Clonotypic analysis of anti-acetylcholine receptor antibodies from experimental autoimmune myasthenia gravis-sensitive Lewis rats and experimental autoimmune myasthenia gravis-resistant Wistar Furth rats

A single immunization of Lewis rats with purified acetylcholine receptor (AChR) emulsified in adjuvant typically stimulates the production of oligoclonal AChR-reactive antibodies (as demonstrated by IEF) dominated by the IgG2a subclass, of moderate but clonotypically heterogeneous relative Ag-binding avidity, and capable of inducing symptoms of experimental autoimmune myasthenia gravis. Although similar immunization of Wistar Furth rats produces AChR-reactive antibodies with similar characteristics of clonotypic heterogeneity, avidity, and isotype expression, no detectable signs of AChR-dependent muscle impairment is observed. This contrasts the ability to induce impaired AChR function upon the passive transfer of pre-formed Lewis anti-AChR antibodies into naive Wistar Furth rats, suggesting that disease resistance in this model is not conferred at the level of the AChR itself. Moreover, if more aggressive immunization protocols are used (i.e., multiple injections of AChR), a transient breakthrough of AChR-dependent muscle dysfunction can be induced directly in the Wistar Furth strain indicating that the potential for the production of disease-causing antibodies does exist in the Wistar Furth repertoire. IEF analysis of Wistar Furth anti-AChR antibodies has revealed that hyperimmunization results in modified antibody clonotype expression that might explain changing expression of disease symptoms; however, explanations for the apparent "resistance" of Wistar Furth rats to disease induction are likely to be complex.     

Scoparia dulcis, a traditional antidiabetic plant, protects against streptozotocin induced oxidative stress and apoptosis in vitro and in vivo

Oxidative stress is implicated in the pathogenesis of diabetic complications. The experiments were performed on normal and experimental male Wistar rats treated with Scoparia dulcis plant extract (SPEt). The effect of SPEt was tested on streptozotocin (STZ) treated Rat insulinoma cell lines (RINm5F cells) and isolated islets in vitro. Administration of an aqueous extract of Scoparia dulcis by intragastric intubation (po) at a dose of 200 mg/kg body weight significantly decreased the blood glucose and lipid peroxidative marker thiobarbituric acid reactive substances (TBARS) with significant increase in the activities of plasma insulin, pancreatic superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in streptozotocin diabetic rats at the end of 15 days treatment. Streptozotocin at a dose of 10 mug/mL evoked 6-fold stimulation of insulin secretion from isolated islets indicating its insulin secretagogue activity. The extract markedly reduced the STZ-induced lipidperoxidation in RINm5F cells. Further, SPEt protected STZ-mediated cytotoxicity and nitric oxide (NO) production in RINm5F cells. Treatment of RINm5F cells with 5 mM STZ and 10 mug of SPEt completely abrogated apoptosis induced by STZ, suggesting the involvement of oxidative stress. Flow cytometric assessment on the level of intracellular peroxides using fluorescent probe 2'7'-dichlorofluorescein diacetate (DCF-DA) confirmed that STZ (46%) induced an intracellular oxidative stress in RINm5F cells, which was suppressed by SPEt (21%). In addition, SPEt also reduced (33%) the STZ-induced apoptosis (72%) in RINm5F cells indicating the mode of protection of SPEt on RIN m5Fcells, islets, and pancreatic beta-cell mass (histopathological observations). Present study thus confirms antihyperglycemic effect of SPEt and also demonstrated the consistently strong antioxidant properties of Scoparia dulcis used in the traditional medicine.     

Effect of long-term thyroxine administration on the endocrine epithelium of rat pancreas]

The effect of prolonged thyroxine administration (0.001 mg/g BW) on pancreatic islets has been studied on 64 Wistar male rats by means of radioautographic, morphometric and electron microscopic methods. The phase response in the amount of the DNA-synthesising cells of the middle class islets has been revealed: the initial increase (5 days) is followed by a decrease (30 days) and then by a return to the control levels (60 days). The level of metabolism in sulphur-containing proteins has decreased in both A- and B-cells. After 30 days of the experiment, B/A cell volume ratio has been shown to increase. Electron microscopic studies have revealed ultrastructural reorganization of B-cells from "resting" B-cells into "dark" B-cells at increased excretion of secretory material.     

In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

Disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal rats like those in human and Wistar Furth rat hereditary macrothrombocytopenias

Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May-Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar rat bone marrow was fixed and processed for transmission electron microscopy after VCR administration alone, after 5-fluorouracil (5-FU) administration alone, or after 5-FU followed by intravenous injection of 0.1–1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5-FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5-FU caused formation of large membrane complexes in the cytoplasm of Wistar rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5-FU and VCR, and was maximal (～two-third of megakaryocytes) at VCR dosages of 0.75–1.0 mg/kg. The majorityof megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted α-granule proteins, and an increased incidence of emperipolesis. Rats administered 5-FU alone did not demonstrate these abnormalities, with the exception of an increase in dense compartments. Platelets from rats treated with VCR aloene or 5-FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface-connected canalicular system (SCCS). The membrane complexes in platelets of 5-FU-, VCR-treated Wistar rats as well as untreated Wistar Furth rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5-FU-, VCR-treated rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte α-granules. © 1995 Wiley-Liss, Inc.     

Intragraft gene expression profile associated with the induction of tolerance by allochimeric MHC I in the rat heart transplantation model

The MHC class I allochimeric protein containing donor‐type epitopes on recipient‐type heavy chains induces indefinite survival of heterotopic cardiac allografts in rats. We analyzed gene expression profile of heart allograft tissue. Mutated peptide [α1h1/u]‐RT1.Aa that contains donor‐type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient‐type (ACI, RT1a) was delivered into ACI recipients of WF hearts at the time of transplantation in addition to a 3 days course of oral cyclosporine. Microarray analysis was performed using Affymetrix Rat 230 2.0 Microarray. Allochimeric molecule treatment caused upregulation of genes involved in structural integrity of heart muscle, downregulation of IL‐1β a key modulator of the immune response, and downregulation of partitioning defective six homolog gamma PAR6, which is involved in T cell polarity, motility, and ability to scan dendritic cells (DC). These indicate that the immunosuppressive function of allochimeric molecule and/or the establishment of allograft tolerance depend on the induction of genes responsible for the heart tissue integrity, the suppression of cytokine pathway(s), and possibly the impairment of T cells mobility and their DC scanning ability. These novel findings may have important clinical implications for inhibition of chronic rejection in transplant recipients. genesis 48:8–19, 2010. © 2009 Wiley‐Liss, Inc.     

Expression of Protein Kinase C Isoforms in Pancreatic Islets and Liver of Male Goto-Kakizaki Rats,a Model of Type 2 Diabetes

Protein kinase C (PKC) is a family of protein kinases controlling protein phosphorylation and playing important roles in the regulation of metabolism. We have investigated expression levels of PKC isoforms in pancreatic islets and liver of diabetic Goto-Kakizaki (GK) rats with and without insulin treatment to evaluate their association with glucose homeostasis. mRNA and protein expression levels of PKC isoforms were assessed in pancreatic islets and liver of Wistar rats and GK rats with or without insulin treatment. PKCα and PKCζ mRNA expressions were down-regulated in islets of GK compared with Wistar rats. PKCα and phosphorylated PKCα (p-PKCα) protein expressions were decreased in islets of GK compared with insulin-treated GK and Wistar rats. PKCζ protein expression in islets was reduced in GK and insulin-treated GK compared with Wistar rats, but p-PKCζ was decreased only in GK rats. Islet PKCε mRNA and protein expressions were lower in GK compared with insulin-treated GK and Wistar rats. In liver, PKCδ and PKCζ mRNA expressions were decreased in both GK and insulin-treated GK compared with Wistar rats. Hepatic PKCζ protein expression was diminished in both GK rats with and without insulin treatment compared with Wistar rats. Hepatic PKCε mRNA expression was down-regulated in insulin-treated GK compared with GK and Wistar rats. PKCα, PKCε, and p-PKCζ expressions were secondary to hyperglycaemia in GK rat islets. Hepatic PKCδ and PKCζ mRNA expressions were primarily linked to hyperglycaemia. Additionally, hepatic PKCε mRNA expression could be under control of insulin.     

Effects of 3-isobutyl-1-methylxanthine on secretory response, cAMP accumulation and DNA synthesis of islets from postnatal and adult Wistar rats

A possible role for cyclic adenosine-3'-5'-monophosphate (cAMP) in islet cell replication was examined in collagenase-isolated pancreatic islets from Wistar rats of different age and different metabolic state (non-pregnant, pregnant, days 15.5-17.5). Islets obtained from pregnant rats released significantly more insulin in response to 10 mmol/l glucose (culture for 24 h) and their DNA synthesis (incorporation of [3H]thymidine into islet DNA) was doubled compared to islets from non-pregnant controls. Islets obtained from 4-6 days old rats showed a maximal stimulation of DNA synthesis after exposure to 0.1 mmol/l IBMX (3-isobutyl-1-methylxanthine) whereas the cAMP accumulation and the insulin biosynthesis measured in a subsequent short-term incubation were dose-dependent stimulated up to 1.0 mmol/l IBMX. In islets of 12 days old rats or 3 months old rats, however, IBMX did not stimulate DNA synthesis or insulin release measured during culture, although the cAMP content per islet was significantly enhanced after culture in the presence of IBMX.     

Correlations between the presence of islet cell surface antibodies in human serum and the cytotoxic effects on rat islet beta-cells

Isolated pancreatic islets from 7-10 days old Wistar rats were incubated in freshly prepared human serum. The cytotoxic insulin leakage from the islets was correlated with the presence in the sera of islet cell surface antibodies (ICSA) determined by indirect immunofluorescence. Such cytotoxic ICSA were found to be common in Type-I diabetics as well as in nondiabetic subjects who had contact with laboratory rats.     

Rat islet isolation yield and function are donor strain dependent

Effective rat islet isolation is pertinent for successful islet transplantation and islet studies in vitro. To determine which rat strain yields the highest number of pure and functional islets, four commonly used rat strains were compared with regard to islet yield, islet purity and islet function. Secretory responses were assessed by stimulation with glucose, and by stimulation with glucose plus 3-isobutyl-1-methylxanthine (IBMX). We show that rat islet function and isolation yield are donor strain dependent. Albino Oxford (AO) rats donated twice as many islets than Wistar, Lewis and Sprague Dawley (SD) rats. Stimulation with glucose plus IBMX resulted in an average five-fold increase of the stimulation index of AO, Lewis, Wistar and SD rats compared to stimulation with glucose only. AO islets had improved secretory responses after a one-week culture period, but required the addition of IBMX to glucose to elicit a distinguished stimulated insulin secretion after 2 days of culture. Islets from SD rats showed inferior results with regard to purity immediately after isolation and with regard to function after short- and after long-time culture. Because Lewis islets possessed the highest secretory response to glucose (without IBMX) immediately after isolation, Lewis rats may be preferred as islet donors for immediate use. The addition of IBMX to glucose for in vitro functional testing is recommended because it elicits high insulin secretory responses of islets regardless of the rat strain. AO rats are preferred for culture experiments since the number of experimental animals is reduced two-fold compared to Lewis, Wistar and SD rats.     

Opposite actions of caffeine and creatine on muscle relaxation time in humans.

The effect of creatine and caffeine supplementation on muscle torque generation and relaxation was investigated in healthy male volunteers. Maximal torque (T(max)), contraction time (CT) from 0.25 to 0.75 of T(max), and relaxation time (RT) from 0.75 to 0.25 of T(max) were measured during an exercise test consisting of 30 intermittent contractions of musculus quadriceps (2 s stimulation, 2 s rest) that were induced by electrical stimulation. According to a double-blind randomized crossover design, subjects (n = 10) performed the exercise test before (pretest) and after (posttest) creatine supplementation (Cr, 4 x 5 g/day, 4 days), short-term caffeine intake (Caf, 5 mg x kg(-1) x day(-1), 3 days), creatine supplementation + short-term caffeine intake (Cr+Caf), acute caffeine intake (ACaf, 5 mg/kg) or placebo. Compared with placebo, Cr shortened RT by approximately 5% (P < 0.05). Conversely, Caf increased RT (+ approximately 10%, P < 0.05), in particular as RT increased because of fatigue. RT was not significantly changed by either Cr+Caf or ACaf. T(max) and CT were similar during all experimental conditions. Initial T(max) was approximately 20% of voluntary maximal isometric contraction force, which was not different between treatments. It is concluded that Caf intake (3 days) prolongs muscle RT and by this action overrides the shortening of RT due to creatine supplementation.     

Cellular basis of allograft rejection in vivo. V. Examination of the mechanisms responsible for the differing efficacy of monoclonal antibody to CD4+ T cell subsets in low- and high-responder rat strains

MRC OX35, an anti-CD4 mAb, was used to treat high responder Wistar Furth (W/F) (RT1u) and low responder DA (RT1a) rats which had been grafted with directly vascularized hearts from PVG (RT1c) rats across a full MHC plus non-MHC incompatibility. Four doses of mAb at 7 mg/kg given in the first 2 wk postgrafting induced indefinite graft survival (greater than 150 days) in DA hosts, but only delayed rejection to 18 to 42 days in W/F as compared to rejection times of 6 to 8 days in untreated rats. The extension of MRC OX35 treatment to 6 wk in W/F rats induced indefinite graft survival in three of six rats. During treatment MRC OX35 therapy only partially depleted CD4+ cells, and all circulating CD4+ cells were coated with MRC OX35. The capacity of naive CD4+ and CD8+ cells from W/F and DA to be activated to PVG alloantigen was compared both in vitro in an MLC assay and in vivo by an adoptive transfer assay of their capacity to restore rejection of PVG heart grafts in irradiated syngeneic hosts. CD4+ cells from both W/F and DA proliferated in MLC and restored graft rejection. W/F CD8+ cells both proliferated in MLC and restored rejection, but DA CD8+ cells neither proliferated nor reconstituted rejection. Examination of lymphocytes from MRC OX35 treated hosts with long-surviving grafts showed that they were neither depleted of CD4+ T cells nor did they lack the capacity to proliferate to PVG Ag in MLC, this response being similar to that to third-party Ag or by naive lymphocytes. Compared to first-set rejection, PVG skin graft rejection was delayed 2 to 3 days in W/F and 10 to 12 days in DA rats with long-surviving grafts after MRC OX35 therapy, whereas they rejected third-party skin grafts in first-set tempo. These studies show that differences in graft survival in anti-CD4 treated low and high responder strains may be due to the inherent capacity of CD8+ cells to be activated to effect rejection independent of CD4+ cells in W/F but not in DA. In those hosts that accept grafts, there is no evidence of clonal deletion, but there appears to be a form of unresponsiveness akin to that induced in adult rats by other immunosuppressive therapies that protects the graft from rejection.     

Role of non-RT1 genes in the response of rat lymphocytes to Mycoplasma arthritidis T cell mitogen, concanavalin A and phytohemagglutinin

A comparison of splenic cells from various inbred rat strains indicated that DA, Lewis, Buffalo, August, Wistar Furth, and (LEW X BN)F1 all responded well to the Mycoplasma arthritidis T cell mitogen, phytohemagglutinin and concanavalin A, but cells from BN and MAXX rats were very weakly or nonresponsive. Cells from congenic strains expressing nonresponder background genes, and responder haplotypes at RT1 (BN.1L(LEW), RT1; BN.1A(DA), RT1av1) failed to respond significantly to the mitogens. Rats expressing responder background genes but the nonresponder haplotype at RT1 at RT1 (WF.1N-(MAXX), RT1n) exhibited high responses to all mitogens. The controlling role of non-RT1 genes was confirmed by testing tissue-typed (DA X BN)F2 progeny and (DA X BN)F1 X DA and (DA X BN)F1 X BN progeny. No association was seen between the expression of a/a, a/n, or n/n at RT1 and the degree of response to the mitogens. In contrast, as the proportion of DA non-RT1 genes increased, so did the degree of mitogenic responsiveness. Similar results were obtained by using a partially purified preparation of the mycoplasma T cell mitogen. The results indicated that in the (DA X BN)F1 hybrids, responsiveness to all mitogens was recessive: this contrasts with the (LEW X BN)F1 hybrids in which responsiveness was dominant. Finally, we showed that both responder and nonresponder splenic cells were capable of binding the M. arthritidis mitogen. The data contrast with those obtained with nonresponder mouse strains the cells of which failed to bind mitogen due to the absence of the E alpha chain of the I-E-coded molecule.     

Reduced glucose‐induced insulin secretion in low‐protein‐fed rats is associated with altered pancreatic islets redox status

In the present study, we investigated the relationship between early life protein malnutrition‐induced redox imbalance, and reduced glucose‐stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal‐protein‐diet (17%‐protein, NP) or to a low‐protein‐diet (6%‐protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H₂O₂), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn‐superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre‐incubated with H₂O₂ and/or N‐acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H₂O₂ production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre‐incubated with H₂O₂ (100 μM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N‐acetylcysteine.     

Vitrification of mouse islets of Langerhans: comparison with a more conventional freezing method

The possibility of cryopreservation of islets of Langerhans by vitrification using a mixture of cryoprotectants was investigated and the results were compared with a more conventional freezing method using Me2SO as cryoprotectant. Isolated mouse islets were divided into three groups: (1) control islets cultured for 6 days, (2) islets which were cryopreserved by vitrification after 2 days of culture, and (3) islets frozen in 1.5 M Me2SO after 2 days of culture. After warming, islets from groups 2 and 3 were cultured for 4 days. The thus treated islets were investigated with respect to insulin secretion in the presence of 2.5 or 25 mM glucose, survival during postwarming culture, morphology, and capability to reverse streptozotocin-induced diabetes. The insulin secretion in islets from all groups could be stimulated by a factor 5 or more by an increase in the concentration of glucose from 2.5 to 25 mM. The secretion of insulin in the presence of 2.5 mM glucose was similar in all groups of islets. The secretion of insulin in the presence of 25 mM glucose was slightly but not significantly lower in the cryopreserved islets than in the control noncryopreserved islets. The survival of islets during postwarming culture was comparable after cryopreservation with both methods, and islets from both groups could lower serum glucose in streptozotocin diabetic mice. We conclude that islets cryopreserved by the vitrification method are functional in vitro and in vivo. This method is quick, simple, and cheap because the use of complicated freezing equipment is avoided.     

Decreased serglycin proteoglycan size is associated with the platelet alpha granule storage defect in Wistar Furth hereditary macrothrombocytopenic rats. Serglycin binding affinity to type I collagen is unaltered

The Wistar Furth (WF) rat has a hereditary defect in platelet formation that resembles gray platelet syndrome of man with a large mean platelet volume and platelet alpha granule deficiency. The alpha granule abnormality is suggestive of a defect in granule packaging and/or stability. Proteoglycans are hypothesized to play a role in granule packaging. Therefore, we have analyzed the structure of the platelet proteoglycan, serglycin, in platelets of WF and normal Wistar rats. Normal and Wistar Furth rats were injected with ³⁵S-sulfate to label platelet proteoglycans via synthesis by the megakaryocytes, and platelets were isolated 3 days later. We found that WF rat platelets have only one-third of the normal proteoglycan mass per unit platelet volume, and the proteoglycans are smaller in hydrodynamic size with shorter glycosaminoglycan chains than those of Wistar rats. However, WF rat platelet proteoglycans showed no defect in binding to collagen on affinity coelectrophoresis gels. We conclude that the structure of WF rat platelet proteoglycans is abnormal, and speculate that this abnormality may contribute to abnormal packaging of the alpha granule contents. Leakage of alpha granule contents into the marrow by platelets and megakaryocytes could perturb the marrow matrix, and promote the development of myelofibrosis noted in gray platelet syndrome. J. Cell. Physiol. 172:87–93, 1997. © 1997 Wiley-Liss, Inc.     

Functional beta cell regeneration in the islets of pancreas in alloxan induced diabetic rats by (−)-epicatechin

(?)-Epicatechin (1), a naturally occuring flavonoid compound was found to have reversed the diabetogenic action of alloxan in albino rats (2). (?)-Epicatechin administration in doses of 30 mg/kg (i.p) twice daily for 4–5 days in alloxan induced (150 mg/kg, i.p.) diabetic albino rats (either sex), has brought down the high blood sugar levels to normal values. Concurrent histological studies of the pancreas of these animals showed regeneration of the beta-cell population of the islets which were earlier necrosed by alloxan. Immunoreactive insulin (IRI) studies showed that the regenerated beta-cells of the islets of pancreas are functional in nature.     

Resistance to experimental autoimmune myasthenia gravis in genetically inbred rats. Association with decreased amounts of in situ acetylcholine receptor-antibody complexes

Genetically related susceptibility for experimental autoimmune myasthenia gravis was investigated in nine inbred strains of rats immunized with heterologous acetylcholine (AChR) from Torpedo californica. Wistar Munich and Fischer strain animals consistently developed severe, fatal disease associated with impaired neuromuscular transmission and increased sensitivity to low doses of curare. A lower incidence of disease was induced in Wistar Kyoto, ACI, Brown Norway, Buffalo, and Lewis strain animals. In contrast, Wistar Furth and Copenhagen strain animals were resistant to experimental autoimmune myasthenia gravis, electrophysiologic responses were normal, and animals were insensitive to curare. All strains of animals manifested equivalent amounts of serum antibody to AChR and total muscle AChR was reduced to the same extent in both resistant and susceptible animals. In contrast, the amount of antibody-bound AChR was greater in susceptible Wistar Munich animals than the amount observed in resistant Wistar Furth animals. These data suggest that impaired neurotransmission is correlated with the extent of antibody binding to the AChR. The discordance in the amount of antibody bound to the AChR of resistant and susceptible animals may result from heritable differences in antibody properties. Cross-breeding experiments with Wistar Munich and Wistar Furth animals show that resistance for development of experimental autoimmune myasthenia gravis is recessive and indicate that disease susceptibility is linked to one or two genetic loci.     

Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury

Hypoxia/reoxygenation (H/R)‐induced injury is the key factor associated with islet graft dysfunction. This study aims to examine the effect of mesenchymal stem cells (MSCs) on islet survival and insulin secretion under H/R conditions. Islets from rats were isolated, purified, cultured with or without MSCs, and exposed to hypoxia (O₂ ≤ 1%) for 8 h and reoxygenation for 24 and 48 h, respectively. Islet function was evaluated by measuring basal and glucose‐stimulated insulin secretion (GSIS). Apoptotic islet cells were quantified using Annexin V‐FITC. Anti‐apoptotic effects were confirmed by mRNA expression analysis of hypoxia‐resistant molecules, HIF‐1α, HO‐1, and COX‐2, using semi‐quantitative retrieval polymerase chain reaction (RT‐PCR). Insulin expression in the implanted islets was detected by immunohistological analysis. The main results show that the stimulation index (SI) of GSIS was maintained at higher levels in islets co‐cultured with MSCs. The MSCs protected the islets from H/R‐induced injury by decreasing the apoptotic cell ratio and increasing HIF‐1α, HO‐1, and COX‐2 mRNA expression. Seven days after islet transplantation, insulin expression in the MSC‐islets group significantly differed from that of the islets‐alone group. We proposed that MSCs could promote anti‐apoptotic gene expression by enhancing their resistance to H/R‐induced apoptosis and dysfunction. This study provides an experimental basis for therapeutic strategies based on enhancing islet function. Copyright © 2010 John Wiley & Sons, Ltd.