Retinal safety of near infrared radiation in photovoltaic restoration of sight.

Photovoltaic restoration of sight requires intense near-infrared light to effectively stimulate retinal neurons. We assess the retinal safety of such radiation with and without the retinal implant. Retinal damage threshold was determined in pigmented rabbits exposed to 880nm laser radiation. The 50% probability (ED50) of retinal damage during 100s exposures with 1.2mm diameter beam occurred at 175mW, corresponding to a modeled temperature rise of 12.5°C. With the implant, the same temperature was reached at 78mW, close to the experimental ED50 of 71mW. In typical use conditions, the retinal temperature rise is not expected to exceed 0.43°C, well within the safety limits for chronic use.

Pulsed electron avalanche knife (PEAK) for intraocular surgery.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

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.

Allogeneic bone marrow cells that facilitate complete chimerism and eliminate tumor cells express both CD8 and T-cell antigen receptor-alphabeta.

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)

Chronic antigen-specific immune-system activation may potentially be involved in the loosening of cemented acetabular components.

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.

Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation.

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.

Expression of insulin-like growth factor-I and transforming growth factor-beta in hypokalemic nephropathy in the rat.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Set domain-dependent regulation of transcriptional silencing and growth control by SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9.

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.

Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation.

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.

The characterization of macrophages and osteoclasts in tissues harvested from revised total hip prostheses.

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.

Comparison of chimeric acid and non-chimeric tolerance using posttransplant total lymphoid irradiation: cytokine expression and chronic rejection.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Monocyte-derived dendritic cell precursors facilitate tolerance to heart allografts after total lymphoid irradiation.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The intravenous injection of donor dendritic cell precursors derived from blood monocytes facilitates long-term acceptance of heart allografts.

Nature of glomerular dysfunction in pre-eclampsia.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

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.

Subsets of transgenic T cells that recognize CD1 induce or prevent murine lupus: role of cytokines.

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.

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.

RANTES chemokine expression in transplant-associated accelerated atherosclerosis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mechanisms of tolerance to rat heart allografts using posttransplant TLI. Changes in cytokine expression.

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.

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.

Cardia allograft unresponsiveness using a posttransplant strategy : characterization of the graft infiltrate.

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.