Lacto-N-fucopentaose III, a pentasaccharide, prolongs heart transplant survival.

BACKGROUND: Lacto-N-fucopentaose III (LNFPIII) is a pentasaccharide containing the Lewis(x) trisaccharide that is found on schistosome eggs and in breast milk. LNFPIII conjugates suppress host immune responses and have therapeutic efficacy in mouse models of psoriasis and type 1 diabetes. METHODS: We used nonvascularized neonatal ear-heart transplantation and heterotopic vascularized heart transplantation models to evaluate immunosuppressive effects of LNFPIII and subsequently analyzed the mechanism. RESULTS: We found that administration of LNFPIII conjugates prolonged median graft survival by 80% when 1-day-old DBA/2 hearts were transplanted into ears of B6 mice. A similar graft prolongation was observed in a fully vascularized heterotopic heart transplantation model (DBA/2 into B6), No prolongation was observed with carrier protein (human serum albumin [HSA] or dextran) alone. We found increased programmed death ligand 1 (PD-L1) expression on F4/80 macrophages, CD4+ T cells, and CD11b+ CD11c+ (myeloid) dendritic cells, and increased arginase1 and Ym1 expression, typical of alternatively activated macrophages, in the draining (cervical) lymph node cells. We found accumulation of Foxp3+ regulatory T cells (Tregs) in the lymph nodes draining donor hearts, suggesting a possible role of Treg induction in graft prolongation. Anti-PD-L1 antibody treatment abrogated LNFPIII-mediated the graft survival benefit and Treg accumulation. LNFPIII-treated macrophages had increased PD-L1 expression and significantly prolonged DBA/2 allograft survival when injected intraperitoneally into B6 recipient mice. CONCLUSIONS: LNFPIII prolongs fully allogeneic graft survival in both vascularized and nonvascularized allograft transplantation models. The mechanism of graft prolongation seems to involve both alternatively activated PDL-1 macrophages and recruitment of Foxp3+ Treg cells.

A simplified approach to human islet quality assessment.

BACKGROUND: The successful treatment of patients with type 1 diabetes by islet transplantation is affected by a multitude of factors of which infusion of the highest quality tissue is essential. The current standard pretransplant quality assessments lack sensitivity, accuracy, and objectivity in the determination of islet viability and potency. We hypothesized that a multiparametric approach focused on islet cell metabolic state, mitochondrial integrity, and in vitro glucose-stimulated insulin secretion (GSIS) could provide data predictive of in vivo function. The objective of this study was to validate a novel set of islet quality assays and develop a simplified islet quality scoring system for both basic research and clinical applications. METHODS: A series of 42 human islet preparations were screened using standard and novel methods, which included determination of yield, viability by fluorescent microscopy, GSIS, percentage of islet loss in culture, quantification of adenine nucleotides, flow cytometric measurement of viability, apoptosis, and mitochondrial membrane potential (MMP). In vivo functional potency was tested by minimal model transplant in streptozotocin-induced diabetic NOD.scid mice. RESULTS: Functionally potent islet preparations showed significantly greater numbers of cells with polarized MMP, higher ATP-to-ADP ratios, and increased glucose-induced insulin secretion. The MMP, ATP-to-ADP ratio, and GSIS data were combined into a single islet scoring formula that showed more than 86% accuracy in predicting in vivo functional potency. CONCLUSIONS: Our study demonstrates that a multiparametric approach using objective assessments focused on islet cell mitochondrial integrity and in vitro function can provide data predictive of in vivo function.

Antisense to transforming growth factor-beta(1) facilitates the apoptosis of macrophages in rat vein grafts.

BACKGROUND: The success of peripheral vein grafts is limited by intimal hyperplasia. Transforming growth factor (TGF)-beta(1) has effects on cell proliferation, apoptosis and extracellular matrix synthesis. We have previously observed positive changes in vessel healing with antisense to TGF-beta(1). METHODS: Adenovirus was used to transduce rat femoral artery vein grafts with antisense to TGF-beta(1) (Ad-AST) or the sequence encoding the bioactive form of TGF-beta(1) (Ad-BAT). Grafts were harvested at 1, 2, 4 and 12 weeks and formalin fixed for immunohistochemical studies of the cell markers proliferating cellular nuclear antigen (proliferation) and active caspase 3 (apoptosis). In situ DNA fragmentation assays were also performed to confirm active caspase 3 results. RESULTS: Ad-AST treatment significantly (p = 0.05) increased apoptosis of macrophages inside the internal elastic lamina. In addition, Ad-AST treatment significantly increased the cellularity of the graft at early time points and reduced it at later time points (p = 0.01). CONCLUSION: The low levels of TGF-beta(1) in Ad-AST treatment promote apoptosis of macrophages and provide an environment that is more conducive to the proliferation or infiltration of cells that contribute to healthy vessels.

Transforming growth factor-beta1 antisense treatment of rat vein grafts reduces the accumulation of collagen and increases the accumulation of h-caldesmon.

BACKGROUND: The main cause of occlusion and vein graft failure after peripheral and coronary arterial reconstruction is intimal hyperplasia. Transforming growth factor beta-1 (TGF-beta1) is a pleiotropic cytokine known to have powerful effects on cell growth, apoptosis, cell differentiation, and extracellular matrix synthesis. METHODS: To investigate the role of TGF-beta1 in intimal hyperplasia, we used adenovirus to deliver to superficial epigastric vein messenger RNA (mRNA) antisense to TGF-beta1 (Ad-AST) or the sequence encoding the bioactive form of TGF-beta1 (Ad-BAT). Infection with "empty" virus was used as a control (Ad-CMVpLpA). The treated vein was then used for an interposition graft into rat femoral artery. Grafts were harvested at 1, 2, 4, and 12 weeks and formalin-fixed for histologic studies or placed in liquid nitrogen for mRNA studies. RESULTS: Ad-AST treatment resulted in an overall reduction of TGF-beta1 expression (P = .001), and Ad-BAT treatment resulted in an overall increase in TGF-beta1 expression (P = .007). Histologic analysis showed Ad-AST caused reduced collagen build up in the neointima at 12 weeks (P = .0001). Immunohistochemical staining for h-caldesmon at 12 weeks indicated Ad-AST increased smooth muscle cells throughout the vessel wall compared with Ad-CMVpLpA (P = .0024) or Ad-BAT (P = .04). Ad-AST also resulted in reduced CD68-positive cells in the media/adventitia (P = .005 vs Ad-CMVpLpA, P = .01 vs Ad-BAT). To further understand how Ad-AST was influencing the build up of collagen, we performed quantitative polymerase chain reaction on complimentary DNA (cDNA) from homogenates of the vein grafts. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) was increased at 1 week by Ad-BAT (P = .048 vs Ad-CMVpLpA) and decreased by Ad-AST at all time points (P

Validation of large particle flow cytometry for the analysis and sorting of intact pancreatic islets.

BACKGROUND: Accurate quantification of total islet yield is an essential step prior to transplantation and for research. The standard method of manually determining an islet equivalent (IEQ) count is subjective and prone to error. We evaluated Complex Object Parametric Analyzer and Sorter (COPAS) large particle flow cytometry for the determination of islet equivalent counts and purities of islet preparations. METHODS: Initial validation of the sensitivity and accuracy of the COPAS flow cytometer was performed by analysis and sorting of uniform polystyrene microspheres with sizes similar to islets. Human and Rhesus monkey islets were stained with the zinc-specific fluorescent dye Newport Green to discriminate islet from nonislet tissue. Islet sizes were extrapolated from standard curves obtained using microspheres from which individual islet volumes were calculated. IEQ counts on six islet preparations were performed by the standard manual method and compared with results obtained by automated COPAS flow cytometry. RESULTS: The COPAS flow cytometer was highly accurate in the detection and measurement of both polystyrene microspheres and islets. IEQ counts determined by COPAS flow cytometry were consistent with manual counts although subject to error when assessing preparations with significant numbers of islets embedded within acinar tissue. Size-specific islet sorting with retention of morphology and dithizone staining was also shown using the COPAS flow cytometer. CONCLUSIONS: COPAS large particle flow cytometry provides a novel automated approach for quantification of intact islets and determination of islet equivalent yield. In addition, the ability to analyze and sort islets based upon user defined criterion opens unique avenues for experimentation.

Prevention of chronic allograft nephropathy with vitamin D.

Chronic allograft nephropathy (CAN) is the leading cause of late allograft loss in kidney transplantation. Interstitial fibrosis and glomerulosclerosis are characteristic of CAN. Transforming growth factor beta-1 (TGFbeta-1) is associated with both of these histologic findings in the transplant setting. Recent studies have suggested that vitamin D signaling pathways may interact with and regulate TGFbeta-1 mediated events. We examined the efficacy of 1,25-dihydroxyvitamin D(3), the active metabolite of vitamin D [1,25-(OH)(2)D(3)], the active metabolite of vitamin D, as monotherapy to prolong allograft survival and preserve renal function in a rat model of CAN, the Fisher 344 to Lewis model. Recipients went without treatment or were treated with cyclosporine A (CSA; 10 days) or 1,25(OH)(2)D(3) (1000, 500 or 250 ng/kg/day). Grafts were harvested at the time of rejection or at 24 weeks post-transplant. A portion of the graft was processed for histology and immunohistochemistry and a second portion was analyzed for protein expression by western blotting. Not only did 1,25-(OH)(2)D(3) treatment significantly prolong graft survival, but it also prevented histological changes associated with CAN. 1,25-(OH)(2)D(3) treatment significantly decreased Smad 2 expression. This TGFbeta signaling molecule is likely involved in fibrosis. Moreover, 1,25-(OH)(2)D(3) treatment increased Smad 7 expression, an important feedback molecule in the TGFbeta-1 signaling pathway. This suggests that 1,25-(OH)(2)D(3) interacts with TGFbeta-1 in limiting histological injury in this model of CAN. Furthermore, 1,25-(OH)(2)D(3), treatment increased expression of matrix metalloproteinase 2 (MMP-2), thus directly affecting levels of another important matrix molecule. Taken together our data suggests that 1,25-(OH)(2)D(3) mitigates CAN in this model by altering TGFbeta-1 and matrix-regulating molecules.

ATR blockade reduces IFN-gamma production in lymphocytes in vivo and in vitro.

BACKGROUND: Type 1 angiotensin II (Ang II) receptor (AT(1)R) signaling induces proinflammatory responses. Recent studies suggest that T lymphocytes express AT(1)R; yet the effects of Ang II binding to AT(1)R on T cells are poorly understood. We examined the effect of AT(1)R blockade on release of the proinflammatory cytokine, interferon-gamma (IFN-gamma) by human lymphocytes in vivo and in vitro. METHODS: We used an AT(1)R blocker losartan in a randomized clinical trial in kidney transplant recipients over a 12-month period [AT(1)R blocker (N= 11) and control (N= 10)]. Peripheral blood lymphocytes, isolated from both cohorts, were analyzed by enzyme-linked immunosorbent spot assays (ELISPOT) analyses and real-time reverse transcription-polymerase chain reaction (RT-PCR) to enumerate IFN-gamma producing T cells and IFN-gamma mRNA levels. The effects of AT(1)R blockade in vitro were assessed using human alloreactive T cells and an IFN-gamma producing human cytotoxic T-lymphocyte line. Alloreactive T cells were treated with losartan or candesartan and enzyme-linked immunosorbant assay (ELISA) was used to measure IFN-gamma protein release. The cytotoxic T-lymphocyte line also was AT(1)R blocker-treated prior to determining IFN-gamma producing cells by intracellular cytokine staining. RESULTS: The AT(1)R blocker cohort had a significant decrease in IFN-gamma producing peripheral blood lymphocytes (P< or = 0.05 for each time point) and IFN-gamma mRNA levels (P= 0.01 vs. control patients). Losartan also decreased IFN-gamma production (P < 0.001) in purified alloreactive T cells in vitro as did candesartan. Moreover, Ang II amplified IFN-gamma generation (P < 0.05) in alloreactive T cells while AT(1)R blocker treatment inhibited Ang II's effect (P < 0.04). AT(1)R blocker treatment furthermore also inhibited IFN-gamma production in the cytotoxic T-lymphocyte line. CONCLUSION: AT(1)R blockers may have a clinically relevant immunomodulatory role by blocking IFN-gamma production in T cells.

Antisense to transforming growth factor-beta1 messenger RNA reduces vein graft intimal hyperplasia and monocyte chemotactic protein 1.

BACKGROUND: Autogenous vein grafts are commonly used for arterial reconstructive procedures. Their success is limited by the development of intimal hyperplasia (IH), a fibroproliferative disease that predisposes the grafts to occlusive stenosis. Mesenchymal cell proliferation and the deposition of an extracellular matrix characterize neointimal development. Increasing evidence suggests that, regardless of blood vessel type, IH results from complex interactions among vessel wall cells, infiltrating leukocytes, and cytokines. Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with powerful effects on inflammatory cell chemotaxis; smooth muscle cell, fibroblast, and endothelial cell proliferation; and extracellular matrix synthesis. METHODS: Epigastric vein to common femoral artery interposition grafts were placed in male Lewis rats and harvested at 1, 2, 4, and 12 weeks after surgery. We used replication-defective adenoviruses to deliver a control reporter gene for the enzyme beta-galactosidase (Ad-GAL), empty virus (Ad-CMVpLpA), or the sequence encoding the antisense strand of TGF-beta1 (Ad-AST). The vein graft was transduced passively in medium containing 10 7 plaque-forming units per milliliter of Ad-GAL, Ad-CMVpLpA, or Ad-AST for 20 minutes at room temperature. The adenovirus-treated grafts were compared with grafts treated with medium without virus (sham). RESULTS: The Ad-GAL control grafts showed beta-galactosidase activity from 3 days to 4 weeks. Twenty percent of cells were positive out to 2 weeks, at which time the number of cells positive for beta-galactosidase activity began to decline. Treatment with Ad-AST resulted in a significant reduction vs sham, Ad-CMVpLpA, and Ad-GAL in TGF-beta1 messenger RNA, total TGF-beta1 protein, and bioactive TGF-beta1 protein. Neointimal area was significantly reduced in the Ad-AST group vs Ad-GAL at 4 weeks, vs Ad-CMVpLpA at 4 and 12 weeks, and vs sham at 2 and 4 weeks. The medial/adventitial layer was significantly thicker in the Ad-AST group than the Ad-GAL group at 12 weeks. In addition, we studied the effect of Ad-AST on monocyte chemotactic protein 1 (MCP-1). Although the reduction in TGF-beta1 resulted in a reduction of MCP-1 messenger RNA in whole-graft homogenates and MCP-1 protein-positive staining in histologic sections from the perianastomotic region, no reduction in the number of ED1-positive cells (monocytes and macrophages) was observed. CONCLUSIONS: Perioperative antisense TGF-beta1 treatment of the vein to be used in arterial reconstructions resulted in a prolonged diminution of IH; this emphasizes the importance of TGF-beta1 in neointimal thickening and indicates that ex vivo gene therapy can reduce the vessel's predisposition to IH. CLINICAL RELEVANCE: The main cause of occlusion and graft failure after peripheral and cardiac arterial reconstruction is IH. The study of the mechanisms and mediators of IH, including TGF-beta1, should lead to future gene therapies to prevent or limit IH. The clinical effect of such treatments would be enormous, because they would increase graft longevity, thereby enhancing quality of life and enabling patients to live without the threat of limb loss or recurrent heart attack.

Donor genomics influence graft events: the effect of donor polymorphisms on acute rejection and chronic allograft nephropathy.

BACKGROUND: Organs procured from deceased donors emanate from individuals with diverse genetic backgrounds. Donor organs, therefore, may vary in their response to injury and immune stimuli in a genetically determined manner. We assessed polymorphisms from 244 renal allograft donors to better understand the impact of donor polymorphisms on selected transplant outcomes. METHODS: Donor genomic DNA restriction fragment length polymorphisms were assayed for evidence of common cytokine [interleukin (IL)-2, IL-6, IL-10, tumor necrosis factor (TNF)-alpha, TGF-beta, interferon (IFN)-gamma] and chemokine (CCR2, CCR5) polymorphisms. Associations between donor polymorphisms and graft events were determined using chi-square, linear regression, and Kaplan-Meier analyses. RESULTS: Several genotypic polymorphisms demonstrated a modest association with acute rejection, including the transforming growth factor (TGF)-beta T/C codon 10 (P= 0.027) and the CCR5 G/A 59029 (P= 0.039) genes by chi-square analysis. Notably, the presence of the T allele in the IFN-gamma gene (+874) demonstrated a highly significant association with biopsy-proven chronic allograft nephropathy (P < 0.008). This association remained highly significant in a multiple linear regression model that incorporated biopsy-proven acute rejection as a covariate. CONCLUSION: These data suggest that many of the donor polymorphisms studied in this analysis may influence a recipient's immune response to a renal allograft. However, their greatest impact may be demonstrated in long-term outcomes.

Modified technique of ureteroureterostomy in rat kidney transplantation.

Different strain combinations of rats are available to study immunological and transplant-related problems in the models of kidney transplantation. Although numerous modifications of surgical techniques for ureteric reconstruction are evaluated in order to reduce complications and to extend long-term survival, ureteric complications still occur frequently, especially when the difference in diameter of both donor and host ureters is disproportionate. Instead of using the current nonsplinted ureteroureterostomy (method A), a versatile and rapid technical modification (method B) was developed to perform reconstruction of ureters with disproportionate diameters. The overall incidence of ureteric complications was 80% (8/10) using method A, whereas this rate was significantly reduced to 15% (3/20) using method B (P < 0.001). Our modification proves the feasibility of nonsplinted ureteroureterostomy in a technical, highly demanding rat model of kidney transplantation with an acceptable rate of ureteric complication, considering the disproportionate difference in diameter between the host and donor ureters.

Macrophage depletion reduces monocyte chemotactic protein-1 and transforming growth factor-beta1 in healing rat vein grafts.

OBJECTIVE: We previously showed that treatment with liposomally encapsulated dichloromethylene bisphosphonate reduces intimal hyperplasia development and macrophage accumulation in a rat epigastric vein to femoral artery model of intimal hyperplasia. Our objective in this study was to determine the effect of liposomally encapsulated dichloromethylene bisphosphonate on the expression of two cytokines essential to neointimal development, monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-beta). METHODS: We injected rats both 2 days preoperatively and 2 weeks postoperatively with liposomally encapsulated dichloromethylene bisphosphonate (Lip-Clod), liposomally encapsulated phosphate-buffered saline solution (Vector), or phosphate-buffered saline solution (PBS), and harvested the grafts at 1 and 4 weeks. In the perianastomotic region, MCP-1 and TGF-beta protein expression in the total graft cross-section and in the neointima was determined with immunohistochemistry. In whole-graft lysates, MCP-1 and TGF-beta protein were determined with an enzyme-linked immunosorbent assay, and messenger RNA expression was determined with reverse transcription quantitative polymerase chain reaction. RESULTS: Lip-Clod treatment reduced intimal hyperplasia when compared with Vector or PBS treatment. These reductions were significant (P<.05) at both time points. When compared with the PBS treatment, at 1 week but not at 4 weeks Lip-Clod reduced both MCP-1 and TGF-beta protein (P< or =.01 and P< or =.006) in the perianastomotic region of vein grafts. In whole-graft lysates, no significant difference was seen in MCP-1 protein at either time point; however, TGF-beta protein expression was significantly reduced at both 1 and 4 weeks (P=.02 and P=.004). Message analysis in whole-graft lysates at 1 week showed that MCP-1 message expression increased in the Lip-Clod group compared with the PBS group (P=.02), but no significant differences among groups for TGF-beta message levels. Results with Vector were often intermediate to results with Lip-Clod and PBS. CONCLUSION: The major effect of Lip-Clod treatment on TGF-beta and MCP-1 protein levels in the perianastomotic region is observed at 1 week, and macrophage depletion with Lip-Clod inhibits graft neointimal hyperplasia and TGF-beta protein expression in whole-graft lysates at 1 and 4 weeks. These results support the concept that the infiltrating macrophages contribute a significant portion of the cytokines that facilitate intimal hyperplasia and that reducing these cytokines early after grafting influences the development of intimal hyperplasia at later time points. CLINICAL RELEVANCE: All vascular surgeons have patients who have undergone a technically satisfying vein graft, only to have the bypass fail during the first year due to perianastomotic intimal hyperplasia (IH). We hypothesize that vein graft IH is analogous to aberrant wound healing. Central to wound healing is the recruitment of macrophages with their cytokines. This work raises the question whether clinical strategies designed to either decrease macrophages or the cytokines released by macrophages at the time of vein graft placement will be efficacious for limiting the development of IH.

Xenogeneic transplantation of porcine islets: an overview.

The extreme demand for human organs or tissues for transplantation has driven the search for viable alternatives. Pigs are considered a possible source of tissue for a number of reasons including shared physiology, plentiful supply, short gestation, and, more recently, the generation of transgenic animals. Porcine islets show promise as a source of islets for the treatment of type 1 diabetes mellitus. Porcine islets regulate glucose levels in the same physiologic range as humans, and porcine insulin has been used for years as an exogenous source of insulin for glucose control. In this review, we discuss the advantages and disadvantages of the use of adult or neonatal porcine islets, the immunologic challenges facing transplantation of xenogeneic islets, and the concerns regarding transmission of infectious agents between species. Porcine islets isolated from both adult and neonatal pigs are capable of restoring euglycemia in experimental animal models of diabetes. Adult islets are more difficult to isolate, whereas neonatal islets have great proliferation potential but require several weeks to function posttransplantation. Xenogeneic islets are susceptible to complement-mediated lysis after the binding of preformed natural antibodies and cellular immunity involving both macrophages and CD4+ T cells. In addition, the potential for transmission of porcine endogenous retroviruses, porcine cytomegalovirus, and porcine lymphotropic herpesvirus type 1 are all concerns that must be addressed. Despite the challenges facing xenotransplantation, the extreme need for donor organs and tissues continues to drive progress toward overcoming the unique issues associated with transplantation between species.

Beta-galactosidase-tagged adventitial myofibroblasts tracked to the neointima in healing rat vein grafts.

OBJECTIVE: Myofibroblasts are present transiently in normal healing wounds. However, they have been found to persist in the stroma of neoplasms, fibrotic conditions and other pathological settings. In rat vein grafts, we have observed the prolonged presence of myofibroblasts. Our aim was to determine the origin of myofibroblasts in vein grafts. METHODS: Epigastric vein to femoral artery grafts were microsurgically placed in male Lewis rats and harvested. Neointimal development, cellular death and proliferation, and cell phenotypes were analyzed using immunohistochemistry and light and electron microscopy. To follow cellular movement in the vessel wall, vein grafts were transfected with replication-defective adenovirus containing the gene encoding beta-galactosidase (n = 50), and harvested at 1, 2, 3, 4, 5, 6, 7, 14 and 28 days. Grafts were analyzed after X-gal staining. RESULTS: Myofibroblasts were detected in the outer adventitia at 4 days, in the media at 1 week and in the developing neointima at 2 weeks. Cells tagged using adenoviral beta-galactosidase demonstrated adventitia to neointima cell migration. CONCLUSIONS: Although there may be other sources of myofibroblasts in this model, the adventitia has been shown to be an origin of myofibroblasts which subsequently migrate through the vessel wall to the neointima during graft remodeling and contribute to neointimal formation.

Pancreatic precursors and differentiated islet cell types from murine embryonic stem cells: an in vitro model to study islet differentiation.

Embryonic stem (ES) cells differentiating in vitro reproduce many facets of early embryonic development, including the expression of developmentally regulated transcription factors and the differentiation of multipotent precursor cells. ES cells were evaluated for their ability to differentiate into pancreatic and islet lineage-restricted stages including pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic precursor cells, early endocrine cell progenitors, and islet hormone-producing cells. Following growth and differentiation in nonselective medium containing serum, murine ES cells spontaneously differentiated into cells individually expressing each of the four major islet hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. PDX1 immunostaining cells appeared first, before hormone-positive cells had emerged. Hormone-positive cells appeared within focal clusters of cells coexpressing PDX1 and the nonclassical hormone markers peptide YY (YY) and islet amyloid polypeptide (IAPP) in combination with the definitive hormones, characteristic of endocrine cells appearing during early pancreaticogenesis. This system allows the investigation of many facets of islet development since it promotes the appearance of the complete range of islet phenotypes and reproduces important developmental stages of normal islet cytodifferentiation in differentiating ES cell cultures.

Vitamin D as immunomodulatory therapy for kidney transplantation.

Vitamin D (1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]) has been studied in the past for its immunosuppressive properties, and, in that context, it may also have potential utility as an immunomodulatory agent for transplantation. A number of studies have demonstrated that 1alpha,25-(OH)(2)D(3) or its analogs regulate immune cell proliferation, differentiation, and responsiveness. A burgeoning number of studies have also explored using 1alpha,25-(OH)(2)D(3) and its analogs directly as therapy in animal models of kidney transplantation with success in prolonging allograft function and preventing acute rejection. Some of these in vivo effects may well be caused by alterations in immune cell function, but it is also possible that exogenous 1alpha,25-(OH)(2)D(3) and its analogs are altering the intragraft milieu as well, specifically through changes in the TGF-beta signaling cascade. Such provocative data and the availability of newer 1alpha,25-(OH)(2)D(3) analogs that may limit side effects (e.g. hypercalcemia) have created interest in examining this secosteroid clinically in kidney transplantation.

A retrospective evaluation of 1,25-dihydroxyvitamin D(3) and its potential effects on renal allograft function.

BACKGROUND/AIMS: 1,25-Dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) plays an important role in regulating immunologic responsiveness in addition to its effects on bone metabolism. This is potentially beneficial in the transplant setting. Animal studies have demonstrated the utility of 1,25-(OH)(2)D(3) in prolonging allograft survival. Therefore, we evaluated the effects of 1,25-(OH)(2)D(3) (oral calcitriol) in human renal transplant recipients. METHODS: A case-control study was undertaken assessing the effects of calcitriol on transplant function. The effect of calcitriol on renal function was analyzed using general linear mixed modeling of the change in slope of serum creatinine (Scr) prior to and following the start of calcitriol therapy. RESULTS: There was a significant increase in baseline Scr (p < 0.001) prior to starting calcitriol. Following initiation of calcitriol, there was a deceleration in the rate of loss of graft function (p = 0.031 at day 300 of therapy). Graft survival was also prolonged in calcitriol-treated patients compared to a control population with evidence of chronic allograft nephropathy but no calcitriol therapy (p < 0.03). Overall, there were no adverse or harmful effects related to calcitriol therapy. 1,25-(OH)(2)D(3) therapy was associated with (1) a deceleration in the rate of loss of renal function in transplant recipients with more than one year of allograft function, and (2) no significant change in allograft function early after transplantation. CONCLUSION: These data suggest that short- and long-term prospective trials evaluating 1,25-(OH)(2)D(3) or 1,25-(OH)(2)D(3) analogs in human kidney transplantation are warranted. Such trials may help us elucidate mechanism and duration of action, as well as safety issues related to these novel immunomodulatory agents.

Cyclooxygenase-2 expression is up-regulated in obstructed human ureter.

PURPOSE: Prostanoids produce significant effects on ureteral function and are synthesized by cyclooxygenase (COX) enzymes. COX is found in the 2 isoforms COX-1 (a constitutive form) and COX-2 (an inducible form). Due to the side effects associated with COX-1 inhibition there is great interest in selective COX-2 inhibition. We determined if COX-2 messenger (m)RNA and protein expression are regulated during ureteral obstruction. MATERIALS AND METHODS: mRNA analysis was performed using excess ureteral segments from 6 patients undergoing reconstructive procedures for chronic ureteral obstruction and 8 (normal ureter) undergoing donor nephrectomy after providing informed consent. All ureteral segments were snap frozen in liquid nitrogen and stored at -70C. RNA was isolated from the segments using phenol extraction and complementary DNA was synthesized by reverse transcription with murine leukemia virus reverse transcriptase (Promega Corp., Madison, Wisconsin). Semiquantitative reverse transcriptase-polymerase chain reaction was performed using specific COX-2 gene primers with ribosomal S26 primers serving as the housekeeping gene. The polymerase chain reaction product was quantified by agarose gel electrophoresis and phospho-imaging. The ratio of COX-2-to-S26 mRNA was compared. Additional segments were homogenized and total protein was extracted, separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. These membranes were Western blotted for COX-2 and glyceraldehyde-3-phosphate dehydrogenase (housekeeping protein) with specific primary and secondary antibodies. RESULTS: The mean ratio of COX-2-to-S26 mRNA plus or minus standard error at 20, 22 and 24 cycles of amplification was 0.22 +/- 0.04 in the 8 normal ureters compared with 1.01 +/- 0.21 in the 6 obstructed ureters (unpaired Student's t test p = 0.004). Similarly the mean ratio of COX-2-to-glyceraldehyde-3-phosphate dehydrogenase protein on immunoblotting was 0.15 +/- 0.02 in the 8 normal ureters compared with 0.59 +/- 0.10 in the 6 obstructed ureters (p = 0.003). CONCLUSIONS: These data indicate that COX-2 mRNA and protein levels are up-regulated in chronically obstructed human ureters. Using selective COX-2 inhibitors may be useful for treating prostanoid induced effects associated with ureteral obstruction.