The immune response to staphylococcal antigens in mice depleted of macrophages by Cl2MDP-liposomes

To investigate the role of macrophages in the induction of the production of antibody to staphylococcal antigens, we used Cl2MDP (clodronate) liposomes as a tool for local macrophage depletion. Macrophage depletion caused in mice by intraperitoneal (i.p.) injection of Cl2MDP liposomes was associated with a reduction in the clearance of Staphylococcus aureus Cowan 1 bacteria from the tissues of infected animals and with a marked decrease in the bactericidal activity of macrophages escaping from the lethal effect of clodronate. Despite the functional defect of macrophages, the mice treated with Cl2MDP liposomes two days before the injection of alpha-toxin (toxoid) or whole heat-killed S. aureus Cowan 1 bacteria, demonstrated an enhancement in the production of anti-staphylococcal alpha-toxin IgM and anti-collagen-binding protein IgG. A similar enhancement of antistaphylococcal antibody synthesis was observed in mice after receiving phosphate buffered saline (PBS) encapsulated in liposomes.     

Humoral antibody and cell-mediated cytotoxicity responses of mice to rat skin xenografts

Aspects of both the humoral and cell-mediated immune responses of mice to rat skin xenografts were studied sequentially and quantitated. Antirat lymphocytotoxic and hemagglutinating antibodies were first detectable at 7 and 6 days, respectively, after primary grafting and their appearance in serum corresponded to the time of graft rejection. Lymphocytotoxic titers were low after primary grafting but increased greater than 4-fold after secondary grafting. Cytotoxicity of mouse spleen and axillary lymph node cells for 51Cr-labeled rat lymphocyte target cells was first detectable 5 days after primary grafting but was quite low showing a peak of only 7% specific 51Cr release 6 days after grafting. In contrast, the cytotoxicity of mouse spleen and lymph node cells for allogeneic target cells after primary skin allografting was significantly greater. It is suggested that the magnitude of the cell-mediated immune response to skin xenografts is less than the response to allografts and that the quicker and more vigorous rejection of skin xenografts is due to a larger participation of humoral antibody in the rejection process.     

Prolonged discordant xenograft survival and delayed xenograft rejection in a pig-to-baboon orthotopic cardiac xenograft model

OBJECTIVE: Our objectives were to study delayed xenograft rejection and the effectiveness of pretransplantation total lymphoid irradiation combined with immunosuppression on rejection in a pig-to-baboon cardiac xenograft model. METHODS: Baboons were treated with pretransplantation total lymphoid irradiation, cyclosporine A (INN: ciclosporin), and methotrexate. Orthotopic pig-to-baboon cardiac transplantations were performed after depletion of circulating xenoreactive natural antibody by pretransplantation donor organ hemoperfusion. Tissue samples were collected for immunologic and immunopathologic evaluation. RESULTS: Pig cardiac xenografts survived more than 18 and 19 days without evidence of hyperacute rejection. Immunologic analysis of serum samples demonstrated that circulating xenoreactive natural antibody levels did not return to pretransplantation levels. The production of xenoreactive natural antibodies from the recipient's splenocytes was inhibited completely. Histologic examination of xenografts showed the feature of acute vascular rejection. Immunohistochemical studies demonstrated infiltration of cardiac xenografts by large numbers of macrophages, small numbers of natural killer cells, and a few T cells. The infiltrating macrophages also showed expression of interleukin-1 and tumor necrosis factor. Diffuse deposition of immunoglobulin G, C1Q, C3, and fibrin on xenograft vasculature was observed. Interleukin-2 expression was not found in rejected cardiac xenografts. Xenograft endothelial cells also showed evidence of activation (expression of cytokines interleukin-1 and tumor necrosis factor). CONCLUSIONS: This study demonstrates prolonged discordant cardiac xenograft survival and delayed xenograft rejection in a pig-to-baboon model. The delayed xenograft rejection is mediated by both humoral and cellular mechanisms. Pretransplantation total lymphoid irradiation combined with cyclosporine A and methotrexate can inhibit xenoreactive natural antibody production but not elicited antipig antibody production and the xenoreactivity of macrophages.     

The establishment of a three dimensional PCR screening system and quality evaluation of human YAC library

To determine whether pregnancy provides an improved milieu for fetal/neonatal pancreas/islet transplantation, we studied neonatal pancreatic implants into non-obese diabetic (NOD) female mice during early gestation. We monitored maternal glycemic status, birthweight of the offspring, and graft histology to assess the efficacy of transplantation. One hundred and thirteen twelve-week-old NOD female mice were randomized into four groups as follows: (1) non-pregnant NOD mice received a sham operation; (2) non-pregnant NOD mice received neonatal pancreatic transplants; (3) pregnant NOD mice received a sham operation; and (4) pregnant NOD mice received neonatal pancreatic transplants. Pancreas segments from 3 neonatal NOD mice were placed via an incision 1 to 2 mm distal to the ear-skull junction of each of the recipients. Maternal blood glucose and glycated hemoglobin were determined between days 18 and 20 after the surgery. Pups were weighed within 5 to 6 hours after delivery. Pregnant NOD that received transplants (n = 29) had lower glucose and glycated hemoglobin (GHb) than sham operated pregnant controls (n = 26) (4.9 +/- 0.05 versus 9.0 +/- 5.0 mmol/L, p < 0.001 for glucose and 2.0 > or = 0.2 versus 3.0 > or = 1.2%, p < 0.008 for GHb) at 18 to 20 days of gestation. Controlling for litter size showed a decrease in birthweight for offspring of transplant recipients versus offspring of pregnant controls (1.59 +/- 0.08 versus 1.65 +/- 0.08 g, p < 0.002). Histological scoring of transplanted tissue at day 21 indicated that the lymphocytic infiltration in the pregnant group was significantly less than the control group (2.9 +/- 1.2 versus 4.9 +/- 0.2, p < 0.0001). We conclude that the pregnant NOD mouse provides a useful transplant model, that pregnancy provides an opportunity to increase beta-cell mass with transplanted tissue, and that pancreatic transplantation decrease birthweight and macrosomia in the offspring of NOD mice.     

Defensiveness and perception of external inspiratory resistive loads in asthma

This paper introduces a model which incorporates fetal thymus organ culture (FTOC) from NOD mice to replicate thymic development of diabetogenic T cells. NOD fetal pancreas organ culture (FPOC) co-cultured with 13-16 day NOD FTOC for an additional 14-21 days produced less insulin than FPOC cultured alone. Insulin production from the FTOC of non-diabetic strains C57BL/6 and BALB/c was not inhibited by co-culture with FTOC from their syngeneic counterparts. Sections of the NOD co-cultures showed peri-islet infiltration with lymphocytes. Insulin reduction by FTOC/FP co-culture was prevented by co-culture of the NOD FT with FT from immunologically incompetent C.B-17 SCID/SCID mice. Co-culture of NOD FP with NOD FT prior to the development of T cells prevented generation of diabetogenic FTOC. Thus, early exposure of NOD T cell precursors to the thymic stromal elements of C.B-17 SCID/SCID FT or to islet antigens can negatively select for diabetogenic T cells or activate immuno-regulatory cells that can suppress diabetogenic T cell activity. The addition of blocking F(ab')2 fragments of anti-CD3epsilon monoclonal antibody to NOD FTOC/FP co-cultures prevented insulin reduction, implicating a role for TcR-mediated recognition in this "in vitro IDDM" model. The addition of activating whole anti-CD3epsilon caused the complete ablation of insulin production in FTOC/FP co-cultures from all strains tested. Transfer of unprimed syngeneic FTOC cells to prediabetic NOD mice prevented the onset of IDDM while transfer of islet-cell primed FTOC/FP cells slightly increased disease incidence. These data suggest that while diabetogenic T cells are present in the FT, they are normally suppressed, even after organ culture. However, these cells can induce the destruction of islet cells, in vitro and in vivo, if they are appropriately activated with pancreatic tissue.     

Evidence of human non-alpha-galactosyl antibodies involved in the hyperacute rejection of pig lungs and their removal by pig organ perfusion

BACKGROUND: Human natural xenoantibodies represent a major hurdle to the clinical application of pig lungs in transplantation by initiating hyperacute rejection within minutes to hours. OBJECTIVE: The object was to compare pig organ perfusion and specific depletion of anti-alpha-galactosyl xenoantibodies for prevention of hyperacute rejection in the pig to human lung combination. METHODS: Large White pig (20-25 kg) left lungs were removed and continuously ventilated and reperfused ex vivo either with (1) whole human blood previously perfused in situ through pig right lung (group I), liver (group II), or spleen (group III) or with (2) human plasma in vitro immunoabsorbed on columns containing alpha-galactosyl disaccharide (Gal-alpha-(1-3)Gal-beta-(CH2)3NH2; B disaccharide) (group IV). Each study group included 6 animals. RESULTS: The in situ and in vitro preperfusions depleted anti-alpha-galactosyl xenoantibodies and all in situ perfused pig organs showed histologic signs of hyperacute rejection. After the ex vivo reperfusion, group I xenografts had a significantly (P < .001) longer functional and histologic survival than did xenografts in groups II, III, and IV. Human blood reperfusing group I xenografts had a significantly (P < 0.05) lower (1) decline of clotting factors and total circulating immunoglobulins, (2) total and membrane attack complex (C5b,6,7,8,9) complement activation, and (3) hemolysis. By Western blot analysis, the in situ lung preperfusion removed antibodies against non-alpha-galactosyl proteins of low molecular weight that were not eliminated by the alpha-galactosyl column. CONCLUSIONS: Results demonstrate that specific depletion of anti-alpha-galactosyl antibodies alone incompletely protects pig lungs from hyperacute rejection. It is speculated that the more complete prevention of this rejection afforded by pig lung preperfusion relates to the removal of other, non-alpha-galactosyl antibodies.     

Mucosal addressin is required for the development of diabetes in nonobese diabetic mice

Immune responses are best initiated in the environment of lymphoid tissues wherein circulating lymphocytes enter by interacting with endothelial adhesion molecules. In type 1 diabetes, immune responses against pancreatic islets develop, but the environment in which this occurs remains unidentified. To determine whether lymphocyte homing to lymphoid organs is involved in the pathogenesis of diabetes in nonobese diabetic (NOD) mice, we blocked the function of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1), which is a vascular addressin-mediating lymphocyte homing into mucosal lymphoid tissues, in these mice. While ineffective if started later, a blockade started at 3 wk of age reduced the incidence of diabetes from 50% to 9% (p < 0.01). This finding is associated with Peyer's patch atrophy, a marked decrease of naive (CD44(low) CD45RB(high)) T lymphocytes, and a reduction in the relative numbers of memory (CD44(high)) T lymphocytes in the spleen. The potential of these spleen cells to cause diabetes was diminished. Anti-MAdCAM-1 treatment also inhibited both lymphocyte entry into the pancreas and diabetes development in NOD/SCID recipients after the transfer of lymphocytes derived from the mesenteric lymph nodes of young, but not of diabetic, NOD donors. Therefore, MAdCAM-1 may be required during two distinct steps in an early phase of diabetes development: for the entry of naive lymphocytes into the lymphoid tissues in which diabetes-causing lymphocytes are originally primed, and for the subsequent homing of these lymphocytes into the pancreas. The role of MAdCAM-1 as a mucosal vascular addressin suggests that mucosal lymphoid tissues are involved in the initiation of pathologic immune responses in NOD mice.     

Transfer of thyroiditis, with syngeneic spleen cells sensitized with the human thyrotropin receptor, to naive BALB/c and NOD mice

In previous studies we have induced TSH binding-inhibiting Igs and thyroiditis in BALB/c mice and thyroiditis alone in NOD mice immunized with the extracellular domain of the human TSH receptor produced as a maltose-binding protein fusion in bacteria (MBP-ECD). In this study, our aim was to determine whether thyroiditis can be transferred to syngeneic naive recipients with in vivo and in vitro primed spleen cells. Groups of 6-week-old female BALB/c and NOD mice were immunized ip with MBP-ECD in an adjuvant of alum plus attenuated Bordetella pertussis toxin, on days 0 (100 micrograms), 14, 28, and 35 (50 micrograms). These mice (in vivo primed) and nontreated age- and sex-matched controls were killed on day 43, and their spleens and thyroids were removed, the latter to verify the induction of thyroiditis in the antigen-treated mice. Splenocytes were disrupted mechanically and cultured at 3 x 10(6)/ml in RPMI supplemented with 20 micrograms/ml MBP-ECD for 48-64 h. After this in vitro priming, some of the splenocytes received no further treatment, but a portion was fractionated into a CD4+-enriched population. Groups of 6-week-old female BALB/c and NOD mice were immunized into the tail vein with 100-200 microliters PBS containing approximately 10(5)-10(7) unfractionated T cells (both in vivo primed and not) and CD4+-enriched (in vivo primed) splenocyte populations. The animals were killed 16 days later, and their thyroids were examined histologically and by immunohistochemistry. In addition, levels of antibody to the MBP-ECD priming antigen were assessed by enzyme-linked immunosorbent assay in the antigen- and spleen-treated mice. In the donor animals, in vivo priming resulted in an extensive lymphocytic infiltration of the thyroids in both BALB/c and NOD mice and follicular destruction in the latter. There was no evidence of thyroiditis in all 9 BALB/c mice and all 4 NOD mice who received unfractionated T cells from mice that had not been primed in vivo. In contrast, transfer of MBP-ECD in vivo primed unfractionated T cells resulted in thyroiditis in 9 of 13 BALB/c mice and 5 of 6 NOD mice; similarly, the equivalent CD4+-enriched population produced extensive thyroiditis in 2 of 3 BALB/c mice and all three NOD mice. The most striking difference between the antigen- and spleen-treated mice was in the quantity of the infiltrate, which was much greater in the latter and extended throughout the thyroid glands of these animals. In common with mice treated directly with the MBP-ECD antigen, the infiltrates of both BALB/c and NOD recipient mice contained large numbers of activated T cells expressing the receptor for interleukin-2, and macrophages and dendritic cells were plentiful, particularly in the BALB/c mice, in which B cells and interleukin-10-positive T cells were also present. The most abundant infiltrates, containing numerous CD8+ T cells and follicular destruction, were observed in NOD mice receiving primed unfractionated T cells or CD4+-enriched T cells. In contrast to the donors, none of the recipient animals had circulating antibodies to the MBP-ECD antigen. In conclusion, we have shown that it is possible to transfer thyroiditis with spleen cells from mice primed in vivo with a human TSH receptor preparation. Furthermore, the thyroiditogenic activity appears to reside in the CD4+ population.     

Soluble and cell surface ICAM-1 as markers for disease activity in multiple sclerosis

Infiltration of a transplanted organ by host lymphoid cells is the hallmark of acute rejection. However, after intestinal transplantation, physiological lymphocyte migration may lead to host cell infiltration of the graft even in the absence of rejection. It is unclear whether this lymphocyte migration also involves the intraepithelial compartment of the graft or whether infiltration there is indicative of acute rejection. We demonstrate here that host cell infiltration of the intestinal mucosa occurs both during acute rejection of a small bowel allograft and, to a lesser extent, when rejection is prevented by immunosuppression with FK506. The infiltrating host cells consisted of CD3+ T cells with a predominant CD4-CD8+ phenotype resembling intraepithelial lymphocytes (IELs). Functional studies showed that the nonspecific cytolytic activity of IELs was not affected by acute rejection or by immunosuppression with FK506. These findings indicate that host cell infiltration of the intestinal mucosa does not connote an ongoing acute rejection. Furthermore, the decreased mucosal barrier function during acute rejection of intestinal allgrafts is probably not due to impaired cytolytic activity of IELs.     

Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine

BACKGROUND: Type 1 diabetes is characterized by the presence of an autoimmune memory, responsible for the destruction of even syngeneic islet grafts. This recurrence of autoimmunity is partly responsible for the need of extensive immunosuppression in pancreas and islet transplantation in type 1 diabetic patients. The aim of the study was to evaluate the capacity of a 20-epi-analog of vitamin D3, KH1060, both alone and in combination with cyclosporine (CsA) to prevent diabetes recurrence in syngeneic islet grafts in nonobese diabetic (NOD) mice. METHODS: Spontaneously diabetic NOD mice grafted with syngeneic islets (n=500) under the kidney capsule were treated with KH1060, CsA, or a combination of both drugs from the day before transplantation until recurrence or 60 days after transplantation. RESULTS: Vehicle-treated mice showed a recurrence of diabetes in 100% of cases (n=17) within 4 weeks. Treatment with high doses of CsA (15 mg/kg/day) or KH1060 (1 microg/kg/2 days) significantly prolonged islet survival (60 days and 50 days, respectively, versus 9.5 days in controls; P<0.001 and P<0.0001). Mice treated with subtherapeutical doses of both drugs combined (KH1060 0.5 microg/kg/2 days + CsA 7.5 mg/kg/day) had significant prolongation of graft survival (48 days; P<0.001) and more importantly, four of five mice that were still normoglycemic 60 days after transplantation showed no recurrence after discontinuation of all treatment. Histology of the grafts of control and combination-treated mice demonstrated that graft infiltration and islet destruction were less severe in grafts of combination-treated mice. Cytokine mRNA analysis in the grafts 6 days after transplantation revealed a clear suppression of interleukin-12 and T helper 1 cytokines and higher levels of interleukin-4 in combination-treated mice. CONCLUSIONS: KH1060, an analog of 1,25(OH)2D3, delays autoimmune disease recurrence after syngeneic islet transplantation in NOD mice, both alone and especially in combination with CsA, possibly restoring tolerance to beta cells in 30% of cases.     

Endogenous immune response to glutamic acid decarboxylase (GAD67) in NOD mice is modulated by adjuvant immunotherapy

We have shown that immunization of non-obese diabetic (NOD) mice with adjuvants (CFA or BCG) prevents the onset of diabetes by induction of regulatory cells. Since autoimmune responses to glutamic acid decarboxylase (GAD) are up-regulated in insulin-dependent diabetes mellitus (IDDM), in this study GAD67-specific antibody, T cell proliferation and lymphokine production patterns were analysed in the adjuvant-treated mice to characterize the regulatory mechanisms underlying the protection. We used both spontaneous diabetes and syngeneic islet transplantation models in NOD mice. Protection against spontaneous diabetes and prevention of syngeneic islet graft rejection by CFA or BCG treatment was found to be accompanied by the production of long lasting and high titre anti-GAD67 antibody of IgG1 isotype in the sera. Upon in vitro stimulation with GAD67, draining lymph node and spleen cells from CFA-immunized NOD mice or syngeneic islet-grafted and BCG-protected NOD mice produced much more IL-4, whereas there was no significant change in IFN-gamma production. The strong early T cell proliferative response to GAD67 in CFA or BCG-immunized NOD mice was followed by a low or unresponsiveness state. Taken together, these results suggest a shift in Th1/Th2 balance in the GAD67-specific endogenous immune response to a change in Th2 levels after adjuvant treatment. We postulate that the protective effect of CFA or BCG is due to the diversion of GAD-specific endogenous cellular immune response to a non-pathogenic humoral response.     

Acute rejection of marrow grafts in patients transplanted from a partially mismatched related donor: clinical and immunologic characteristics

Bone marrow transplantation (BMT) from a partially mismatched related donor (PMRD) provides a treatment option for patients lacking a matched sibling donor. T lymphocyte depletion of the graft reduces the risk of severe graft-versus-host disease, but may increase the risk of graft failure. We evaluated the pattern of acute graft rejection in eight patients receiving PMRD BMT with respect to the conditioning therapy, diagnosis, age and sex of donor and recipient, degree of HLA mismatch, and peripheral blood immunophenotype at the time of graft failure. All grafts were partially depleted of T lymphocytes. Marrow grafts infused into patients who experienced acute rejection did not differ significantly in nucleated cell dose, degree of T lymphocyte depletion, T cell dose, or CFU-GM/kg infused, from those received by 31 patients who showed durable engraftment. In three of four patients who rejected their grafts, and had sufficient peripheral blood cells for immunophenotyping, a CD3+CD8+ T lymphocyte phenotype was predominant at the time of acute rejection. In one patient rejection was associated with a predominant population of CD3+CD4+ T lymphocytes. Rejection was significantly associated with chronic myelogeneous leukemia and in patients mismatched by more than two antigens.     

Functional consequences of a decreased potassium affinity in a potassium channel pore. Ion interactions and C-type inactivation

We have shown previously that the inactivation of macrophages in nonobese diabetic (NOD) mice results in the prevention of diabetes; however, the mechanisms involved remain unknown. In this study, we found that T cells in a macrophage-depleted environment lost their ability to differentiate into beta cell-cytotoxic T cells, resulting in the prevention of autoimmune diabetes, but these T cells regained their beta cell-cytotoxic potential when returned to a macrophage-containing environment. To learn why T cells in a macrophage-depleted environment lose their ability to kill beta cells, we examined the islet antigen-specific immune response and T cell activation in macrophage-depleted NOD mice. There was a shift in the immune balance, a decrease in the T helper cell type 1 (Th1) immune response, and an increase in the Th2 immune response, due to the reduced expression of the macrophage-derived cytokine IL-12. As well, there was a deficit in T cell activation, evidenced by significant decreases in the expression of Fas ligand and perforin. The administration of IL-12 substantially reversed the prevention of diabetes in NOD mice conferred by macrophage depletion. We conclude that macrophages play an essential role in the development and activation of beta cell-cytotoxic T cells that cause beta cell destruction, resulting in autoimmune diabetes in NOD mice.     

Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease

Glutamic acid decarboxylase (GAD)65 is a pancreatic beta cell autoantigen implicated as a target of T cells that initiate and sustain insulin-dependent diabetes mellitus (IDDM) in humans and in non-obese diabetic (NOD) mice. In an attempt to establish immunological tolerance toward GAD65 in NOD mice, and thereby to test the importance of GAD in IDDM, we generated three lines transgenic for murine GAD65 driven by a major histocompatibility complex class I promoter. However, despite widespread transgene expression in both newborn and adult mice, T cell tolerance was not induced. Mononuclear cell infiltration of the islets (insulitis) and diabetes were at least as bad in transgenic mice as in nontransgenic NOD mice, and in mice with the highest level of GAD65 expression, disease was exacerbated. In contrast, the same transgene introduced into mouse strain, FvB, induced neither insulitis nor diabetes, and T cells were tolerant to GAD. Thus, the failure of NOD mice to develop tolerance toward GAD65 reflects at minimum a basic defect in central tolerance, not seen in animals not predisposed to IDDM. Hence, it may not be possible experimentally to induce full tolerance toward GAD65 in prediabetic individuals. Additionally, the fact that autoimmune infiltration in GAD65 transgenic NOD mice remained largely restricted to the pancreas, indicates that the organ-specificity of autoimmune disease is dictated by tissue-specific factors in addition to those directing autoantigen expression.     

IDDM patients neither show humoral reactivities against endogenous retroviral envelope protein nor do they differ in retroviral mRNA expression from healthy relatives or normal individuals

Xenotransplantation of encapsulated islets of Langerhans is a possibility to overcome problems of human organ donor shortage in islet transplantation. Preexisting natural xenoantibodies are known to play a major role in the rejection of vascularized xenografts. Only little is known about the mechanism of rejection of non-vascularized cellular xenotransplants. In this study we introduce a method for the characterization of xenograft rejection of encapsulated islets by FACS analysis of peritoneal cells. Pig islets were transplanted intraperitoneally into non-diabetic Lewis rats either encapsulated or non-encapsulated. Animals receiving empty capsules and sham-operated animals served as controls. After 7 days a peritoneal lavage was performed. The total cell number and the viability of the cells were determined. Cells were analysed after staining with a panel of antibodies for the detection of T-lymphocytes, B-lymphocytes, macrophages, MHC class II molecules. Total cell number was highest after microencapsulated transplantation (149.4+/-30.1x10(6)) compared with empty capsules (41.4+/-19.7x10(6)) and non-encapsulated porcine islets (18.1+/-3.3x10(6)). The percentage of CD 3 positive T-lymphocytes rose to 44.5+/-11.5% in case of microencapsulated xenografts compared with 19.2+/-8.2% for non-encapsulated xenografts and 4.9+/-2.4% for empty controls. B-lymphocytes were detected in only small amounts. MHC class II expression on macrophages as activation marker was significantly increased after encapsulated transplantation (60.2+/-8.9% vs 15.2+/-7.0% for free islets and 4.9+/-1.2% for empty controls). The discrepancy between the macrophage activation due to encapsulated xenogeneic islets in comparison to empty capsules made from the same material clearly indicates that the reaction is not only material related but that a recognition of the encapsulated islet takes place despite the effective inhibition of a direct cell-to-cell contact. This recognition occurs on a T-cell level as well as on the macrophage level. 7 days after transplantation the reaction towards encapsulated xenografts is even more intense than to non-encapsulated xenografts. This might be due either to the time course of the rejection process or to a prolongation of the activation because antigen elimination is hindered by the capsule.     

The pattern of disulfide linkages in the extracellular loop regions of connexin 32 suggests a model for the docking interface of gap junctions

This study evaluated the contribution of acute parenchymal rejection and interferon (IFN)-gamma to the development of graft arterial disease (GAD) in totally allogeneic murine cardiac transplants. BALB/c (H-2d) hearts were transplanted into wild-type C57BL/6 (B6, H-2b) or B6 IFN-gamma-deficient (GKO) recipient mice. Assessing the role of acute parenchymal rejection in the GAD process involved two different immunosuppression protocols using anti-CD4 and -CD8 monoclonal antibodies (MAbs): virtually complete long-term immunosuppression (denoted as complete immunosuppression) was achieved by administering both MAbs 6, 3, and 1 day before transplantation and weekly thereafter; in contradistinction, a single, early, transient episode of rejection (transient rejection) was attained by administering MAbs beginning 4 days after transplant and then at weekly intervals. The extent and duration of T cell depletion under these two regimens were evaluated using flow cytometric analysis of peripheral blood lymphocytes. After a single injection of MAbs, peripheral blood CD4+ and CD8+ T cell depletion was approximately 98% at 1 week and approximately 88% at 2 weeks. After three injections (analogous to days 6, 3, and 1 before transplant), peripheral blood CD4+ and CD8+ T cell depletion was >98% at 2 weeks and approximately 87% at 4 weeks. Functioning cardiac allografts were removed at 8 and 12 weeks after transplant and analyzed by hematoxylin and eosin, elastic tissue, and immunohistochemical stains, and the severity of parenchymal rejection versus GAD was scored. With complete immunosuppression (antibody before and after transplant), BALB/c allografts showed little parenchymal rejection or GAD, suggesting that persistent depletion of T cells blocked subsequent development of GAD. However, even a single transient acute rejection episode allowed the subsequent development of GAD accompanied by augmented major histocompatibility complex (MHC) class II, VCAM-1, and ICAM-1 expression at 12 weeks; these allografts showed no residual CD4+ or CD8+ T cells. In comparison, allografts undergoing transient rejection in GKO recipients did not develop GAD, despite persistent macrophage and natural killer cell (NK) infiltrates comparable to those seen in wild-type recipients. Moreover, the arterioles of hearts transplanted into GKO recipients showed no or minimal increases in MHC class II, ICAM-1, and VCAM-1 relative to baseline expression. In conclusion, a single episode of allogeneic injury mediated by T cells suffices to evoke subsequent graft arteriosclerosis, even in the absence of additional T-cell-mediated injury, and the process appears to depend on IFN-gamma.     

Role of macrophages in acute murine cytomegalovirus infection

It has been recognized that macrophages play an important role in controlling virus infection in experimental animal models. To evaluate the role of macrophages in acute murine cytomegalovirus infection, macrophages in the spleen and the liver were eliminated by an intravenous injection of liposomes containing a cytolytic agent, dichloromethylene diphosphonate. The depletion of macrophages led to a significant increase of virus titer in the spleen and lungs in both susceptible BALB/c and resistant C57BL/6 mice during the first three days after intravenous infection. In the spleen, the increase of virus titer in macrophage-depleted BALB/c mice was much greater than that in NK cell-depleted mice. These results suggest that macrophages contribute to protection mainly by the mechanisms which are independent of NK cells during the first three days after infection. The increase of virus titer in macrophage-depleted C57BL/6 mice was as great as that in NK cell-depleted mice because of the high contribution of NK cells to protection in C57BL/6 mice. In the liver in both strains of mice, the effects of macrophage depletion on virus titer were not as much as those in the spleen and lungs. Furthermore, the local depletion of peritoneal macrophages resulted in a great increase of virus titer in the spleen at three days after intraperitoneal infection. We conclude that macrophages greatly contribute to decreasing the virus load in some organs possibly through either or both intrinsic and extrinsic mechanisms in the early phase of primary infection with murine cytomegalovirus.     

Correction of diabetic nod mice with insulinomas implanted within Baxter immunoisolation devices

Insulin replacement by injection is clearly not a cure for Insulin Dependent Diabetes Mellitus (IDDM). Replacement of the destroyed islets by pancreas or islet allograft transplantation can achieve the good metabolic control required to prevent diabetic complications, but tissue supply is limited. The problem of islet supply to treat the 1 million IDDM patients in the USA could be overcome by using immortalized islet beta-cells as a donor source. However, before either allogeneic or xenogeneic immortalized beta-cells are used, some major problems have to be overcome: control of immortalized cell growth, allograft or xenograft rejection and recurrence of autoimmunity. To tackle these problems we have used a cell impermeable immunoisolation device containing mouse insulinoma cells. Transplantation of devices with insulinomas from NOD mice carrying the Rat-insulin promoter regulated SV40 T-Antigen transgene (RIP-TAg), normalized the blood glucose levels of diabetic NOD mice. Insulinomas from allogeneic CBA/NOD-RIP-TAg mice were also capable of normalizing diabetic NOD mice. Not only were non-fasting blood glucoses normalized but when given an intraperitoneal injection of glucose, the corrected mice had a near normal clearance of glucose from the blood. When the devices were removed from normalized mice they became diabetic again, demonstrating that the immunoisolation device was capable of protecting against both alloimmune and autoimmune destruction. The results with allogeneic mouse beta-cells suggest the possibility that immortalized human beta-cells could be an effective source of tissue to correct diabetes in IDDM patients without the use of immunosuppression.