Pierre Wattré

Increased Level of Interferon-α in Blood of Patients with Insulin-Dependent Diabetes Mellitus: Relationship with Coxsackievirus B Infection

The activation of the interferon (IFN)-alpha system and its relationship with coxsackievirus B (CVB) infection has been analyzed in 56 patients with insulin-dependent diabetes mellitus (IDDM; 25 children and 31 adults). Elevated levels of IFN-alpha were found in plasma of 70% of patients (39/56), and a positive detection of IFN-alpha mRNA in blood cells by reverse transcriptase-polymerase chain reaction (RT-PCR) was observed in 75% of patients (42/56). Enterovirus (EV) RNA assayed by seminested RT-PCR was detected in the blood of 50% of IFN-alpha-positive patients but not in any IFN-alpha-negative patients. The results of genotype analysis of amplified EV RNA sequences (5 CVB2, 8 CVB3, and 8 CVB4) were concordant with the results of CVB-neutralization tests. The comparison between IFN-alpha, EV RNA, and serology suggested that the proportion of CVB infection associated with IFN-alpha positivity might be higher than is predicted from the investigation of EV RNA. Together, the results suggest that, in a majority of cases, a CVB infection is associated with clinical IDDM.

Persistent Infection of Human Pancreatic Islets by Coxsackievirus B Is Associated with Alpha Interferon Synthesis in β Cells

ABSTRACT The interactions of coxsackievirus B3 (CVB3), CVB4E2 (diabetogenic), and CVB4JBV (nondiabetogenic) strains with human pancreatic islets from eight adult brain-dead donors were investigated. Persistent replication of viruses in human islets was proved by detection of viral RNA by in situ hybridization, VP1 capsid protein by immunofluorescence (IF) staining, negative-strand viral RNA by reverse transcription-PCR in extracted RNA from islets, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double IF staining, glucagon-containing α cells and insulin-containing β cells were shown to be susceptible to CVB. The persistence of CVB3 and CVB4 in islet cells was associated with the chronic synthesis of alpha interferon (IFN-α), as evidenced by the detection of IFN-α mRNA and immunoreactive IFN-α with antiviral activity. By double IF staining, IFN-α was detected in insulin-producing β cells only. Experiments with neutralizing anti-coxsackievirus and adenovirus receptor (CAR) antibodies provided evidence that CAR was expressed by α and β cells and that it played a role in the infection of these cells with CVB and the consecutive IFN-α expression in β cells. The viral replication and the expression of IFN-α in islets were not restricted to the CVB4E2 diabetogenic strain and did not depend on the genetic background of the host. The neutralization of endogenous IFN-α significantly enhanced the CVB replication in islet cells and resulted in rapid destruction of islets. Thus, human β cells can harbor a persistent CVB infection, and CVB-induced IFN-α plays a role in the initiation and/or maintenance of chronic CVB infection in human islets.

Detection of Coxsackie B virus RNA sequences in whole blood samples from adult patients at the onset of type I diabetes mellitus

Enteroviruses may be linked to insulin‐dependent diabetes mellitus (IDDM). The prevalence of enteroviral (EV) infection at onset of adult IDDM was investigated by detection of specific EV sequences in peripheral blood using a reverse transcription and a seminested polymerase chain reaction (seminested RT‐PCR). EDTA‐treated whole blood samples taken from 12 newly diagnosed IDDM patients with ketosis or ketoacidosis were examined. The comparison groups were 12 adult patients suffering from metabolic decompensation in the course of IDDM, 12 adult patients with decompensated non‐IDDM, and 15 healthy adults without any presumed EV infection or metabolic disease. EV genome was detected in five of 12 (42%) 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 had EV sequences in whole blood. Subsequent sequencing of the EV PCR products from the six positive patients showed a significant homology with Coxsackie B3 or B4 viruses, and some common patterns were observed among the sequences. The present study demonstrates that Coxsackie B virus RNA sequences can be detected in peripheral blood from patients at the onset or in the course of IDDM and provides evidence for a role for enteroviruses in adult type I diabetes. J. Med. Virol. 52:121–127, 1997.

Differential detection of rhinoviruses and enteroviruses RNA sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis

To define the role of enteroviruses and human rhinoviruses as etiological agents in childhood bronchiolitis, clinical aspirates from 84 infants admitted to hospital with symptoms of obstructive bronchiolitis were tested by picornavirus RT‐PCR assay, adenovirus PCR assay and classical immunofluorescence antigen detection of common respiratory viral agents. Respiratory syncytial viruses (A&B) were detectable in 45 of 84 (53.6%) nasopharyngeal aspirates from infants with bronchiolitis, whereas coronaviruses, influenza viruses, and parainfluenza viruses were not detectable in the same samples. Adenoviruses were detectable by PCR in 11 of 84 (13.1%) nasopharyngeal swabs. By using a picornavirus RT‐PCR assay followed by a differential molecular hybridisation, rhinovirus and enterovirus RNA sequences were detected in 16 of 84 (19%) and in 10 of 84 (11.9%) of the nasopharyngeal swabs tested. Positive human rhinovirus or enterovirus RT‐PCR assay, however, was the only evidence of respiratory infection in 8 of 84 (9.5%) and in 7 of 84 (8.33%) of the studied patients. Respiratory syncytial viruses, human rhinoviruses, adenoviruses, and enteroviruses occur in dual infections detected in 18 of 84 (21.4%) respiratory samples tested. The median duration of stay in hospital was not significantly different between the patients demonstrating a single viral infection and those with a dual viral infection (6.22 ± 2.07 vs. 5.04 ± 0.95 days; P > 0.05). In summary, combination of molecular and classical detection assays of common viruses can be used to demonstrate enterovirus and human rhinovirus respiratory infection in childhood bronchiolitis, and provides an improved approach to obtain new insights into concomitant viral respiratory tract infection in infants. J. Med. Virol. 61:341–346, 2000.

High Levels of sTNFR p75 and TNFα in Dengue-Infected Patients

Soluble forms of the two molecular species of the cell surface TNF receptors (sTNFR p55 and sTNFR p75) can reduce the activity of TNFα but they may also enhance its function by stabilizing the active TNFα oligomer. Considering the pathophysiological importance of sTNFR p75 for the regulation of the bioavailability of TNFα in the body, we determined the serum levels of sTNFR p75 and TNFα in 45 children and 28 adults with laboratory‐confirmed dengue infection by using immunoassays. The serum samples were obtained from day 1 to day 15 after the onset of the disease during the 1989–1990 outbreak of dengue‐3 in Tahiti, French Polynesia. The patients were clinically classified as having dengue hemorrhagic fever (DHF) and graded according to the criteria of the World Health Organization (WHO) into four grades from less severe (grade I) to severe (grade IV). The sera of both children and adult patients of all severity grades contained higher levels of sTNFR p75 than the sera of control subjects. Although high levels of TNFα were also detected in children and adults among grade I, II, III and IV patients, we found no correlation between sTNFR p75 and TNFα. We observed in adults a moderate elevation of sTNFR p75 and TNFα in sera compared with that observed in children. The raised levels of immunoreactive sTNFR p75 and TNFα in all clinical groups of dengue‐infected patients strongly indicate activation of the TNFα system during dengue infection. The balance between sTNFR p75 and TNFα may be altered in dengue infection. Further investigations are needed to understand the role of sTNFR p75 and TNFα in the pathogenesis of DHF and to improve the management of dengue infection.

Antibody-Dependent Enhancement of Coxsackievirus B4 Infectivity of Human Peripheral Blood Mononuclear Cells Results in Increased Interferon-α Synthesis

IgG devoid of neutralizing activity and isolated from donor plasma by chromatography formed immune complexes with coxsackievirus B4 (CVB4) and significantly increased the infection of peripheral blood mononuclear cells with CVB4. The major host cells for CVB4 infection enhanced with IgG are monocytic CD14+ cells. The roles of CVB and adenovirus receptor and Fcgamma receptor II and III have been shown. Increased viral replication and the release of infectious particles were demonstrated when interferon (IFN)-alpha produced by infected cells was first neutralized by use of antibodies. The CVB4 IgG-induced synthesis of IFN-alpha by monocytes reflected entry and uncoating of CVB4 but not of viral replication and required the presence of CVB4 RNA inside the cells. Thus, CVB4 can infect monocytes by an antibody-dependent mechanism through interactions between the virus, antiviral antibodies, and specific receptors that result in IFN-alpha production.

Enteroviruses Can Persist with or without Active Viral Replication in Cardiac Tissue of Patients with End-Stage Ischemic or Dilated Cardiomyopathy

To investigate enterovirus replication versus persistence in end-stage cardiac diseases, endomyocardial biopsies from explanted hearts of 70 patients with idiopathic dilated cardiomyopathy (IDCM), 64 patients with chronic coronary disease (CCD), and 45 donors of healthy hearts (controls) were examined by reverse transcriptase-polymerase chain reaction for genomic and antigenomic enterovirus RNA and by VP1 antigen immunohistochemistry. Enterovirus genome was detected in 25 of 70 patients with IDCM and in 21 of 64 patients with CCDs (35.7 vs. 32.8%, respectively; P=.12). Of the 46 patients positive for genomic RNA, only 3 exhibited antigenomic RNA and VP1 antigen that demonstrated active viral replication, whereas 43 had latent infection characterized by the absence of antigenomic RNA associated with or not with VP1 antigen expression. No viral component was detected in control subjects. The findings demonstrate that a small percentage of patients with end-stage chronic cardiac diseases had active enterovirus replication in their myocardium.

HIGH SERUM PROCALCITONIN LEVEL IN A 4-YEAR-OLD LIVER TRANSPLANT RECIPIENT WITH A DISSEMINATED CANDIDIASIS

High serum concentrations of proc~/lcitonin (proCT), a 116 amino acid peptide, have recently been found during bacterial sepsis in the absence of mature calcitonin[1]. High serum levels of proCT were associated with the severity of bacterial and parasitic infections [2,3] and positively correlated to TNF-c~, IL-6 and IL-8 serum levels in patients with severe sepsis or septic shock [4]. Moreover, evidence has been provided that proCT can be released in humans following endotoxin injection [5]. To assess its importance as a potential marker of infection, we have measured proCT concentrations in a variety of infectious diseases. We report the first case of high serum proCT concentration during a disseminated fungal infection. ProCT concentrations were measured with an immuno-luminometric assay using two monocl0nal antibodies recognizing different epitopes of the molecule and 50 pJ of serum samples which were stored at -20°C until analysis. A 4-year-old gift developed a symptomatic cytomegalovirus (CMV) infection 10 days after receiving hepatic transplant because of a biliary atresia, with fever and alterations of hepatic tests. At the time of this diagnosis, she received an immunosuppressive therapy with cyclosporin, azathioprin and corticosteroids. The CMV infection was confirmed by a biological seroconversion, positive antigenemia, CMV isolation on fibroblast culture, and a pathological study confirming histopathologic impairment. Ganciclovir treatment rapidly permitted a biological improvement, as assessed by a marked decrease in antigenemia, and negative cultures. Pneumocystis carinii pneumonia (PCP) was also diagnosed on day 29, on the basis of pulmonary symptoms and a positive broncho-alveolar lavage examination. The treatment with co-trimoxazole was rapidly effective and was maintained for 3 weeks. On day 31, as shown in Table 1, proCT level in serum was moderately elevated: 4 ng/ml (normal values below 0.1 ng/ml). Five days later, the proCT level was 103 ng/ml; at this time a Candida albicans infection was documented. One ascitic fluid sample and two blood cultures were positive for C. albicans, despite a prophylaxis for systemic fungal infection with liposomal amphotericin B. Under appropriate antifungal therapy, consisting of high amphotericin B dosage associated with intravenous fiucytosine, proCT levels rapidly decreased and the clinical outcome was favourable. PCP or CMV infection could be responsible for the moderate elevation of proCT observed on days 26 and 31. However, since the patient was recovering from CMV and PCP infection when proCT value was high, an overlap between disseminated C. albicans infection and other infectious diseases seems unlikely. Moreover, the only change in the administered therapeutic regimen leading to improvement was anfifungal therapy. This report emphasizes the potential value of proCT as a marker of severe sepsis, not only of bacterial origin as previously described, but also of fungal origin. Surveillance of serum proCT values could be of particular interest in immunodeficient individuals such as transplant recipients. Further studies are required to confirm the importance of procalcitonin production in association with infectious diseases, to assess its potential role in diagnosis and follow-up studies, and to determine the physiopathologic mechanisms leading to proCT production.

Tumor Necrosis Factor Alpha Levels in Plasma and Whole-Blood Culture in Dengue-Infected Patients: Relationship Between Virus Detection and Pre-existing Specific Antibodies

The pathogenesis of dengue hemorrhagic fever (DHF) is not well known, but the role of host factors has been suggested. The level of immunoreactive circulating and cell‐generated tumor necrosis factor alpha (TNFα) was studied in 35 patients with DHF; its relationship with virus isolation and/or genome detection by reverse transcription polymerase chain reaction (RT‐PCR) and specific antibodies were detected by hemagglutination inhibition (HI). Large variation of TNFα plasma levels was obtained in dengue‐infected patients at the same stage of the disease and at the same day after infection. Most of the patients (14 out of 17 patients) who displayed augmented spontaneous in vitro production of TNFα by heparinized whole‐blood culture compared with controls also had elevated levels of TNFα in the plasma. The TNFα values in lipopolysaccharide and phytohemagglutinin heparinized whole‐blood cultures were not higher in patients than in controls, but low TNFα levels were obtained in three out of 30 patients. An inverse correlation was observed between spontaneous in vitro TNFα production and viral replication, which raises the issue of the antiviral effect of TNFα in dengue infection. The results do not support the hypothesis of the role of antibody‐dependent enhancement giving rise to increased viremic titers and production of TNFα in patients. The present study demonstrates the activation of the TNFα‐producing cells in dengue‐infected patients and suggests further investigation to define the mechanism and the role of TNFα in the pathogenesis of dengue virus infection. J. Med. Virol. 54:210–218, 1998.

Detection of enteroviral RNA by polymerase chain reaction in endomyocardial tissue of patients with chronic cardiac diseases

Enteroviruses are suspected to be etiologic agents in myocarditis and cardiomyopathy. The prevalence of enteroviral (EV) heart infection in patients with chronic cardiomyopathy was determined through detection of specific EV genomic sequences using reverse transcription and polymerase chain reaction (RT‐PCR) followed by slot blotting. Endomyocardial biopsies from the explanted hearts of 19 patients with dilated cardiomyopathy (DCM) and 14 patients with chronic coronary disease (CCD) were examined. EV genome was detected in 11 of 19 patients with DCM and in 8 of 14 patients with CDD. Ventricular biopsies from the control group, which included 35 healthy heart patients and 33 patients with myocardial infarction, were negative by EV RT‐PCR. The percentage of patients showing presence of EV‐RNA was almost similar in the DCM (57.9%) and CCD (57.1%) groups. The present study demonstrates that enterovirus RNA sequences persist in the myocardium in a significant proportion of patients suffering from end‐stage ischaemic and dilated cardiac diseases and supports the hypothesis of a possible direct link between EV infection and the pathogenesis of chronic heart disease.