Philip Huie

RANTES chemokine expression in cell-mediated transplant rejection of the kidney

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.

Predominance of NK1.1^+TCRαβ^+ or DX5^+TCRαβ^+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease : "natural suppressor" cells

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.

Cellular profile and cytokine production at prosthetic interfaces: STUDY OF TISSUES RETRIEVED FROM REVISED HIP AND KNEE REPLACEMENTS

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.

Photovoltaic retinal prosthesis with high pixel density

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/mm2, two orders of magnitude below the ocular safety limit. Neural responses were elicited by illuminating a single 70 μm bipolar pixel, demonstrating the possibility of a fully-integrated photovoltaic retinal prosthesis with high pixel density.

Tissue Damage by Pulsed Electrical Stimulation

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 mum to 1 mm, pulse duration from 6 mus 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 mum, and scaled as 1/r2 for electrodes smaller than 200 mum. 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 mus. 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.

Heterogeneity in cellular and cytokine profiles from multiple samples of tissue surrounding revised hip prostheses

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.

Photovoltaic restoration of sight with high visual acuity.

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.

The chick chorioallantoic membrane as a model tissue for surgical retinal research and simulation.

Purpose: 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. Methods: 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. Results: The CAM’s ease of use, low cost, and anatomic structure make it a convenient model for surgical retinal and retinal vascular modeling. Conclusion: 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.

Loosening and osteolysis of cemented joint arthroplasties : A biologic spectrum

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.

Photovoltaic retinal prosthesis : implant fabrication and performance

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.