Photodiodes convert light into pulsed electric current, with charge injection maximized by applying a common biphasic bias waveform. The resulting prosthesis will provide stimulation with a frame rate of up to 50 Hz in a central 10 degrees visual field, with a full 30 degrees field accessible via eye movements. Pixel sizes are scalable from 100 to 25 microm, corresponding to 640-10,000 pixels on an implant 3 mm in diameter.

    View details for DOI 10.1088/1741-2560/4/1/S09

    View details for Web of Science ID 000245612700010

    View details for PubMedID 17325419

  • Progress towards a high-resolution retinal prosthesis OPHTHALMIC TECHNOLOGIES XVII Butterwick, A., Vankov, A., Huie, P., Vijayraghavan, K., Loudin, J., Palanker, D. 2007; 6426

    View details for DOI 10.1117/12.701787

    View details for Web of Science ID 000246519000018

  • Intravenous Tamm-Horsfall protein polyps: report of a case in association with a hematoma that mimicked a renal neoplasm. American journal of kidney diseases Higgins, J. P., Huie, P., Rigaud, G., Sibley, R. K. 2006; 48 (5): e67-71


    Tamm-Horsfall protein (THP) is a glycoprotein produced only in the thick ascending limb of the loop of Henle. Its primary physiological function is unknown, but it may have a role in host defense against infectious organisms. THP is the primary scaffolding protein in all varieties of tubular casts. Under certain conditions, THP may be extruded from tubular lumens into the interstitium and lymphatic channels. It even may be found within lymph nodes sampled for staging of neoplastic conditions. THP deposits were described in lumens of large veins. The pathogenetic basis of this finding is not known, but obstruction of renal outflow was suggested, and several cases were associated with macroscopic hematuria. We report a case of intravenous THP polyposis in which, in addition to abundant hemorrhage, there was formation of a hematoma. This measured 12 cm in diameter and caused clinical concern for the possibility of renal cell carcinoma. Although the cause of the hematoma was not apparent, the association with striking intravenous polyps of THP is noteworthy because this represents the first association of intravenous THP polyps with a large intraparenchymal hematoma.

    View details for PubMedID 17059985

  • Gene transfer to rabbit retina with electron avalanche transfection INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Chalberg, T. W., Vankov, A., Molnar, F. E., Butterwick, A. F., Huie, P., Calos, M. P., Palanker, D. V. 2006; 47 (9): 4083-4090


    Nonviral gene therapy represents a promising treatment for retinal diseases, given clinically acceptable methods for efficient gene transfer. Electroporation is widely used for transfection, but causes significant collateral damage and a high rate of cell death, especially in applications in situ. This study was conducted in the interest of developing efficient and less toxic forms of gene transfer for the eye.A novel method for nonviral DNA transfer, called electron avalanche transfection, was used that involves microsecond electric plasma-mediated discharges applied via microelectrode array. This transfection method, which produces synchronized pulses of mechanical stress and high electric field, was first applied to chorioallantoic membrane as a model system and then to rabbit RPE in vivo. Gene transfer was measured by using luciferase bioluminescence and in vivo fluorescent fundus photography. Safety was evaluated by performing electroretinograms and histology.In chorioallantoic membrane, electron avalanche transfection was approximately 10,000-fold more efficient and produced less tissue damage than conventional electroporation. Also demonstrated was efficient plasmid DNA transfer to the rabbit retina after subretinal DNA injection and transscleral electron avalanche transfection. Electroretinograms and histology showed no evidence of damage from the procedure.Electron avalanche transfection is a powerful new technology for safe DNA delivery that has great promise as a nonviral system of gene transfer.

    View details for DOI 10.1167/iovs.06-0092

    View details for Web of Science ID 000240050700055

    View details for PubMedID 16936128

  • Plasma-mediated transfection of RPE OPHTHALMIC TECHNOLOGIES XVI Palanker, D., Chalberg, T., Vankov, A., Huie, P., Molnar, F. E., Butterwick, A., Calos, M., Marmor, M., Blumenkranz, M. S. 2006; 6138

    View details for DOI 10.1117/12.649624

    View details for Web of Science ID 000237708800038

  • Dynamic range of safe electrical stimulation of the retina OPHTHALMIC TECHNOLOGIES XVI Butterwick, A. F., Vankov, A., Huie, P., Palanker, D. V. 2006; 6138

    View details for DOI 10.1117/12.650652

    View details for Web of Science ID 000237708800018

  • Glucose permeability of human, bovine, and porcine corneas in vitro OPHTHALMIC RESEARCH Myung, D., Derr, K., Huie, P., Noolandi, J., Ta, K. P., Ta, C. N. 2006; 38 (3): 158-163


    To measure glucose flux across human, bovine, and porcine corneas and to determine the diffusion coefficient of each type of cornea.Diffusion of glucose across human (n = 8), bovine (n = 7), and pig corneas (n = 8) was measured using a modified blind well chamber apparatus (Boyden chamber). Dialysis membranes (n = 7) and nonporous Mylar membranes (n = 7) were used as positive and negative controls, respectively. Glucose concentrations were measured at 30-min intervals with a commercially available glucose meter.The diffusion of glucose through corneas in vitro was calculated by a simple Fickian diffusion model. The diffusion coefficient of glucose is highest for the human cornea (D(HC) = 3.0 +/- 0.2 x 10(-6) cm(2)/s) followed by porcine corneas (D(PC) = 1.8 +/- 0.6 x 10(-6) cm(2)/s) and bovine corneas (D(BC) = 1.6 +/- 0.1 x 10(-6) cm(2)/s) (p < 0.05). The diffusion coefficients of all tested corneas were significantly higher (p < 0.05) than that of dialysis membrane (D(DM) = 3.4 +/- 0.2 x 10(-7) cm(2)/s).The glucose diffusion coefficients of human, bovine, and porcine corneas are on the order of 10(-6). Human corneas have higher permeability to glucose than either porcine or bovine corneas.

    View details for DOI 10.1159/000090726

    View details for Web of Science ID 000238052500005

    View details for PubMedID 16401912

  • Optical spectroscopy noninvasively monitors response of organelles to cellular stress JOURNAL OF BIOMEDICAL OPTICS Schuele, G., Vitkin, E., Huie, P., O'Connell-Rodwell, C., Palanker, D., Perelman, L. T. 2005; 10 (5)


    Fast and noninvasive detection of cellular stress is extremely useful for fundamental research and practical applications in medicine and biology. We discovered that light scattering spectroscopy enables us to monitor the transformations in cellular organelles under thermal stress. At the temperatures triggering expression of heat shock proteins, the refractive index of mitochondria increase within 1 min after the onset of heating, indicating enhanced metabolic activity. At higher temperatures and longer exposures, the organelles increase in size. This technique provides an insight into metabolic processes within organelles larger than 50 nm without exogenous staining and opens doors for noninvasive real-time assessment of cellular stress.

    View details for DOI 10.1117/1.2075207

    View details for Web of Science ID 000233711300006

    View details for PubMedID 16292941

  • Design of a high-resolution optoelectronic retinal prosthesis. Journal of neural engineering Palanker, D., Vankov, A., Huie, P., Baccus, S. 2005; 2 (1): S105-20


    It has been demonstrated that electrical stimulation of the retina can produce visual percepts in blind patients suffering from macular degeneration and retinitis pigmentosa. However, current retinal implants provide very low resolution (just a few electrodes), whereas at least several thousand pixels would be required for functional restoration of sight. This paper presents the design of an optoelectronic retinal prosthetic system with a stimulating pixel density of up to 2500 pix mm(-2) (corresponding geometrically to a maximum visual acuity of 20/80). Requirements on proximity of neural cells to the stimulation electrodes are described as a function of the desired resolution. Two basic geometries of sub-retinal implants providing required proximity are presented: perforated membranes and protruding electrode arrays. To provide for natural eye scanning of the scene, rather than scanning with a head-mounted camera, the system operates similar to 'virtual reality' devices. An image from a video camera is projected by a goggle-mounted collimated infrared LED-LCD display onto the retina, activating an array of powered photodiodes in the retinal implant. The goggles are transparent to visible light, thus allowing for the simultaneous use of remaining natural vision along with prosthetic stimulation. Optical delivery of visual information to the implant allows for real-time image processing adjustable to retinal architecture, as well as flexible control of image processing algorithms and stimulation parameters.

    View details for PubMedID 15876646

  • Optical monitoring of thermal effects in RPE during heating OPHTHALMIC TECHNOLOGIES XV Schuele, G., Huie, P., Yellachich, D., Molnar, F. E., O'Conell-Rodwell, C., Vitkin, E., Perelman, L. T., Palanker, D. 2005; 5688: 194-200

    View details for DOI 10.1117/12.591068

    View details for Web of Science ID 000229038000024

  • Towards high-resolution optoelectronic retinal prosthesis OPHTHALMIC TECHNOLOGIES XV Palanker, D., Huie, P., Vankov, A., Asher, A., Baccus, S. 2005; 5688: 223-233

    View details for DOI 10.1117/12.590964

    View details for Web of Science ID 000229038000028

  • Migration of retinal cells through a perforated membrane: Implications for a high-resolution prosthesis INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Palanker, D., Huie, P., Vankov, A., Aramant, R., Seiler, M., Fishman, H., Marmor, M., Blumenkranz, M. 2004; 45 (9): 3266-3270


    One of the critical difficulties in design of a high-resolution retinal implant is the proximity of stimulating electrodes to the target cells. This is a report of a phenomenon of retinal cellular migration into a perforated membrane that may help to address this problem.Mylar membranes with an array of perforations (3-40 microm in diameter) were used as a substrate for in vitro retinal culture (chicken, rats) and were also transplanted into the subretinal space of adult RCS rats. A membrane was also constructed with a seal on one side to restrict the migration.Retinal tissue in vitro grew within 3 days through perforations of greater than 5 microm in diameter when the membranes were positioned on the photoreceptor side, but no migration occurred if the implant was placed on the inner retinal surface. Histology with light microscopy and transmission electron microscopy (TEM) demonstrated that migrating cells retain neuronal structures for signal transduction. Similar growth of RCS rat retinal cells occurred in vivo within 5 days of implantation. A basal seal kept the migrating tissue within a small membrane compartment.Retinal neurons migrate within a few days into perforations (> 5 microm in diameter) of a membrane placed into the subretinal space. This may provide a means of gaining close proximity between electrodes in a retinal prosthetic chip and target cells, and thus allow a greater density of stimulating elements to subserve higher resolution. Further studies are needed to explore the long-term stability of the retinal migration.

    View details for DOI 10.1167/iovs.03-1327

    View details for Web of Science ID 000223500900055

    View details for PubMedID 15326150

  • The chick chorioallantoic membrane as a model tissue for surgical retinal research and simulation RETINA-THE JOURNAL OF RETINAL AND VITREOUS DISEASES Leng, T., MILLER, J. M., Bilbao, K. V., Palanker, D. V., Huie, P., Blumenkranz, M. S. 2004; 24 (3): 427-434


    We describe the use of chick chorioallantoic membrane (CAM) as a model system for the study of the precision and safety of vitreoretinal microsurgical instruments and techniques.The CAM was prepared for experimentation with and without its inner shell membrane (ISM) attached for in vivo and in vitro experiments that simulated medical and surgical interventions on the retina.The CAM's ease of use, low cost, and anatomic structure make it a convenient model for surgical retinal and retinal vascular modeling.While CAM has been used extensively in the past for ocular angiogenesis studies, we describe the tissue as a useful tool for a variety of other applications, including (1) testing of novel surgical tools and techniques for cutting and coagulating retina and its vasculature, (2) testing vessel cannulation and injection techniques, (3) angiographic studies, and (4) endoscopic surgery.

    View details for Web of Science ID 000222156800014

    View details for PubMedID 15187666

  • Attracting retinal cells to electrodes for high-resolution stimulation OPHTHALMIC TECHNOLOGIES XIV Palanker, D. V., Huie, P., Vankov, A., Freyvert, Y., Fishman, H., Marmor, M. F., Blumenkranz, M. S. 2004; 5314: 306-314

    View details for DOI 10.1117/12.529757

    View details for Web of Science ID 000223299200039

  • Non-invasive monitoring of the thermal stress in RPE using light scattering spectroscopy OPHTHALMIC TECHNOLOGIES XIV Schuele, G., Huie, P., Vankov, A., Vitkin, E., Fang, H., Hanlon, E. B., Perelman, L. T., Palanker, D. 2004; 5314: 95-99

    View details for DOI 10.1117/12.529415

    View details for Web of Science ID 000223299200012

  • Electro-adhesive forceps for tissue manipulation OPHTHALMIC TECHNOLOGIES XIV Vankov, A., Huie, P., Blumenkranz, M., Palanker, D. 2004; 5314: 270-274

    View details for DOI 10.1117/12.529723

    View details for Web of Science ID 000223299200035

  • Expression of nitric oxide, peroxynitrite, and apoptosis in loose total hip replacements JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Puskas, B. L., Menke, N. E., Huie, P., Song, Y., Ecklund, K., Trindade, M. C., Smith, R. L., Goodman, S. B. 2003; 66A (3): 541-549


    Nitric oxide (NO) is an effector molecule associated with inflammation, immune function, bone metabolism, and the induction of apoptosis. This study examined the role of NO, peroxynitrite (ONOO(-)), and apoptosis in cases of revision total hip replacements (THRs). We hypothesized that apoptosis and excess production of NO contribute to the inflammatory reaction to orthopedic biomaterial wear debris that is associated with loosening and osteolysis. Periprosthetic membranous specimens were collected from revised cemented acetabular components with simple loosening and ballooning osteolysis. Synovial samples from patients undergoing primary THR were used as controls. The presence of macrophages (CD68(+)) and levels of inducible nitric oxide synthase (INOS), endothelial nitric oxide synthase (EcNOS), ONOO(-) (Nitro, assayed by the amount of nitrated tyrosine residues), and apoptosis (TUNEL staining) were examined using immunohistochemistry. Increased expression for INOS, EcNOS, and ONOO(-) in both the loose/osteolytic and the loose/non-osteolytic groups was observed when compared to the synovium group. There were no significant differences between the loose/osteolytic group and loose/non-osteolytic group for these biologic markers. TUNEL staining showed a significant increase in apoptosis in the loose/osteolytic group compared to the loose/non-osteolytic group and synovial tissues. These findings suggest that NO and NO-derived molecules, such as ONOO(-), may be involved in sustaining the foreign-body reaction to wear debris. NO and ONOO(-) may prove to be useful markers of prosthetic loosening whereas apoptosis may be a marker distinguishing ballooning from simple osteolysis.

    View details for DOI 10.1002/jbm.a.10010

    View details for Web of Science ID 000185104800014

  • Towards a neurotransmitter-based retinal prosthesis using an inkjet print-head BIOMEDICAL MICRODEVICES Noolandi, J., Peterman, M. C., Huie, P., Lee, C., Blumenkranz, M. S., Fishman, H. A. 2003; 5 (3): 195-199
  • In vitro epithelialization of a synthetic polymer for generation of corneal onlay/keratoprosthesis Pilyugina, S. A., Lavoie, A., Huie, P., Derr, K., SMITH, A. J., Noolandi, J., Waymouth, R. M., Ta, C. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2003: U321-U321
  • Building thick photoresist structures from the bottom up JOURNAL OF MICROMECHANICS AND MICROENGINEERING Peterman, M. C., Huie, P., Bloom, D. M., Fishman, H. A. 2003; 13 (3): 380-382
  • The role of the TH1 and TH2 immune responses in loosening and osteolysis of cemented total hip replacements. Journal of biomedical materials research. Part A Arora, A., Song, Y., Chun, L., Huie, P., Trindade, M., Smith, R. L., Goodman, S. 2003; 64 (4): 693-697


    The mechanisms underlying the development of osteolysis and aseptic loosening have an impact on the longevity of total hip replacements (THRs). This study examines the specific roles of lymphocytes in the TH1 and TH2 subsets in osteolysis and aseptic loosening of THR. Tissue from periprosthetic regions from patients with loose, cemented acetabular components were used to determine the TH1 and TH2 cytokine profile. Twelve tissue specimens from patients with radiographic signs of osteolysis, and nine tissue specimens from patients with no signs of osteolysis were harvested during revision surgery. Immunohistochemistry using primary antibodies against CD3, interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10 was performed on frozen sections to determine the percentage of positive cells for each of the sections. No statistically significant differences in the percentage of positive cells expressing cytokines characteristic of the TH1 pathway (IFN-gamma, IL-2) or TH2 pathway (IL-4, IL-10) were found when comparing osteolytic and non-osteolytic tissues. However, significant numbers of T cells (averaging about 10% of the total cells) and TH1 and TH2 immune cytokines (averaging 3-5% of cells) implicate a possible role for immune processes at the prosthetic interface.

    View details for PubMedID 12601781

  • The role of the TH1 and TH2 immune responses in loosening and osteolysis of cemented total hip replacements JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Arora, A., Song, Y., Chun, L., Huie, P., Trindade, M., Smith, R. L., Goodman, S. 2003; 64A (4): 693-697
  • Microcontact printing on human tissue for retinal cell transplantation ARCHIVES OF OPHTHALMOLOGY Lee, C. J., Huie, P., Leng, T., Peterman, M. C., Marmor, M. F., Blumenkranz, M. S., Bent, S. F., Fishman, H. A. 2002; 120 (12): 1714-1718


    To demonstrate that microcontact printing, a modern materials fabrication technique, can be used to engineer the surface of human tissue and to show that inhibitory molecules can be used to pattern the growth of retinal pigment epithelial cells or iris pigment epithelial cells on human lens capsule for transplantation.Photolithographic techniques were used to fabricate photoresist-coated silicon substrates into molds. Poly(dimethylsiloxane)stamps for microcontact printing were made from these molds. The poly(dimethylsiloxane) stamps were then used to "wet-transfer" growth inhibitory molecules to the surface of prepared human lens capsules that were obtained during cataract surgery. Human retinal pigment epithelial and rabbit iris pigment epithelial cells were grown on a lens capsule substrate in the presence and absence of a patterned array of inhibitory factors.We found that human lens capsule could be microprinted with a precision similar to that obtained on glass or synthetic polymers. Retinal pigment epithelial cells and iris pigment epithelial cells cultured onto an untreated lens capsule showed spreading and formed into fusiform-appearing cells. In contrast, cells cultured on a lens capsule with a hexagonal micropattern of growth inhibitory molecules retained an epithelioid form within the inhibitory hexagons.Inhibitory growth molecules can be micropatterned onto human lens capsule, and these micropatterns can control the organization of retinal pigment epithelial cells or iris pigment epithelial cells cultured onto the lens capsule surface.Microprinting on autologous human tissue may facilitate efforts to effectively organize cell cultures and transplantations for the replacement of vital ocular tissues such as the retinal pigment epithelium in age-related macular degeneration.

    View details for Web of Science ID 000179737900014

    View details for PubMedID 12470147

  • Intravascular drug delivery with a pulsed liquid microjet ARCHIVES OF OPHTHALMOLOGY Fletcher, D. A., Palanker, D. V., Huie, P., Miller, J., Marmor, M. F., Blumenkranz, M. S. 2002; 120 (9): 1206-1208


    Occlusions of the retinal veins and arteries, associated with diseases such as hypertension and arteriosclerosis, are a major cause of severe and irreversible loss of vision. Treatments for retinal vascular diseases have been unsatisfactory owing in part to the difficulty of delivering drugs to the site of disease within the eye. In this article, we demonstrate that a new device, the vapor bubble-driven pulsed liquid microjet, can deliver drugs into the lumen of small vessels such as those found in the retina. A 15- micro m-diameter liquid jet traveling at more than 60 m/s was shown to penetrate and deliver fluid through the wall of a blood vessel that was 60 micro m in diameter. Perforation of the wall of the blood vessel did not extend beyond the jet diameter.

    View details for Web of Science ID 000178022300013

    View details for PubMedID 12215096

  • Effects of the pulsed electron avalanche knife on retinal tissue ARCHIVES OF OPHTHALMOLOGY Palanker, D. V., Marmor, M. F., Branco, A., Huie, P., MILLER, J. M., Sanislo, S. R., Vankov, A., Blumenkranz, M. S. 2002; 120 (5): 636-640


    To evaluate the precision of retinal tissue dissection by the pulsed electron avalanche knife (PEAK) and to assess possible toxic effects from this device.To demonstrate precision of cutting, bovine retina (in vitro) and rabbit retina (in vivo) were incised with the PEAK. Samples were examined by scanning electron microscopy and histologic examination (light microscopy). To evaluate possible toxic effects in rabbit eyes, 30 000 pulses were delivered into the vitreous 1 cm above the retina. Histologic examinations and electroretinography were performed at intervals up to 1 month after exposure.Cuts in postmortem bovine retina showed extremely sharp edges with no signs of thermal damage. Full-thickness cuts in living attached rabbit retina were similarly sharp and were typically less than 100 microm wide. No signs of retinal toxic effects were detected by histologic examination or electroretinography.The PEAK is capable of precise cutting through retinal tissue, and there are no demonstrable retinal toxic effects from its use. The precision and tractionless nature of PEAK cutting offers advantages over mechanical tools and laser-based instrumentation. We believe this new device will prove useful in a variety of vitreoretinal surgical applications.

    View details for Web of Science ID 000175503400015

    View details for PubMedID 12003616

  • Male infertility, impaired spermatogenesis, and azoospermia in mice deficient for the pseudophosphatase Sbf1 JOURNAL OF CLINICAL INVESTIGATION Firestein, R., Nagy, P. L., Daly, M., Huie, P., Conti, M., Cleary, M. L. 2002; 109 (9): 1165-1172


    Pseudophosphatases display extensive sequence similarities to phosphatases but harbor amino acid alterations in their active-site consensus motifs that render them catalytically inactive. A potential role in substrate trapping or docking has been proposed, but the specific requirements for pseudophosphatases during development and differentiation are unknown. We demonstrate here that Sbf1, a pseudophosphatase of the myotubularin family, is expressed at high levels in seminiferous tubules of the testis, specifically in Sertoli's cells, spermatogonia, and pachytene spermatocytes, but not in postmeiotic round spermatids. Mice that are nullizygous for Sbf1 exhibit male infertility characterized by azoospermia. The onset of the spermatogenic defect occurs in the first wave of spermatogenesis at 17 days after birth during the synchronized progression of pachytene spermatocytes to haploid spermatids. Vacuolation of the Sertoli's cells is the earliest observed phenotype and is followed by reduced formation of spermatids and eventual depletion of the germ cell compartment in older mice. The nullizygous phenotype in conjunction with high-level expression of Sbf1 in premeiotic germ cells and Sertoli's cells is consistent with a crucial role for Sbf1 in transition from diploid to haploid spermatocytes. These studies demonstrate an essential role for a pseudophosphatase and implicate signaling pathways regulated by myotubularin family proteins in spermatogenesis and germ cell differentiation.

    View details for DOI 10.1172/JCI200212589

    View details for Web of Science ID 000175488000007

    View details for PubMedID 11994405

  • A biodegradable matrix facilitates the use of lens capsule as a substrate for subretinal cell transplantation Bilbao, K. V., Leng, T., Fung, A. E., Huie, P., Sanislo, S. R., Marmor, M. F., Blumenkranz, M. S., Fishman, H. A. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2002: U972-U972
  • Directed ganglion cell growth and stimulation with microcontact printing as a prototype visual prosthesis interface Leng, T., Huie, P., Mehenti, N. Z., Peterman, M. C., Lee, C. J., Marmor, M. F., Sanislo, S. R., Beni, S. F., Blumenkranz, M. S., Fishman, H. A. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2002: U1279-U1279
  • The artificial synapse chip: A novel interface for a retinal prosthesis based on neurotransmitter stimulation and nerve regeneration Fishman, H. A., Peterman, M. C., Leng, T., Huie, P., Lee, C. J., Bloom, D. M., Sanislo, S. R., Marmor, M. F., Bent, S. F., Blumenkranz, M. S. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2002: U803-U803
  • Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation BLOOD Zeng, D. F., Hoffmann, P., Lan, F. S., Huie, P., Higgins, J., Strober, S. 2002; 99 (4): 1449-1457


    The "conventional" NK1.1(-) T cells from mouse blood and marrow were compared with regard to surface receptors, cytokine secretion, and function. Most blood NK1.1(-) CD4(+) and CD8(+) T cells expressed the naive CD44(int/lo)CD62L(hi)CD45RB(hi) T-cell phenotype typical of those in the peripheral lymphoid tissues. In contrast, most marrow NK1.1(-) CD4(+) and CD8(+) T cells expressed an unusual CD44(hi)CD62L(hi)CD45RB(hi) phenotype. The blood NK1.1(-) CD4(+) T cells had a naive T-helper cytokine profile and a potent capacity to induce lethal graft versus host (GVH) disease in a C57BL/6 donor to a BALB/c host bone marrow transplantation model. In contrast, the marrow NK1.1(-) CD4(+) T cells had a Th0 cytokine profile and failed to induce lethal GVH disease, even at 20-fold higher numbers than those from the blood. NK1.1(-) CD8(+) T cells from the blood but not the marrow induced lethal GVH disease. Nevertheless, the marrow NK1.1(-) CD8(+) T cells induced potent antitumor activity that was augmented by marrow NK1.1(-) CD4(+) T cells and facilitated hematopoietic progenitor engraftment. The inability of marrow CD4(+) and CD8(+) T cells to induce GVH disease was associated with their inability to expand in the blood and gut of allogeneic recipients. Because neither the purified marrow CD4(+) or CD8(+) T cells induced GVH disease, their unique features are desirable for inclusion in allogeneic bone marrow or hematopoietic progenitor transplants.

    View details for Web of Science ID 000173787600050

    View details for PubMedID 11830499

  • Pulsed liquid microjet for intravascular injection OPHTHALMIC TECHNOLOGIES XII Palanker, D., Fletcher, D., Miller, J., Huie, P., Marmor, M., Blumenkranz, M. 2002; 4611: 72-75
  • Novel interface to biological systems for retinal prosthetics BIOMEMS AND BIONANOTECHNOLOGY Peterman, M. C., Lee, C., Leng, T., Huie, P., Fishman, H. A. 2002; 729: 149-154
  • Pulsed Electron Avalanche Knife (PEAK) for intraocular surgery INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Palanker, D. V., MILLER, J. M., Marmor, M. F., Sanislo, S. R., Huie, P., Blumenkranz, M. S. 2001; 42 (11): 2673-2678


    To develop a better and more economical instrument for precise, tractionless, "cold" cutting during intraocular surgery. The use of highly localized electric fields rather than laser light as the means of tissue dissection was investigated.A high electric field at the tip of a fine wire can, like lasers, initiate plasma formation. Micrometer-length plasma streamers are generated when an insulated 25 micron (microm) wire, exposed to physiological medium at one end, is subjected to nanosecond electrical pulses between 1 and 8 kV in magnitude. The explosive evaporation of water in the vicinity of these streamers cuts soft tissue without heat deposition into surrounding material (cold cutting). Streamers of plasma and the dynamics of water evaporation were imaged using an inverted microscope and fast flash photography. Cutting effectiveness was evaluated on both polyacrylamide gels, on different tissues from excised bovine eyes, and in vivo on rabbit retina. Standard histology techniques were used to examine the tissue.Electric pulses with energies between 150 and 670 microJ produced plasma streamers in saline between 10 and 200 microm in length. Application of electric discharges to dense (10%) polyacrylamide gels resulted in fracturing of the gel without ejection of bulk material. In both dense and softer (6%) gels, layer by layer shaving was possible with pulse energy rather than number of pulses as the determinant of ultimate cutting depth. The instrument made precise partial or full-thickness cuts of retina, iris, lens, and lens capsule without any evidence of thermal damage. Because different tissues require distinct energies for dissection, tissue-selective cutting on complex structures can be performed if the appropriate pulse energies are used; for example, retina can be dissected without damage to the major retinal vessels.This instrument, called the Pulsed Electron Avalanche Knife (PEAK), can quickly and precisely cut intraocular tissues without traction. The small delivery probe and modest cost make it promising for many ophthalmic applications, including retinal, cataract, and glaucoma surgery. In addition, the instrument may be useful in nonophthalmic procedures such as intravascular surgery and neurosurgery.

    View details for Web of Science ID 000171433300037

    View details for PubMedID 11581215

  • Predominance of NK1.1(+)TCR alpha beta(+) or DX5(+)TCR alpha beta(+) T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: "Natural suppressor" cells JOURNAL OF IMMUNOLOGY Lan, F. S., Zeng, D. F., Higuchi, M., Huie, P., Higgins, J. P., Strober, S. 2001; 167 (4): 2087-2096


    We developed a nonmyeloablative host conditioning regimen in a mouse model of MHC-mismatched bone marrow transplantation that not only reduces radiation toxicity, but also protects against graft-vs-host disease. The regimen of fractionated irradiation directed to the lymphoid tissues and depletive anti-T cell Abs results in a marked change in the residual host T cells, such that NK1.1+ or DX5+asialo-GM1+ T cells become the predominant T cell subset in the lymphoid tissues of C57BL/6 and BALB/c mice, respectively. The latter "natural suppressor" T cells protect hosts from graft-vs-host disease after the infusion of allogeneic bone marrow and peripheral blood cells that ordinarily kill hosts conditioned with sublethal or lethal total body irradiation. Protected hosts become stable mixed chimeras, but fail to show the early expansion and infiltration of donor T cells in the gut, liver, and blood associated with host tissue injury. Cytokine secretion and adoptive transfer studies using wild-type and IL-4(-/-) mice showed that protection afforded by NK1.1+ and DX5+asialo-GM1+ T cells derived from either donors or hosts conditioned with lymphoid irradiation is dependent on their secretion of high levels of IL-4.

    View details for Web of Science ID 000170949600034

    View details for PubMedID 11489992

  • Chronic antigen-specific immune-system activation may potentially be involved in the loosening of cemented acetabular components JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Farber, A., Chin, R., Song, Y., Huie, P., Goodman, S. 2001; 55 (3): 433-441


    Previous studies have attempted to determine whether aseptic loosening and osteolysis are caused by a T cell-mediated type IV hypersensitivity reaction or a nonspecific foreign body reaction involving phagocytic macrophages. The purpose of this study was to examine the role of the B7-CD28 costimulatory pathway (which is indicative of an activated immune response) in loosening and osteolysis of total joint replacements (TJRs). We harvested periprosthetic tissues from 24 loose, cemented, all polyethylene, acetabular components in patients undergoing revision total hip replacement surgery for aseptic loosening. Prostheses were classified radiographically as to whether ballooning, scalloping osteolysis was present or not. Monoclonal antibodies were used to identify macrophages, antigen presenting cells (APCs) expressing B7-1 or B7-2, total T lymphocytes, and T cells expressing CD28 or CTLA-4. The large numbers of positive cells, including macrophages, T cells, and APCs in both groups are substantially higher than previously reported. Macrophages constituted the predominant cell type, the majority of which were APCs. B7-1 was expressed by 18.3% of all cells, and B7-2 was expressed by 61.0% of cells. Despite the fact that there were no statistically significant differences in expression of proteins in the B7-CD28 pathway between the osteolytic and nonosteolytic groups, the magnitude of positive staining suggests that the process of aseptic loosening (not osteolysis) may involve proteins of the B7-CD28 pathway, particularly B7-2. One possible antigenic stimulus is protein-coated particulate wear debris from prosthetic materials.

    View details for Web of Science ID 000167677200021

    View details for PubMedID 11255198

  • Allogeneic bone marrow cells that facilitate complete chimerism and eliminate tumor cells express both CD8 and T-cell antigen receptor-alpha beta BLOOD Lan, F. S., Zeng, D. F., Huie, P., Higgins, J. P., Strober, S. 2001; 97 (11): 3458-3465


    Nonmyeloablative host conditioning regimens have been used in clinical allogeneic bone marrow and hematopoietic progenitor transplantation to effectively treat lymphohematopoietic tumors and reduce early toxicity. However, severe graft-versus-host disease (GVHD) remains a major problem. The goal of the current study was to determine whether specific subsets of cells in allogeneic bone marrow transplants can effectively treat the BCL(1) B-cell lymphoma in nonmyeloablated BALB/c mouse hosts given a single dose of sublethal (450 cGy) total body irradiation, without inducing severe GVHD. The experimental results show that high doses of whole bone marrow cells from major histocompatiblity complex (MHC)-mismatched donors eliminate both normal and malignant host-type lymphohematopoietic cells without causing injury to nonlymphohematopoietic host tissues. The CD8(+)T-cell antigen receptor-alphabeta+ (TCRalphabeta+) T cells within the marrow transplants mediated the killing of the tumor cells via both perforin- and FasL-dependent pathways. Cells present in marrow transplants from either CD8-/- or TCRalpha-/- donors failed to eliminate malignant and normal host lymphohematopoietic cells. Addition of small numbers of blood mononuclear cells to the marrow inoculum caused lethal GVHD. Thus, the resident allogeneic bone marrow CD8(+) TCRalphabeta+ T cells had the unique capacity to eliminate the host lymphohematopoietic cells without nonlymphohematopoietic tissue injury. (Blood. 2001;97:3458-3465)

    View details for Web of Science ID 000168927900020

    View details for PubMedID 11369637

  • Evaluation of toxicity in vitreoretinal application of the Pulsed Electron Avalanche Knife (PEAK). Sanislo, S. R., Palanker, D. V., Miller, J., Marmor, M. F., Huie, P., Vankov, A., Branco, A., Blumenkranz, M. S. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2001: S429-S429
  • Hepatocyte growth factor receptor in acute tubular necrosis JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY Rabkin, R., Fervenza, F., Tsao, T., Sibley, R., Friedlaender, M., Hsu, F., Lassman, C., Hausmann, M., Huie, P., Schwall, R. H. 2001; 12 (3): 531-540


    In acute tubular necrosis, there are early transient increases in circulating and local bioactive hepatocyte growth factor (HGF) levels and renal HGF receptor (c-MET) gene expression. It has therefore been suggested that endogenous HGF may play a role in initiating renal repair. To test this hypothesis, changes in the levels, activity, and anatomic distribution of c-MET protein were characterized in relation to the onset and localization of DNA synthesis in kidneys of rats with ischemia-induced acute tubular necrosis. Whole-kidney c-MET protein levels were significantly increased in the injured kidneys 12 h after injury and rose to a maximum after 1 d, exceeding the control values by sevenfold. Eight days after injury, c-MET levels, although decreasing, were still elevated above control values. An increase in the levels of activated c-MET, i.e., tyrosine-phosphorylated c-MET, was also evident as early as 12 h after injury. Histologic analyses demonstrated that the increase in c-MET immunoreactivity was most marked in the most severely damaged nephron segments in the outer medulla. In injured proximal tubules, the receptor was redistributed from an apical location to an intracellular location. DNA synthesis was increased in the injured kidneys, especially in the outer medulla, where the increase in c-MET protein levels was most prominent. The increase in DNA synthesis was first detected 12 h after the initial increase in activated c-MET levels. It is concluded that the early increases in the levels of c-MET protein and activated receptor support the hypothesis that HGF participates in the initiation of renal regeneration. In addition, the persistent elevation of c-Met protein levels suggests that prolonged and even late treatment with HGF may be of therapeutic value

    View details for Web of Science ID 000167221400014

    View details for PubMedID 11181801

  • Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation HUMAN IMMUNOLOGY Sarwal, M. M., Jani, A., Chang, S., Huie, P., Wang, Z., Salvatierra, O., CLAYBERGER, C., Sibley, R., KRENSKY, A. M., Pavlakis, M. 2001; 62 (1): 21-31


    Differentiating etiologies of transplant dysfunction without biopsy and optimizing therapy for acute rejection by predicting steroid resistance will reduce patient morbidity. Granulysin is a cytolytic molecule released by CTL and NK cells and coexpressed with effectors of acute allograft rejection, like perforin and granzymes. Granulysin mRNA and protein expression were studied in peripheral blood lymphocytes (PBL; n = 61 total, n = 10 with intercurrent infections) and biopsy tissue from adult and children renal transplant recipients (n = 97) by competitive quantitative-reverse transcriptase-PCR (QC-RT-PCR) and immunohistochemistry. Differences in cell phenotypes were studied in steroid sensitive and resistant acute rejection biopsies. Granulysin was studied in phytohemagglutinin (PHA) stimulated cell lines (donor PBL and CD45RO(+) T cells) by FACS, Western blotting, and RT-PCR after pretreating with cyclosporine A (CSA), azathioprine, mycophenolic acid, and steroids. Granulysin mRNA was significantly increased in patient PBL and transplant biopsies during acute rejection (p < 0.0001) and infection (p < 0.001). Rejecting biopsies alone (n = 53) had mononuclear cell granulysin staining. Steroid resistant biopsies (n = 25) had denser granulysin staining (>2 cells/high power field) and CD45RO(+) lymphocytes, when compared with steroid sensitive (n = 28) rejecting tissue. Granulysin levels were unchanged after azathioprine and mycophenolic acid treatment, decreased after treating activated PBL with steroids and cyclosporine A (CSA), and paradoxically, increased (p < 0.05) after treating CD45RO(+) CTL with CSA. Elevated PBL granulysin is a peripheral marker for acute rejection and infection and dense granulysin staining a tissue marker for steroid resistance. Memory CTL abound in steroid resistant grafts and may have a markedly different response to CSA immunotherapy, suggesting a possible mechanism for steroid resistance.

    View details for Web of Science ID 000167037400003

    View details for PubMedID 11165712

  • Expression of insulin-like growth factor-I and transforming growth factor-beta in hypokalemic nephropathy in the rat KIDNEY INTERNATIONAL Tsao, T., Fawcett, J., Fervenza, F. C., Hsu, F. W., Huie, P., Sibley, R. K., Rabkin, R. 2001; 59 (1): 96-105


    Potassium deficiency (KD) in the rat retards body growth but stimulates renal enlargement caused by cellular hypertrophy and hyperplasia, which is most marked in the outer medulla. If hypokalemia persists, interstitial infiltrates appear and eventually fibrosis. Since early in KD insulin-like growth factor-I (IGF-I) levels in the kidney are elevated, suggesting that it may be an early mediator of the exaggerated renal growth, and as transforming growth factor-beta (TGF-beta) promotes cellular hypertrophy and fibrosis, we examined the renal expression of these growth factors in prolonged KD.Rats were given a K-deficient diet or were pair fed or ad libitum fed a K-replete diet for 21 days. Growth factor mRNA levels were measured in whole kidney and protein expression localized by immunohistochemistry.KD rats weighed less than pair-fed controls, while the kidneys were 49% larger. Their serum IGF-I and kidney IGF-I protein levels were depressed, as were their IGF-I mRNA levels in liver, kidney, and muscle. These changes can largely be attributed to decreased food intake. In contrast, kidney IGF binding protein-1 (IGFBP-1) mRNA and TGF-beta mRNA levels were increased significantly. Histology of outer medulla revealed marked hypertrophy and adenomatous hyperplasia of the collecting ducts and hypertrophy of the thick ascending limbs of Henle with cellular infiltrates in the interstitium. Both nephron segments immunostained strongly for IGF-I and IGFBP-1, but only the nonhyperplastic enlarged thick ascending Henle limb cells immunostained for TGF-beta, which was strongly positive. Prominent interstitial infiltrates with ED1 immunostained monocytes/macrophages were present.These findings are consistent with a sustained role for IGF-I in promoting the exaggerated renal growth of KD and appear to be mediated through local trapping of IGF-I by the overexpressed IGFBP-1, which together with IGF-I can promote renal growth. The selective localization of TGF-beta to hypertrophied nonhyperplastic nephron segments containing IGF-I raises the possibility that TGF-beta may be serving to convert the mitogenic action of IGF-I into a hypertrophic response in these segments. It is also conceivable that TGF-beta may be a cause of the tubulointerstitial infiltrate. Finally, the low circulating IGF-I levels likely contribute to the impaired body growth.

    View details for Web of Science ID 000166294700012

    View details for PubMedID 11135062

  • On contrast parameters and topographic artifacts in near-field infrared microscopy JOURNAL OF APPLIED PHYSICS Palanker, D. V., Simanovskii, D. M., Huie, P., Smith, T. I. 2000; 88 (11): 6808-6814
  • Reduction of aortic wall motion inhibits hypertension-mediated experimental atherosclerosis ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Tropea, B. I., Schwarzacher, S. P., Chang, A., Asvar, C., Huie, P., Sibley, R. K., Zarins, C. K. 2000; 20 (9): 2127-2133


    Hypertension is a well-known risk factor for coronary artery disease and carotid and lower extremity occlusive disease. Surgically induced hypertension in hypercholesterolemic animals results in increased aortic wall motion and increased plaque formation. We tested the hypothesis that reduction in aortic wall motion, despite continued hypertension, could reduce plaque formation. New Zealand White rabbits (n=26) underwent thoracic aortic banding to induce hypertension and were fed an atherogenic diet for 3 weeks. In 13 rabbits, a segment of aorta proximal to an aortic band was externally wrapped to reduce wall motion. All animals were fed an atherogenic diet for 3 weeks. Four groups were studied: 1, coarctation control (no wrap, n=7); 2, coarctation with loose wrap (n=6); 3, coarctation with firm wrap (n=7); and 4, control (noncoarcted, n=6). Wall motion, blood pressure, and pulse pressure were measured at standard reference sites proximal and distal to the coarctation by use of intravascular ultrasound. Quantitative morphometry was used to measure intimal plaque. Mean arterial pressure and cyclic aortic wall motion were equally increased proximal to the aortic coarctation in all 3 coarcted rabbit groups compared with the control group (P:<0.001). Wall motion in the segment of aorta under the loose and firm wraps was no different from the control value. The external wrap significantly reduced intimal thickening in the 4 groups by the following amounts: group 1, 0.30+/-0.03 mm(2); group 2, 0.06+/-0.02 mm(2); group 3, 0. 04+/-0.02 mm(2); and group 4, 0.01+/-0.01 mm(2) (P:<0.001). Localized inhibition of aortic wall motion in the lesion-prone hypertensive aorta resulted in significant reduction in intimal plaque formation. These data suggest that arterial wall cyclic motion may stimulate cellular proliferation and lipid uptake in experimental atherosclerosis.

    View details for Web of Science ID 000089333600019

    View details for PubMedID 10978259

  • Set domain-dependent regulation of transcriptional silencing and growth control by SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9 MOLECULAR AND CELLULAR BIOLOGY Firestein, R., Cui, X. M., Huie, P., Cleary, M. L. 2000; 20 (13): 4900-4909


    Mammalian SET domain-containing proteins define a distinctive class of chromatin-associated factors that are targets for growth control signals and oncogenic activation. SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9, contains both SET and chromo domains, signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. In this report we demonstrate that SUV39H1 represses transcription in a transient transcriptional assay when tethered to DNA through the GAL4 DNA binding domain. Under these conditions, SUV39H1 displays features of a long-range repressor capable of acting over several kilobases to silence basal promoters. A possible role in chromatin-mediated gene silencing is supported by the localization of exogenously expressed SUV39H1 to nuclear bodies with morphologic features suggestive of heterochromatin in interphase cells. In addition, we show that SUV39H1 is phosphorylated specifically at the G(1)/S cell cycle transition and when forcibly expressed suppresses cell growth. Growth suppression as well as the ability of SUV39H1 to form nuclear bodies and silence transcription are antagonized by the oncogenic antiphosphatase Sbf1 that when hyperexpressed interacts with the SET domain and stabilizes the phosphorylated form of SUV39H1. These studies suggest a phosphorylation-dependent mechanism for regulating the chromatin organizing activity of a mammalian su(var) protein and implicate the SET domain as a gatekeeper motif that integrates upstream signaling pathways to epigenetic regulation and growth control.

    View details for Web of Science ID 000087576400041

    View details for PubMedID 10848615

  • Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation JOURNAL OF IMMUNOLOGY Ling, X. F., Kamangar, S., Boytim, M. L., Kelman, Z., Huie, P., Lyu, S. C., Sibley, R. K., Hurwitz, J., CLAYBERGER, C., KRENSKY, A. M. 2000; 164 (12): 6188-6192


    Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents. A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein. A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry. Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo. Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro. These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.

    View details for Web of Science ID 000087508500014

    View details for PubMedID 10843669

  • On image formation in near-field infrared microscopy SCANNING AND FORCE MICROSCOPIES FOR BIOMEDICAL APPLICATIONS II Simanovskii, D. M., Palanker, D. V., Huie, P., Smith, T. I. 2000; 1 (16): 182-188
  • Breaking the femtogram barrier using scanning near-field infrared microscopy (SNIM) BIOMEDICAL SPECTROSCOPY: VIBRATIONAL SPECTROSCOPY AND OTHER NOVEL TECHNIQUES Erramilli, S., Hong, M. K., Huie, P. 2000; 1 (12): 197-201
  • The characterization of macrophages and osteoclasts in tissues harvested from revised total hip prostheses JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Chun, L., Yoon, J., Song, Y., Huie, P., Regula, D., Goodman, S. 1999; 48 (6): 899-903


    The differentiation and maturation of macrophages and osteoclasts at the prosthetic interface in cases of implant loosening are poorly understood. Using histochemical and immunohistochemical staining methods, we compare macrophage differentiation in tissues from revised hip replacements in patients with specific clinical-radiological appearances. Periprosthetic tissues were harvested from 12 cemented acetabular and 12 cemented femoral components in 24 patients undergoing revision hip replacement. The prostheses were all radiographically and clinically loose. Six acetabular and six femoral components demonstrated radiographic ballooning osteolysis. Serial 6 microm frozen sections of the periprosthetic tissues were processed with hematoxylin and eosin for general tissue morphology, and analyzed for the presence of tartrate resistant acid phosphatase (TRAP, an osteoclast marker). Immunoperoxidase staining using monoclonal antibodies to CD68 (macrophages and osteoclasts) and CD51 (the alpha chain of the vitronectin receptor, an osteoclast marker) was also performed. Approximately 8-30% of the total cells in the tissues were positive for TRAP and the vitronectin receptor, and comprised a subset of the CD68 positive macrophages and macrophage polykaryons. However, there were no statistically significant differences between specific groups (femoral vs. acetabular, osteolysis vs. no osteolysis) for the numbers or percentages of macrophages or osteoclast-like cells. Once prosthetic loosening has occurred, few differences in the macrophage-osteoclast profile of tissues from different periprosthetic locations, with and without osteolysis, are noted. This suggests a final common biologic pathway for periprosthetic bone resorption, once implant loosening has transpired.

    View details for Web of Science ID 000083772200020

    View details for PubMedID 10556857

  • Comparison of chimeric and non-chimeric tolerance using posttransplant total lymphoid irradiation - Cytokine expression and chronic rejection TRANSPLANTATION Hayamizu, K., Lan, F. S., Huie, P., Sibley, R. K., Strober, S. 1999; 68 (7): 1036-1044


    Previous studies showed that an intravenous infusion of donor blood cells facilitates tolerance to ACI heart allografts in Lewis rat hosts given posttransplant total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG). The object of the current study was to compare tolerance induction using donor cells that do or do not induce chimerism.Normal peripheral blood mononuclear cells (PBMC), granulocyte colony-stimulating factor (G-CSF)-mobilized PBMC, and bone marrow (BM) cells from ACI donors were tested for their capacity to prolong ACI heart allograft survival in Lewis hosts. Chimerism, anti-donor cell reactivity, and cytokine gene expression in grafts were determined.Intravenous injections of equal numbers of all three donor cells markedly prolonged graft survival (median: >164 to >175 days) as compared to uninjected controls (median: 53 days). Chimerism among T and B cells in the blood was determined by immunofluorescent staining in hosts bearing long-term (> 150 days) grafts. Although no chimerism was detected in hosts given normal or G-CSF-mobilized PBMC, chimerism was detected at variable levels in all hosts given BM cells. Vigorous anti-donor reactivity in the mixed leukocyte reaction was present only in non-chimeric hosts. Long-term grafts from hosts given normal ACI PBMC developed chronic rejection, but those from hosts given ACI BM cells did not. The latter hosts showed the lowest levels of intragraft cytokine mRNA.Chimeric tolerance is more robust than non-chimeric tolerance in the model of posttransplant TLI, ATG, and donor cell infusion, and is associated with less chronic rejection.

    View details for Web of Science ID 000083163800023

    View details for PubMedID 10532547

  • Components of an SCE ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle GENES & DEVELOPMENT Freed, E., Lacey, K. R., Huie, P., Lyapina, S. A., Deshaies, R. J., Stearns, T., Jackson, P. K. 1999; 13 (17): 2242-2257


    Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.

    View details for Web of Science ID 000082647200006

    View details for PubMedID 10485847

  • Monocyte-derived dendritic cell precursors facilitate tolerance to heart allografts after total lymphoid irradiation TRANSPLANTATION Hayamizu, K., Huie, P., Sibley, R. K., Strober, S. 1998; 66 (10): 1285-1291


    Previous studies have shown that posttransplant total lymphoid irradiation, anti-thymocyte globulin, and an intravenous donor blood cell infusion induce tolerance to ACI heart allografts in Lewis rat hosts.In the current study, fresh ACI monocytes and dendritic cell precursors, derived from short-term culture of the latter cells in granulocyte macrophage colony-stimulating factor, were tested for their capacity to prolong heart allograft survival in this model.The experimental results show that significant prolongation of graft survival was achieved after injection of the fresh donor monocytes or 2-day or 6-day cultured cells. The 2-day cultured cells were most effective, and more than 60% of hosts maintained graft survival for more than 160 days. Ten-day cultured cells and fresh splenic dendritic cells failed to prolong graft survival. Studies of cell surface markers showed that the 2-day cultured cells had up-regulated class II major histocompatibility complex and CD80, but not CD86 molecules. On the other hand, the 10-day cultured cells and splenic dendritic cells showed intense expression of all three markers. The latter cells stimulated vigorous proliferative and cell-mediated lympholysis responses in the mixed leukocyte reaction, but the fresh and 2-day cultured cells were weak stimulators.The intravenous injection of donor dendritic cell precursors derived from blood monocytes facilitates long-term acceptance of heart allografts.

    View details for Web of Science ID 000077336800004

    View details for PubMedID 9846510

  • Nature of glomerular dysfunction in pre-eclampsia KIDNEY INTERNATIONAL Lafayette, R. A., Druzin, M., Sibley, R., Derby, G., Malik, T., Huie, P., Polhemus, C., Deen, W. M., Myers, B. D. 1998; 54 (4): 1240-1249


    Pre-eclampsia is characterized by hypertension, proteinuria and edema. Simultaneous studies of kidney function and structure have not been reported. We wished to explore the degree and nature of glomerular dysfunction in pre-eclampsia.Physiologic techniques were used to estimate glomerular filtration rate (GFR), renal plasma flow and afferent oncotic pressure immediately after delivery in consecutive patients with pre-eclampsia (PET; N = 13). Healthy mothers completing an uncomplicated pregnancy served as functional controls (N = 12). A morphometric analysis of glomeruli obtained by biopsy and mathematical modeling were used to estimate the glomerular ultrafiltration coefficient (Kf). Glomeruli from healthy female kidney transplant donors served as structural controls (N = 8).The GFR in PET was depressed below the control level, 91 +/- 23 versus 149 +/- 34 ml/min/1.73 m2, respectively (P < 0.0001). In contrast, renal plasma flow and oncotic pressure were similar in the two groups (P = NS). A reduction in the density and size of endothelial fenestrae and subendothelial accumulation of fibrinoid deposits lowered glomerular hydraulic permeability in PET compared to controls, 1.81 versus 2.58 x 10(-9) m/sec/PA. Mesangial cell interposition also curtailed effective filtration surface area. Together, these changes lowered the computed single nephron Kf in PET below control, 4.26 versus 6.78 nl/min x mm Hg, respectively.The proportionate (approximately 40%) depression of Kf for single nephrons and GFR suggests that hypofiltration in PET does not have a hemodynamic basis, but is a consequence of structural changes that lead to impairment of intrinsic glomerular ultrafiltration capacity.

    View details for Web of Science ID 000076096900022

    View details for PubMedID 9767540

  • Donor blood monocytes but not T or B cells facilitate long-term allograft survival after total lymphoid irradiation TRANSPLANTATION Hayamizu, K., Zeng, D., Huie, P., Garcia-Ojeda, M. E., Bloch, D. A., Fong, L., Engleman, E. G., Sibley, R. K., Strober, S. 1998; 66 (5): 585-593


    Previous studies showed that a combination of posttransplant total lymphoid irradiation (TLI), rabbit antithymocyte globulin (ATG), and a single donor blood transfusion induced tolerance to ACI heart allografts in Lewis rats. All three modalities were required to achieve tolerance. The objective of the current study was to determine the subset(s) of cells in the donor blood that facilitated long-term allograft survival.Lewis hosts received TLI, ATG, and donor cell infusion after heart transplantation. Graft survival, mixed leukocyte reaction (MLR), and intragraft cytokine mRNA were studied.The intravenous injection of 25 x 10(6) ACI peripheral blood mononuclear cells (PBMC) significantly prolonged graft survival as compared with that of Lewis hosts given TLI and ATG alone. Injection of highly enriched blood T cells or splenic B cells adjusted for the number contained in 25 x 10(6) PBMC failed to induce significant graft prolongation. Unexpectedly, depletion of monocytes (CD11b+ cells) from PBMC resulted in the loss of graft prolongation activity. Enriched populations of monocytes obtained by plastic adherence were more efficient in prolonging graft survival than PBMC on a per cell basis. Hosts with long-term grafts (>100-day survival) showed evidence of immune deviation, because the MLR to ACI stimulator cells was vigorous, but secretion of interferon-gamma in the MLR was markedly reduced. In situ hybridization studies of long-term grafts showed markedly reduced levels of interferon-gamma mRNA as compared with rejecting grafts.Infusion of donor monocytes facilitated graft prolongation via immune deviation.

    View details for Web of Science ID 000075996500006

    View details for PubMedID 9753336

  • Imaging single living cells with a scanning near-field infrared microscope based on a free electron laser NUCLEAR INSTRUMENTS & METHODS IN PHYSICS RESEARCH SECTION B-BEAM INTERACTIONS WITH MATERIALS AND ATOMS Hong, M. K., Jeung, A. G., Dokholyan, N. V., Smith, T. I., Schwettman, H. A., Huie, P., Erramilli, S. 1998; 144 (1-4): 246-255
  • Inducible nitric oxide synthase messenger RNA levels in hip periprosthetic tissue: A preliminary study JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Pearson, M. L., Goodman, S. B., Huie, P., Sibley, R. K. 1998; 40 (3): 419-424


    Nitric oxide (NO) is a ubiquitous molecule that has been associated with inflammation, arthritis, autoimmune disease, bone resorption, and other biological processes. Elucidating the role of NO at the bone-implant interface may further our understanding of the biological processes of osseointegration, loosening, and osteolysis. This study demonstrates the use of a molecular biological technique to investigate the possible role of NO in prosthetic loosening and periprosthetic bone resorption following total hip arthroplasty. Periprosthetic tissue from 12 patients undergoing revision hip arthroplasty was harvested and total ribonucleic acid (RNA) was extracted. In six of the 12 patients, multiple samples from different anatomic locations along the same interface were studied. To estimate the amount of NO present in the tissues in vivo, the level of inducible NO synthase (iNOS) messenger RNA (mRNA) was determined using a ribonuclease (RNase) protection assay. Inducible NOS mRNA was detected in every tissue sample: there was no correlation between iNOS mRNA levels and clinical loosening or osteolysis. Analysis of multiple tissue samples from the same prosthetic component revealed that the levels of iNOS mRNA vary greatly, confirming the heterogeneous nature of the interface.

    View details for Web of Science ID 000073207100012

    View details for PubMedID 9570074

  • Backleak, light junctions, and cell-cell adhesion in postischemic injury to the renal allograft JOURNAL OF CLINICAL INVESTIGATION Kwon, O., NELSON, W. J., Sibley, R., Huie, P., Scandling, J. D., Dafoe, D., Alfrey, E., Myers, B. D. 1998; 101 (10): 2054-2064


    Postischemic injury in recipients of 3-7-d-old renal allografts was classified into sustained (n = 19) or recovering (n = 20) acute renal failure (ARF) according to the prevailing inulin clearance. Recipients of optimally functioning, long-standing allografts and living donors undergoing nephrectomy served as functional (n = 14) and structural controls (n = 10), respectively. Marked elevation above control of fractional clearance of dextrans of graded size was consistent with transtubular backleak of 57% of filtrate (inulin) in sustained ARF. No backleak was detected in recovering ARF. To explore a structural basis for backleak, allograft biopsies were taken intraoperatively, 1 h after reperfusion in all recipients, and again on day 7 after transplant in a subset (n = 10). Electron microscopy revealed disruption of both apical and basolateral membranes of proximal tubule cells in both sustained and recovering ARF, but cell exfoliation and tubule basement membrane denudation were negligible. Histochemical analysis of membrane-associated adhesion complexes confirmed an abnormality of proximal but not distal tubule cells, marked in sustained ARF but not in recovering ARF. Staining for the zonula occludens complex (ZO-1) and adherens complex (alpha, beta, and gamma catenins) revealed diminished intensity and redistribution of each cytoskeletal protein from the apico-lateral membrane boundary. We conclude that impaired integrity of tight junctions and cell-cell adhesion in the proximal tubule provides a paracellular pathway through which filtrate leaks back in sustained allograft ARF.

    View details for Web of Science ID 000073808800004

    View details for PubMedID 9593761

  • Cellular profile and cytokine production at prosthetic interfaces - Study of tissues retrieved from revised hip and knee replacements JOURNAL OF BONE AND JOINT SURGERY-BRITISH VOLUME Goodman, S. B., Huie, P., Song, Y., Schurman, D., Maloney, W., Woolson, S., Sibley, R. 1998; 80B (3): 531-539


    The tissues surrounding 65 cemented and 36 cementless total joint replacements undergoing revision were characterised for cell types by immunohistochemistry and for cytokine expression by in situ hybridisation. We identified three distinct groups of revised implants: loose implants with ballooning radiological osteolysis, loose implants without osteolysis, and well-fixed implants. In the cemented series, osteolysis was associated with increased numbers of macrophages (p = 0.0006), T-lymphocyte subgroups (p = 0.03) and IL-1 (p = 0.02) and IL-6 (p = 0.0001) expression, and in the cementless series with increased numbers of T-lymphocyte subgroups (p = 0.005) and increased TNF alpha expression (p = 0.04). For cemented implants, the histological, histochemical and cytokine profiles of the interface correlated with the clinical and radiological grade of loosening and osteolysis. Our findings suggest that there are different biological mechanisms of loosening and osteolysis for cemented and cementless implants. T-lymphocyte modulation of macrophage function may be an important interaction at prosthetic interfaces.

    View details for Web of Science ID 000073647500031

  • Subsets of transgenic T cells that recognize CD1 induce or prevent murine lupus: Role of cytokines JOURNAL OF EXPERIMENTAL MEDICINE Zeng, D. F., Dick, M., Cheng, L. R., Amano, M., Dejbakhsh-Jones, S., Huie, P., Sibley, R., Strober, S. 1998; 187 (4): 525-536


    T cells with T cell receptor (TCR) transgenes that recognized CD1 on syngeneic B cells stimulated B cells to secrete immunoglobulins in vitro. The CD4+, CD8+, or CD4-CD8- T cells from the spleen of the TCR transgenic BALB/c donors induced lupus with anti-double stranded DNA antibodies, proteinuria, and immune complex glomerulonephritis in irradiated BALB/c nude mice reconstituted with nude bone marrow. Injection of purified CD4-CD8- T cells from the marrow of transgenic donors prevented the induction of lupus by the transgenic T cells. Transgenic T cells that induced lupus secreted large amounts of interferon (IFN)-gamma and little interleukin (IL)-4, and those that prevented lupus secreted large amounts of IL-4 and little IFN-gamma or IL-10.

    View details for Web of Science ID 000072160200009

    View details for PubMedID 9463403

  • Loosening and osteolysis of cemented joint arthroplasties - A biologic spectrum CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Goodman, S. B., Huie, P., Song, Y., Lee, K., Doshi, A., Rushdieh, B., Woolson, S., Maloney, W., Schurman, D., Sibley, R. 1997: 149-163


    The purpose of this study was to characterize the cell types (using immunohistochemistry) and cytokine expression (using in situ hybridization) of tissues surrounding well fixed and loose cemented prostheses undergoing revision. Clinical and radiographic data were gathered prospectively for a series of cemented total joint replacements undergoing revision. Three groups were identified: (1) loose implants with osteolysis (10 specimens), (2) loose implants without osteolysis (11 specimens), and (3) well fixed implants (7 specimens). At surgery, a specimen was harvested from the bone cement interface. Immunohistochemical staining was performed using monoclonal antibodies to identify macrophages and lymphocyte subgroups. Human antisense probes were selected to identify the mRNA for specific cytokines using in situ hybridization. The percentage of positively staining cells was determined for each antibody or probe using a grid counting technique. Tissues from loose cemented prostheses with osteolysis contained significantly greater numbers of macrophages and T lymphocytes compared with tissues from loose and well fixed cemented prostheses without osteolysis. The number of interleukin-1 and interleukin-6 positive cells was highest in specimens with osteolysis and lowest in specimens from well fixed prostheses. These cytokines modulate the growth and differentiation of cells in the immune system and the monocyte and macrophage system and mediate the remodeling of bone and mesenchymal tissues. Specific cell populations and cytokine profiles appear to be involved in periprosthetic osteolysis; this information may be useful in planning strategies for prevention and treatment.

    View details for Web of Science ID A1997WT70700017

    View details for PubMedID 9137186

  • Osteoarthritis: Differential expression of matrix metalloproteinase-9 mRNA in nonfibrillated and fibrillated cartilage Tsuchiya, K., Maloney, W. J., Vu, T., Hoffman, A. R., Huie, P., Sibley, R., Schurman, D. J., Smith, R. L. WILEY-BLACKWELL. 1997: 94-100


    Expression of matrix metalloproteinase-9 mRNA in osteoarthritic and normal cartilage was analyzed using reverse transcription-polymerase chain reaction and in situ hybridization. Fifty-four osteoarthritic cartilage samples were obtained from 24 patients undergoing total knee arthroplasty. Sixteen normal cartilage samples were obtained from non-osteoarthritic knees of four autopsy cases. With normal cartilage, reverse transcription-polymerase chain reaction analysis for matrix metalloproteinase-9 mRNA showed that chondrocytes exhibited only a trace signal. In analysis of osteoarthritic cartilage, chondrocytes of moderately and severely fibrillated cartilage exhibited a 73-fold and 110-fold increase in matrix metalloproteinase-9 mRNA signal, respectively, relative to normal cartilage. Chondrocytes of nonfibrillated osteoarthritic cartilage exhibited a 6-fold increase (p < 0.02) in matrix metalloproteinase-9 mRNA signal relative to normal cartilage. Analysis of matrix metalloproteinase-9 mRNA expression in fresh-frozen sections of normal and osteoarthritic cartilage by in situ hybridization confirmed these results. This study showed that reverse transcription-polymerase chain reaction provides a sensitive index of mRNA levels in normal and osteoarthritic cartilage samples and suggests that increased expression of matrix metalloproteinase-9 precedes fibrillation of cartilage in the development of osteoarthritis.

    View details for Web of Science ID A1997WL95000013

    View details for PubMedID 9066532

  • Analysis of biological tissues using scanning near-field infrared microspectroscopy ACCELERATOR-BASED INFRARED SOURCES AND APPLICATIONS Jeung, A., Erramilli, S., Hong, M. K., Huie, P., Smith, T. 1997; 3153: 117-124
  • The fibrous tissue interface surrounding well-fixed, revised, cementless acetabular components for hip replacement Goodman, S. B., Huie, P., Song, Y., OConnor, M., Woolson, S. T., Maloney, W. J., Schurman, D. J., Sibley, R. AMERICAN SOCIETY TESTING AND MATERIALS. 1997: 21-32
  • RANTES chemokine expression in transplant-associated accelerated atherosclerosis JOURNAL OF HEART AND LUNG TRANSPLANTATION Pattison, J. M., Nelson, P. J., Huie, P., Sibley, R. K., KRENSKY, A. M. 1996; 15 (12): 1194-1199


    The pathogenesis of transplantation-associated accelerated atherosclerosis is poorly understood, but it is likely to be an alloimmune response involving infiltration of the vessel wall by T lymphocytes and monocytes leading to smooth muscle cell proliferation and extracellular matrix deposition. RANTES is a chemokine that selectively chemoattracts T lymphocytes, NK cells, monocytes, and eosinophils. The expression of RANTES in accelerated atherosclerosis was investigated by in situ hybridization and immunohistochemistry.Coronary arteries from six patients undergoing accelerated atherosclerosis were obtained at the time of retransplantation. Normal coronary arteries from two patients with idiopathic dilated cardiomyopathy were used as controls. Messenger RNA for RANTES was localized with digoxigenin-labeled complementary DNA probes. RANTES protein was detected by use of a monoclonal antibody and a three-step horseradish peroxidase method.RANTES mRNA and protein were detected in the lymphocytes, macrophages, myofibroblasts, and endothelial cells of arteries undergoing accelerated atherosclerosis but not in normal coronary arteries.In view of its in vitro biologic activity and in vivo expression pattern, RANTES may be a pivotal mediator of the cellular infiltrate seen in graft atherosclerosis. This information may help in the design of novel diagnostic and therapeutic approaches to this increasingly important disease process.

    View details for Web of Science ID A1996WA94600004

    View details for PubMedID 8981204

  • Coexistence of TH1- and TH2-type cytokine profiles in anti-CD2 monoclonal antibody-induced tolerance TRANSPLANTATION Krieger, N. R., Most, D., Bromberg, J. S., Holm, B., Huie, P., Sibley, R. K., Dafoe, D. C., Alfrey, E. J. 1996; 62 (9): 1285-1292


    Anti-CD2 monoclonal antibody OX34 has been shown to suppress immunity in rodents in vitro and in vivo. To evaluate the effects of OX34 on vascularized allografts, Lewis (RT1(1)) hearts were transplanted heterotopically into Wistar Furth (RT1(u)) rats. A single 5 mg/kg intraperitoneal dose of OX34 administered at transplantation induced indefinite graft survival (mean survival time >140.3+/-12.3 vs. 12.7+/-0.7 control, P=0.001). The mixed lymphocyte response was partially inhibited at 60 days after transplant, returning to normal at 100 days. Donor-specific tolerance was confirmed by acceptance of second donor (>100 days, n=2) and rejection of third-party (mean survival time: 7.5+/-0.5 days, n=2) hearts. Immunohistochemical staining of allograft tissue from tolerant animals demonstrated abundant CD2+, CD4+, and CD8+ graft-infiltrating cells. To elucidate further the nature of these cells, we compared the expression of interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-gamma mRNA in allografted tissue from tolerant, acutely rejecting (AR), isografted, and naive animals using nonisotopic in situ hybridization. A significant increase in IL-2, IL-4, IL-10, and IFN-gamma mRNA was observed in graft-infiltrating cells of both tolerant and AR animals. IL-10 mRNA expression 4 days after transplant was significantly elevated in the OX34-treated compared to AR recipients. These data demonstrate that a single dose of OX34 at engraftment induces tolerance to vascularized allografts. Expression of both T helper 1 and T helper 2 cytokine mRNA profiles (IL-2/IFN-gamma and IL-4/ IL-10, respectively) are up-regulated locally in graft-infiltrating cells of AR and tolerant animal allografts.

    View details for Web of Science ID A1996VU11700019

    View details for PubMedID 8932273

  • Mechanisms of tolerance to rat heart allografts using posttransplant TLI - Changes in cytokine expression TRANSPLANTATION Zeng, D. F., Ready, A., Hute, P., Hayamizu, K., Holm, B., Yin, D. P., Sibley, R. K., Strober, S. 1996; 62 (4): 510-517


    Lewis rats were rendered tolerant to ACI heart allografts using a regimen of posttransplant total lymphoid irradiation (TLI), rabbit antithymocyte or antilymphocyte globulin (RATG or RALG), and a single donor blood transfusion. All three treatment modalities were required to induce tolerance. The mechanism of the maintenance of tolerance was investigated by comparing the secretion of cytokines in the MLR, and the expression of cytokine mRNA in the allografts of tolerant and nontolerant Lewis rats. Although, the 3H-thymidine incorporation and secretion of IL-2 was frequently comparable in the MLR from tolerant and nontolerant rats, the secretion of IFN-gamma was markedly reduced in the tolerant rats. This was reflected in a markedly reduced frequency of cells expressing IFN-gamma mRNA in the allografts of tolerant as compared with nontolerant hosts. The frequency of cells expressing IL-2 and IL-10 mRNA was also reduced, but no significant difference was observed for cells with IL-4 mRNA. Spleen cells from nontolerant rats rapidly rejected ACI allografts in irradiated adoptive hosts, but spleen cells from tolerant rats did not. Evaluation of the cytokine mRNA expression at early and late time points in the allografts of adoptive hosts showed a pattern similar to that of the primary hosts. Thus, the tolerant state was associated with a maintenance or elevation of IL-4 expression and a marked reduction of IFN-gamma expression. Previous reports have shown that TLI alone induced this shift in the early recovery phase after irradiation.

    View details for Web of Science ID A1996VE75100014

    View details for PubMedID 8781618

  • Heterogeneity in cellular and cytokine profiles from multiple samples of tissue surrounding revised hip prostheses JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Goodman, S. B., Knoblich, G., OConnor, M., Song, Y., Huie, P., Sibley, R. 1996; 31 (3): 421-428


    Previous studies have attempted to define the biologic properties of the bone-implant interface using a single specimen harvested from the periprosthetic tissues. The purpose of this study was to examine the heterogeneity in cellular and cytokine profiles of multiple samples taken from the tissues surrounding revised hip prostheses. Clinical and radiographic data for nine patients undergoing surgical revision was gathered prospectively. Three tissue samples were taken systematically from the acetabular and/or femoral bed. Morphologic characteristics of the tissues were assessed using hematoxylin and eosin-stained sections. Immunohistochemical staining was performed using monoclonal antibodies to identify macrophages (EMB11 and CD68); activated macrophages (Leu M3); total T lymphocytes (Leu 4 and T11); T-helper lymphocytes (Leu 3A and CD4); cytotoxic/suppressor T lymphocytes (Leu 2A and CD3); and fibroblasts (5B5). In situ hybridization was used to identify the mRNA for specific proteins: interleukin (IL)1 alpha and -beta, IL-2, IL-6, transforming growth factor beta, tumor necrosis factor alpha (TNF alpha), platelet-derived growth factor alpha (PDGF alpha), and interferon gamma. A quantitative assessment was performed for each section by calculating the percentage of positively staining cells using a light microscope and grid-counting technique. A random effect analysis of variance was calculated to determine both the variance between samples within each patient and the variance between different patients. Standard deviations contributed by sampling variance and patient variance were calculated and an F test was applied. Tissue samples taken from different regions of the bone-prosthesis interface showed marked heterogeneity in cellular and cytokine profiles. Critical F values indicating a statistically significant degree of variance between different tissue samples were exceeded for macrophages, cytotoxic/suppressor T lymphocytes, and T-helper lymphocytes. The cytokine profile was significantly different for IL-2, PDGF alpha, and TNF alpha. This tissue heterogeneity may be due to different mechanical and biologic environments along the bone-prosthesis interface. Thus, caution must be exercised in defining the biologic properties of the tissue surrounding revised prostheses according to a single biopsy.

    View details for Web of Science ID A1996UU78600017

    View details for PubMedID 8806069

  • Cardiac allograft unresponsiveness using a posttransplant strategy: Characterization of the graft infiltrate Krieger, N. R., Quezada, V. R., Huie, P., Holm, B., Sibley, R. K., Dafoe, D. C., Alfrey, E. J. ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS. 1996: 86-92


    We evaluated a combined posttransplant strategy using antilymphocyte serum (ALS) at time of engraftment followed by low dose total lymphoid irradiation (TLI) and donor bone marrow cell (BMC) inoculation administered either intrathymically (IT) or intravenously (IV) in the vigorously rejecting strain combination DA into Lew recipients. Allograft survival was significantly prolonged with administration of ALS in combination with TLI and IT (105 +/- 28.6 days) or IV (106.8 +/- 28.6 days) BMC compared to administration of ALS combined with either TLI (17.8 +/- 0.4 days) or BMC (9.0 +/- 0.0 days), or TLI combined with BMC (1 1.5 +/- 0.5 days) (P < 0.000 1, experimental vs control animals). There was no difference in survival between those animals who underwent IT or IV BMC inoculation. Third-party (WF) BMC inoculation did not significantly prolong allograft survival (10.0 +/- 1.0 days). A mild to moderate cellular infiltrate was present in allograft tissue after 100 days. To further characterize these cells, cytokine mRNA expression in allograft tissue (> 100 days posttransplant) was evaluated using nonisotopic in situ hybridization. A similar cytokine profile was demonstrated in allograft tissue compared to naive and isograft tissue, except for a slight increase in IL-2 (P < 0.02, control vs IV BMC; P = NS, other groups). In summary, unresponsiveness was induced in a high-responder strain combination using a combined posttransplant strategy of ALS, TLI, and donor antigen either IT or IV. The cytokine profile of the graft infiltrating cells was similar to that of normal tissue. Unresponsiveness may be the result of functional inactivation of these cells.

    View details for Web of Science ID A1996UU47100017

    View details for PubMedID 8661178

  • Untitled - Reply JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME Goodman, S., Song, Y., Knoblich, G., Huie, P., Regula, D., Aspenberg, P., Lidgren, L. 1996; 78A (5): 793-793
  • Differential localization of allograft nitric oxide synthesis: Comparison of liver and heart transplantation in the rat model IMMUNOLOGY Kuo, P. C., Alfrey, E. J., Krieger, N. R., Abe, K. Y., Huie, P., Sibley, R. K., Dafoe, D. C. 1996; 87 (4): 647-653


    Nitric oxide (NO) is a free radical with a diversity of cellular origins and potential functions. Within the realm of solid organ transplantation, NO has been the focus of much attention. Discordant reports have documented both suppression and potentiation of the alloimmune response. In addition to questions regarding its functional role, little is known of the cellular origins of NO in acute rejection of vascularized allografts. To address this question, acute rejection models of rat heterotopic heart and orthotopic liver transplantation were chosen. When compared with naive controls and isografted animals, acute rejection in both heart and liver transplantation was associated with elevated systemic levels of the NO metabolite, nitrite. This was accompanied by increased graft content of iNOS protein as determined by immunoblot analysis of protein extracts. Expression of iNOS mRNA was localized with in situ hybridization. In both heart and liver transplantation, iNOS mRNA was found in the inflammatory infiltrate accompanying acute rejection. In addition, hepatocytes also expressed iNOS mRNA in the rejecting liver allograft. In contrast, cardiac myocytes in the rejecting heart allograft did not stain for iNOS mRNA. These results indicate that organ-specific, differential cellular expression of iNOS occurs in the acutely rejecting allograft. Transcriptional regulation of iNOS may vary among various organs according to the local cellular milieu. In addition, there may be a variable allograft specific response to acute rejection which may modify the associated immunologic biology.

    View details for Web of Science ID A1996UD90500020

    View details for PubMedID 8675222

  • Hypertension-enhanced monocyte adhesion in experimental atherosclerosis JOURNAL OF VASCULAR SURGERY Tropea, B. I., Huie, P., Cooke, J. P., Tsao, P. S., Sibley, R. K., Zarins, C. K. 1996; 23 (4): 596-605


    Hypertension is a known clinical risk factor for atherosclerosis. In experimental atherosclerosis, monocyte adhesion to the endothelial surface is enhanced and is considered to be an important early stage in plaque formation. We tested the hypothesis that hypertension enhances monocyte adhesion in experimental atherosclerosis.Twenty-two New Zealand White rabbits were fed an atherogenic diet for 3 weeks to induce plaque formation. Aortic coarctation was created in eight rabbits by wrapping a Dacron band around the midportion of the descending thoracic aorta (stenosis group), whereas six rabbits underwent banding without aortic constriction (no stenosis group). Eight rabbits served as nonoperated controls. Monocyte binding to the aortic endothelial surface was counted with epifluorescent microscopy on standard aortic segments proximal and distal to the band. Immunohistochemistry was performed for the following antibodies: VCAM-1, RAM11, CD11b, and factor VIII.Mean blood pressure was 89 +/- 3 mm Hg in the aorta proximal to the stenosis, compared with 64 +/- 4 mm Hg in the no stenosis group and 74 +/- 3 mm Hg in the control group (p < 0.01). The mean aortic blood pressure gradient across the stenosis was 16 +/- 2 mm Hg in the stenosis group, whereas the aortic blood pressure gradient was 0.2 +/- 0.6 mm Hg in the no stenosis group and -0.3 +/- 0.4 mm Hg in the control group (p < 0.001). Monocyte adhesion to the aortic endothelial surface proximal to the stenosis was increased twofold compared with adhesion to the aorta distal to the stenosis and compared with the proximal aorta in the control group (p < 0.02). The proximal-to-distal aortic ratio of monocyte binding was enhanced in the stenosis group (2.2) compared with the no stenosis (0.76) and control (0.83) groups (p < 0.01). The intima area of the aorta proximal to the stenosis was significantly increased compared with the proximal aortas in the no stenosis and control groups (p < 0.01). RAM11, CD11b, and endothelial VCAM-1 expression were enhanced in the hypertensive region proximal to the stenosis.In the hypertensive region in the aorta proximal to the stenosis, monocyte adhesion and endothelial VCAM-1 expression were increased, with intimal thickening and accumulation of macrophages. These findings suggest that hypertension may promote atherosclerotic plaque formation by enhancing monocyte adhesion.

    View details for Web of Science ID A1996UJ12600010

    View details for PubMedID 8627894

  • Double negative (CD4(-)CD8(-)alpha beta(+)) T cells which promote tolerance induction and regulate autoimmunity IMMUNOLOGICAL REVIEWS Strober, S., Cheng, L. R., Zeng, D. F., Palathumpat, R., DEJBAKHSHJONES, S., Huie, P., Sibley, R. 1996; 149: 217-230

    View details for Web of Science ID A1996UC52100010

    View details for PubMedID 9005216

  • Cellular localization and effect of nitric oxide synthesis in a rat model of orthotopic liver transplantation Kuo, P. C., Alfrey, E. J., Abe, K. Y., Huie, P., Sibley, R. K., Dafoe, D. C. LIPPINCOTT WILLIAMS & WILKINS. 1996: 305-312


    Nitric oxide (NO) is a multifunctional free radical with a variety of described biochemical and physiological roles. The immunologic relationships between organ transplantation and NO synthesis are unknown. While a number of in vitro and in vivo models have demonstrated an immunomodulatory role for NO, results suggest both an immunosuppressive and immunostimulatory function. In order to better delineate the role of NO in liver transplantation, the Kamada model of rat OLT with strain combinations simulating acute rejection and spontaneous hyporesponsiveness was chosen. In this setting, both acute rejection and spontaneous hyporesponsiveness were associated with increased levels of plasma NO metabolites and allograft expression of the enzyme, NO synthase (iNOS). The extent of expression was significantly greater with acute rejection. Using in situ hybridization, iNOS mRNA was localized to both infiltrating inflammatory cells and hepatocytes in the context of acute rejection. In contrast, iNOS mRNA expression was isolated to the hepatocytes in the hyporesponsive state. To specifically delineate the role of hepatocyte-derived NO, NO synthesis was ablated in the spontaneous hyporesponsiveness model and resulted in significant elevation of serum transaminase values with accompanying histologic evidence of increased periportal inflammatory infiltration. Our results suggest that the site of NO production varies according to the immunologic status of the liver allograft, and hepatocyte-derived NO may be protective in the hyporesponsive state.

    View details for Web of Science ID A1996TT97000024

    View details for PubMedID 8600641

  • RANTES chemokine expression in diseased and normal human tissues CYTOKINE VONLUETTICHAU, I., Nelson, P. J., Pattison, J. M., VANDERIJN, M., Huie, P., Warnke, R., Wiedermann, C. J., Stahl, R. A., Sibley, R. K., KRENSKY, A. M. 1996; 8 (1): 89-98


    RANTES is a member of a large family of cytokines, called chemokines, which are thought to play a regulatory role in inflammatory processes. We have made recombinant human RANTES protein which was used to generate a panel of anti-RANTES monoclonal antibodies. Following characterization, select anti-RANTES monoclonal antibodies were used for immunohistologic staining of a large panel of normal, diseased and fetal tissue sections. Diseased tissues included eleven lymphomas and eight renal tumors. Most tissues were also tested in parallel for RANTES mRNA by in situ hybridization using RANTES mRNA specific oligomeric probes. As expected, most normal adult tissues contain few, if any, RANTES positive cells. In contrast, RANTES expression dramatically increases in inflammatory sites. In addition, megakaryocytes, some tumours, and select fetal tissues express high levels of RANTES message and protein. These results indicate a wider expression of RANTES than previously appreciated and suggest multiple physiologic roles for this soluble factor.

    View details for Web of Science ID A1996TT98200012

    View details for PubMedID 8742071



    We evaluated the postischemic renal injury in 22 patients undergoing renal transplantation. Renal tissue obtained 45 to 60 minutes after reperfusion of the allograft was stained with specific antibodies against the delta subunit of Na+/K(+)-ATPase, fodrin and ankyrin. The distribution of each cytoskeletal protein was analyzed by laser confocal microscopy. Subsequent allograft function was assessed on two occasions, 1 to 3 and 36 hours post-reperfusion, respectively. Recipients were divided into two groups: those who achieved a normal GFR on post-transplant day 3 (group 1, N = 12) and those with persistent hypofiltration (group 2, N = 10). Patients of both groups exhibited impaired sodium reabsorption and isosthenuria one to three hours postoperatively, but these abnormalities persisted on day 3 only in group 2 subjects with persistent hypofiltration. Abnormalities of Na+/K(+)-ATPase, ankyrin and fodrin were confined to proximal tubule cells and were marked only in the subjects of group 2. They consisted of redistribution of each cytoskeletal protein from the basolateral membrane to the cytoplasm. We conclude that postischemic injury to a renal allograft results in a loss of polarity of proximal tubule cells. We propose that ensuing impairment of proximal sodium reabsorption could activate tubuloglomerular feedback, thereby contributing to the protracted hypofiltration that characterizes this form of postischemic, acute renal failure.

    View details for Web of Science ID A1995RV92600045

    View details for PubMedID 8569093

  • VASODILATOR UNRESPONSIVENESS OF ACUTE POSTISCHEMIC RENAL-ALLOGRAFT FAILURE Tse, E., VINOT, O., Huie, P., Sibley, R. K., Wang, B. Y., Cooke, J. P., Dafoe, D., Alfrey, E., Scandling, J., Myers, B. D. AMER SOC NEPHROLOGY. 1995: 478-478


    Immune regulation requires antigen recognition, signaling, activation, secretion of cytokines, and effector function by lymphocytes. Although there is redundancy in the activation and function of the immune response, some cytokines simultaneously promote and suppress different pathways of immunity. In the experiments reported here we compare cytokine gene expression within liver allografts from tolerant rats with normal and isografted liver tissue. We also compare the secretion of interferon-gamma (IFN-gamma) in the supernatant from mixed lymphocyte cultures by using peripheral blood lymphocytes stimulated against donor antigen.Orthotopic liver transplantations were performed using the cuff technique without hepatic artery revascularization. Nonisotopic in situ hybridization (ISH) was used to detect and localize messenger RNA to specific cells within tissue. Antisense DNA probes were generated to interleukin-2 (IL-2), IL-4, IL-10, and IFN-gamma. One-way mixed lymphocyte cultures were set up against irradiated donor splenocytes, and the supernatant was collected to measure IFN-gamma by enzyme-linked immunosorbent assay.Expression of IFN-gamma and IL-10 was up-regulated in tolerant animals versus normal or isografted liver (p = 0.0002 and 0.0001, IFN-gamma and IL-10, respectively). In situ hybridization localized the expression of messenger RNA predominantly to the cytoplasm of the hepatocytes. Levels of IFN-gamma were higher in the supernatant from proliferating peripheral lymphocytes against donor antigen from tolerant animals versus naive control animals.Expression of IFN-gamma and IL-10 is up-regulated in hepatocytes from allograft tissue after orthotopic liver transplantation. We believe that the up-regulation of IL-10 cross-regulates the effector function of IFN-gamma and supports cytokine-mediated immune dysregulation, which may be a mechanism of tolerance after orthotopic liver transplantation in rats.

    View details for Web of Science ID A1995RM96500038

    View details for PubMedID 7638757



    Particulate wear debris from joint replacements has been implicated in the etiology of periprosthetic bone resorption. However, the effect of high-density-polyethylene or cobalt-chromium-alloy particles on osteoclastic bone resorption in vivo has not been studied previously, to our knowledge. Therefore, we examined the effect of these particles on tissue ingrowth, net bone formation (per cent trabecular bone), and osteoclastic bone resorption (osteoclasts per unit of bone surface) with use of a bone-harvest chamber that had a transverse one-millimeter channel for tissue ingrowth. After an initial six-week period for incorporation of the chamber into the proximal part of the tibia of rabbits, the contents of the channel were harvested repeatedly at three-week intervals. The carrier solution, 1 per cent sodium hyaluronate, was implanted first. In subsequent implantations, the hyaluronate was mixed with high-density-polyethylene or cobalt-chromium particles at concentrations of 10(8) particles per milliliter. The tissue harvested from the chambers that contained no particles was composed of longitudinally oriented trabecular bone in a fibrovascular stroma. Particulate high-density polyethylene evoked a moderate foreign-body reaction and a chronic inflammatory response and decreased net bone formation. When cobalt-chromium particles had been implanted, the tissue exhibited a more florid foreign-body reaction and a chronic inflammatory response, often in a nodular arrangement, in a background of dense connective tissue. Bone was sparse, and areas of cell necrosis and hyaline degeneration were noted. Histomorphometric analyses were carried out to determine the amount of net bone formation and osteoclastic bone resorption in the presence or absence of high-density-polyethylene or cobalt-chromium particles. The amount of bone was greatest in the control specimens, moderately decreased in the presence of high-density-polyethylene particles, and greatly decreased in the presence of cobalt-chromium particles. The number of osteoclasts in Howship lacunae per unit of trabecular bone surface was increased in the presence of high-density polyethylene, indicating that these particles stimulate osteoclastic bone resorption.

    View details for Web of Science ID A1995RJ67100008



    Cytokines are short-acting protein modulators of many physiologic processes including graft rejection. An understanding of the production, action, and interaction of cytokines may lead to better appreciation of the complex mechanism of graft rejection. The potential would then exist for more selective and less-toxic means of modulating the immune response. A rat hind limb allograft model with major immunohistoincompatibility was used to study the local mRNA expression of IL-1 alpha, IL-2, IL-6, gamma interferon (gamma INF), platelet-derived growth factor-alpha (PDGF-alpha), basic fibroblast growth factor (FGF), and transforming growth factor-beta (TGF-beta) during acute allograft rejection. A 14-day postoperative course of immunosuppressive therapy with FK506 or rapamycin was administered. In situ hybridization was performed on serial full-thickness skin punch biopsies of the untreated rejecting limb allograft and compared with tissue from treated allografts, isografts, and to normal limb tissue. A sequential pattern of cytokine mRNA expression was demonstrated which progressed in a time-dependent manner and paralleled observed clinical rejection. Maximal cytokine mRNA expression correlated with peak graft rejection. Cellular expression of IL-1 alpha, IL-2, IL-6, gamma-INF, FGF, and TGF-beta mRNA was suppressed with FK506 to below isograft levels, and clinical rejection was not observed with the doses, routes, and schedules used. Rapamycin was ineffective in suppressing cytokine expression, and allograft rejection was not prevented. Isografts demonstrated no evidence of rejection. The in situ hybridization technique demonstrates a time-dependent, selective expression of cytokines within rejecting allograft tissue, and the modification of this response with immunosuppressive therapy. Down-regulation of cytokine expression is associated with clinical allograft survival.

    View details for Web of Science ID A1995RB42900020

    View details for PubMedID 7539555



    Cardiac allograft vascular disease is characterized by accelerated and diffuse intimal proliferation involving both the microvasculature and epicardial vessels. Because in vivo documentation of this complication is now possible with intracoronary ultrasound imaging, we can examine the relationship of intimal proliferation to markers of immunity and endothelial activation. We hypothesize that alterations of microvascular cell surface markers likely mirror changes in the epicardial vessels that may be important in the pathophysiology of intimal proliferation.Forty-three heart transplant patients were examined by intracoronary ultrasound more than 1 year after transplantation, and these images were analyzed to obtain mean intimal thickness and intimal thickness class (I through IV), calculated from the mean thickness and circumferential involvement. Right ventricular endomyocardial biopsies obtained at the time of intracoronary ultrasound were examined by immunohistochemistry to detect microvascular expression of histocompatibility leukocyte antigen (HLA) classes I and II (HLA ABC, DR, DP, and DQ); endothelial-specific antigen detected by the monoclonal antibody E 1.5; intercellular adhesion molecules (ICAM-1); CD4+ and CD8+ lymphocytes and macrophages (CD 14+). Microvascular antigen expression was graded 1 through 5 on the basis of the diffuseness of positive staining. The number of each inflammatory cell phenotype present per high-power field was counted. By ANOVA, scores for HLA DR, HLA DQ, and E1.5 expression were lower in intimal thickness classes II, III, and IV compared with class I. This inverse relationship was significant by linear regression analysis of mean intimal thickness. Inflammatory cells were not significantly correlated with intimal thickening. Rejection incidence was higher, and time since transplantation longer, in intimal thickness classes II, III, and IV compared with class I.Transplant coronary artery intimal proliferation is associated with alteration of microvascular endothelial cell surface markers. These changes in cell surface antigen expression could provide the substrate for coronary artery intimal proliferation and narrowing.

    View details for Web of Science ID A1995QL45800006

    View details for PubMedID 7882470


    View details for Web of Science ID A1994PM60300142

    View details for PubMedID 7940898



    RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemotactic cytokine (a chemokine) for memory T lymphocytes, monocytes, and eosinophils. RANTES expression was studied in renal allograft biopsy specimens. Although RANTES was not expressed in samples taken one hour after transplantation, or in native renal biopsy specimens from patients with cyclosporin nephrotoxicity, it was expressed during cell-mediated transplant rejection. RANTES mRNA was detected in infiltrating mononuclear cells and renal tubular epithelium, and RANTES protein was localised to mononuclear cells, tubular epithelium, and vascular endothelium. This suggests RANTES has a role in allograft rejection.

    View details for Web of Science ID A1994MT33200011

    View details for PubMedID 7507196

  • THE BCL-2 ONCOGENE IN HODGKINS-DISEASE ARISING IN THE SETTING OF FOLLICULAR NON-HODGKINS-LYMPHOMA BLOOD LeBrun, D. P., Ngan, B. Y., Weiss, L. M., Huie, P., Warnke, R. A., Cleary, M. L. 1994; 83 (1): 223-230


    Expression of the bcl-2 proto-oncogene on chromosome 18 is deregulated by the 14; 18 chromosomal translocation, an abnormality that is consistently associated with follicular non-Hodgkin's lymphomas (NHL). Because bcl-2 is believed to function by prolonging cell survival rather than by increasing proliferation, the presence of t(14; 18) in Hodgkin's disease (HD) would have profound implications for the pathogenesis of this neoplasm. We evaluated 32 cases of HD for t(14; 18) by polymerase chain reaction (PCR). These results were correlated with expression of bcl-2 oncogenic protein by Hodgkin cells and with the presence of Epstein-Barr virus (EBV), as determined by immunohistochemistry or in situ hybridization. PCR provided evidence of t(14; 18) in only 2 HD cases (6%), both of which were associated with a prior history of follicular lymphoma, and both of which were among the 7 cases (22%) with strong bcl-2 expression in Hodgkin cells. In at least 1 of the cases, the translocation involved identical chromosomal breakpoints in both types of lymphoma. Furthermore, 7 additional cases of combined follicular NHL and HD showed strong bcl-2 staining in Hodgkin cells. Although EBV was detected in 6 of 30 cases, it was not associated with t(14; 18) and usually not with strong bcl-2 expression. These results suggest that a small proportion of HD cases might evolve from follicular NHL, possibly through molecular events superimposed on the t(14; 18). High-level bcl-2 expression in Hodgkin cells is a potentially useful but not definitive marker for these cases.

    View details for Web of Science ID A1994MQ09800029

    View details for PubMedID 8274737

  • MONOCYTE HAPTOTAXIS INDUCED BY THE RANTES CHEMOKINE CURRENT BIOLOGY Wiedermann, C. J., Kowald, E., Reinisch, N., Kaehler, C. M., VONLUETTICHAU, I., Pattison, J. M., Huie, P., Sibley, R. K., Nelson, P. J., KRENSKY, A. M. 1993; 3 (11): 735-739


    Soluble mediators and inducible cell-surface molecules coordinate the ordered cascade of events giving rise to inflammation. The specific mechanisms underlying the attraction of antigen-specific cells into a site of inflammation remain sketchy, however. In particular, it is unclear how chemoattractants cause rapidly moving immune cells to adhere to the blood vessel wall and to enter inflamed tissues.Here we show that RANTES, a potent chemo-attractant for monocytes and T lymphocytes, is inducibly expressed within an inflamed organ, binds to endothelial cells, and promotes haptotaxis, the migration of cells induced by surface-bound gradients.These findings lead us to propose a model for the role of RANTES in the migration of antigen-specific immune cells into an inflammatory site.

    View details for Web of Science ID A1993MR82300002

    View details for PubMedID 15335836



    The effect of rapamycin (RPM) on the extent of arterial intimal thickening was determined in rat recipients of orthotopic femoral artery allografts or in rats that had undergone balloon catheter injury to carotid arteries. In untreated rats, neointima comprised approximately 50% of the arterial wall area in both models. Although treatment of allograft recipients for 40 days with 1.5 mg/kg/day RPM was ineffective, a dose of 6 mg/kg/day (days 0-7) followed by 3 mg/kg/day (days 8-39) reduced intimal thickening by 98% (P < 0.0001). The higher RPM dose reduced T cell and macrophage infiltration significantly and decreased the expression of IL-2 receptor, class II Ag, and mRNAs for growth factors and cytokines. Treatment with 1.5 mg/kg/day RPM (days 0-13) after balloon-catheter injury reduced intimal thickening by 45% (P = 0.0254) and substantially decreased macrophage infiltration and expression of class II Ag in the adventitia. Within the neointima, however, mRNAs for platelet-derived growth factor-alpha, basic fibroblast growth factor, and transforming growth factor-beta were still expressed. In summary, we have shown that RPM inhibits not only the vascular response to injury caused by allograft rejection, but also the response to balloon catheter injury. This new information is important to our understanding of: (1) the fundamental processes responsible for intimal thickening regardless of the cause of vascular injury, (2) mechanisms of action of RPM that explain its effects on the response to very different types of vascular injury, and (3) the potentially diverse therapeutic applications of drugs, like RPM, that inhibit the actions of both immune and nonimmune cytokines and growth factors.

    View details for Web of Science ID A1993LK58900037

    View details for PubMedID 8516827


    View details for Web of Science ID A1993KN62100294

    View details for PubMedID 7679842


    View details for Web of Science ID A1993KN62100039

    View details for PubMedID 8438246



    Immunohistochemistry and in situ hybridization with a synthetic oligonucleotide probe were used to compare the topographical distribution of BCL-2 proto-oncogenic protein with that of its messenger RNA (mRNA) in normal lymphoid tissues, follicular lymphomas, and lymphoma-derived cell lines. In normal lymph nodes, BCL-2 protein was most abundant in the small lymphocytes of primary lymphoid follicles and the mantle zones of secondary follicles, virtually absent within germinal centers, and of variable abundance in many interfollicular cells. In contrast, the distribution of BCL-2 mRNA was roughly reciprocal to that of the protein with intense hybridization signal in germinal centers and almost none in mantle zones. Discordant BCL-2 RNA and protein levels were also observed in tonsillar epithelial cells and cortical thymocytes. Concordant and abundant expression of BCL-2 mRNA and protein was detected in biopsy tissues and cell lines from t(14;18)-carrying lymphomas. The contrasting distributions of BCL-2 protein and RNA in normal lymphoid tissues suggest that translational and posttranslational control mechanisms play a significant role in regulating BCL-2 protein levels in germinal center cells, epithelial cells, and cortical thymocytes. Concordant BCL-2 mRNA and protein levels in follicular lymphomas suggest that translational control mechanisms may be disrupted as part of the sequence of genetic changes that transforms normal lymphoid cells into neoplastic follicular lymphoma cells.

    View details for Web of Science ID A1993KH51200003

    View details for PubMedID 8422456



    Glomerular permselectivity and dynamics were evaluated serially in 14 nephrotic patients with membranous glomerulopathy (MG). Analysis of transglomerular dextran sieving, before and again after proteinuria remitted, revealed persistent depression by 60-80% of glomerular pore density and the two-kidney ultrafiltration coefficient, Kf. The glomerular filtration rate was lowered by half on each occasion. Morphometric examination of glomeruli in a second group of 16 nephrotic patients with MG revealed a low prevalence of glomerulosclerosis (5 +/- 3%) and a twofold increase in filtration surface due to marked glomerular hypertrophy. Presumably, widening by threefold of the basement membrane and/or epithelial podocytes accounted for the computed reduction in ultrafiltration capacity. There was no correlation between glomerular structure and the subsequent course of MG over the ensuing 24-96 mo. Rather, a twofold expansion of the interstitial compartment predicted those who went on to exhibit progressive renal insufficiency. We conclude that increasing resistance to water flow by walls of patent and perfused glomerular capillaries is the proximate cause of progressive renal insufficiency in MG.

    View details for Web of Science ID A1992KF37700085

    View details for PubMedID 1282782



    Lenticles are dome-shaped circles or ovals of cuticle with a dark rim. They occur with a precise segmental arrangement in the larvae and pupae of lycaenid and hesperiid butterflies. In Calpodes ethlius (Lepidoptera, Hesperiidae) each lenticle is secreted by a pair of large polyploid epidermal cells. The dark rim or annulus is formed from a ring-shaped cell. The dome, which consists of an epicuticle with a perforate intermediate layer like a pepper-pot, is formed by a central goblet cell. Between the perforate intermediate layer and the cell surfaces there is a cavity that contains material presumed to be secretion. Both cells have elaborate basal plasma membrane reticular systems and the apical microvilli associated with an extensive smooth endoplasmic reticulum that is typical of lipid secreting cells. In addition, there is a plasma membrane reticular system in the ring cell and between it and the goblet cell that contains the endings of nerves having neurosecretory vesicles. Lenticles thus have a structure appropriate for an innervated organ of lipid secretion. However, in their development, lenticles arise from bristles that are presumed to be sensory. Lenticles or their precursors are segmentally arranged in the five larval instars and the pupa, but the pattern changes at each moult. The cells that form a lenticle at one moult have a rest period at the next one when they only secrete surface cuticle. Many lenticles are paired in their cycle of development, with only one of the pair making a lenticle at a particular moult. For example, the dorsal and lateral lenticles alternate in position between anterior and posterior. The second and fourth instar segments have anterior and the third and fifth instars have posterior lenticles. In the first instar the cells that will make lenticles for the second and third instars both make bristles. Lenticles are thus formed by cells that not only change their response to ecdysone qualitatively by switching from bristle to lenticle but also alternate in their later responses, switching back and forth at alternate moults between the formation of a lenticle and the secretion of surface cuticle.

    View details for Web of Science ID A1984SP55300010

    View details for PubMedID 6740650



    The basal surface in transporting epithelia is infolded in a way that encourages the formation of standing gradients. Many insect cells have a similar infolded reticular system (RS) although they are clearly not transporting epithelia. These cells are like one another metabolically in that they sequester lipid from hemolymph lipophorins (lipid transporting proteins). Dietary lipids enter the hemolymph from the midgut RS which may be an adaptation for lipophorin loading. The plasma membrane reticular system of tissues metabolizing lipids (fat body, wax glands, oenocytes, lenticles) may be an adaptation for lipophorin reception and unloading. Cationic ferritin (pI 8.5) shows all RSs are covered by a lamina functioning as a negatively charged sieve. The basal plasma membrane leading to the RS is also negatively charged. The RS is a container with charged entrances that would be expected to affect the composition of the contents. Midgut cells release lipid particles into their RS. The particles are positively charged since in tracer studies they associate with anionic but not cationic ferritin. Lipophorins are anionic. The electrostatic binding of lipid to lipophorin would make it less anionic and more likely to leave the RS when loaded, thus carrying lipid to the hemolymph. Conversely, at the destination RS, loaded lipophorin would penetrate more easily than unloaded. A change in charge with unloading would be expected to alter the equilibrium between entering and leaving lipophorin, causing protein concentration in the RS of lipid receiving tissues as has been observed in the fat body. Reticular systems may thus be reaction vessels for interactions between carrier proteins and their load.

    View details for Web of Science ID A1983RW03200004

    View details for PubMedID 6665784



    The Champy-Maillet OsKI reaction has been used upon Golgi complexes to show two kinds of staining. It stains material being processed as it passes along the secretory pathway of the rough endoplasmic reticulum (RER) and Golgi cisternae (GC) up to crystallization in secretory vesicles. It also stains separately the environment within parts of the GC. This GC staining may occur in all compartments (transition vesicles, saccules, condensing vacuoles), but it is characteristically missing from any one of them. The unstained cisternae may be explained if outer saccules are made from either stained or unstained transition vesicles, both of which occur. The presence of empty, unstained transition vesicles is dictated by the surface to volume ratios of microvesicles in relation to saccules. Most transition vesicles must return their membrane to the endoplasmic reticulum, but from time to time it is presumed that they fuse to make a saccule. Saccules, stained and unstained, then mature through the stack. OsKI reactions with tissues and test molecules suggest that in the RER and GC the stain detects labile--S . S--bridges before they lock the tertiary configuration of proteins.

    View details for Web of Science ID A1983RB01000006

    View details for PubMedID 6190856



    Four groups of intracellular structures can be recognized according to bismuth and uranyl staining and phosphorus content. (1) Those which contain phosphorus and stain strongly with uranyl acetate but not with bismuth (ribosomes, heterochromatin and mature ribosomal precursor granules), presumably because of their nucleic acid content. (2) Those which contain phosphorus and stain with uranyl acetate and bismuth (interchromatin granules, immature ribosomal precursor granules and mitochondrial granules), presumably because at least some of their phosphate is available to react with bismuth. (3) Those which contain little phosphorus but which stain strongly with bismuth and weakly with uranyl acetate (Golgi complex beads), perhaps because some ligand in addition to phosphate reacts with bismuth, and (4) those which do not contain phosphorus and stain with neither uranyl acetate nor bismuth (portasomes). Uranyl staining correlates strongly with the phosphorus content of nucleic acids, proteins and inorganic deposits. Bismuth will stain some phosphorylated molecules but not all. Thus only some phosphates stain with bismuth.

    View details for Web of Science ID A1982QF16200002

    View details for PubMedID 6189262

  • EPIDERMAL FEET IN INSECT MORPHOGENESIS NATURE Locke, M., Huie, P. 1981; 293 (5835): 733-735

    View details for Web of Science ID A1981MM83600038

    View details for PubMedID 7197325



    Epidermal cells in insect integumental epithelia develop branched cytoskeletal extensions or feet at their base that are similar in appearance to the processes put out by cells in tissue culture. We have developed a procedure to show the feet that gives an effect as if thousands of cells randomly arranged in the epithelium had each been injected with lead salt visualized as black lead sulphide. The procedure depends upon the fact that after brief glutaraldehyde fixation, tannic acid only penetrates some cells where it mordants lead ions and binds osmium. Individual cells visualized in this manner show their outlines as if they are separate in a tissue culture although they are part of a closely packed epithelium. The feet are metamorphic structures formed after pupal commitment and are necessary for metamorphic changes in segment shape. In Calpodes larvae the feet are orientated axially in the direction of the segmentally repeating gradient and may extend for several cell diameters. They extend under the influence of low titres of 20-hydroxyecdysone such as those occurring in the intermoult. When stimulated by high titres like those in pre-pupae, the feet contract at the same time as the segments shorten to pupal proportions. We believe that cell processes like the epidermal feet are ubiquitous but that they have often been overlooked because of the difficulty of demonstrating the outlines of single cells that are united in epithelia.

    View details for Web of Science ID A1981MX01700014

    View details for PubMedID 7330857



    The fifth stadium of Calpodes has two phases of epidermal cell development corresponding to preparation for intermoult and for moult syntheses. Both phases begin with a period of elevated RNA synthesis and the elaboration of a multilobed nucleolus. The apparent number of nucleoli changes from about two to eight and back to two again within the few hours of elevated RNA synthesis. The nucleolar changes are preceded by elevated titres of haemolymph ecdysteroid. During the two periods of activity, alveoli in the matrix of the nucleoli contain particles believed to be ribosomal precursors. The staining properties of these granules differ according to size in a way that suggests a developmental sequence. Mature granules are about 20 nm in diameter and do not stain with bismuth. They are found at the periphery of the nucleolus, in the nucleoplasm, at the approaches to and within the nucleopores. Perichromatin granules, believed to be m-RNA precursor packages, are up to 60 nm in diameter, do stain with bismuth and are found at the periphery of chromatin, in nucleoplasm and distorted at the approaches to the nuclear pores to fit within the central channel. During these periods of heightened activity the nuclear envelope contains microvesicles that may be free or attached to either nuclear or cytoplasmic surfaces. The structure is appropriate for the microvesicular transnuclear envelope movement of molecules such as the ecdysteroid believed to initiate the nuclear changes.

    View details for Web of Science ID A1980JG40700013

    View details for PubMedID 7361299

  • Ultrastructural Methods in Cuticle Research Cuticle Techniques in Arthropods Locke, M., Huie, P. edited by Miller, T. A. Springer-Verlag. 1980: 91-144


    The apical plasma membranes of Calpodes epidermal cells have small fattened areas or plaques with an extra density upon their cytoplasmic face. The plaques are typically at the tips of microvilli. The are present during the deposition of fibrous cuticle and the cuticulin layer. Since the plaques are close (less than 15nm) to the sites where these kinds of cuticle first appear, they are presumed to have a role in their synthesis and/or deposition and orientation. When fifth stage larval cuticle deposition ceases prior to pupation, the plaques are lost as the area of the apical plasma membrane is reduced. The plaques pass from the surface into pinocytosis vesicles and multivesicular bodies where they are presumably digested. The loss of plaques occurs as the blood level of moulting hormone reaches a peak at the critical period after which the prothoracic glands are no longer needed for pupation. Apolysis or separation of the epidermis from the old cuticle is the stage when plaques are absent, the old ones have been lost but the new ones have yet to form. After the critical period, the epidermis prepared for pupation with a phase of elevated RNA synthesis at the end of which plaques and microvilli reform in time to secrete the new cuticulin layer and later the fibrous cuticle of the pharate pupa. There is a new generation of plaques for each moult and succeeding intermoult and each generation is involved in two kinds of cuticle deposition before involution and redifferentiation.

    View details for Web of Science ID A1979HB22100007

    View details for PubMedID 473162


    View details for Web of Science ID A1977DR35500012

    View details for PubMedID 71764



    Insects and other arthropods have bead-like structures in Golgi complexes from all cell types. They are arranged in rings at the base of transition vesicles located near the smooth surface of the rough endoplasmic reticulum making the forming face of the Golgi complex and are only seen easily after staining in bismuth salts. Procedures used to demonstrate the beads in arthropod Golgi complexes do not selectively stain any structures where they would be expected to occur in several mouse and tadpole tissues. However, a faint pattern similar to the arthropod GC beads can be made out in the large GCs concerned in the formation of acrosomes during mouse spermatogenesis. Uranyl staining shows particles of about the same size and spacing as the beads of arthropod GCs. We conclude that vertebrate GCs may have beads that differ from arthropods in their staining properties.

    View details for Web of Science ID A1976CV19600013

    View details for PubMedID 65806



    The region between the rough endoplasmic reticulum (ER) and the Golgi complex has been studied in a variety of insect cell types in an attempt to find a marker for the exit gate or gates from the ER. We have found that the smooth surface of the rough endoplasmic reticulum near Golgi complex transitional elements has beadlike structures arranged in rings at the base of transition vesicles. They occur in all insect cell types and a variety of other organisms. The beads can be seen only after staining in bismuth salts. They are 10-12 nm in diameter and are separated from the membrane and one another by a clear halo giving them a center to center spacing of about 27 nm. The beads are not sensitive to nucleases under conditions which disrupt ribosomes or remove all Feulgen staining material from the nucleus. Under conditions similar to those used to stain tissue, bismuth does not react in vitro with nucleic acids. The component of the beads that stains preferentially with bismuth is therefore probably not nucleic acid.

    View details for Web of Science ID A1976BZ77800011

    View details for PubMedID 59729


    View details for Web of Science ID A1975W109200016

    View details for PubMedID 47196



    The smooth surface of the rough endoplasmic reticulum that makes the forming face of the Golgi complex has beadlike structures arranged in rings at the base of transition vesicles. The beads can only be seen easily after staining in bismuth salts. They are 10 to 12 nanometers in diameter and occur in a variety of cell types and organisms.

    View details for Web of Science ID A1975AE48300024

    View details for PubMedID 49926


    View details for Web of Science ID A1972O399700005

    View details for PubMedID 4119828

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