Elsevier

Virus Research

Volume 124, Issues 1–2, March 2007, Pages 193-203
Virus Research

Effects on isolated human pancreatic islet cells after infection with strains of enterovirus isolated at clinical presentation of type 1 diabetes

https://doi.org/10.1016/j.virusres.2006.11.004Get rights and content

Abstract

Enterovirus (EV) infections have been associated with the pathogenesis of type 1 diabetes (T1D). They may cause β-cell destruction either by cytolytic infection of the cells or indirectly by triggering the autoimmune response. Evidence for EV involvement have been presented in several studies, EV-IgM antibodies have been reported in T1D patients, EV-RNA has been found in the blood from T1D patients at onset, and EV have been isolated from newly diagnosed T1D. Our aim was to study infections with EV isolates from newly diagnosed T1D patients in human pancreatic islets in vitro. Two of them (T1 and T2) originated from a mother and her son diagnosed with T1D on the same day, the other two (A and E) were isolated from a pair of twins at the time of diagnosis of T1D in one of them. Isolated human pancreatic islets were infected and viral replication, viability and degree of cytolysis as well as insulin release in response to high glucose were measured. All four EV isolates replicated in the islet cells and virus particles and virus-induced vesicles were seen in the cytoplasm of the β-cells. The isolates varied in their ability to induce cytolysis and to cause destruction of the islets and infection with two of the isolates (T1 and A) caused more pronounced destruction of the islets. Infection with the isolate from the healthy twin boy (E) was the least cytolytic. The ability to secrete insulin in response to high glucose was reduced in all infected islets as early as 3 days post infection, before any difference in viability was observed. To conclude, strains of EV isolated from T1D patients at clinical presentation of T1D revealed β-cell tropism, and clearly affected the function of the β-cell. In addition, the infection caused a clear increase in the number of dead cells.

Introduction

Type 1 diabetes (T1D) is a chronic multi-factorial disease in which the insulin producing β-cells in the pancreas are selectively destroyed. The prevailing view has long been that T1D is an autoimmune disease with genetic susceptibility linked to HLA DR3, DR4 and to DQβ alleles (Gale et al., 2001). Due to lack of concordance in identical twins, an environmental trigger is also thought to be involved, which has been suggested to be a virus. Several lines of epidemiological evidence suggest that enterovirus (EV) infections might cause or trigger T1D (Yoon et al., 1979, King et al., 1983, Banatvala et al., 1985, Yoon, 1990, Frisk et al., 1992, Dahlquist et al., 1995, Chehadeh et al., 2000, Helfand et al., 1995, Hyoty et al., 1995, Hiltunen et al., 1997, Frisk and Diderholm, 1997, Yin et al., 2002a, Nairn et al., 1999, Lonnrot et al., 2000a, Roivainen et al., 1998, Salminen et al., 2003, Lonnrot et al., 2000b, Champsaur et al., 1982). The basic evidence for the involvement of EV in the aetiology of T1D has been presented in a number of studies (King et al., 1983, Hyoty et al., 1995, Frisk and Diderholm, 1997, Yin et al., 2002a, Nairn et al., 1999, Lonnrot et al., 2000a, Lonnrot et al., 2000b). EV have also been isolated from newly diagnosed T1D patients in a few cases (Yoon et al., 1979, Vreugdenhil et al., 2000, Hindersson et al., 2005) and some of the isolated virus strains have been shown to cause diabetes in animal models (Yoon, 1995). There are instances where the diagnosis of T1D in one member of a family has been rapidly followed by its appearance in other members of the same family (Nelson et al., 1977, Phillips and Pauli, 1981). We have previously published a case report where EV was isolated from a mother and from her son diagnosed with T1D the same day (Hindersson et al., 2005). Such clustering of T1D cases in time within a family is a strong indication of an infectious agent as a trigger or cause of the disease.

EV are prime candidates for constituting an environmental risk factor because they induce strong immune responses and they can infect the β-cells leading to local inflammation (Ylipaasto et al., 2004). The genus EV, a member of the picornavirus family are divided into five major groups: Human enterovirus-A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus (King, 2000), as many as 97 different EV serotypes are recognised and this number is still increasing. EV is transmitted by faecal, oral or by the respiratory route from one person to another. EV infections have been associated with a wide range of clinical manifestations including meningitis, encephalitis, paralysis, skin disease, common cold-like symptoms but mostly the infection is asymptomatic. EV infection usually starts from the respiratory tract or from the gastrointestinal mucosa, from where it might be spread to specific tissues and organs.

Upon infection, EV induces shut off of almost all host cell translation and induces a severe cytopathic effect (CPE) in infected cells. The shut off of host cell translation has been thought to result from the cleavage of the eukaryotic translation factor eIF4G (Ehrenfeld, 1982, Lamphear et al., 1995). The cleavage of eIF4G blocks the initiation of cap-dependent translation (Etchison et al., 1982). EV-infected cells reveal typical signs of CPE such as rounding up, accumulation of membranous vesicles (Dales et al., 1965) condensation of chromatin (Tolskaya et al., 1995, Romanova et al., 2005) and detachment from the basal surface of the culture dish or from other cells. Broadly speaking, there are two types of cell death; necrosis and apoptosis: It has been suggested that EV trigger the apoptotic pathway and that they before cell death has ensued terminated the process and initiate the alternative pathway (Agol et al., 2000).

It has been known for several years that the secretory pathway is disrupted in cells infected with picornaviruses (Barco and Carrasco, 1998), and this is characterized by the appearance of large number of membrane vesicles in the cytoplasm (Bienz et al., 1987, Rust et al., 2001, Suhy et al., 2000).

Different strains of EV have earlier been shown to infect isolated human pancreatic islets in vitro and such infections could either result in cell lysis or in a persistent infection depending on the viral strain (Chehadeh et al., 2000, Paananen et al., 2003, Frisk and Diderholm, 2000, Roivainen et al., 2000, Roivainen et al., 2002). However, the pathophysiological mechanism of virus induce T1D is still a matter of debate. Direct apoptosis/necrosis induced by the EV and indirect autoimmune responses induced by cytokines in the infected pancreatic islet cells remain attractive but unconfirmed hypothesis to explain the induction of T1D. Generally, viral proteins in infected cells interact with cellular proteins and interfere with cellular protein production to enable viral replication and propagation. Therefore, understanding the virus–host relationship is important if we are to understand the cellular responses against viral infections and the pathological mechanisms behind virus-induced T1D.

EV infections have for decades been associated with T1D, our successful isolation of four strains of EV from members of T1D families at clinical presentation of T1D together with the knowledge that such infections in non-β-cells interferes with the cell protein processing, made us design the following tasks (i) to study the ability of the isolated EV strains to induce cytolysis in isolated human islets; (ii) to study the ability of such an infection to affect insulin release in response to high glucose; (iii) to what extent these isolates would cause morphological changes of the islet and/or of the islet cells.

Section snippets

Patients

In an otherwise healthy family comprising mother, father and two sons, the mother and a 10-year-old son developed symptoms of increasing thirst and frequent urination. At a visit to the local physician, both were diagnosed with diabetes. Both were referred to the university hospital, T1D was immediately diagnosed in the son and he was put on insulin the same day. In the case of the mother it took some time before the type of diabetes was clarified, but it was clearly also diagnosed as T1D. The

Results

Two different GAD65 antibody assays were used and the Radioimmunoassay (RIA) results (detecting only IgG antibodies) obtained with the acute and the convalescent serum samples from the mother and the son revealed that the former had high levels of such antibodies (>198 IU) whereas no such antibodies was detected in the son's serum samples. When the same samples were analysed from the twin boys, the acute sample from both were totally negative while the antibody levels in the convalescent sample

Discussion

We have isolated three EV strains from T1D patients and one relative at time of clinical presentation of the disease. It has been shown before that T1D can aggregate in families suggesting that genetic predispositions are risk factors for T1D. However, only 10% of children diagnosed with T1D have an affected family member and among first-degree relatives of individuals with T1D, the risk of developing the disease is 5–6%, thus the genetic susceptibility can only partly explain why an individual

Conclusion

All four isolates from the T1D families belonged to the genera EV (echovirus 21 and coxsakievirus B5) and all of them could infect isolated human islets. All four caused islet destruction and a higher rate of cell death than seen in the controls 7 days post infection. In addition, three of them also caused rearrangement of cellular membranes in the β-cells. The most destructive virus was the A isolate, and it originated from the twin boys with T1D, the least destruction was the isolate from his

Acknowledgements

The authors thank Mrs. Kerstin Flink for skilful technical assistance, and the personnel of the β-Cell Transplant, Uppsala University, Uppsala and Dr. Anna-Karin Berg, Uppsala University, Uppsala, Sweden for performing the semi-nested EV PCR. The Family Ernfors fund, Gillbergska foundation, Novo Nordisk Foundation, The Swedish Diabetes foundation, Ronald McDonald fund and the Swedish Medical Research Council (K97-12XC-12445-01A, K98-12XC-12445-02B, 2005-2051) are also acknowledged for financial

References (51)

  • A. Olsson et al.

    Inflammatory gene expression in Coxsackievirus B-4-infected human islets of Langerhans

    Biochem. Biophys. Res. Commun.

    (2005)
  • W.R. Phillips et al.

    Simultaneous onset of insulin dependent diabetes mellitus in siblings

    Lancet

    (1981)
  • L.I. Romanova et al.

    Variability in apoptotic response to poliovirus infection

    Virology

    (2005)
  • B. Verheyden et al.

    Quantitative RT-PCR ELISA to determine the amount and ratio of positive- and negative strand viral RNA synthesis and the effect of guanidine in poliovirus infected cells

    J. Pharm. Biomed. Anal.

    (2003)
  • V.I. Agol et al.

    Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle

    J. Virol.

    (2000)
  • A. Barco et al.

    Co-expression of human eIF-4G and poliovirus 2Apro in Saccharomyces cerevisiae: effects on gene expression

    J. Gen. Virol.

    (1998)
  • S. Bopegamage et al.

    Coxsackie B virus infection of mice: inoculation by the oral route protects the pancreas from damage, but not from infection

    J. Gen. Virol.

    (2005)
  • W. Chehadeh et al.

    Persistent infection of human pancreatic islets by coxsackievirus B is associated with alpha interferon synthesis in beta cells

    J. Virol.

    (2000)
  • G. Dahlquist et al.

    Indications that maternal coxsackie B virus infection during pregnancy is a risk factor for childhood-onset IDDM

    Diabetologia

    (1995)
  • G. Frisk et al.

    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

    Int. J. Exp. Diabetes Res.

    (2000)
  • G. Frisk et al.

    Coxsackie B virus IgM in children at onset of type 1 (insulin-dependent) diabetes mellitus: evidence for IgM induction by a recent or current infection

    Diabetologia

    (1992)
  • E.A. Gale et al.

    Reanalysis of twin studies suggests that diabetes is mainly genetic

    BMJ

    (2001)
  • M. Goto et al.

    Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture

    Transplantation

    (2004)
  • R.F. Helfand et al.

    Serologic evidence of an association between enteroviruses and the onset of type 1 diabetes mellitus. Pittsburgh Diabetes Research Group

    J. Infect. Dis.

    (1995)
  • M. Hiltunen et al.

    Islet cell antibody seroconversion in children is temporally associated with enterovirus infections. Childhood Diabetes in Finland (DiMe) Study Group

    J. Infect. Dis.

    (1997)
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