Coxsackie B4 virus induces short-term changes in the metabolic functions of mouse pancreatic islets in vitro

Mouse pancreatic islets cultured in vitro were infected with a tissue culture-adapted or a mouse pancreas-adapted strain of Coxsackie B4 (CB4) virus. The effects of the viruses on the islets were assessed by examination of their biochemical functions. It was found that the mouse pancreas-adapted strain of CB4 induced a 'leakage' of insulin from islets incubated at a basal (2 mmol l-1) glucose concentration, both at two and four days following infection. However, at a stimulatory concentration of glucose (20 mmol l-1) the rate of insulin secretion appeared to be normal in these islets. At two days the rate of total protein synthesis in islets infected with mouse pancreas-adapted CB4, incubated at high glucose concentration, was reduced; at four days the degree of inhibition was more severe, the rate at basal glucose concentration falling to half that of the control islets and at the stimulatory glucose concentration to a quarter of the control islets. (Pro)insulin biosynthesis was also inhibited, the rate being reduced to less than half the mean control value in islets infected with mouse pancreas-adapted CB4 virus at 20 mmol l-1 glucose at two days; at four days the rate was greatly reduced at both 2 and 20 mmol l-1 glucose. It is concluded from this study that only certain strains of CB4 virus can infect mouse pancreatic islets in vitro and that infection with strains of virus tropic for the islets leads to an impairment of metabolic functions of the B-cells, and is not necessarily lytic.     

柯萨奇病毒B3型诱导的免疫偏离与心肌炎发病关系的研究

目的 研究2种近交系小鼠在柯萨奇病毒B3型(CVB3)感染后辅助性T细胞(Th)免疫偏离对心肌炎发病的影响。方法 用CVB3腹腔感染BALB/c和C57BL/62种近交系小鼠,感染后7d通过检测小鼠血清肌酸激酶(CK)活性,观察心脏外观变化以及心脏石蜡切片H.E染色观察心脏病理改变,比较2种小鼠心肌炎的发病情况;通过体外感染心肌细胞观察病毒复制情况以及体内心脏组织病毒载量的分析,比较2种小鼠对病毒感染和复制的差异;通过检测感染小鼠细胞因子白细胞介素-4(IL-4)、IL-12和γ干扰素(IFN-γ)的表达,抗CVB3VP1抗体的亚型以及T-bet和Gata-3的表达,比较2种小鼠Th免疫偏离的情况。结果 CVB3在体外和体内都可以感染BALB/c和C57BL/6小鼠心肌细胞,但仅BALB/c小鼠感染后可发生明显的病毒性心肌炎,C57BL/6小鼠则不能;BALB/c小鼠感染后表现为Th1型免疫反应而C57BL/6小鼠则偏向于Th2型免疫反应。结论 CVB3感染2种品系小鼠表现为不同的心肌炎发生率,与其诱导了不同类型的免疫偏离密切相关。     

Tissue Culture of Isolated Human Pancreatic Islets Infected With Different Strains of Coxsackievirus B4: Assessment of Virus Replication and Effects on Islet Morphology and Insulin Release

The aim was to study whether different strains of Coxsackievirus B4 (CBV-4) are able to infect human pancreatic islet cells in vitro and cause morphological and functional damages. Isolated islets maintained in tissue culture were infected with five well- characterised strains of CBV-4. Aliquots of the culture medium were analysed with regard to virus replication and insulin content. Infected and uninfected islets were examined by light microscopy to determine the degree of virus-induced cytopathic effect (CPE). The results showed that the islet cells were susceptible to infection by all the strains of CBV-4 although the outcome of the infection differed. The virus titres obtained at 48 and 72 hours post infection differed significantly between all the CBV-4 strains (p < 0.001), indicating different ability to replicate in islet cells. Pronounced to weak CPE, which was partly due to the origin (donor) of the islets, was induced by four of the five CBV-4 strains. One strain (VD2921) replicated without causing CPE despite high virus titres. One (V89-4557) of the CBV-4 strains always revealed pronounced CPE. Infection by this strain also caused functional impairment that significantly affected insulin response to high glucose at 48 hours post infection (p < 0.001). Replication of another CBV-4 strain (JVB) in the islet cells significantly increased the release of insulin compared to non-infected control cells (p < 0.001) indicating damage of the β-cells leading to leakage of insulin.     

Experimental diabetes in mice infected with Coxsackie viruses

The influence of Coxsackie B4 and AI3 viruses on the pancreas of mice (resistant and susceptible to diabetes) was studied. Glucose intolerance and changes in the synthesis of immunoreactive insulin were detected in all the treated groups of animals. Biochemical changes were more prominent in male DBA/2 mice, infected with Coxsackie B4 virus, in FI (CBA X C57Bl/6) hybrids and in female DBA/2 mice infected with Coxsackie AI3 virus and alloxan.     

Diabetes in Mice after Coxsackie B4 Virus Infection

The intraperitoneal inoculation of CD₁ mice with Coxsackie virus B₄ resulted in the raising of blood sugar levels to diabetic values 12 days after the administration of the virus. Serum insulin remained inappropriately low. Light microscopy changes in the islets of Langerhans showed mononuclear cell infiltration of the islets and marked degranulation of the β cells. The acinar tissue appeared to be little changed. It is concluded that Coxsackie B₄ virus may cause a diabetic state compatible with islet cell damage.     

Coxsackie virus in Southern California; isolation of a strain from stools of a patient

Thirty-three stool specimens from 29 patients were examined for Coxsackie virus by the inoculation of suckling mice. Such a virus, designated "California I," was obtained from two stool specimens collected on successive days from a patient with so-called nonparalytic poliomyelitis. Neutralizing antibodies for the California I strain of Coxsackie virus could not be demonstrated in serum obtained from the patient early in the illness, but were present in convalescent serum.Serum from the patient's daughter, who previously had had a similar illness, neutralized the strain of virus isolated from the father. In pathologic examination of the skeletal muscles of mice infected with the California I virus, lesions typical of those produced by Coxsackie virus, group A, were noted. California I strain of the virus was not neutralized by immune serum prepared from several other strains of Coxsackie virus.     

COXSACKIE VIRUS IN SOUTHERN CALIFORNIA—Isolation of a Strain from Stools of a Patient

Thirty-three stool specimens from 29 patients were examined for Coxsackie virus by the inoculation of suckling mice. Such a virus, designated “California I,” was obtained from two stool specimens collected on successive days from a patient with so-called nonparalytic poliomyelitis.Neutralizing antibodies for the California I strain of Coxsackie virus could not be demonstrated in serum obtained from the patient early in the illness, but were present in convalescent serum.Serum from the patient''s daughter, who previously had had a similar illness, neutralized the strain of virus isolated from the father.In pathologic examination of the skeletal muscles of mice infected with the California I virus, lesions typical of those produced by Coxsackie virus, group A, were noted.California I strain of the virus was not neutralized by immune serum prepared from several other strains of Coxsackie virus.     

Expression of Innate Immunity Genes and Damage of Primary Human Pancreatic Islets by Epidemic Strains of Echovirus: Implication for Post-Virus Islet Autoimmunity

Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.     

Susceptibility of cell lines of primate and nonprimate origin to certain human viruses

A comparative study was made of the susceptibility of 11 cell lines of human and animal origin, the WI-38 cell strain and fresh cultures of human thyroid, monkey kidney and hamster embryo tissues to certain human viruses. The animal cell lines were derived from monkey, rabbit, mouse, pig and calf tissues. The viruses used were strains of influenza A₂ and B viruses, parainfluenza viruses types 1, 2 and 3, RS virus, adenoviruses types 3, 4 and 21, poliovirus type 1 and Coxsackie A type 21 and Coxsackie B type 3 viruses. Cell lines derived from nonprimate tissues were generally less susceptible than cell cultures of human and simian origin. The combined use of fresh cultures of human thyroid and monkey kidney tissues and of a human cell line seems to provide a satisfactory indicator system for the viruses employed in this study.     

Enteroviral Syndromes in Toronto, 1964

Virological or serological investigations of 72 children in Toronto and environs, who were hospitalized between January and October 1964 with a variety of syndromes, revealed evidence of enteroviral infection in 29 subjects. Coxsackie B2 was the dominant enterovirus, being isolated from feces and/or cerebrospinal fluid (CSF) of three children with aseptic meningitis, three with pleurodynia, one with myalgia and one with pericarditis; four additional patients showed rising antibody titres to this virus. Coxsackie B1 virus, which has not been isolated in Toronto since 1950, was recovered from feces of three patients with pleurodynia, CSF of one patient with myalgia, and peritoneal fluid of a child with primary peritonitis; one patient with pericarditis showed a rising antibody titre to Coxsackie B1 virus. Coxsackie B3, B4 and Echo 23 viruses were associated with one case each of pleurodynia. Coxsackie B5 virus infected five patients with aseptic meningitis, and one each with pericarditis and myocarditis.     

Alpha-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice

Most patients at risk for developing type 2 diabetes are hyperinsulinemic. Hyperinsulinemia may be a response to insulin resistance, but another possible abnormality is insulin hypersecretion. BTBR mice are insulin resistant and hyperinsulinemic. When the leptin(ob) mutation is introgressed into BTBR mice, they develop severe diabetes. We compared the responsiveness of lean B6 and BTBR mouse islets to various insulin secretagogues. The transamination product of leucine, alpha-ketoisocaproate (KIC), elicited a dramatic insulin secretory response in BTBR islets. The KIC response was blocked by methyl-leucine or aminooxyacetate, inhibitors of branched-chain amino transferase. When dimethylglutamate was combined with KIC, the fractional insulin secretion was identical in islets from both mouse strains, predicting that the amine donor is rate-limiting for KIC-induced insulin secretion. Consistent with this prediction, glutamate levels were higher in BTBR than in B6 islets. The transamination product of glutamate, alpha-ketoglutarate, elicited insulin secretion equally from B6 and BTBR islets. Thus formation of alpha-ketoglutarate is a requisite step in the response of mouse islets to KIC. alpha-Ketoglutarate can be oxidized to succinate. However, succinate does not stimulate insulin secretion in mouse islets. Our data suggest that alpha-ketoglutarate may directly stimulate insulin secretion and that increased formation of alpha-ketoglutarate leads to hyperinsulinemia.     

Both T and B cells shed infectious mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers. Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice. These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.     

Lipopolysaccharides impair insulin gene expression in isolated islets of Langerhans via Toll-Like Receptor-4 and NF-κB signalling

Background

Type 2 diabetes is characterized by pancreatic β-cell dysfunction and is associated with low-grade inflammation. Recent observations suggest that the signalling cascade activated by lipopolysaccharides (LPS) binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. In this study, we tested the hypothesis that LPS alters insulin gene expression via TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in islets.

Methodology/Principal Findings

A 24-h exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of pancreas-duodenum homebox-1 (PDX-1) and mammalian homologue of avian MafA/l-Maf (MafA). Accordingly, LPS exposure also decreased glucose-induced insulin secretion. LPS repression of insulin, PDX-1 and MafA expression, as well as its inhibition of insulin secretion, were not observed in islets from TLR4-deficient mice. LPS inhibition of β-cell gene expression in rat islets was prevented by inhibition of the NF-κB pathway, but not the p38 mitogen-activated protein kinase (p38 MAPK) pathway.

Conclusions/Significance

Our findings demonstrate that LPS inhibit β-cell gene expression in a TLR4-dependent manner and via NF-κB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function.     

Effects of overexpression of pancreatic derived factor (FAM3B) in isolated mouse islets and insulin-secreting betaTC3 cells

PANcreatic DERived factor (PANDER, FAM3B) is a recently discovered islet-specific cytokine. We have previously shown that, in vitro, truncated recombinant PANDER isoforms (20 and 21 kDa) are cytotoxic to beta-cell lines but the effects of full-length PANDER on islet biology remain unclear. In this study, we used adenovirus (Ad-PANDER) to overexpress full-length cDNA of PANDER in islets and betaTC3 cells. BetaTC3 cells were infected with Ad-PANDER or control vector. After 48 h, cell viability was significantly decreased as evaluated by MTT assay. The number of dead cells was significantly increased as indicated by the fluorescent intensity of the propidium iodide-stained cells (160 +/- 13 vs. control 100 +/- 7%, P = 0.001). Flow cytometric Tunel assay showed that overexpressing PANDER induced a significant fourfold increase in beta-cell apoptosis (19.4 +/- 6.3 vs. control 4.1 +/- 0.8%, P < 0.05). There was a significant increase in the number of annexin V-positive (apoptotic) cells and propidium iodide-positive (dead) cells in mouse islets infected with Ad-PANDER compared with control cells infected with Ad-LacZ. Addition of 4 nM recombinant PANDER protein to betaTC3 cells or infection of Ad-PANDER did not affect Akt and STAT1 phosphorylation, Bcl-2, Fas, and NF-kappaB protein levels. However, activation of caspase-3 was observed in betaTC3 and islets infected with Ad-PANDER. Overexpression of PANDER in mouse islets or addition of recombinant PANDER decreased insulin secretion induced by carbachol plus glucose or high potassium but not that by glucose alone. Culture with recombinant PANDER did not affect glucose-induced NAD(P)H elevation in mouse islets. In conclusion, Ad-PANDER infection is as effective as truncated recombinant PANDER to induce betaTC3 cell and mouse islet apoptosis.     

Impaired beta-oxidation increases vulnerability to influenza A infection

Influenza A virus (IAV) infection casts a significant burden on society. It has particularly high morbidity and mortality rates in patients suffering from metabolic disorders. The aim of this study was to relate metabolic changes with IAV susceptibility using well-characterized inbred mouse models. We compared the highly susceptible DBA/2J (D2) mouse strain for which IAV infection is lethal with the C57BL/6J (B6) strain, which exhibits a moderate course of disease and survives IAV infection. Previous studies showed that D2 has higher insulin and glucose levels and is predisposed to develop diet-induced type 2 diabetes. Using high-resolution liquid chromatography–coupled MS, the plasma metabolomes of individual animals were repeatedly measured up to 30 days postinfection. The biggest metabolic difference between these strains in healthy and infected states was in the levels of malonylcarnitine, which was consistently increased 5-fold in D2. Other interstrain and intrastrain differences in healthy and infected animals were observed for acylcarnitines, glucose, branched-chain amino acids, and oxidized fatty acids. By mapping metabolic changes to canonical pathways, we found that mitochondrial beta-oxidation is likely disturbed in D2 animals. In noninfected D2 mice, this leads to increased glycerolipid production and reduced acylcarnitine production, whereas in infected D2 animals, peroxisomal beta-oxidation becomes strongly increased. From these studies, we conclude that metabolic changes caused by a distortion of mitochondrial and peroxisomal metabolism might impact the innate immune response in D2, leading to high viral titers and mortality.     

Association of Virus with Cases of Rubella Studied in Toronto: Propagation of the Agent and Transmission to Monkeys

Using an interference test with indicator virus Echo 11, a virus has been isolated in nine of 18 specimens from cases of typical rubella. The virus will interfere with the development of cytopathology in green monkey kidney cells with viruses Echo 11, Coxsackie B1 and B4, Poliovirus I and III (Sabin strains) and simian virus SV4. In four of five paired sera this virus was neutralized by convalescent but not by the acute phase serum, tested by interference inhibition. No cytopathology was observed in unstained cultures or in sequential cultures stained with acridine orange or fluorescent antibody. The virus was destroyed by exposure to 56° C. for 30 minutes and 15% ether at 4° C. for 24 hours, but survived with some reduction in titre at 4° C. for 24 hours. Green monkeys infected by this virus developed a macular rash, lymphadenopathy and modest rise in white blood cell count.     

Beta-cell PDE3B regulates Ca2+-stimulated exocytosis of insulin

cAMP signaling is important for the regulation of insulin secretion in pancreatic beta-cells. The level of intracellular cAMP is controlled through its production by adenylyl cyclases and its breakdown by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE3B is involved in the regulation of nutrient-stimulated insulin secretion. Here, aiming at getting deeper functional insights, we have examined the role of PDE3B in the two phases of insulin secretion as well as its localization in the beta-cell. Depolarization-induced insulin secretion was assessed and in models where PDE3B was overexpressed [islets from transgenic RIP-PDE3B/7 mice and adenovirally (AdPDE3B) infected INS-1 (832/13) cells], the first phase of insulin secretion, occurring in response to stimulation with high K(+) for 5 min, was significantly reduced ( approximately 25% compared to controls). In contrast, in islets from PDE3B(-/-) mice the response to high K(+) was increased. Further, stimulation of isolated beta-cells from RIP-PDE3B/7 islets, using successive trains of voltage-clamped depolarizations, resulted in reduced Ca(2+)-triggered first phase exocytotic response as well as reduced granule mobilization-dependent second phase, compared to wild-type beta-cells. Using sub-cellular fractionation, confocal microscopy and transmission electron microscopy of isolated mouse islets and INS-1 (832/13) cells, we show that endogenous and overexpressed PDE3B is localized to insulin granules and plasma membrane. We conclude that PDE3B, through hydrolysis of cAMP in pools regulated by Ca(2+), plays a regulatory role in depolarization-induced insulin secretion and that the enzyme is associated with the exocytotic machinery in beta-cells.     

Roles of macrophages in measles virus infection of genetically modified mice

Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.     

In Vivo Monocyte Tropism of Pathogenic Feline Immunodeficiency Viruses

Virus-infected monocytes rarely are detected in the bloodstreams of animals or people infected with immunodeficiency-inducing lentiviruses, yet tissue macrophages are thought to be a major reservoir of virus-infected cells in vivo. We have identified feline immunodeficiency virus (FIV) clinical isolates that are pathogenic in cats and readily transmitted vertically. We report here that five of these FIV isolates are highly monocytotropic in vivo. However, while FIV-infected monocytes were numerous in the blood of experimentally infected cats, viral antigen was not detectable in freshly isolated cells. Only after a short-term (at least 12-h) in vitro monocyte culture were FIV antigens detectable (by immunocytochemical analysis or enzyme-linked immunosorbent assay). In vitro experiments suggested that monocyte adherence provided an important trigger for virus antigen expression. In the blood of cats infected with a prototype monocytotropic isolate (FIV subtype B strain 2542), infected monocytes appeared within 2 weeks, correlating with high blood mononuclear-cell-associated viral titers and CD4 cell depletion. By contrast, infected monocytes could not be detected in the blood of cats infected with a less pathogenic FIV strain (FIV subtype A strain Petaluma). We concluded that some strains of FIV are monocytotropic in vivo. Moreover, this property may relate to virus virulence, vertical transmission, and infection of tissue macrophages.