Bone marrow-derived hematopoietic stem and progenitor cells infiltrate allogeneic and syngeneic transplants.

Lineage (CD3e, CD11b, GR1, B220 and Ly-76) negative hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) infiltrate islet allografts within 24 h posttransplantation. In fact, lineage(negative) Sca-1(+) cKit(+) ("LSK") cells, a classic signature for HSCs, were also detected among these graft infiltrating cells. Lineage negative graft infiltrating cells are functionally multi-potential as determined by a standard competitive bone marrow transplant (BMT) assay. By 3 months post-BMT, both CD45.1 congenic, lineage negative HSCs/HPCs and classic "LSK" HSCs purified from islet allograft infiltrating cells, differentiate and repopulate multiple mature blood cell phenotypes in peripheral blood, lymph nodes, spleen, bone marrow and thymus of CD45.2 hosts. Interestingly, "LSK" HSCs also rapidly infiltrate syngeneic islet transplants as well as allogeneic cardiac transplants and sham surgery sites. It seems likely that an inflammatory response, not an adaptive immune response to allo-antigen, is responsible for the rapid infiltration of islet and cardiac transplants by biologically active HSCs/HPCs. The pattern of hematopoietic differentiation obtained from graft infiltrating HSCs/HPCs, cells that are recovered from inflammatory sites, as noted in the competitive BMT assay, is not precisely the same as that of intramedullary HSCs. This does not refute the obvious multi-lineage potential of graft infiltrating HSCs/HPCs.

Prevention of nonimmunologic loss of transplanted islets in monkeys.

The nonimmunologic loss of islets in the pre-, peri-, and early post-islet transplant periods is profound. To determine the potential role that transplantation of only a marginal mass of functioning beta cells may play in triggering late nonimmunologic graft loss, we studied the effect of treatment with alpha-1-antitrypsin (AAT) in the autologous cynomolgus islet transplant model. A marginal mass of autologous islets, that is islets prepared from 70% to 80% of the pancreas, was transplanted at 1600-4100 IEQ/kg into subtotal pancreatectomized, streptozotocin-treated and insulin-deficient diabetic hosts. In this marginal mass islet transplant model, islet function is insidiously lost over time and diabetes recurs in all untreated monkeys by 180 days posttransplantation. Short-term treatment with AAT, an acute phase reactant, in the peri-transplant period serves to terminate inflammation through effects upon expression of TGFß, NFκB and AKT and favorably altering expression of cell death and survival pathways, as detected by a system biology approach and histology. These effects enabled functional expansion of the islet mass in transplanted hosts such that graft function improves rather than deteriorating over time.

Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term triple therapy.

Preclinical studies in nonhuman primates (NHP) are particularly useful to evaluate the safety and efficacy of new therapeutic proteins developed for use in clinical transplantation. We hypothesized that a treatment that selectively destroys activated cytopathic donor reactive T cells while sparing resting and immunoregulatory T cells in a mouse model might also produce long-term drug-free engraftment and tolerance without the hazards of lymphopenia in the challenging nonhuman primate islet allograft model. Short-term treatment with a regimen consisting of rapamycin, and IL-2.Ig plus mutant antagonist-type IL-15.Ig cytolytic fusion proteins (triple therapy) posttransplantation results in prolonged, drug-free engraftment of cynomolgus islet allografts. Moreover slow progressive loss of islet function in some recipients was not associated with obvious pathologic evidence of rejection.

Expression of CD39 by human peripheral blood CD4+ CD25+ T cells denotes a regulatory memory phenotype.

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4+ Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood-derived human CD4+ CD25+ CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4+ CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4+ CD25+ CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell-populations to allow tracking of these in health and disease, as in renal allograft rejection.

Effect of ischemia and temperature on fetal mouse pancreas. Insulin production in vitro, and function after isotransplantation.

The effects of ischemia at varying temperatures on the survival of fetal islet endocrine cells was investigated by placing 17-day-old fetal mouse pancreata in organ culture after 2, 4, or 6 h at either 4 degrees C, 22 degrees C, or 37 degrees C. Insulin secretion by the cells in vitro, the content of insulin in the cultured pancreata, and the ability of the cultured islets to reverse diabetes in syngeneic streptozotocin-diabetic mice were assessed. Fetal pancreas subjected to 2-6 h of ischemia at either 4 degrees C or 22 degrees C showed neither loss of insulin secretory capacity in vitro nor loss of ability to produce large functional grafts, and behaved identically to tissue not subjected to deliberate ischemia. In contrast, after 2 h of ischemia at 37 degrees C, although some grafts functioned, their insulin content was reduced despite apparently normal prior insulin production in vitro, but 4 or 6 h at 37 degrees C resulted in total loss of functional islet tissue. However, despite retention of functional capacity and the ability to produce large grafts with high insulin content after cold or room temperature ischemia, some loss of insulin storage capacity in vitro was noted by islets subjected to ischemic periods longer than 2 h even at 4 degrees C. Thus, fetal pancreas can withstand prolonged periods of ischemia provided its temperature is reduced, and functional recovery can be demonstrated after transplantation.

Effect of culture conditions on fetal mouse pancreas in vitro and after transplantation in syngeneic and allogeneic recipients.

Organ culture of fetal mouse pancreas under conditions designed to either maximally preserve islet cell survival or reduce immunogenicity was used to test the efficacy of islet graft function in diabetic syngeneic or fully allogeneic recipients. Organ culture in either 90% O2, or at low temperature (22 degrees C), or in a combination of 90% O2 and 22 degrees C for 14 days not only failed to reduce immunogenicity in fully allogeneic recipients but also diminished endocrine cell survival in grafts in syngeneic recipients. Monitoring of in vitro insulin secretion provided a better guide to future graft function than did the insulin content of the cultured tissue. It is concluded that culture conditions that have been shown to reduce immunogenicity (high O2 concentration and low temperature) are not synergistic when used together and are also potentially damaging to endocrine cells.

Organ culture of fetal mouse and fetal human pancreatic islets for allografting.

In organ culture of fetal human and fetal murine pancreas under "conventional" conditions (10% CO2 in air), the islet cells of both species survive, proliferate and function but the acinar tissue rapidly degenerates. Fetal mouse islet cells also survive in 90% O2 and 10% CO2 but nonendocrine cells, including fibroblasts and macrophages, degenerate. Fetal mouse islets grown in 90% O2 show diminished immunogenicity when transplanted into recipients differing across the entire MHC, but a reduced allograft response by the host is frequently still present in the absence of immunosuppression. Fetal human islets, grown in 10% CO2 in air, produce insulin in vitro for prolonged periods, and as xenografts, differentiate under the kidney capsule of athymic mice, suggesting that under appropriate conditions both in vitro and in vivo, the fetal human islets can survive. However, fetal human pancreatic cells of all types are highly susceptible to high oxygen concentrations and are rapidly killed. Because of the susceptibility of fetal human pancreas to oxygen, conditions for the culture of fetal human islets for allotransplantation may need to be modified from those tolerated by fetal mouse islets. Fetal human islets may be a useful source of transplant material in human insulin-dependent diabetes, but it is likely that tissue matching and immunosuppression may be required in addition to modification of islet immunogenicity by prior organ culture.

Detrimental effect of chronic diabetes on growth and function of fetal islet isografts in mice.

We investigated (1), whether long-term (more than 6 months) streptozotocin-induced diabetes in mice had a detrimental effect on the function of pancreatic islet isografts; and (2), whether there was an effect on graft function in chronically diabetic mice of continuous pretransplant insulin infusion. BALB/c female mice that had been diabetic for more than 6 months were each transplanted with 1/2 of a 17-day fetal mouse pancreas that had been in organ culture for 14 days. All animals were grafted with same batch of tissue. One group of animals received continuous intraperitoneal infusion of regular insulin via an Alzet 2002 osmotic pump at the rate of 0.5 U/day for 14 days prior to grafting. Matched, chronically diabetic animals with pumps containing diluent alone, acutely diabetic animals of the same age, and acutely diabetic younger animals were used as controls. At 20 weeks after transplantation the grafts were removed and their insulin content measured. Following transplantation and removal of the pumps, all acutely diabetic animals returned to euglycemia within 6 weeks. The chronically diabetic animals which received diluent alone took 11 weeks to reach euglycemia compared to 7 weeks for their littermates that had received insulin. Graft insulin content was decreased from 16,300 +/- 4100 ng in the acutely diabetic animals to 9600 +/- 5200 ng in the chronically diabetic, non--insulin-treated group. The chronically insulin-treated group, however, had grafts with 16,400 +/- 5100 ng. Our studies suggest that there is a detrimental effect of chronic diabetes on graft insulin content that is ameliorated by pretransplant insulin therapy.

Evidence that macrophages are required for T-cell infiltration and rejection of fetal pig pancreas xenografts in nonobese diabetic mice.

BACKGROUND: Host macrophages are abundant within fetal pig pancreas xenografts undergoing rejection, but their role is unknown. Therefore, we examined the effect of host macrophage depletion on xenograft rejection. METHODS: Nonobese diabetic (NOD) mice were given clodronate-loaded liposomes intravenously to deplete macrophages. Controls received phosphate-buffered saline (PBS) or PBS-liposomes. General immune status was assessed after 2, 3, and 7 days by (1) fluorescence-activated cell sorter analysis of peripheral blood, spleen, and lymph node cells, (2) immunohistochemistry on spleens, and (3) mixed lymphocyte reaction. Organ-cultured fetal pig pancreas was transplanted under the kidney capsule of NOD mice 3 days after clodronate or PBS injection. Grafts were assessed histologically at 4, 5, 6, and 8 days after transplantation. RESULTS: Splenic macrophages and peripheral blood monocytes were depleted 2 days after clodronate treatment but had recovered within 11 days. T cell, B cell, and dendritic cell numbers were normal in spleen, peripheral blood, and lymph nodes of clodronate-treated mice, and T cells and antigen-presenting cells from these mice functioned normally in mixed lymphocyte reaction. Clodronate treatment markedly reduced graft infiltration by macrophages, T cells, and eosinophils at 4, 5, and 6 days after transplantation, and was associated with maintenance of endocrine cell viability and insulin expression. However, all grafts were rejected 8 days after transplantation, concordant with reappearance of splenic macrophages. CONCLUSIONS: Short-term, specific depletion of macrophages markedly delayed cellular infiltration and rejection of xenografts. The results provide the first evidence that macrophages promote T-cell infiltration and rejection of fetal pig pancreas xenografts in NOD mice.

Long-term islet allograft function in the absence of chronic immunosuppression: a case report of a nonhuman primate previously made tolerant to a renal allograft from the same donor.

UNLABELLED: Development of mixed chimerism by donor bone marrow transplantation (DBMT) has led to long-term tolerance of solid organ allografts in nonhuman primates. As an initial attempt to extend this approach to cellular transplant, islet transplant from the same donor was attempted in the recipient previously made tolerant to a kidney allograft. METHODS: After the conditioning with ATG, total body irradiation, thymic irradiation, and splenectomy, DBMT was performed followed by 4 weeks of cyclosporine. Kidney transplantation and native nephrectomies were subsequently performed on day 89. After 2.8 years of DBMT, diabetes was induced by streptozocin (STZ) and islets from bone marrow and kidney donor were transplanted without immunosuppression. RESULTS: After DBMT, the recipient developed chimerism and no evidence of kidney rejection for more than 1000 days. STZ induced diabetes was reversed after the islet transplantation. Islet biopsies demonstrated insulin staining without rejection. Although the recipient became diabetic 300 days after islet transplantation, viable transplanted islets were found in the liver and under the kidney capsule without any evidence of rejection. CONCLUSION: Tolerance with a nonmyeloablative conditioning can allow successful pancreatic islet transplantation without immunosuppression. Because no histological evidence of rejection was identified, recurrent diabetes is presumed to be inadequate islet mass.

High and low diabetes incidence nonobese diabetic (NOD) mice: origins and characterisation.

The Nonobese Diabetic mouse (NOD mouse) is an established model of autoimmune diabetes mellitus. While all colonies of NOD mice are derived from a single diabetic female detected during the breeding of a cataract-prone strain of mice, some of the dispersed colonies have been separated for many generations and express varying levels of diabetes. It is unclear to what extent this is due to environmental factors such as diet factor or a result of the varied origins of the colonies. Here we compare the incidence of diabetes and severity of insulitis in two divergent lines of NOD mice that differ in incidence of disease, but are maintained in the same environment. F1 crosses were performed and the progeny found to express the disease incidence of the low incidence line. This finding is consistent with either a dominant resistance gene(s) being responsible for reduced penetrance of disease or a transmissible environmental agent reducing the severity of the autoimmune process.

Cytokines and autoimmune beta cell destruction in NOD mouse fetal pancreas isografts in cyclophosphamide-induced diabetes.

The role of cytokines in a model of cyclophosphamide (CP)-accelerated beta cell destruction in fetal pancreas isografts transplanted into NOD mice was studied. One group of prediabetic NOD mice was injected with CP at a dose of 300 mg/kg i.p. and 7 days later isografts of organ cultured fetal pancreas (FP) were transplanted under the kidney capsule of these and untreated control mice. The mice were killed at several time points post-transplantation and the histological appearance of the host pancreas used to evaluate the disease progress in the grafts since previous studies had shown good correlation between isograft and native pancreas pathology. Intragraft cytokine gene expression was monitored by reverse-transcriptase polymerase chain reaction (RT-PCR) at the same time points and the expression levels between the experimental groups compared to normal kidney tissue. In comparison to isografts from non-CP injected mice, isografts from CP-treated mice showed increased expression of IFN-gamma, TNF-alpha, TNF-beta, IL-5, and eotaxin but no increase in IL-10 expression. The enhanced transcription of these cytokines correlated with massive infiltration of immune cells and ongoing beta cell destruction in the host pancreas of the CP-treated recipients.

Inherent beta-cell dysfunction induced by transgenic expression of allogeneic major histocompatibility complex class I antigen in islet cells.

Insulin-dependent diabetes mellitus (IDDM) is generally believed to be an autoimmune disease resulting from T-cell dysfunction that produces beta-cell damage, but it is conceivable that some forms of IDDM are not immunologically mediated. The effect of the expression of a foreign transgenic MHC class I antigen (H-2Kb), restricted to pancreatic islet beta-cells, was tested in vitro and in nude (athymic) mice to determine whether beta-cell dysfunction was due to non-immune mechanisms. The models used clearly excluded immune involvement in beta-cell damage. Fetal pancreas from transgenic and littermate control mice was maintained in organ culture for up to 18 days and insulin secretion into the medium assessed. For the initial 3-4 days in vitro, fetal control and transgenic pancreas secreted similar amounts of insulin, but thereafter insulin secretion by the transgenic tissue decreased in comparison with the controls. When the cultured pancreas was transplanted into nude mice, the transgenic issue produced smaller grafts than the control pancreas, but there was wide variation in graft size. Expression of H-2Kb antigens in beta-cells of nude transgenic mice also resulted in early-onset diabetes. The insulin content in the pancreas of young H-2Kb transgenic euthymic mice, (previously shown not to have insulitis), was reduced but glucagon content was normal. The reduction in in vivo insulin production was similar chronologically to the reduced insulin production by transgenic islets in vitro. These data confirm the non-immune loss of beta-cell function in MHC-transgenic mice and they may be a model for atypical Type I diabetes.

The effect of a depleting anti-CD4 monoclonal antibody on T cells and fetal pig islet xenograft survival in various strains of mice.

The effect of a cell-depleting anti-CD4 monoclonal antibody (mAb), GK1.5, was studied in a number of strains of inbred mice. Young adult female NOD/Lt, CBA and BALB/c mice were transplanted with organ cultured fetal pig pancreas and given 0.3 mg of the mAb (as ascites) on days -1, 0 and +1. The grafts were mostly rejected within 13 days in CBA mice but BALB/c and NOD recipients still had essentially intact grafts with the NOD mice showing evidence of early rejection. By 28 days posttransplantation the BALB/c recipients still had well-preserved grafts with minimal infiltration, but NOD and CBA mice had generally rejected their grafts totally. Peritransplant mAb treatment reduced CD4+ T cells in the spleen and they showed only incomplete recovery by 28 days. To further analyse the effect of anti-CD4 treatment, these strains as well as C57BL/6 mice were given a single dose (0.3 mg) of GK1.5 either as ascites or as affinity purified mAb. There was no obvious difference in effect between the ascites and the purified mAb within a strain but the various strains showed consistent differences in their blood, spleen and lymph node lymphocytes and in their response to the mAb. C57BL/6 mice differed from the other strains in having fewer T cells but more B cells in the blood, spleen and lymph nodes and a low CD4/CD8 ratio. Recovery of CD4+ T cells was most rapid in NOD mice and this together with the relatively high number of these cells may account for the ability of these mice to reject grafts despite immunosuppression that can allow prolonged graft survival in other strains. This study emphasizes the need to examine various strains of mice when making general statements about the efficacy of immunosuppression in transplantation and stresses the need to be aware of the frequent use of 'permissive' strains in reports where excellent graft survival is reported.

The role of cytokines during rejection of foetal pig and foetal mouse pancreas grafts in nonobese diabetic mice.

The rejection of discordant foetal pig islet xenografts in nonimmunosuppressed nonobese diabetic (NOD) mice is dominated by polymorphonuclear cell infiltration whereas allografts are almost exclusively infiltrated by mononuclear cells. To determine if this variation is due to different proinflammatory factors generated at the graft site, we analysed graft-site mRNA expression of various cytokines, and the eosinophil attractant chemokine, eotaxin, in a renal subcapsular islet transplant model using organ cultured foetal pig (xenograft) and foetal BALB/c (allograft) pancreas in prediabetic NOD mice. Using semiquantitative RT-PCR on samples recovered at multiple time points during the first 15 post-transplantation days from mice transplanted with either allogeneic or xenogeneic tissue, we found increased expression of IL-2, IL-4. TNF-beta and IL-10 mRNAs at the peak of the cellular infiltrate (on day 5) in both xenografts and allografts but, in contrast to the allografts, no enhanced transcription of IFN-gamma mRNA in the rejecting xenografts. When an allograft and a xenograft were placed at the opposite pole of the same kidney the histological appearance of the rejecting allograft site resembled the xenograft site with significant numbers of eosinophils in both, and enhanced expression of eotaxin and iNOS. Additionally, the xenograft response, unlike the allograft response, was marked by an early increased expression of TNF-alpha and IL-S (day 3) and an almost complete absence of IFN-gamma expression. The results suggest a distinct cell-mediated mechanism for rejection of foetal pancrease xenografts compared to the rejection of foetal pancreas allografts.

A comparison between islet transplantation and parenteral insulin in the control of diabetes and prevention of renal complications in mice.

The control of diabetes and the prevention of renal complications were studied in mice that received different treatment regimes for six months. Transplantation of syngeneic cultured fetal pancreas completely reversed streptozotocin-induced diabetes (mean FBG 5.1 +/- 0.4 mmole/liter six months after transplantation, versus 5.8 +/- 0.2 mmole/liter in normal mice). The mean fasting blood glucose (FBG) level of insulin-treated mice was lower than the mean FBG level of untreated diabetic mice (9.0 +/- 1.2 mmole/liter versus 11.5 +/- 1.3 mmole/liter, P less than 0.05) but exceeded the FBG level of transplanted mice (P less than 0.001) or normal controls (P less than 0.001). There were no significant differences between the mean level of glycosylated hemoglobin (HbA1c) of normal (4.8 +/- 0.3%), transplanted (4.5 +/- 0.3%), or insulin-treated mice (5.3 +/- 0.4%), but the HbA1c level in the untreated diabetic group was increased (7.0 +/- 0.5%; P less than 0.001). Six months after transplantation, the thickness of the glomerular capillary basement membrane (GCBM) was not different in the transplanted group and normal controls (156.4 +/- 5.7 nm versus 157.3 +/- 12.6 nm); the GCBM was thicker in the insulin-treated mice than in the transplanted mice (179.8 +/- 4.2 nm versus 156.4 +/- 5.7 nm; P less than 0.02), but thinner than in untreated diabetic mice (179.8 +/- 4.2 nm versus 202.2 +/- 4.4 nm; P less than 0.001). It is concluded that islet transplantation, in contrast to good control as judged by normalization of HbA1c levels achieved with parenteral insulin, prevents GCBM thickening in experimental diabetes.

Trace-element contents of human fetal liver of 12-22 weeks gestation.

There are some papers in the literature on the trace element contents of fetal livers of 20-wk gestation time and over. However, there is very little information on this subject for fetal livers of less than 20-wk gestation. We have initiated a program on the measurement of trace elements in fetal livers of 12-22-wk gestation, using thick-target X-ray fluorescence analysis.The liver samples were obtained from freshly aborted fetuses. After removing blood from the samples, they were chopped into small pieces and freeze dried. The resulting material was ground into fine powder and compressed into 3-mm thick pellets, with boric acid backing. A similar pellet was also made of NBS-Bovine Liver-which was used as the standard for calculating the absolute concentrations of different trace elements.The measurements were carried out using a commercial Wave-Length-Dispersive XRF-System. Different X-ray tubes were used for different sets of elements in order to maximize the detection sensitivity. The results are compared with those of fetal liver of longer gestation and adult livers.

Effect of diabetes on trace-element concentration of blood.

The effect of diabetes on trace elements concentration in blood of experimental animals has been studied by thin-target X-ray fluorescence analysis. Balb/C young adult mice, 6-8 wk old, were used in the study. About 100-200 µL venous blood was taken from each mouse for trace element analysis. The measurements were carried out on a commercial Wave-Length-Dispersive XRF System, with different X-ray tubes being used for maximizing the detection sensitivity of different groups of elements.Later on, the mice were made diabetic by an intravenous injection of Streptozotocin (250 mg/kg). Then, 2 and 3 wk after the injection, 100 µL of venous blood was drawn from each of the mice and analyzed for trace element concentration. In this way we were able to study the changes in blood trace elements caused by diabetes.The results and advantages of using experimental animals, under controlled conditions, to study trace element variations caused by different diseases, are discussed in the paper.