Immunomodulatory effects of vitamin D: implications for GVHD.

GVHD remains a major source of morbidity and mortality after allogeneic BMT. GVHD is mediated by alloreactive T cells derived from the hematopoietic graft that target host tissues. Pre-clinical models have shown that presentation of alloantigens by host DCs results in the activation of donor-derived T cells that mediate GVHD. Strategies that interfere with the Ag-presenting capacity of DCs after allogeneic transplantation may decrease the risk of developing GVHD. Vitamin D is a hormone essential for calcium metabolism that shows immunomodulatory properties. We showed that correction of vitamin D deficiency appeared to mitigate manifestations of GVHD. In pre-clinical studies, we have shown that vitamin D inhibits DC maturation, polarizes T-cell populations toward the expression of Th2 as compared with Th1 cytokines, and blunts allogeneic T-cell proliferation in response to DC stimulation. Exposure to vitamin D resulted in increased expression of IDO, an enzyme responsible for tryptophan metabolism that is upregulated in tolerizing DCs. These data suggest that exposure to vitamin D results in immature DC populations that bias toward tolerizing rather than stimulatory T-cell populations. Vitamin D may therefore have a role in the prevention of GVHD.

Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its two receptor tyrosine kinases, Flk-1/KDR and Flt-1, may play an important role in mediating the revascularization of transplanted pancreatic islets. METHODS: Using semiquantitative multiplex reverse-transcribed polymerase chain reaction we determined the gene expression of VEGF and its receptors in cultured and transplanted rat islets. RESULTS: After exposure of islet cells to hypoxia in vitro, increases were found in the gene expression of the VEGF120 and VEGF164 isoforms, with simultaneous increases in VE-cadherin, Flk-1/KDR, and Flt-1. In vivo studies consisted of analysis of islet grafts transplanted into both normal and diabetic recipients. Expression of both VEGF120 and VEGF164 in grafts was up-regulated for the first 2-3 days after transplantation, with the response being more prolonged in the diabetic rats. These increases were followed by reduced expression of VEGF on days 5, 7, and 9. Increases in the expression of VE-cadherin in islet grafts in normal and diabetic recipients tended to parallel VEGF expression, with the increases in both probably being caused by hypoxia. The early increases of VEGF expression were followed by a rise in the expression of VEGF receptors, which probably represents the early stages of angiogenesis. Graft expression of Flk-1/KDR and Flt-1 was enhanced at 3 and 5 days in the normoglycemic recipients, while in the diabetic recipients increases were found later on days 5, 7, and 14. CONCLUSIONS: The delayed expression of VEGF receptors in the diabetic recipients could reflect impaired angiogenesis caused by the diabetic milieu; this delay could contribute to the less outcomes of grafts transplanted into a hyperglycemic environment.

Effects of diabetes and hypoxia on gene markers of angiogenesis (HGF, cMET, uPA and uPAR, TGF-alpha, TGF-beta, bFGF and Vimentin) in cultured and transplanted rat islets.

AIMS/HYPOTHESIS: The vascularisation of newly transplanted islets originates from the recipients. Because islets transplanted into a diabetic do less well than those transplanted into a euglycaemic environment, we examined the hypothesis that gene expression of angiogenic factors in grafts is delayed in diabetes. These factors include hepatocyte growth factor (HGF) and its receptor c-MET, and urokinase plasminogen activator (uPA) and its receptor uPAR, basic fibroblast growth factor (bFGF), TGF-alpha and TGF beta-1. METHODS: Isolated rat islets were studied in vitro under normoxic and hypoxic culture conditions and gene expression was determined with semi-quantitative multiplex RT-PCR. We found that HGF but not c-MET expression was induced by hypoxia in vitro. Using syngeneic Lewis rats, gene expression was also studied in grafts on days 1, 3, 5, 7 and 14 after transplantation. RESULTS: In grafts of normoglycaemic rats, HGF expression was enhanced on day 3 and maintained whereas expression of c-MET fell and remained down until day 14. Expression of uPA was up at day 3 and remained high; expression of uPAR was also up at day 3 but then fell to control levels at day 14. Expression of bFGF, TGF-alpha and TGF beta-1 persisted throughout. Vimentin, a marker of fibroblasts, had increased expression at day 1 which was further enhanced in subsequent days. In the grafts of diabetic recipients the expression of HGF, uPA and uPAR were delayed, being clearly expressed at day 5 rather than day 3. Vimentin expression was similarly delayed. CONCLUSION/INTERPRETATION: This apparent delay in angiogenesis provides a potential mechanism for the less favourable outcomes of islets transplanted into diabetic recipients.

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells.

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.

Essential role of vif in establishing productive HIV-1 infection in peripheral blood T lymphocytes and monocyte/macrophages.

The role of vif during the establishment of human immunodeficiency virus type 1 (HIV-1) infection of peripheral blood T lymphocytes and monocyte/macrophages was investigated using vif mutants of three HIV-1 proviral DNAs. Vif was found to be essential for the establishment of productive HIV-1 infection in peripheral blood T lymphocytes after cell-free infection with HXB2 and DFCI-HD, a vpr-positive, vpu-positive, nef-positive derivative of HXB2. A chimeric HIV-1 provirus in which the T-cell line-tropic env sequences in DFCI-HD were replaced with the macrophagetropic env of the ADA strain was constructed for studies on the role of vif during the establishment of HIV-1 infection in primary monocyte/macrophages. These studies showed that vif is also essential for the initiation of productive HIV-1 infection in primary monocyte/macrophage cultures after cell-free virus transmission. The DFCI-HD-ADA virus was shown to replicate in the CD4+ T-cell line Molt 4 clone 8 but not in other T-cell or monocytic cell lines, as previously shown for another macrophagetropic strain YU-2 (1), suggesting that this cell line may be useful for future studies on at least some macrophagetropic strains of HIV-1. The finding that vif is essential for the establishment of productive HIV-1 infection in primary T lymphocytes and monocyte/macrophages suggests that vif may be required for HIV-1 transmission and disease pathogenesis during natural infections and thus may be a good target for prophylactic or therapeutic intervention.

Changes in the cytopathic effects of human immunodeficiency virus type 1 associated with a single amino acid alteration in the ectodomain of the gp41 transmembrane glycoprotein.

A human immunodeficiency virus type 1 mutant with a single amino acid change (designated 596 W/M) in the ectodomain of the gp41 transmembrane envelope glycoprotein replicated in T-cell lines and in CD4-positive peripheral blood mononuclear cells identically to the wild-type virus. However, the cytopathic effects associated with infection by the mutant virus were altered, with a marked attenuation of syncytium formation and a significant delay in single-cell lysis relative to those of the wild-type virus-infected culture. The 596 W/M mutant is apparently defective in a function that is dispensable for virus entry but that contributes to the efficiency of induction of viral cytopathic effects.

Normalization of hyperglycemia in diabetic rats by intraportal transplantation of cryopreserved islets from four donors.

Techniques for freezing rat islets have been examined by the intensive use of the supravital stains fluorescein diacetate and ethidium bromide. By the use of a simple scoring system, the effect of the cooling rate, treatment with dimethyl sulfoxide (DMSO), rate of thawing, and postthaw culture were examined. These studies showed the most effective method to be a 24-h culture of islets, followed by partial incubation with 20% DMSO at 0 degrees C, followed by seeding at -8 degrees C in an alcohol bath. The islets were then cooled at a rate of -0.25 degrees C/min to -40 degrees C followed by quenching in liquid nitrogen at -196 degrees C. Rapid thawing at 37 degrees C was then followed by a 24-h culture. Islets from four Lewis rat donors were cryopreserved, counted, and transplanted intraportally into streptozocin-induced diabetic Lewis rats. Corresponding control transplants were performed with islets from four donors only cultured for 48 h. The results showed that reversal of hyperglycemia in severely diabetic rats was obtained at 5, 5, 6, 6, 6, or 8 days with cryopreserved islets from four donors, compared to reversal of diabetes at 1, 4, 5, 6, 7, and 12 days with islets from four donors subjected to culture alone. The new cryopreservation technique has several small modifications over previously described methods and results in a significant improvement in islet survival.

The relevance of pressure changes in transplanted hearts and kidneys in immunosuppressed rats.

The pressure within rat cardiac and renal allografts has been observed to rise during rejection. We wished to see if this pressure rise could be prevented or reversed with immunosuppression. Transplants were performed from Lewis or DA donors to Lewis recipients. The rats received either a heterotopic cardiac transplant or an orthotopic renal transplant and were then treated with different immunosuppressive protocols. Intramyocardial pressure was recorded using a fine-gauge needle connected to an air pressure manometer. In the case of the renal transplants, a pressure transducer was used as well and the two methods compared. Intramyocardial and intrarenal pressures rose dramatically in unimmunosuppressed recipients of DA allografts. No such rise was seen in isografted organs, although the pressures recorded remained significantly higher than those found in untransplanted hearts and kidneys. Cyclosporine 20 mg/kg/day was effective in suppressing rejection in both models, and inhibited any rise in intraorgan pressure. Cyclosporine 10 mg/kg/day was less effective, and with 2 mg/kg/day allograft function was considerably impaired, one-third of the cardiac grafts being rejected by 16 days. In both models intraorgan pressures became raised. The addition of methylprednisolone 16 mg/kg i.p. on days 7 and 8 to this low dose regimen of cyclosporine 2 mg/kg/day rapidly reversed the rise in pressure and restored graft function to normal. Intraorgan pressure levels therefore accurately reflected the state of function of transplanted hearts and kidneys. When a manometer and a transducer were compared as a means of measuring the pressure in the renal transplants, the manometer method was found to be superior.

Intraorgan pressure changes in rejecting kidney, liver, and heart transplants in the rat.

Intraorgan pressure was measured in DA kidneys, livers, and hearts after transplantation into Lewis rats. Recordings were obtained by passing a fine needle into the graft and measuring the resistance to the flow of saline. Rats with organ isografts (Lewis-to-Lewis) were used as controls, and a further control group comprised Lewis rats that had undergone left ureteric ligation. Following transplantation a rise in pressure was recorded from some of the isografts, it returned to base line levels by the second or third day. Similar changes were observed in the organ allografts, except that a second, much greater (P less than .05) rise soon followed. This was observed from day 3 onward in the case of the kidney and liver transplants, and from day 4 in the cardiac transplants. Control experiments showed that ligation of the ureter (as performed in all the kidney transplants) did not influence intrarenal pressure. It is concluded that a rise in intraorgan pressure is an early feature of rejection, and that pressure monitoring might be useful in patients with cardiac or liver transplants--in addition to those with kidney transplants in whom the test has been shown to be of value.