Biochemical changes induced by Coxsackie B4 virus in short-term culture of mouse pancreatic islets

Isolated mouse pancreatic islets were infected in vitro with two strains of Coxsackie B4 virus — a tissue culture - adapted strain and a mouse pancreas-adapted strain. Within 48 h of infection changes had occurred in the biochemical activities of islets infected with the mouse pancreas-adapted strain of virus. Basal insulin release was increased two-fold in these islets, while glucose-induced insulin secretion remained unchanged. Insulin biosynthesis was greatly reduced at a sti, mulatory concentration of glucose (20 raM), thus leading to a reduced insulin content in these islets. These effects are of importance because they demonstrate that certain strains of Coxsackie B4 virus, like encephalo-myocarditis virus, may selectively alter B-cell function in vitro.     

Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me₂SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME₂SO, 1M ME₂SO + 1M EG, and 1M ME₂SO + 0.5M EG). Next, fresh islets, 2M ME₂SO islets, and 1M ME₂SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME₂SO (1M ME₂SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME₂SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME₂SO + 0.5M EG), compared to islets preserved with 2M ME₂SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.     

A mixed epidemic of poliomyelitis and Bornholm's disease (pleurodynia)

Summary During a mixed epidemic of poliomyelitis and Bornholm's disease in the summer of 1951, evidence was obtained of the involvement of at least 6 different immunological types of Coxsackie virus, among which the Albany A₂ type dominated. Poliomyelitis virus was isolated from the stools of 6 out of 20 patients suffering from paralytic poliomyelitis; Coxsackie virus from 1, and both poliomyelitis and Coxsackie virus from 2 out of these 20 patients. During the whole year, Coxsackie virus was recovered from the stools of patients suffering from paralytic poliomyelitis, aseptic meningitis, pleurodynia and summer grippe in approximately equal percentages (11 to 14%), but during the epidemic months from July to October, 25% of the patients with poliomyelitis, and 16% of the patients with pleurodynia gave positive results for Coxsackie virus. The sparing or the enhancing effect of Coxsackie virus infection on the development of paralysis in patients with dual infections is discussed. Aided by a grant from the National Health Research Council T.N.O.     

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.     

Synthesis and antiviral properties of novel indole-based thiosemicarbazides and 4-thiazolidinones

A novel series of indolylthiosemicarbazides (6a–6g) and their cyclization products, 4-thiazolidinones (7a–7g), have been designed, synthesized and evaluated, in vitro, for their antiviral activity against a wide range of DNA and RNA viruses. Compounds 6a, 6b, 6c and 6d exhibited notable antiviral activity against Coxsackie B4 virus, at EC₅₀ values ranging from 0.4 to 2.1 μg/mL. The selectivity index (ratio of cytotoxic to antivirally effective concentration) values of these compounds were between 9 and 56. Besides, 6b, 6c and 6d also inhibited the replication of two other RNA viruses, Sindbis virus and respiratory syncytial virus, although these EC₅₀ values were higher compared to those noted for Coxsackie B4 virus. The SAR analysis indicated that keeping the free thiosemicarbazide moiety is crucial to obtain this antiviral activity, since the cyclization products (7a–7g) did not produce any antiviral effect.     

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.     

Ocular application of the kinin B1 receptor antagonist LF22-0542 inhibits retinal inflammation and oxidative stress in streptozotocin-diabetic rats

Purpose

Kinin B₁ receptor (B₁R) is upregulated in retina of Streptozotocin (STZ)-diabetic rats and contributes to vasodilation of retinal microvessels and breakdown of the blood-retinal barrier. Systemic treatment with B₁R antagonists reversed the increased retinal plasma extravasation in STZ rats. The present study aims at determining whether ocular application of a water soluble B₁R antagonist could reverse diabetes-induced retinal inflammation and oxidative stress.

Methods

Wistar rats were made diabetic with STZ (65 mg/kg, i.p.) and 7 days later, they received one eye drop application of LF22-0542 (1% in saline) twice a day for a 7 day-period. The impact was determined on retinal vascular permeability (Evans blue exudation), leukostasis (leukocyte infiltration using Fluorescein-isothiocyanate (FITC)-coupled Concanavalin A lectin), retinal mRNA levels (by qRT-PCR) of inflammatory (B₁R, iNOS, COX-2, ICAM-1, VEGF-A, VEGF receptor type 2, IL-1β and HIF-1α) and anti-inflammatory (B₂R, eNOS) markers and retinal level of superoxide anion (dihydroethidium staining).

Results

Retinal plasma extravasation, leukostasis and mRNA levels of B₁R, iNOS, COX-2, VEGF receptor type 2, IL-1β and HIF-1α were significantly increased in diabetic retinae compared to control rats. All these abnormalities were reversed to control values in diabetic rats treated with LF22-0542. B₁R antagonist also significantly inhibited the increased production of superoxide anion in diabetic retinae.

Conclusion

B₁R displays a pathological role in the early stage of diabetes by increasing oxidative stress and pro-inflammatory mediators involved in retinal vascular alterations. Hence, topical application of kinin B₁R antagonist appears a highly promising novel approach for the treatment of diabetic retinopathy.     

Rash Associated with Coxsackie A9 Infection

Coxsackie A9 virus was identified by the authors during the fall of 1965 in Montreal in six children with fever and exanthem. Three of the six children were siblings. The exanthem was centrally distributed as described by Lerner et al. and consisted of discrete maculopapules 3 to 4 mm. in diameter. The viral agent was recovered and identified in tissue culture in five cases, while in the sixth Coxsackie type-A lesions were produced in suckling mice. Serological confirmation was obtained in two patients from whom sera were available. In contrast, no exanthem was observed in three older patients with a diagnosis of aseptic meningitis associated with Coxsackie A9 virus. In only one of 16 patients with Coxsackie B virus infection was an exanthem observed during the same period.The true incidence of Coxsackie A9-associated exanthems is difficult to determine because of the benign nature of the disease.     

Coxsackie B virus infection and β cell autoantibodies in newly diagnosed IDDM adult patients

Background: Environmental agents such as viruses have been identified as potentially important determinants of insulin-dependent diabetes mellitus (IDDM). Enterovirus infections, Coxsackievirus B especially, could be linked to the β cell damaging process and to the onset of clinical IDDM.Objectives: Enteroviral (EV) infection and β cell autoimmunity were studied in adult patients at the onset of IDDM.Study design: A total of 14 newly diagnosed-IDDM patients with ketosis or ketoacidosis were compared to, anteriorly diagnosed IDDM patients with metabolic decompensation, non-IDDM patients with metabolic decompensation and healthy adults. EV infection was studied by genomic RNA detection in whole blood using a RT-PCR assay. In order to assess the level of β cell autoantibodies at the time of the initial metabolic decompensation, serum specimens from IDDM patients were tested for GAD65 antibodies and islet cell antibodies (ICAs).Results: Coxsackie B3 or B4 virus genome was detected and genotyped in five of 14 (35.7%) newly diagnosed IDDM patients and in one of 12 (8%) patients in the course of IDDM. By contrast, none of the 12 non-IDDM patients and none of the 15 healthy adults was positive for enterovirus RNA detection in whole blood. Positive GAD65 antibodies and ICAs assays were not significantly correlated to a positive EV-RNA detection.Conclusion: The present study demonstrates that Coxsackie B virus RNA sequences can be detected in the peripheral blood from adult patients at the onset or in the course of IDDM and suggests that a Coxsackie B virus infection could initiate or accelerate β cell autoimmune damaging process.     

Antiviral activity and metal ion-binding properties of some 2-hydroxy-3-methoxyphenyl acylhydrazones

Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC₅₀ values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC₅₀ ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).     

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.     

Modulation of Glucose Metabolism by Balanced Deep-Sea Water Ameliorates Hyperglycemia and Pancreatic Function in Streptozotocin-Induced Diabetic Mice

The aim of this study was to determine the effects of balanced deep-sea water (BDSW) on hyperglycemia and glucose intolerance in streptozotocin (STZ)-induced diabetic mice. BDSW was prepared by mixing DSW mineral extracts and desalinated water to yield a final hardness of 1000–4000 ppm. Male ICR mice were assigned to 6 groups; mice in each group were given tap water (normal and STZ diabetic groups) or STZ with BDSW of varying hardness (0, 1000, 2000, and 4000 ppm) for 4 weeks. The STZ with BDSW group exhibited lowered fasting plasma glucose levels than the STZ-induced diabetic group. Oral glucose tolerance tests showed that BDSW improves impaired glucose tolerance in STZ-induced diabetic mice. Histopathological evaluation of the pancreas showed that BDSW restores the morphology of the pancreatic islets of Langerhans and increases the secretion of insulin in STZ-induced diabetic mice. Quantitative real-time PCR assay revealed that the expression of hepatic genes involved in gluconeogenesis, glucose oxidation, and glycogenolysis was suppressed, while the expression of the genes involved in glucose uptake, β-oxidation, and glucose oxidation in muscle were increased in the STZ with BDSW group. BDSW stimulated PI3-K, AMPK, and mTOR pathway-mediated glucose uptake in C₂C₁₂ myotubes. BDSW increased AMPK phosphorylation in C₂C₁₂ myotubes and improved impaired AMPK phosphorylation in the muscles of STZ-induced diabetic mice. Taken together, these results suggest that BDSW is a potential anti-diabetic agent, owing to its ability to suppress hyperglycemia and improve glucose intolerance by modulating glucose metabolism, recovering pancreatic islets of Langerhans and increasing glucose uptake.     

Effects on Myzus persicae (Sulz.) and transmission of beet yellows virus of applying certain trace elements to sugar beet

Applications of lithium chloride (LiCl), zinc sulphate (ZnSO₄) or nickel sulphate (NiSO₄) to the roots of sugar-beet plants in the glasshouse encouraged settling on the leaves of adult apterae from a clone of Myzus persicae (Sulz.); conversely, treatment with boric acid (H₂B₂O₇) inhibited aphid settling. Larviposition of M. persicae was increased by NiSO₄ and tin chloride (SnCl₂). Viruliferous M. persicae transmitted beet yellows virus (BYV) more efficiently to plants treated with LiCl or H₂B₂O₇ than to those treated with copper sulphate (CuSO₄), ZnSO₄ or SnCl₂. The sulphate and chloride anions of the applied chemicals appeared to have little effect on M. persicae and virus transmission. It is suggested that applications of trace elements to sugar beet affected M. persicae and virus transmission by changing the concentrations of trace elements in the aphids' diet and by altering the metabolism of the leaf tissues in the host plant.     

Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice

Background aimsThe success of islet transplantation for diabetes depends on the availability of an adequate number of allogeneic or autologous islets. Postnatal stem cells are now considered for the generation of physiologically competent, insulin-producing cells. Our group showed earlier that it is possible to generate functional islets from human dental pulp stem cells by using a serum-free cocktail in a three-step protocol.MethodsWe compared the yield of generated islet-like cell clusters (ICCs) from stem cells from pulps of human exfoliated deciduous teeth (SHED) and dental pulp stem cells from permanent teeth (DPSCs). ICCs derived from SHED were packed in immuno-isolatory biocompatible macro-capsules and transplanted into streptozotocin (STZ)-induced diabetic mice. Non-diabetic and diabetic controls were transplanted with macro-capsules with or without islets.ResultsSHED were superior to DPSCs. STZ diabetic mice alone and mice transplanted with empty macro-capsules exhibited hyperglycemia throughout the experiment, whereas mice transplanted with macro-capsules containing ICCs were restored to normoglycemia within 3–4 weeks, which persisted for >60 days.ConclusionsOur results demonstrate for the first time that ICCs derived from SHED reverse STZ diabetes in mice without immunosuppression and offer an autologous and non-controversial source of human tissue that could be used for stem cell therapy in diabetes.     

Various phenotypes of diabetes mellitus at ultimate outcome of acutely developed diabetic state induced by viral infection

For 4 to 8 years we followed up 3 diabetic patients in whom the onset of diabetes seemed to be closely related to the well-documented Epstein-Barr virus infection (Case 1) or Coxsackie B4 virus infection (Case 2, 3). Although all developed acute ketosis-prone diabetes in the convalescent stage of the viral infections, the subsequent clinical courses were quite different from each other. Case 1 has remained consistently insulin-dependent and associated with positive islet cell antibody, gastric parietal cell antibody, thyroglobulin hemoagglutinating antibody and thyroidal microsomal hemoagglutinating antibody. Case 2 restored normal glucose tolerance. Case 3 has become noninsulin-dependent diabetes mellitus after a 6 year interval. Thus, it is reasonably presumed that virus could be responsible for the occurrence of different phenotypes of diabetes.     

Vitamin-B12 Status of Patients on Long-term Metformin Therapy

Vitamin-B₁₂ malabsorption has been found in 21 (30%) of 71 diabetic patients taking long-term metformin therapy in addition to dietary management. The patients with evidence of B₁₂ malabsorption had significantly lower haemoglobin levels (and significantly higher serum folic acid levels) than those with normal B₁₂ absorption. Steatorrhoea was found in only one patient. Stopping metformin therapy resulted in reversion of B₁₂ absorption to normal in most patients examined. Four patients with B₁₂ malabsorption were found to have pathologically low serum B₁₂ levels. The causes and implications of these findings are discussed and it is concluded that all patients on long-term metformin therapy should have annual serum B₁₂ estimations.     

Direct evidence for the response of B and T cells to pokeweed mitogen

Chicken spleen cells containing chromosomally marked thymus derived (T) and bursa-derived (B₂) cells were evaluated for their ability to respond to pokeweed mitogen (PWM), concanavalin A (Con A), and to anti-immunoglobulin serum during a 4-day culture period. The results indicate that soluble PWM induces a proliferative response of B₂ cells in addition to a predominant T cell response. The PWM-induced B₂ cell proliferative response was clearly detected only at 4 days after culture initiation. Soluble Con A did not induce detectable proliferation of B₂ cells and stimulated T cells exclusively. In contrast, anti-immunoglobulin serum was a specific stimulant for B₂ cells under the culture conditions used.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

Metabolic Response to Nonglucidic Nutrient Secretagogues and Enzymatic Activities in Pancreatic Islets of Adult Rats after Neonatal Streptozotocin Administration

In islets from adult rats injected with streptozotocin during the neonatal period, both a nonmetabolized analog of L-leucine and 3-phenylpyruvate augmented ¹⁴CO₂ output from islets either prelabeled with L-[U-¹⁴C]glutamine or exposed to D-[2-¹⁴C]glucose and D-[6-¹⁴C]glucose in a manner qualitatively comparable to that found in islets from control rats. The islets of diabetic rats differed, however, from those of control rats by their unresponsiveness to both the L-leucine analog and a high concentration of D-glucose in terms of increasing ³HOH generation from [2-³H]glycerol, an impaired sparing action of the hexose upon ¹⁴CO₂ output from islets prelabeled with [U-¹⁴C]palmitate, and, most importantly, by a decreased rate of D-[2-¹⁴C]glucose and D-[6-¹⁴C]glucose oxidation when either incubated at a high concentration of the hexose (16.7 mM) or stimulated by nonglucidic nutrient secretagogues at a low concentration of D-glucose (2.8 mM). In islet homogenates, the activity of glyceraldehyde phosphate dehydrogenase, glutamate decarboxylase, and NADP-malate dehydrogenase was lower in diabetic than control islets. Such was not the case for glutamatealanine transaminase, glutamate-aspartate transaminase, or glutamate dehydrogenase. The neonatal injection of streptozotocin thus affected, in the adult rats, the activity of several islet enzymes. Nevertheless, the metabolic data suggest that an impaired circulation in the glycerol phosphate shuttle, as observed in response to stimulation of the islets by either a high concentration of D-glucose or nonglucidic nutrient secretagogues, represents an essential determinant of the preferential impairment of glucose-induced insulin release in this model of non-insulin-dependent diabetes.     

Proiactin Binding in Rat Langerhans Islets

Abstract

Membrane preparations of collagenase-dispersed Langerhans islets of female Wistar rats exhibit specific binding sites for ¹²⁵I labelled ovine prolactin (¹²⁵I-oPrl). Almost negligible binding was detected in islets of male animals. The binding is a saturable and time-temperature dependent process, equilibrium being reached after 16 h incubation at 0°C. The bound oPrl is not displaceable by hFSH, hLH, bGH or hGH. In contrast with other cell fractions, the 12,000 g pellet accounts for more than 80% of the specific binding of ¹²⁵I-oPrl. Scatchard plots of data obtained in saturation studies indicate a single class of binding sites with K_a = 0.21 × 10¹⁰ M^-1. Protein and phospholipid moieties are essential for the receptor activity, since after trypsin or phospholipase C digestions marked loss of binding was verified. In islets of streptozotocin diabetic rats a marked reduction in the number of binding sites was observed. These findings may suggest that some of the actions of prolactin on endocrine pancreas could be explained by its specific interaction with islet cell membranes.