Exercise stress increases susceptibility to influenza infection.

Exercise stress is associated with an increased risk for upper respiratory tract infection (URTI) while moderate exercise has been associated with a decreased risk. We have shown that exercise stress can increase susceptibility (morbidity, symptom severity and mortality) to HSV-1 respiratory infection, but there is little evidence on the effects of stressful exercise on susceptibility to the principal etiological agents of human respiratory infections, including influenza viruses. This study examined the effects of stressful exercise on susceptibility to influenza virus (A/Puerto Rico/8/34 (H1N1)). Mice were assigned to one of two groups: exercise (Ex) or control (Con). Exercise consisted of a treadmill run to volitional fatigue ( approximately 120 min) performed on three consecutive days. Fifteen minutes after the last bout of exercise or rest, mice (n=20-21/group) were intranasally inoculated with a standardized dose of influenza virus (0.25 HAU). Mice were monitored daily for morbidity (time to sickness), symptom severity and mortality (time to death) for 21 days. Exercise stress was associated with an increase in susceptibility to infection (morbidity, mortality and symptom severity on days 6 and 7; P<0.05). These data from a controlled influenza virus challenge model add significantly to the growing body of evidence that severe exercise can increase susceptibility to URTI.

Quercetin reduces susceptibility to influenza infection following stressful exercise.

Exercise stress is associated with increased risk for upper respiratory tract infection. We have shown that exercise stress can increase susceptibility to infection. Quercetin, a flavonoid present in a wide variety of fruits and vegetables, has been reported to inhibit infectivity and replication of a broad spectrum of viruses and may offset the increase in susceptibility to infection associated with stressful exercise. This study examined the effects of quercetin feedings on susceptibility to the influenza virus A/Puerto Rico/8/34 (H1N1) following stressful exercise. Mice were randomly assigned to one of four treatment groups: exercise-placebo, exercise-quercetin, control-placebo, or control-quercetin. Exercise consisted of a run to fatigue (approximately 140 min) on a treadmill for 3 consecutive days. Quercetin (12.5 mg/kg) was administered via gavage for 7 days before viral challenge. At 30 min after the last bout of exercise or rest, mice (n=23-30) were intranasally inoculated with a standardized dose of influenza virus (0.04 hemagglutinating units). Mice were monitored daily for morbidity (time to sickness), symptom severity, and mortality (time to death) for 21 days. Exercise stress was associated with an increased susceptibility to infection [morbidity, mortality, and symptom severity on days 5-7 (P<0.05)]; quercetin offset the increase in susceptibility to infection [morbidity, mortality, and symptom severity on days 5-7 (P<0.05)] that was associated with stressful exercise. These data suggest that short-term quercetin feedings may prove to be an effective strategy to lessen the impact of stressful exercise on susceptibility to respiratory infection.

Effect of interferon alpha and cell cycle progression on translation mediated by the hepatitis C virus 5' untranslated region: a study using a transgenic mouse model.

The effect of interferon alpha (IFN alpha) and the progression of the cell cycle on translation mediated by the 5' untranslated region (5'UTR) of hepatitis C virus (HCV) was evaluated in a transgenic mouse model containing the beta-galactosidase (beta-gal) gene under the control of the mouse albumin promoter and HCV 5'UTR. The transgene was exclusively expressed in the liver and specifically in hepatocytes around the periportal area. IFN alpha significantly suppressed the expression of both the beta-gal gene product and its enzymatic activity at 6 h after the treatment of the mice. The mRNA level of the transgene and endogenous albumin gene expression were not affected, so this suppression was considered to be specific to 5'UTR-directed translation. Phosphorylation of the Stat1 protein was observed in the liver extract 20 min after the treatment, thus confirming a specific known effect of IFN alpha in vivo. We suggest that suppression of 5'UTR-directed translation may be one of the mechanisms whereby IFN alpha exerts its anti-viral activity. We further investigated whether the restriction of 5'UTR-directed translation in periportal hepatocytes may be explained by the proliferative state of the cell. Transgene expression was slightly enhanced in the liver 48 h after partial hepatectomy when a substantial number of hepatocytes entered cell cycle progression. However, 5'UTR-directed translation could not be detected in hepatocellular carcinoma lesions in transgenic mice that were induced to develop such tumours. We suggest that the state of differentiation of the cell, and not its proliferative capacity, is important for supporting HCV expression. This animal model may be a useful tool to dissect the control of HCV expression and to search for ways to block viral replication.

Enhanced antiviral benefit of combination therapy with lamivudine and alpha interferon against WHV replication in chronic carrier woodchucks.

Cell culture studies in our laboratory previously demonstrated synergistic antiviral activity for the combinations of lamivudine and a novel recombinant hybrid human alpha B/D interferon (rHu alpha B/D IFN) against hepatitis B virus (HBV) replication. Based on these results, a study was designed to determine if an enhanced antiviral effect with this drug combination could be demonstrated in vivo using the woodchuck hepatitis virus (WHV)/woodchuck experimental model of chronic HBV infection. Both antiviral agents have been shown to be effective against WHV replication in WHV chronic carriers during previous studies by our laboratories. Two combination treatment regimens were compared to matched monotherapies in a placebo-controlled trial. The first used simultaneous administration of rHu alpha B/D IFN and lamivudine for 24 weeks. The other combination treatment regimen used a staggered dosing schedule of 12 weeks of administration of lamivudine alone, followed by 12 weeks of simultaneous dosing with both drugs, followed by 12 weeks of therapy with rHu alpha B/D IFN alone. Both treatment regimens with combinations of lamivudine and rHu alpha B/D IFN were more effective at reducing WHV replication in chronically infected wood-chucks than the corresponding monotherapies. Both combination treatments produced antiviral effects that were at least equal to that expected for additive activity based on estimations generated by Bliss Independence calculations. The staggered treatment regimen reduced viraemia and intrahepatic WHV replication significantly more than that expected for additive interactions, indicating synergistic antiviral effects. These studies demonstrate that combination therapy of chronic WHV infection has enhanced antiviral benefit over corresponding monotherapies and indicate that combination treatment of chronic HBV infection can be superior to therapies using a single antiviral agent.

Ineffectiveness of echinacea for prevention of experimental rhinovirus colds.

The purpose of this study was to assess the effectiveness of echinacea for the prevention of experimental rhinovirus colds. Infection occurred in 44 and 57% and illness occurred in 36 and 43% of the echinacea- and placebo-treated subjects, respectively. This preparation of echinacea had no significant effect on either the occurrence of infection or the severity of illness.

Quantitative immunodetection of viral oncoprotein levels in human papillomavirus-transformed human cells.

Human papillomaviruses (HPV) comprise a genus in the species Papovaviridae, which consist of small, naked icosahedral viruses containing circular dsDNA. HPV types 16 and 18 infect squamous epithelium of the genitalia and may infect as many as 45% of the female population in developed countries (1).

Effect of recombinant human interferon alpha B/D (rHu-IFN-alpha B/D) in combination with acyclovir in experimental HSV-1 encephalitis.

The efficacy of recombinant human interferon alpha B/D in experimental HSV-1 encephalitis was investigated in the murine system. Recombinant Hu-IFN-alpha B/D significantly reduced the mortality in a mouse encephalitis model (about 30%, P = 0.021), whereas natural mouse interferon was inactive. Combination of acyclovir with Hu-IFN-alpha B/D had an additive effect.

IFN-tau exhibits potent suppression of human papillomavirus E6/E7 oncoprotein expression.

The effects of interferon-tau (IFN-tau) on tumor suppressor factors and virus oncoprotein expression were compared with two other type I IFN in human papillomavirus (HPV-16)-transformed cells. Nontumorigenic human keratinocytes, HuKc/HPV-16d-2C (d-2C), treated with recombinant human IFN-alpha2a (Roferon), a human recombinant alpha IFN hybrid, alpha B/D (IFN-alphaB/D), or ovine IFN-tau were evaluated for their effects on the levels of E6 and E7 expression. IFN-tau was comparable to IFN-alpha2a in decreasing intracellular levels of E6 and E7, and IFN-alphaB/D was more effective than IFN-a2a in suppressing E7 levels. All three IFN were capable of increasing the cellular concentration of wild-type p53 tumor suppressor with the magnitude IFN-tau > IFN-alpha2a > IFN-alphaB/D. Increases in p53 concentrations correlated with the observed decreases in E6 mRNA and protein levels. The antiviral effects observed in this study reveal that IFN-tau has potent antipapillomavirus activity. Sequences/structures unique to IFN-tau could allow for alternative IFN/receptor interactions and may explain the differences in biologic function.

Selective inhibition of human papillomavirus-induced cell proliferation by (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine.

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) is a nucleoside phosphonate analog which in its active diphosphorylated form is known to inhibit herpesvirus DNA polymerase. In this study, we have demonstrated that, in a dose-dependent manner, this compound irreversibly suppressed proliferation of cells infected with human papillomavirus (HPV), which does not possess a viral DNA polymerase. To elucidate the mechanism of cell growth inhibition, cell cycle indicator-regulator expression, thymidine incorporation, transcript levels of apoptosis factors, and anabolic products of HPMPC following drug treatment were evaluated. HPMPC treatment reduced WAF1 (p21) levels independent of those of p53, while proliferating cell nuclear antigen increased. However, in comparison to controls, HPMPC-treated cells displayed a decrease in thymidine incorporation, indicating an inhibition of host DNA polymerase activity. In normal primary keratinocytes, HPMPC predominantly accumulated in the form of the choline adduct HPMPCp-choline. However, in HPV type 16-transformed keratinocytes, HPMPCpp was the most abundant anabolic product, with little HPMPCp-choline having formed. The data imply that an unrecognized viral factor is modulating the conversion of nucleotides, including HPMPC, to the triphosphorylated form.

Mycoplasma penetrans and other mycoplasmas in urine of human immunodeficiency virus-positive children.

Urine samples from children with human immunodeficiency virus (HIV) infection and healthy controls were examined for mycoplasmas by culture. Standard biochemical assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and PCR (16S and 16S-23S spacer rRNA region) were used for identification of isolates. Mycoplasmas were identified from 13 (87%) of 15 HIV-positive patients and 3 (20%) of 15 HIV-negative control patients. The frequency and type of mycoplasma varied with the severity of HIV infection. Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma fermentans, and Mycoplasma genitalium were isolated from patients with severe immunodeficiency. Mycoplasma hominis and Ureaplasma urealyticum were isolated more frequently from children in the early stages of HIV infection and from HIV-negative patients. Mycoplasma penetrans was isolated from one (50%) of two patients in Centers for Disease Control and Prevention (CDC) group B and from five (55.5%) of nine pediatric patients with AIDS (CDC group C). This is the first report that indicates that "AIDS-associated" mycoplasmas are more common in HIV-infected children than in HIV-negative controls.

Immunogenicity and safety of recombinant Helicobacter pylori urease in a nonhuman primate.

Groups of squirrel monkeys (Saimiri spp.), predetermined to be free of Helicobacter infections in the gastric mucosa, were immunized orally with 0.5-4.5 mg of Helicobacter pylori recombinant urease (rUrease) and 25-500 micrograms of Escherichia coli heat-labile enterotoxin (LT) adjuvant. Oral immunization with rUrease resulted in a markedly elevated serum immunoglobulin G (IgG) antibody response with peak levels at 45 days after immunization. No significant gastric inflammation or cytotoxicity was evident in rUrease immunized monkeys as determined by light and electron microscopy. Twenty-five micrograms of LT was a sufficient and safe adjuvant dosage, whereas higher dosages resulted in diarrhea and lethargy. Animals developed a serum IgG antibody response to LT that did not impede the production of anti-rUrease antibody levels. The results of this investigation indicate that rUrease is immunogenic in a nonhuman primate.

Differential effect of recombinant human and mouse interferons on replication of herpes simplex virus type 1 in mouse cells.

Pretreatment of murine (BALB/3T3) cells with either murine or recombinant hybrid human B/D interferon (IFN) blocked the release of infectious herpes simplex virus type 1 (HSV-1) from treated cells. The block in replication was not due to an effect on attachment of HSV-1 to the target cells or to toxic effects of IFN. Immunoblot analyses showed that murine IFN significantly reduced the expression of virus-specific proteins in IFN-treated cells. In contrast, B/D IFN had no major effect on the expression of viral proteins in treated cells. In support of the above observation, electron microscopy of virus-infected cells displayed formation of nucleocapsids within the nucleus of IFN-treated cells. However, the expression of glycoproteins B and D was reduced in B/D IFN-treated cells. These results suggested that murine IFN blocked HSV-1 replication at an early stage whereas B/D IFN inhibited HSV-1 replication at a late stage in virus morphogenesis.

Functional inactivation but not structural mutation of p53 causes liver cancer.

Structural mutations in the p53 gene are seen in virtually every form of human cancer. To determine whether such mutations are important for initiating tumorigenesis, we have been studying hepatocellular carcinoma, in which most cases are associated with chronic hepatitis B virus infections. Using a transgenic mouse model where expression of a single HBV gene product, the HBx protein, induces progressive changes in the liver, we show that tumour development correlates precisely with p53 binding to HBx in the cytoplasm and complete blockage of p53 entry into the nucleus. Analysis of tumour cell DNA shows no evidence for p53 mutation, except in advanced tumours where a small proportion of cells may have acquired specific base substitutions. Our results suggest that genetic changes in p53 are late events which may contribute to tumour progression.

HPV replication in experimental models: effects of interferon.

Preclinical evaluation of the effectiveness of interferon (IFN) therapy on human papillomaviruses (HPV) has been hampered by the inability to propagate these viruses in cell culture. Nonetheless, interferon is used extensively in the treatment of HPV infections. Alpha interferons in particular have found a place in the treatment of anogenital disease, plantar warts, and laryngeal papillomas. While their is significant clinical evidence to suggest that interferon is useful in therapy of disease, the cellular mechanism(s) (i.e., antiviral, antiproliferative, immunomodulatory) by which IFN is able to control HPV-induced pathology is not well understood. This review focuses on experimental animal and cell culture models which are currently being used to help identify the antiviral, antiproliferative and immunomodulatory effects of IFN on HPV infection.

Lipopolysaccharide-induced suppression of erythrocyte binding and phagocytosis via Fc gamma RI, Fc gamma RII, Fc gamma RIII, and CR3 receptors in murine macrophages.

In this study we have investigated the ability of lipopolysaccharide (LPS) to suppress binding and phagocytosis of erythrocytes via various receptors on mouse macrophages. Thioglycollate-elicited peritoneal macrophages were treated in vitro with LPS and the ability to bind and phagocytose radiolabeled sheep red blood cells was determined. We show that LPS can directly suppress phagocytosis of immunoglobulin G-opsonized and nonopsonized sheep red blood cells (SRBCs) by inflammatory macrophages. Suppression was dose dependent and was observed after 4 h of exposure. This effect lasted for at least 24 h following the removal of LPS. LPS suppressed the binding, rate, and absolute level of phagocytosis via Fc receptors. Phagocytosis via all Fc receptors (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) was suppressed by LPS. Furthermore, suppression was not limited to Fc receptor-mediated phagocytosis because binding and uptake of C3bi-opsonized SRBCs to CR3 receptors was also decreased following LPS treatment. LPS did not exert its effects via the production of interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha, or interferon alpha/beta, because antibodies to these cytokines did not abrogate the effect. The ability of LPS to suppress binding and phagocytosis of microorganisms may contribute to the toxic effects of LPS during gram-negative sepsis by preventing or delaying elimination of bacteria by host macrophages.

Inhibition of growth, transformation, and expression of human papillomavirus type 16 E7 in human keratinocytes by alpha interferons.

We used a model system of normal human keratinocytes (HKc) and HKc immortalized with human papillomavirus type 16 DNA (HKc/HPV16) to investigate the effects of alpha interferons (IFN-alpha) on the growth of HPV16-immortalized human epithelial cells, on HPV16-mediated immortalization of normal HKc, and on HPV16 gene expression. Normal HKc and HKc/HPV16 were treated with several recombinant human IFN-alpha subtypes (IFN-alpha B, IFN-alpha D, and IFN-alpha B/D). These IFN-alpha subtypes inhibited proliferation of both normal HKc and HKc/HPV16 in a dose-dependent fashion; however, although 1,000 to 10,000 U of IFN-alpha per ml were required to inhibit growth of normal HKc, HKc/HPV16 were substantially growth inhibited by 100 U/ml. In addition, 100 U of IFN-alpha B/D per ml inhibited transformation of normal HKc by HPV16 DNA. Northern (RNA) blot analysis showed no effect of IFN-alpha on the mRNA levels of the HPV16 E6 and E7 open reading frames. However, immunofluorescence studies of the HPV16 E6 and E7 proteins with anti-E6 and anti-E7 monoclonal antibodies showed significant inhibition of E7 protein expression in cells treated with IFN-alpha, whereas E6 protein expression was not altered. The inhibition of E7 protein expression in cells treated with IFN-alpha was further confirmed by Western immunoblot analysis. These results suggest that IFN-alpha may inhibit HPV16-mediated transformation of HKc and proliferation of HKc/HPV16 through an inhibition of HPV16 E7 protein expression.

Poly I:C-induced antiviral and cytotoxic activities are mediated by different mechanisms.

Macrophages play an important role in host defenses against tumors and virus infections by killing tumor or virus infected cells (extrinsic cytotoxicity) and by limiting virus replication within themselves (intrinsic antiviral activity). Since common macrophage products may be involved in both extrinsic cytotoxicity and intrinsic antiviral activity, we decided to investigate the mechanisms by which Poly I:C-activated macrophages resist infection with HSV-1 and inhibit the growth of tumor cells. The ability of macrophages to resist infection with HSV-1 or to inhibit growth of tumor cells was assessed following treatment with Poly I:C in the presence of antibodies to various cytokines or in the presence of inhibitors/scavengers of toxic macrophage products. Only antibodies to IFN-beta were able to abrogate the protective effects of Poly I:C in macrophages infected with HSV-1, suggesting that the antiviral activity induced by this immunomodulator was mediated by the production of IFN-beta, which acted in an autocrine manner. In contrast, anti-TNF-alpha, anti-IFN-alpha/beta anti-IFN-beta antibodies and inhibitors of nitric oxide and C1q production were all able to partially abrogate Poly I:C-induced cytostatic activity, suggesting that multiple mechanisms are involved in macrophage cytostasis. Our results indicate the Poly I:C-induced intrinsic antiviral and extrinsic cytotoxic activities are mediated by different mechanisms.