Neuroendocrine immuno-ontogeny of the pathogenesis of autoimmune disease in the nonobese diabetic (NOD) mouse

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which insulin-producing beta cells of the pancreatic islets of Langerhans are destroyed. The nonobese diabetic (NOD) mouse is one of the rare spontaneous models that enable the study of prediabetic pancreatic events. The etiology of the autoimmune attack in human and animal T1D is still unknown, but genetic and environmental factors are involved in both cases. Although several autoantigens have been identified and defective immune-system regulation is implicated, this information does not satisfactorily explain the generally accepted beta-cell specificity of the disease or how so many and diverse environmental factors intervene in its pathogenesis. Based on data obtained from evaluating glucose homeostasis in a variety of situations, particularly stress and cytokine administration, in young prediabetic NOD mice, the author hypothesizes that the islet of Langerhans is a major actor, and its altered regulation through environmentally induced insulin resistance might reveal latent T1D. It is also postulated that T1D pathogenesis might be linked to abnormal pancreas development, probably due to disturbances of glutamic acid decarboxylase (GAD)+ innervation phagocytosis by defective macrophages during the early postnatal period. Also discussed is the role of defective presentation of pancreatic hormones and GAD in the thymus, and its potential repercussion on T-cell tolerance. Observations have demonstrated that the diabetogenic process in the NOD mouse is extremely complex, involving neuroendocrine immune interaction from fetal life onward.     

Antidiabetic properties of Hibiscus rosa sinensis L. leaf extract fractions on nonobese diabetic (NOD) mouse

On fractionation the ethanolic extract of H. rosa sinensis leaves, 5 fractions were obtained. Of these, fraction-3 (F3) and fraction-5 (F5) were chosen for detailed investigation on non obese diabetic (NOD) mouse to study anti-diabetic properties because they were more active than others. Serum glucose, glycosylated hemoglobin, triglyceride, cholesterol, blood urea, insulin, LDL, VLDL, and HDL were estimated. Both fractions F3 and F5 on oral feeding (100 and 200 mg/kg body weight) demonstrated insulinotropic nature and protective effect in NOD mice. These fractions may contain potential oral hypoglycemic agent.     

Glucocorticoids in the nonobese diabetic (NOD) mouse: basal serum levels, effect of endocrine manipulation and immobilization stress.

The NOD mouse is a recognized model for studying immunologically mediated insulin-dependent diabetes mellitus (IDDM). In most colonies, the disease appears with a greater preponderance in females than in males and castration alters the expression of the disease. The prevalence of diabetes may also vary depending upon environmental factors such as stress. Therefore, we measured in the NOD mouse serum glucocorticoid concentrations in basal and stress conditions. We observed in NOD as well as in C57BL/6 mice, taken as controls, a circadian rhythm of corticosterone, with females having higher values than males. After a single restraint stress, female and male NOD mice exhibit a comparable response, whereas after repeated stress, males respond significantly less than females, suggesting an adaptation phenomenon. In contrast, there is no difference in the pattern of corticosterone response of C57BL/6 females and males to both types of stress, but females always respond better than males. Moreover, whatever the stress considered, NOD mice generally exhibit a higher corticosterone response than C57BL/6 mice. The sexual dimorphism in diabetes expression in NOD mice may be related to the levels of corticosterone, a hyperglycemic hormone, in both basal and stress conditions. However, the understanding of corticosteroid effects in this model of type I IDDM is rather complex given their well known anti-inflammatory and immunosuppressive effects in other models of autoimmune diseases.     

Statistical evaluation of multiple-locus linkage data in experimental species and its relevance to human studies: application to nonobese diabetic (NOD) mouse and human insulin-dependent diabetes mellitus (IDDM).

Common, familial human disorders generally do not follow Mendelian inheritance patterns, presumably because multiple loci are involved in disease susceptibility. One approach to mapping genes for such traits in humans is to first study an analogous form in an animal model, such as mouse, by using inbred strains and backcross experiments. Here we describe methodology for analyzing multiple-locus linkage data from such experimental backcrosses, particularly in light of multilocus genetic models, including the effects of epistasis. We illustrate these methods by using data from backcrosses involving nonobese diabetic mouse, which serves as an animal model for human insulin-dependent diabetes mellitus. We show that it is likely that a minimum of nine loci contribute to susceptibility, with strong epistasis effects among these loci. Three of the loci actually confer a protective effect in the homozygote, compared with the heterozygote. Further, we discuss the relevance of these studies for analogous studies of the human form of the trait. Specifically, we show that the magnitude of the gene effect in the experimental backcross is likely to correlate only weakly, at best, with the expected magnitude of effect for a human form, because in humans the gene effect will depend more heavily on disease allele frequencies than on the observed penetrance ratios; such allele frequencies are unpredictable. Hence, the major benefit from animal studies may be a better understanding of the disease process itself, rather than identification of cells through comparison mapping in humans by using regions of homology.     

Evidence for unimpaired pancreatic secretion and hepatic removal of insulin in healthy offspring of type 2 (noninsulin-dependent) diabetic couples

The aim of the present study was to investigate the secretion and the hepatic removal of insulin in a group of 14 unaffected offspring of 14 type 2 (noninsulin-dependent) diabetic couples compared to 14 healthy subjects without family history of diabetes mellitus. The two groups, each consisting of 5 obese and 9 nonobese subjects, were carefully matched for sex, age, and body weight. We examined glucose, insulin, and C-peptide levels, as well as C-peptide to insulin ratios and relations during the oral glucose tolerance test. Glucose concentrations and incremental areas were similar in the two groups, as well as insulin and C-peptide levels and areas. C-peptide to insulin molar ratios, both in fasting state and after glucose load, as well as relations between C-peptide and insulin incremental areas were not different. Our results suggest that the healthy offspring of type 2 diabetic couples have a normal response of beta-cell to oral glucose as well as a normal removal of insulin by the liver.     

Diabetic complications in a new animal model (TSOD mouse) of spontaneous NIDDM with obesity.

The TSOD mouse has been established as an inbred strain with spontaneous development of diabetes mellitus as the first clinical signs of diabetes. Polydipsia and polyuria are observed at about 2 months old only in male mice, after which hyperglycemia and hyperinsulinemia are detected. Following these symptoms obesity gradually develops until about 12 months old. In histopathological examination of the pancreas, severe hypertrophy of pancreatic islets was observed due to proliferation and swelling of B cells. In the kidney, thickening of the basement membrane in glomeruli and an increase of the mesangial area were observed at 18 months old. Motor neuropathy in TSOD mice began to appear at 14 months old and most male mice at 17 months old showed weakness of front and hind paws caused by neuron degeneration in the peripheral nerve. In sensory neuropathy, the threshold in the tail pressure test decreased significantly at 12 months old. Light microscopic and electron microscopic examination of sciatic nerves showed a decrease in the density of nerve fibers by the endoneural fibrosis and loss of these fibers. Degenerative changes of myelinated fibers, separation of myelin sheaths with intralamellar edema and remyelination were frequently observed. In the severely affected nerve fibers, the lamellar structure was completely destroyed and macrophages migrated around the myelin sheath or invaded the intramyelin space. Considering these findings similar to non-insulin dependent diabetes mellitus (NIDDM) in humans, the TSOD mouse should be a useful model for the pathogenic study of diabetic complications, especially of peripheral neuropathy.     

Myenteric plexus of obese diabetic mice (an animal model of human type 2 diabetes)

The myenteric plexus of the gastrointestinal tract was investigated in the obese diabetic mouse, an animal model of human type 2 diabetes. Sections were immunostained by the avidin-biotin complex method, using a general nerve marker, protein gene product 9.5 (PGP 9.5), as well as antibodies to several important neurotransmitters. Computerized image analysis was used for quantification. When diabetic mice were compared with controls, no difference was found in the density of PGP 9.5-immunoreactive (IR) nerve fibres in antrum, duodenum or colon. In antrum and duodenum, diabetic mice showed a decreased number of vasoactive intestinal peptide (VIP)-IR neurons in myenteric ganglia as well a decreased relative volume density in myenteric plexus (though not significantly in antrum, p=0.073). No difference was found regarding VIP-IR nerves in colon. The volume density of nitric oxide synthase (NOS)-IR nerve fibres was decreased in antrum and duodenum of diabetic mice, whereas no difference was found in colon. The density of galanin-IR nerve fibres was decreased in duodenum. Whereas neuropeptide Y (NPY)- and vesicular acetylcholine transporter (VAChT)-IR nerve fibres was increased in density in colon of diabetic mice, no difference was found in antrum and duodenum. Regarding substance P, there was no difference between diabetic and control mice in antrum, duodenum or colon. The present study shows that gut innervation is affected in this animal model of human type 2 diabetes. It is possible that the present findings may have some relevance for the gastrointestinal dysfunctions seen in patients with type 2 diabetes.     

Establishment of a new model of human multiple myeloma using NOD/SCID/gammac(null) (NOG) mice

We developed a new experimental animal model of human multiple myeloma using immunodeficient NOD/SCID/gammac(null) (NOG) mice. A human myeloma cell line, U266, was intravenously inoculated into 20 NOG mice, all of which developed hind leg paralysis and distress around 6 weeks after transplantation. Pathological studies showed that only the bone marrow was infiltrated with U266 cells, and no cells were present in other organs. Osteolytic lesions in cortical bones and loss of trabecular bones were prominent in U266-transplanted NOG mice. In contrast, U266 cells were not detected in CB17scid or NOD/SCID mice 6 weeks after intravenous inoculation. Human IgE, produced by U266 cells, was detected in the serum of U266-transplanted NOG mice by ELISA. The results indicated that this hu-myeloma NOG model might be useful for studying the pathogenesis of myeloma and related osteolytic lesions, and are suggestive of its applicability to the future development of new drugs.     

Pancreatic fate of a (125)I-labelled mouse monoclonal antibody directed against pancreatic B-cell surface ganglioside(s) in control and diabetic rats

The possible use of a mouse monoclonal antibody directed against rat pancreatic B-cell surface ganglioside(s) and labelled with radioactive iodine for selective imaging of the endocrine pancreas by a non-invasive procedure was investigated by following its pancreatic fate in experiments conducted either in vitro by incubation of rat isolated pancreatic islets, acinar tissue and pancreatic pieces or in vivo after intravenous injection of the (125)I-labelled antibodies ([(125)I]gamma-G). Although the binding of [(125)I]gamma-G per microg protein was about one order of magnitude higher in isolated islets than in acinar tissue, no significant difference was detected when comparing pancreatic pieces or isolated islets from control animals and rats rendered diabetic by one or two prior administrations of streptozotocin (STZ rats). Likewise, except in one set of experiments, no significant difference was found between control animals and STZ rats, when measuring the radioactive content of the pancreatic gland, relative to that of plasma, 1-4 days after the intravenous injection of [(125)I]gamma-G. These findings indicate that under the present experimental conditions, the mouse monoclonal antibody labelled with radioactive iodine does not appear to be a promising tool for selective imaging of the endocrine pancreas, e.g. by single photon emission computerized tomography.     

rim2 (recombination-induced mutation 2) is a new allele of pearl and a mouse model of human Hermansky-Pudlak Syndrome (HPS): genetic and physical mapping

A mouse mutation, rim2, is one of a series of spontaneous mutations that arose from the intra-MHC recombinants between Japanese wild mouse-derived wm7 and laboratory MHC haplotypes. This mutation is single recessive and characterized by diluted coat color and hypo-pigmentation of the eyes. We mapped the rim2 gene close to an old coat color mutation, pearl (pe), on Chromosome (Chr) 13 by the high-density linkage analysis. The pearl mutant is known to have abnormalities similar to Hermansky-Pudlak syndrome (HPS), a human hemorrhagic disorder, characterized by albinism and storage pool deficiency (SPD) of dense granules in platelets. A mating cross of C57BL10/Slc-rim2/rim2 and C57BL/6J-pe/pe showed no complementation of coat color. Additionally, characteristics similar to SPD were also observed in rim2. Thus, rim2 appeared to be a new allele of the pe locus and serves as a mouse model for human HPS. We have made a YAC contig covering the rim2/pe locus toward positional cloning of the causative gene. Received: 23 July 1997 / Accepted: 26 August 1997     

The <Emphasis Type="Italic">Idd6.2</Emphasis> diabetes susceptibility region controls defective expression of the <Emphasis Type="Italic">Lrmp</Emphasis> gene in nonobese diabetic (NOD) mice

The identification of genes mediating susceptibility to type 1 diabetes (T1D) remains a challenging task. Using a positional cloning approach based on the analysis of nonobese diabetic (NOD) mice congenic over the Idd6 diabetes susceptibility region, we found that the NOD allele at this locus mediates lower mRNA expression levels of the lymphoid restricted membrane protein gene (Lrmp/Jaw1). Analysis of thymic populations indicates that Lrmp is expressed mainly in immature thymocytes. The Lrmp gene encodes a type 1 transmembrane protein that localizes to the ER membrane and has homology to the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate gene, which negatively regulates intracellular calcium levels. We hypothesize that the observed decrease in expression of the Lrmp gene in NOD mice may constitute a T1D susceptibility factor in the Idd6 region.     

Mouse trisomy 16 as an animal model of human trisomy 21 (Down syndrome): production of viable trisomy 16 diploid mouse chimeras

We have previously proposed that mice trisomic for chromosome 16 will provide an animal model of human trisomy 21 (Down syndrome). However, the value of this model is limited to some extent because trisomy 16 mouse fetuses do not survive as live-born animals. Therefore, in an effort to produce viable mice with cells trisomic for chromosome 16, we have used an aggregation technique to generate trisomy 16 diploid (Ts 16 2n) chimeras. A total of 79 chimeric mice were produced, 11 of which were Ts 16 2n chimeras. Seven of these Ts 16 2n mice were analyzed as fetuses, just prior to birth, and 4 were analyzed as live-born animals. Unlike nonchimeric Ts 16 mouse fetuses which die shortly before birth with edema, congenital heart disease, and thymic and splenic hypoplasia, all but 1 of the Ts 16 2n animals were viable and phenotypically normal. The oldest of the live-born Ts 16 2n chimeras was 12 months old at the time of necropsy. Ts 16 cells, identified by coat color, enzyme marker, and/or karyotype analyses, comprised 50-60% of the brain, heart, lung, liver, and kidney in the 7 Ts 16 2n chimeric fetuses and 30-40% of these organs in the 4 live-born Ts 16 2n animals. Ts 16 cells comprised an average of 40% of the thymus and 80% of the spleen in the Ts 16 2n chimeras analyzed as fetuses, with no evidence of thymic or splenic hypoplasia. However, we observed a marked deficiency to Ts 16 cells in the blood, spleen, thymus, and bone marrow of live-born Ts 16 2n chimeras as compared to 2n 2n controls. These results demonstrate that although the Ts 16 2n chimeras were, with one exception, viable and phenotypically normal, each animal contained a significant proportion of trisomic cells in a variety of tissues, including the brain. Furthermore, our results suggest that although the abnormal development of Ts 16 thymus and spleen cells observed in Ts 16 fetuses is largely corrected in Ts 16 2n fetuses, Ts 16 erythroid and lymphoid cells have a severe proliferative disadvantage as compared to diploid cells in older live-born Ts 16 2n chimeras. Ts 16 2n chimeric mice will provide a valuable tool for studying the functional consequences of aneuploidy and may provide insight into the mechanisms by which trisomy 21 leads to developmental abnormalities in man.     

The baboon model (Papio hamadryas) of fetal loss: maternal weight,age, reproductive history and pregnancy outcome

Background Several risk factors are associated with the incidence of human stillbirths. The prevention of stillbirths in women is a pressing clinical problem. Methods We reviewed 402 pathology records of fetal loss occurring in a large baboon (Papio spp.) colony during a 15‐year period. Clinical histories of 565 female baboons with one or more fetal losses during a 20‐year period were analyzed for weight, age, and reproductive history. Results Fetal loss was most common at term (35.57%) and preterm (28.61%) and less common in the first half of gestation (11.20%) and post‐term (5.22%). Greater maternal weight, older age, history of stillbirth and higher parity were independent predictors for stillbirth. An exponential increase in the incidence of fetal loss was observed beginning at age 14 years in baboons. Conclusions Fetal loss and maternal risk factors associated with stillbirths in baboons were similar to those documented in women.     

Demonstration of the antimyoclonic effect of 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) using a new animal model

Comparatively high doses of muscimol induces myoclonic jerks in a particular strain of mice. 1-Hydroxy-3-amino-pyrrolidone-2 (HA-966) in doses of 1–10 mg/kg caused a dose-dependent blockade of this response of muscimol. Our earlier studies showed that the muscimol-induced myoclinic jerks fulfill certain criteria necessary to be considered as a valid animal model for postanoxic action myoclonus. On this basis it was concluded that HA-966 might prove to be of value in the management of this disorder.     

Altered calcium currents in cultured sensory neurons of normal and trisomy 16 mouse fetuses, an animal model for human trisomy 21 (Down syndrome)

Down syndrome is determined by the presence of an extra copy of autosome 21 and is expressed by multiple abnormalities, with mental retardation being the most striking feature. The condition results in altered electrical membrane properties of fetal dorsal root ganglia (DRG) neurons, as in the trisomy 16 fetal mouse, an animal model of the human condition. Cultured trisomic DRG neurons from human and mouse fetuses present faster rates of depolarization and repolarization in the action potential compared to normal controls and a shorter spike duration. Also, trisomy 16 brain and spinal cord tissue exhibit reduced acetylcholine secretion. Therefore, we decided to study Ca2+ currents in cultured DRG neurons from trisomy 16 and age-matched control mice, using the whole-cell patch-clamp technique. Trisomic neurons exhibited a 62% reduction in Ca2+ current amplitude and reduced voltage dependence of current activation at -30 and -20 mV levels. Also, trisomic neurons showed slower activation kinetics for Ca2+ currents, with up to 80% increase in time constant values. Kinetics of the inactivation phase were similar in both conditions. The results indicate that murine trisomy 16 alter Ca2+ currents, which may contribute to impaired cell function, including neurotransmitter release. These abnormalities also may alter neural development.     

Targeting altered cancer methionine metabolism with recombinant methioninase (rMETase) overcomes partial gemcitabine-resistance and regresses a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer

Pancreatic cancer is a recalcitrant disease. Gemcitabine (GEM) is the most widely-used first-line therapy for pancreatic cancer, but most patients eventually fail. Transformative therapy is necessary to significantly improve the outcome of pancreatic cancer patients. Tumors have an elevated requirement for methionine and are susceptible to methionine restriction. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer to determine the efficacy of recombinant methioninase (rMETase) to effect methionine restriction and thereby overcome GEM-resistance. A pancreatic cancer obtained from a patient was grown orthotopically in the pancreatic tail of nude mice to establish the PDOX model. Five weeks after implantation, 40 pancreatic cancer PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); GEM (100 mg/kg, i.p., once a week for 5 weeks, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); GEM+rMETase (GEM: 100 mg/kg, i.p., once a week for 5 weeks, rMETase: 100 units, i.p., 14 consecutive days, n = 10). Although GEM partially inhibited PDOX tumor growth, combination therapy (GEM+rMETase) was significantly more effective than mono therapy (GEM: p = 0.0025, rMETase: p = 0.0010). The present study is the first demonstrating the efficacy of rMETase combination therapy in a pancreatic cancer PDOX model to overcome first-line therapy resistance in this recalcitrant disease.     

The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity

Human metapneumovirus (hMPV) is a newly described paramyxovirus that is an important cause of acute respiratory tract disease. We undertook to develop a small animal model of hMPV infection, pathogenesis, and protection. Hamsters, guinea pigs, cotton rats, and nine inbred strains of mice were inoculated intranasally with hMPV. The animals were sacrificed, and nasal and lung tissue virus yields were determined by plaque titration. None of the animals exhibited respiratory symptoms. The quantity of virus present in the nasal tissue ranged from 4.6 x 10(2) PFU/gram tissue (C3H mice) to greater than 10(5) PFU/gram (hamster). The amount of virus in the lungs was considerably less than in nasal tissue in each species tested, ranging from undetectable (<5 PFU/g; guinea pigs) to 1.8 x 10(5) PFU/gram (cotton rat). The peak virus titer in cotton rat lungs occurred on day 4 postinfection. hMPV-infected cotton rat lungs examined on day 4 postinfection exhibited histopathological changes consisting of peribronchial inflammatory infiltrates. Immunohistochemical staining detected virus only at the luminal surfaces of respiratory epithelial cells throughout the respiratory tract. hMPV-infected cotton rats mounted virus-neutralizing antibody responses and were partially protected against virus shedding and lung pathology on subsequent rechallenge with hMPV. Viral antigen was undetectable in the lungs on challenge of previously infected animals. This study demonstrates that the cotton rat is a permissive small animal model of hMPV infection that exhibits lung histopathology associated with infection and that primary infection protected animals against subsequent infection. This model will allow further in vivo studies of hMPV pathogenesis and evaluation of vaccine candidates.