The mechanism of chronic coxsackievirus B hepatitis in rabbits.

The pathophysiology of chronic hepatitis in rabbits infected with coxsackievirus B5 (CVB5), (strain Mitchell) was investigated. Three-week-old male New Zealand White rabbits were inoculated intraperitoneally with 1 x 10(5) plaque forming units of virus. Every 3 months for 15 months postinoculation (p.i.) groups of animals were sacrificed for the following tests: interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-beta cytokine levels by enzyme-linked immunosorbent assay (ELISA); splenic natural killer (NK) cell function; sequence of a 154-bp section of the 5' noncoding region; antihepatocyte autoantibodies; histologic examination; in situ polymerase chain reaction (ISPCR) of the liver; neutralizing antibody response to CVB5; and viral cultures of liver, spleen, blood, brain, heart, skeletal muscle, and pancreas samples. Histologic evidence of hepatocyte necrosis was evident at each time point, although few inflammatory cells were seen. Liver samples were positive at each time by ISPCR, with viral nucleic acid localized to hepatocyte cytoplasm. Other cells in the liver did not stain. No hepatocyte autoantibodies were detected, and there was no elevation of intrahepatic cytokine levels compared to uninfected controls. There were no mutations in the virus over time. A vigorous neutralizing antibody response to CVB5, Mitchell was generated, but splenic natural killer (NK) function and numbers of splenic NK cells were significantly decreased. Virus culture was positive at 3 months, but negative at further time points. Cultures were negative at 3 months for the other tissues tested. Thus, CVB5, Mitchell causes a chronic hepatitis in rabbits, with virus limited to hepatocyte cytoplasm and no evidence of autoimmunity.

The in vitro immunomodulatory effects of glyconutrients on peripheral blood mononuclear cells of patients with chronic fatigue syndrome.

In humans, eight monosaccharides are required for the synthesis of glycoproteins. Dietary supplements that supply these crucial sugars are known as glyconutrients. A glyconutrient compound was added to Peripheral Blood Mononuclear Cells (PBMC) isolated from normal controls and patients with the Chronic Fatigue Syndrome (CFS), a disease associated with immune dysregulation. The in vitro immunomodulatory effects were investigated. Cell surface expression of the glycoproteins CD5, CD8, and CD11a were significantly lower in patients with CFS compared to normal controls. Addition of glyconutrient homogenate to PBMC from patients with CFS stimulated with phytohemagglutinin significantly increased the expression of each glycoprotein. Furthermore, natural killer (NK) cell function was reduced in CFS patients. The glyconutrient preparation significantly enhanced NK cell activity versus human herpes virus 6 (HHV-6)-infected H9 cells in an 8 h 51Cr release assay compared to placebo for PBMC from patients with CFS (p< .01). Finally, apoptosis was significantly higher in patients with CFS. The percentage of apoptotic cells was significantly decreased in PBMC from patients with CFS that had been incubated for 48 h with glyconutrients. Thus, glyconutrients improved abnormal immune parameters in vitro in patients with CFS.

The pathogenesis of viral-induced diabetes.

Serologic case-control studies have suggested an association between coxsasckie group B viruses and insulin-dependent diabetes mellitus (IDDM). New investigations have identified enteroviral nucleic acid in the peripheral blood mononuclear cells of newly-diagnosed patients with IDDM. The disease pathogenesis is dependent on several factors. including the genetics of the host, strain of virus, activation status of autoreactive T-cells, upregulation of pancreatic MHC-1 antigens, molecular mimicry between viral and beta cell epitopes and direct islet cell destruction by viral cytolysis. Epitopes (IDDM-E1 and E2) on glutamate decarboxylase 65 (GAD65) are the most common targets for antibody and cellular-mediated autoimmune beta cell destruction.

Demonstration of persistent enterovirus in the pancreas of diabetic mice by in situ polymerase chain reaction.

BACKGROUND: Although Enterovirus (EV) do not persist in the tissue, which is essential to maintain autoimmunity, they have been associated as the cause of chronic autoimmunity in some cases of insulin dependent diabetes mellitus (IDDM). Convincing reports, demonstrating persistent EV infections in the pancreases, are rare. OBJECTIVES: To determine the role of EV in IDDM, a mouse model was tested and i situ polymerase chain reaction (ISPCR) developed. The major problem of ISPCR are the high amounts of non-specific staining. In the current study we developed an ISPCR protocol which minimised non-specific staining and allowed the accurate localisation of the viral RNA in the tissue. STUDY DESIGN: Five mice were infected with coxsackievirus group B4, sacrificed 7 weeks later and the pancreases were harvested. The EV nucleic acid were localised and detected in the pancreases by ISPCR. RESULTS: In the current study non-specific staining of ISPCR, due to DNA repair and diffuse artefacts, were minimised and the EV nucleic acids were localised in the beta cells of the endocrine pancreases in all five diabetogenic mice. CONCLUSION: This study demonstrates an association of viral RNA with the development of diabetes in mice and the usefulness of ISPCR to determine the role of EV in IDDM.

Successful treatment of mycotic hepatic artery pseudoaneurysms with arterial reconstruction and liposomal amphotericin B.

A 55-year-old woman developed end-stage liver disease and the hepatorenal syndrome secondary to cryptogenic cirrhosis. Orthotopic liver transplantation was complicated by bile peritonitis, requiring reoperation and eventual placement of an internal biliary stent. On postoperative day 26, hemobilia was caused by localized rupture of mycotic (Aspergillus fumigatus) hepatic artery pseudoaneurysms with fistulization into the biliary tree. After arterial reconstruction with a reversed autologous saphenous vein graft, the patient was treated successfully with liposomal amphotericin B.

Occurrence of coxsackievirus hepatitis in baby rabbits and protection by a formalin-inactivated polyvalent vaccine.

It was observed that 23-day-old New Zealand white rabbits came down with acute hepatitis demonstrable 3 days after intraperitoneal injection with 12 coxsackievirus group B strains. The model was used to evaluate a polyvalent, formalin-inactivated virus vaccine prepared with prototype strains of coxsackievirus groups B1-6. Seven-day-old animals received one intraperitoneal and two subcutaneous injections containing the vaccine or placebo. The regimen was repeated at 15 days of age. At 23 days of age, groups of rabbits were challenged with 1 x 10(5) plaque-forming units of a clinical strain of group B coxsackievirus and sacrificed 3 days later. The mean neutralizing antibody titer for the 12 strains tested (log2) was 4.5 +/- 1.0 eight days after the second dose of vaccine. In vaccinated animals, elevated liver function tests in the serum, and titer of virus and histopathologic abnormalities in the liver were significantly reduced for each strain tested compared with infected, unvaccinated controls. Cultures of the heart, skeletal muscle, pancreas, blood, and spleen were all negative. Thus, clinical strains of coxsackie group B viruses produced isolated hepatitis in baby rabbits. Prophylaxis with a polyvalent, inactivated-virus vaccine significantly reduced the severity of liver involvement for all 12 clinical strains tested.

The role of natural killer cells in viral infections.

Natural killer (NK) cells are important effectors for the lysis of both neoplastic and virus-infected cells. Lectin-like receptors on human NK cells, such as NKR-PIA and CD94, bind to target cell carbohydrate ligands and initiate the lytic process. In addition, P58 and P70 bind to major histocompatibility class I antigens on targets and mediate negative signals. Models using NK cell-deficient mice have proven useful in elaborating the role of NK cells in the immune defence against multiple viral agents. In addition, studies in humans have suggested a vital role of NK cells in the host defence against human immunodeficiency virus, herpesviruses, hepatitis B and C and other viruses. Several genetic disorders, chronic illnesses and infections have been associated with decreased NK function.

Direct in situ transcriptase polymerase chain reaction for the detection of Enterovirus genome in liver tissues.

Adolescent female mice were inoculated intraperitoneally with coxsackievirus B3 Nancy strain, sacrificed 3 and 5 days later and the livers harvested. A protocol for direct reverse transcriptase in situ PCR (RT-ISPCR) detection of enteroviral RNA in paraffin-embedded liver tissues was developed. The optimal conditions for the assay were determined. The best results were obtained when the tissue was fixed in formalin, prior to being embedded in paraffin, then cut in 5 micron thick sections, and mounted onto silanized slides. After deparaffination the slides were incubated in 1 microgram/m1 Proteinase K for 10 min and cDNA synthesis was carried out. For successful RT-ISPCR 40-50 cycles of amplification were necessary. The optimal concentrations of dNTP, primers and Taq Polymerase for RT-ISPCR were determined by serial dilution assays. Primers were selected from highly conserved sequences in the 5' non-coding region (5'NTR). To detect the viral RNA in the liver, digoxigenin-dUTP was incorporated during amplification, subsequently bound with an antidigoxigenin antibody conjugated to alkaline phosphatase (AP), followed by colorimetric detection with nitroblue tetrazolium salt (NBT) and 5-brom-4chloro-3indolyl-phosphate (BCIP). The result was a blue precipitate in the cytoplasm of hepatocytes from infected mice. Fibroblasts, endothelial cells, lymphocytes and the nuclei of hepatocytes were negative. Thus, RT-ISPCR is a specific method for the detection of enterovirus RNA in the hepatocytes of infected mice, and can be of use for the determination of EV liver disease in man.

In vitro effects of echinacea and ginseng on natural killer and antibody-dependent cell cytotoxicity in healthy subjects and chronic fatigue syndrome or acquired immunodeficiency syndrome patients.

Extracts of Echinacea purpurea and Panax ginseng were evaluated for their capacity to stimulate cellular immune function by peripheral blood mononuclear cells (PBMC) from normal individuals and patients with either the chronic fatigue syndrome or the acquired immunodeficiency syndrome. PBMC isolated on a Ficoll-hypaque density gradient were tested in the presence or absence of varying concentrations of each extract for natural killer (NK) cell activity versus K562 cells and antibody-dependent cellular cytotoxicity (ADCC) against human herpesvirus 6 infected H9 cells. Both echinacea and ginseng, at concentrations > or = 0.1 or 10 micrograms/kg, respectively, significantly enhanced NK-function of all groups. Similarly, the addition of either herb significantly increased ADCC of PBMC from all subject groups. Thus, extracts of Echinacea purpurea and Panax ginseng enhance cellular immune function of PBMC both from normal individuals and patients with depressed cellular immunity.

Use of magnetic beads versus guanidium thiocyanate-phenol-chloroform RNA extraction followed by polymerase chain reaction for the rapid, sensitive detection of enterovirus RNA.

The current study compares the sensitivity of RNA extraction using magnetic beads versus that of a standard extraction method. Streptavadin-coated magnetic beads were labelled with a biotinylated, enterovirus-specific oligonucleotide. RNA was extracted using labelled beads or guanidium thiocyanate-phenol-chloroform from 1, 0.1 and 0.01 TCID50/100 microliters of stock coxsackievirus types A9 and B3, echovirus type 11, enterovirus type 70 and poliovirus type 1. Each strain was tested three times. RNA extraction using magnetic beads was > 50% faster than the standard method. The RNA was amplified using RT-PCR, and the products were detected using agarose gel electrophoresis; 6/15 and 7/15 samples at an initial concentration of 0.01 TCID50/100 microliters were detected using magnetic beads or standard extraction, respectively. Negative-stain electron microscopy was used to determine that 0.01 TCID50/100 microliters of coxsackievirus B3 contained approximately 3 genomes. Thus, use of magnetic beads labelled with an enterovirus-specific oligonucleotide was less toxic, more rapid and as sensitive as the current standard RNA extraction method.

Comparison of procedures for the detection of enteroviruses in murine heart samples by in situ polymerase chain reaction.

A protocol for the in situ polymerase chain reaction (IS-PCR) detection of viral nucleic acid in the heart tissue of four-to-five-week-old CD1 mice infected with coxsackievirus B3 (CBV3) Nancy strain is described. To compare the effects of formalin concentration on the IS-PCR process, two different concentrations (10 and 37%) were employed. Using 37% formalin, 25 PCR cycles were sufficient and a permeabilization step could be omitted. However, postfixation of tissues with 4% paraformaldehyde and 100% ethanol after the deparaffinization, reverse transcriptase and amplification steps was required in order to minimize artefacts. When the tissues were fixed in 10% formalin, postfixation with 4% paraformaldehyde was not required, but a permeabilization step had to be employed and 40 cycles of PCR amplification were needed. To detect the PCR product in the 10% formalin-fixed samples, incubation with 0.3 U/ml of an anti-digoxigenin antibody conjugated to alkaline phosphatase was performed for 90 min. When 37% formalin-fixed samples were used, the concentration of the antibody conjugate had to be increased to 3 U/ml and the exposure time was decreased to 30 min. Enterovirus (EV) nucleic acid was detected in the cytoplasm of myocytes. Thus, IS-PCR was successful in localizing EV nucleic acid in the cytoplasm of myocytes in mice infected with a cardiotropic strain of CBV3. Using this technique, 10% formalin-fixed tissues gave better results than 37% formalin-fixed tissues.

Surveillance of community-acquired viral infections due to respiratory viruses in Rhone-Alpes (France) during winter 1994 to 1995.

Nasal swab from patients with acute flu-like illness were evaluated for the presence of respiratory viruses in the Rhone-Alpes region of France from 1 October 1994 through 2 May 1995. The relative frequencies and seasonal distributions of the specific viruses were assessed. In addition, virus type was correlated with specific clinical signs and symptoms. During the study, 962 samples were collected by 75 medical practitioners participating in the Groupe Regional d'Observation de la Grippe surveillance network. One or more viruses were detected from 348 samples (36.1%), including 108 respiratory syncytial virus (RSV), 64 influenza virus A type H3N2, 47 influenza virus B, 64 coronavirus, 35 rhinovirus, 22 adenovirus, 5 enterovirus, and 3 parainfluenza-fluenza strains. There were 16 mixed infections. RSV infections peaked in the early winter, and influenza viruses A and B infections peaked during the late winter and early spring. There were two peaks of coronavirus infections (late fall and late winter). Other viruses were detected at lower levels throughout the study period. Patients from whom adenovirus was isolated were significantly more likely to have a fever of > 39.5 degrees C than were patients with other detectable viruses (P < 0.001). Furthermore, there was a significant correlation between influenza and cough (P < 0.01) and RSV and bronchiolitis (P < .001). Thus, the current study defined the overall and relative frequencies of respiratory virus detection from nasal swab specimens in patients with an acute flu-like illness in the Rhone-Alpes region of France during a 7-month period. Correlation with clinical signs and symptoms and provisional conclusions regarding seasonality were also determined.

alpha-Interferon treatment of patients with chronic fatigue syndrome.

Thirty patients who fulfilled clinical criteria defined by the CDC for Chronic Fatigue Syndrome were treated with alfa 2a interferon or placebo in a double-blind crossover study. Outcome was evaluated by Natural Killer (NK) cell function, lymphocyte proliferation to mitogens and soluble antigens, CD4/CD8 counts and a 10 item Quality of Life (QOL) survey. Although mean NK function rose from 87.8 +/- 19.6 to 129.3 +/- 20.7 lytic untis (LU; p < .05) with 12 weeks of interferon therapy, there was no significant change in the other immunologic parameters or QOL scores. When the 26 patients who completed the study were stratified according to their baseline NK function and lymphocyte proliferation, 4 groups were identified: 3 patients had normal NK cell function and lymphocyte proliferation when compared to normal, healthy controls, 9 had isolated deficiency in lymphocyte proliferation, 7 had diminished NK function only, and 7 had abnormalities for both parameters. QOL scores were not significantly different for the four groups at baseline. After 12 weeks of interferon therapy, QOL score significantly improved in each of the seven patients with isolated NK cell dysfunction (mean score, 16.3 +/- 7.9) compared to baseline (39.7 +/- 12.1; p < .05). In these patients the mean NK function increased from 35.1 +/- 11.7 to 91.5 +/- 22.7 LU (p < .01). Significant improvement was not recorded for QOL in the other three groups. Thus, therapy with alpha interferon has a significant effect on the QOL of that subgroup of patients with CFS manifesting an isolated decrease in NK function.

Pathogenesis of virus-induced diabetes in mice.

Male CD-1 mice were challenged with a diabetogenic strain (E2) of coxsackievirus B4 (CVB4). At 7 weeks, 6 months, and 1 year after inoculation, mean histopathologic scores, postprandial blood glucose levels, and serum levels of antibody to islet cells were significantly elevated and mean fasting serum insulin levels significantly reduced in infected mice versus uninfected controls (P < .001 for each). At 7 weeks after infection, viral RNA, but not protein or infectious virus, was demonstrated in the pancreases of most infected mice. The pancreases of 4 of 12 and 0 of 10 infected animals were positive for viral RNA at 6 months and at 1 year after infection, respectively. Interferon-gamma-stimulated peritoneal macrophages were cytotoxic to islet cells with and without sera with high titers of islet cell autoantibody (ICA). Thus, islet cell destruction in mice infected with CVB4 strain E2 is associated with chronic islet cell inflammation, elevation of islet cell antibody, and prolonged presence of viral RNA in the pancreas. Stimulated peritoneal macrophages lyse islet cells directly and by an antibody-dependent mechanism.

Efficacy of a polyvalent inactivated-virus vaccine in protecting mice from infection with clinical strains of group B coxsackieviruses.

A polyvalent vaccine developed from formalin-inactivated prototype strains of coxsackieviruses group B1-6 was tested for its ability to protect mice from acute infection with clinical strains. Adolescent male CD1 mice received an intraperitoneal injection, repeated once or twice at 8 day intervals, containing 0.3 ml placebo or vaccine. Eight days after the final dose of vaccine, groups of 4 mice were challenged with 1 x 10(4) plaque-forming units of a test strain of group B coxsackievirus, and killed 3 days later. The mean neutralizing antibody titers for the 29 strains tested (4 mice/strain, log2) were 2.2 +/- 0.6, 3.3 +/- 0.9 or 6.4 +/- 1.7 after 1, 2 or 3 doses of vaccine, respectively. In mice receiving 2 or 3 doses of vaccine, titers of virus were significantly lower in the pancreas and/or blood in 7/14 or 14/15 strains, respectively, compared to unvaccinated infected controls. Uninfected, vaccinated mice failed to develop islet cell autoantibodies, histopathological abnormalities in the pancreas, or evidence of viral RNA in the pancreas 12 weeks after 3 doses of vaccine. Thus, prophylaxis with a polyvalent, inactivated-virus vaccine reduced the severity of acute infection with clinical strains of coxsackie group B viruses. A schedule of 3 doses of vaccine was superior to 1 or 2 doses.

WIN 54954 treatment of mice infected with a diabetogenic strain of group B coxsackievirus.

The therapeutic efficacy of an experimental antiviral agent, WIN 54954, was evaluated in a mouse model in which infection by coxsackievirus B4 (CVB4) strain E2 was followed by diabetes mellitus. Male CD1 mice (age, 5 weeks) were inoculated with 10(4) PFU of CVB4. WIN 54954 was administered orally via gavage tube in a dose of either 5 or 50 mg/kg of body weight per day. Treatment was initiated on the day of inoculation and was continued for 10 days. Control animals received the xanthan gum carrier only. At 3 days postinoculation (p.i.), the mean titer of virus in the pancreas was found to be significantly lower in both the high-dose (P < 0.001) and low-dose (P < 0.05) treatment groups compared with that in the controls. Furthermore, islet histologic abnormalities were significantly less common in the high-dose group (P < 0.02) than in the controls. At 7 weeks p.i., both fasting and 1-h postprandial glucose levels in blood were significantly lower for both the high-dose (P < 0.001) and the low-dose (P < 0.01) treatment groups than in controls. The proportion of mice with persistent viral RNA in the pancreas at this time, as detected by polymerase chain reaction, was significantly reduced in the high-dose treatment group (4 of 11 mice) compared with that in the controls (7 of 8 mice). When mice received 50 mg of WIN 54954 per kg daily beginning at either 48 or 72 h postinoculation, the titers in the pancreas were again significantly reduced at 3 days p.i. compared with those in the controls (P < 0.01 and P < 0.05, respectively). Thus, WIN 54954 effectively reduces virus replication and islet histologic changes acutely and decreases, at 7 weeks, both the metabolic alteration associated with diabetes mellitus and the incidence of detectable viral RNA in the pancreas.

Treatment of Coxsackievirus A9 myocarditis in mice with WIN 54954.

The therapeutic efficacy of an experimental antiviral agent, WIN 54954, was evaluated in murine myocardial infection with coxsackievirus A9 (CVA9). Eight-month-old male Swiss Webster mice were inoculated with 1.5 x 10(4) PFU of CVA9, Boston strain 13. WIN 54954, a broad-spectrum antipicornavirus agent, was administered orally in a dose of 0.25, 2.5, 25, 50, 100, or 200 mg/kg of body weight per day on days 1 to 3 after virus inoculation. Control animals received xanthan gum carrier only. Mice were sacrificed on day 4. Myocardial titers of virus were determined and found to be significantly lower in the four highest dose treatment groups (P less than 0.001 for all groups) compared with controls. Heart weights were also significantly lower compared with controls in these four groups (P less than 0.001 for all groups). When mice received 50 mg of WIN 54954 per kg daily beginning at either 48 or 72 h postinoculation, myocardial titers were once again significantly reduced compared with those of controls (P less than 0.001 for both groups). Neurological toxicity was observed in the 100- and 200-mg/kg/day groups but not in the lower-dose groups. Thus, WIN 54954 effectively reduced myocardial CVA9 replication in a murine model.

Viral myocarditis.

Although most cases of viral myocarditis are subclinical, some patients develop overt symptomatic disease. These patients may present with findings that range from benign myopericarditis to frank heart failure. Furthermore, a growing body of evidence links viral myocarditis with idiopathic dilated cardiomyopathy, sudden death, and chronic arrythmias. The pathogenesis of the disease is currently incompletely understood in humans but is being investigated in animal models. A multifactorial process involving direct viral damage, autoimmunity, and possibly vascular damage is emerging. Breakthroughs in rapid diagnosis, such as the use of DNA probes, are occurring and may soon provide the opportunity for early intervention. Although there is currently no widely accepted standard of treatment, promising new therapeutic modalities are under investigation. These include the use of general immunosuppressive agents, T cell monoclonal antibody, interferon, specific immunization, and synthetic antiviral agents.