Antagonizing leukotriene B4 receptors delays cardiac allograft rejection in mice.

BACKGROUND: Allograft rejection is a cellular immunological/inflammatory response that is, in part, directed by potent proinflammatory mediators. This study was designed to test the hypothesis that leukotriene B4 (LTB4) may have a role in graft rejection and that LTB4 receptor antagonists may be clinically useful in the treatment of allograft rejection. METHODS: We evaluated the potent and selective LTB4 receptor antagonist CP-105696 in a murine heterotopic cardiac allograft model with oral dosing daily for 28 days or in an induction protocol (day -1 to day 3). RESULTS: At a dose of 50 mg/kg/day (28 days), B10.BR (H2k) allografts transplanted into C57Bl/6 (H2b) recipients were significantly protected, as reflected by the mean survival time versus control grafts (27+/-20 days [n=10] vs. 12+/-6 days [n=14]; P=0.0146). Using an induction protocol (day -1 to day 3), CP-105696 at 100 mg/kg/day significantly prolonged allograft survival (33+/-23 days [n=9]; P=0.0026), but CP-105696 at 10 mg/kg/day did not (18+/-16 days [n=8]; P=0.1433). Syngeneic grafts survived indefinitely (n=11). Immunohistological evaluation of allografts at rejection revealed a mononuclear cell infiltrate composed primarily of CD3+ and CD11b+ (Mac-1+) cells, which were infrequent in syngeneic grafts. Allografts from mice treated with CP-105696 at 50 or 100 mg/kg/day demonstrated a selective reduction in beta2-integrin (Mac-1) expression on monocytes/macrophages, as demonstrated by CD11b staining density compared with allograft controls. CONCLUSIONS: The results suggest that LTB4 or other potential ligands for LTB4 receptors may be important mediators of allograft rejection and support the clinical evaluation of LTB4 receptor antagonists in human organ transplantation.

Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

Identification of T cell subsets and class I and class II antigen expression in islet grafts and pancreatic islets of diabetic BioBreeding/Worcester rats.

The BioBreeding/Worcester (BB/Wor) rat develops a spontaneous disorder that closely resembles human insulin-dependent (Type I) diabetes mellitus. The syndrome is preceded by lymphocytic insulitis that destroys pancreatic beta cells. The morphologic features of the spontaneous insulitis lesions are also observed within islets transplanted beneath the renal capsule of diabetes-prone and diabetic animals. This study reports the results of experiments in which immunohistochemical techniques were used to characterize the phenotype of the infiltrating mononuclear cells and detect the expression of class I and class II MHC antigens in native islets and islet transplants in diabetic and diabetes-prone BB/Wor rats. The infiltrates within native pancreatic islets and islet grafts were comprised predominantly of Ia+ cells (dendritic cells and macrophages) CD4+ cells (helper/inducer lymphocytes and macrophages), CD5+ (pan-T) cells and smaller numbers of CD8+ (cytotoxic/suppressor and NK) cells. Pancreatic and graft insulitis were accompanied by markedly enhanced class I antigen expression on islet and exocrine cells. Class II (Ia) antigens were not detected on normal islet cells, islets undergoing insulitis or on islet transplants subjected to immune attack. In islet grafts stained with polymorphic MAbs that distinguish Ia antigens of donor and host origin, Ia antigen expression was limited to infiltrating dendritic cells and macrophages of host origin. It is concluded that the phenotypes of infiltrating mononuclear cells that comprise the insulitis lesion in spontaneous BB/Wor diabetes, and the inflammatory attack on islets transplanted into diabetic BB/Wor rats are the same, that pancreatic islet and graft insulitis occur in the presence of enhanced class I antigen expression but in the absence of class II antigen expression, and that infiltrating Ia+ cells within islet grafts are exclusively of recipient (BB/Wor) origin and may explain the initiation of immune insulitis within grafts derived from donors of incompatible MHC.

Prevention of diabetes in BioBreeding/Worcester rats with monoclonal antibodies that recognize T lymphocytes or natural killer cells.

Diabetes-prone BioBreeding/Worcester (BB/Wor) rats received thrice weekly injections of mAb against antigens expressed on the surface of all T cells (OX19), cytotoxic/suppressor, and NK cells (OX8), helper/inducer cells (W3/25, OX35, OX38), and Ia+ cells (OX6, 3JP, OX17). Treatment with OX8 or OX19 achieved stable reductions of splenic and peripheral blood NK cells and helper/inducer T lymphocytes, respectively, and protected against diabetes. OX19 injections also prevented lymphocytic insulitis, thyroiditis, and the synthesis of autoantibodies to thyroid colloid and smooth muscle antigens. OX8 injections reduced splenic NK-mediated YAC-1 cell lysis, but did not prevent insulitis, thyroiditis, or autoantibody synthesis. Injections of mAb specific for antigens on the surface of helper/inducer cells, and for cells expressing IaE antigens provided marginal protection against diabetes without reductions of phenotypic subsets. These findings suggest that pancreatic beta cell destruction in the spontaneously diabetic BB/Wor rat is mediated by the combined action of NK and helper/inducer cells.

Diabetes mellitus in the BB/W rat. Insulitis in pancreatic islet grafts after transplantation in diabetic recipients.

Spontaneous diabetes mellitus in the BioBreeding/Worcester (BB/W) rat is preceded by lymphocytic insulitis which destroys pancreatic beta cells. Cultured major histocompatibility complex identical pancreatic islets and adrenal cortex derived from diabetes-resistant BB/W donors were transplanted into diabetic recipients with hyperglycemia of variable duration. Islet grafts were the targets of BB/W immune attack and revealed lymphocytic insulitis after transplantation into diabetic recipients even in the absence of insulitis within endogenous pancreatic islets. These findings suggest that the BB/W immune attack on pancreatic beta cells can recur in islet grafts long after the onset of the diabetic syndrome.

Immune attack on pancreatic islet transplants in the spontaneously diabetic BioBreeding/Worcester (BB/W) rat is not MHC restricted.

Spontaneous diabetes mellitus in the BB/W rat is preceded by lymphocytic insulitis that destroys pancreatic beta cells. Cultured pancreatic islets and adrenal cortex from inbred rats of variable MHC were transplanted to RT1/u BB/W rats without allograft rejection. Islet grafts from RT1/u and non-RT1/u rats evidenced lymphocytic insulitis in BB/W recipients that became diabetic or evidenced lymphocytic insulitis within endogenous islets. These findings suggest that BB immune insulitis is not MHC restricted and may be directed against islet transplants from non-RT1/u animals.

Adoptive transfer of autoimmune diabetes mellitus in biobreeding/Worcester (BB/W) inbred and hybrid rats.

Adoptive transfer of diabetes was accomplished by the injection of Con A-activated acutely diabetic BB/W rat spleen cells into immunosuppressed diabetes-resistant BB/W control rats and F1 hybrid offspring produced by BB/W X Lewis, BN, Yashida, and NEDH matings. Immune suppression methods that facilitated adoptive transfer of diabetes included neonatal thymectomy, cyclophosphamide, and splenectomy plus rabbit anti-rat lymphocyte serum injections. The successful transfer of BB/W diabetes to otherwise normal (BB/W X inbred)F1 rats and to diabetes-resistant BB/W animals suggests that antigenically normal pancreatic beta cells were destroyed by the injected effector cells. Diabetes-resistant BB/W control rats also evidenced diabetes after the injection of cyclophosphamide alone. The requirement for immunosuppression suggests that an intact immune system protects against adoptive transfer and diabetes in diabetes-resistant BB/W rats.

Spontaneous autoimmune diabetes mellitus in the BB rat.

The BioBreeding Rat is a recently discovered model of spontaneous diabetes mellitus. Studies to date have revealed the following characteristics of the syndrome: genetic predisposition, equal frequency and severity among males and females, absence of obesity, life sustaining requirement for insulin therapy, lymphocytic insulitis with destruction of pancreatic beta-cells, lymphocytic thyroiditis and the presence of autoantibodies to smooth muscle, thyroid colloid and other cellular antigens. Animals raised in a germ-free environment evidence diabetes with equal frequency and severity. Support for a cell-mediated autoimmune pathogenesis of the diabetic syndrome is derived from the following experiments: administration of antiserum to rat lymphocytes prevents diabetes in susceptible animals and normalizes plasma glucose levels in 36% of diabetic rats; neonatal thymectomy almost completely prevents the occurrence of diabetes. Although the BB rat may not be an appropriate model for studying the vascular complications of diabetes, peripheral nerve functional and ultrastructural defects have been reported and renal glomerular immuneglobulin deposits have been observed in long-term diabetic animals.

Effects of insulin on wound healing in diabetic mice.

In the present study wound healing was examined in normal C57B1/6 male mice, diabetic mice, non-treated; and in diabetic mice treated with insulin. Small dermal wounds were made in the ears of the mice 40 h after the initial injection of insulin or vehicle alone. All animals were biopsied 8 h later. The wounds were examined by light and electron microscopy and wound components (capillaries, fibroblasts, PMN's oedema, collagen) were quantitated by lineal point analysis. The non-treated, diabetic mice demonstrated in inability to heal wounds when compared to controls; whereas, diabetic mice given a multidose insulin regimen demonstrated a response similar to controls. Insulin treatment of diabetic mice reduced the mean level of hyperglycaemia when compared to the non-treated diabetic mice. There was no detectable difference in the healing response with duration of diabetes in either the insulin-treated or non-treated diabetic mice. Although there was a mild reduction in hyperglycaemia, these data support the hypotheses that insulin is a necessary component of an adequate wound healing response.

Wound healing in normal and diabetic Chinese hamsters.

Wound healing was examined in normal and diabetic, non ketotic Chinese hamsters by morphological and morphometric methods. Dermal, perforating wounds were made in the ears of the hamsters and the response to injury was evaluated in tissue biopsies. The response in normal hamsters was characterized by vascular and cellular migration and pronounced infiltration of polymorphonuclear leukocytes into the area closest to the wound (zone 1). The transition region (zone 2) between wounded and non-wounded tissue was infiltrated primarily by fibroblasts and capillaries. In wounds from diabetic hamsters, 8 h after injury, there was less cellular infiltration (fibroblasts 49%, polymorphonuclear leukocytes 48% of control) and vascular proliferation (47% of control). In the late phase of healing (16 h after injury) the vascular (87% of control) and polymorphonuclear leukocyte (103%) responses in diabetic wounds were not significantly different from control in zones 1 and 2. Wounds from diabetic hamsters also showed considerable oedema (143% of control) in zones 1 and 2, which was accompanied by vascular degeneration and necrosis. At 16 h the collagen content of diabetic wounds was also decreased (54% of control). Increased oedema with reduced vascular proliferation and cellular infiltration in the early healing period characterises the response to injury in the diabetic Chinese hamster.

The effect of antisera to insulin, 2-deoxyglucose-induced hyperglycemia, and starvation on wound healing in normal mice.

Wound healing was examined in normal C57BL/6 male mice treated with antiserum to insulin or 2-deoxyglucose (2-DG) and in mice starved for 33 h. Hyperglycemia was induced after antiserum or 2-DG treatment; the blood glucose was lowered in the starved mice when compared with controls. Small dermal wounds were made in the ears of the mice 1 h after the initial injection of antisera or 2-DG. The starved group were wounded after 25 h of fasting. All animals were biopsied 8 h later. The wounds were examined by light microscopy and wound components (capillaries, fibroblasts, PMNs, collagen, and edema) were quantitated by lineal point analysis. Mice treated with antisera to insulin and mice starved for 33 h had an impaired healing response; the mice treated with 2-DG had a response similar to controls. These results suggest that hyperglycemia, per se, or the production of any toxic metabolites from high blood glucose levels could not alone induce the poor healing response. The depressed response in the antiserum-treated and starved mice may be due to the decreased availability of insulin to the wound tissues. These data support the hypotheses that insulin is a necessary component for an adequate wound healing response. In addition to a role in glucose transport and metabolism, insulin may also promote cellular growth.

A proposed cellular mechanism for calcium transport in the intestinal epithelial cell.

Intracellular transport of calcium from the apical to the basal-lateral region of the intestinal epithelial cell was investigated in duodenum from normal fed, fasted, and calcium-loaded rats. The process was followed with time using electron microscopy with potassium pyroantimonate to precipitate calcium. The observations made were subjected to morphometric analysis. The specificity of the method was demonstrated in the villus cell by resistance to microincineration and by absence of deposits following exposure to EGTA. Using this method calcium was seen in cells from calcium-fed rats at the microvillus border, in the Golgi zone, and within the internal compartments of the mitochondria. In cells from fasted rats calcium was not seen. Mitochondria were found largely at the apex of the cell and were free of detectable calcium. By 5 min, in the cells of fasted rats given a calcium load, the calcium had reached the Golgi apparatus and the inner mitochondrial compartment. After 15 min mitochondria were heavily loaded with calcium and had moved to the basal region of the cell. These observations suggest that mitochondria play an important role in absorption of calcium and appear to transport this ion from the apex to the basal region of the cell where entry into the capillaries takes place.

Wound healing: a model for the study of diabetic angiopathy.

Wound healing as a model for diabetic angiopathy has been studied by light and electron microscopy. Biochemical studies of the rate of incorporation of 3H-proline and 3H-thymidine into collagen and DNA, respectively, have confirmed the morphologic observations. In both the normal and the diabetic, there was a marked decrease in the rate of collagen and DNA synthesis, suggesting that most of the cells in the biopsies were stunned by the injury and ceased DNA replication during the initial phase. In control mice this decrease was followed by a modest but significant burst of DNA replication, which peaked at two hours and by the fourth hour had returned to the one-hour level. In the diabetic this burst of DNA replication was absent and no capillary morphogenesis was seen at two, four, and eight hours. At 16 hours, there were only a few abnormal nascent vessels observed in the diabetic and antiserum-treated mice. The peak in the rate of collagen synthesis at four hours correlated well with the condensation of collagen at the wound margin and the fibroblast rough-endoplasmic-reticulum (RER) proliferation. In the diabetic mice, there was a significantly attenuated rate of collagen synthesis for the entire 16-hour period. The lack of DNA replication, capillary morphogenesis, fibroblast RER proliferation, and decreased collagen synthesis in the diabetic mouse can be considered interrelated and significant factors in the diabetic's impaired response to cellular injury. In view of the increased frequency and severity of injury to the circulation of the diabetic and the impaired response to repair such injury, it is likely that wound healing is a promising model for diabetic angiopathy.