Longitudinal observation of enterovirus and adenovirus in stool samples from Norwegian infants with the highest genetic risk of type 1 diabetes.

BACKGROUND: Enterovirus and adenovirus are common in infancy, causing mostly asymptomatic infections. However, even an asymptomatic infection may be associated with increased risk of development of certain chronic non-infectious diseases, as has been suggested for enterovirus and type 1 diabetes. Data on occurrence and course of the infections in infancy are therefore important for designing effective approaches towards study of the association. OBJECTIVES: To estimate the frequency of enterovirus and adenovirus infections in Norwegian infants, to evaluate the duration of the infections, to investigate their association with symptoms, and to establish a robust procedure that will be used to study the association between these viruses and the development of auto-immunity leading to type 1 diabetes. STUDY DESIGN: Parents of infants, recruited for a study on environmental triggers of type 1 diabetes, submitted monthly samples of infant faeces, as well as information on symptoms of infection. The samples were analysed for enterovirus and adenovirus using quantitative real-time PCR, and enterovirus-positive samples were sequenced. RESULTS: Enteroviruses were found in 142/1,255 (11.3%), and adenoviruses in 138/1,255 (11.0%) of stool samples. Approximately half of the infants were exposed to these viruses at least once during the first year of observation (period 3-14 months of age). The presence of adenovirus was associated with fever and with symptoms of cold but not with diarrhoea and vomiting. The enterovirus positivity was not associated with any symptoms. CONCLUSIONS: The prevalence of enterovirus and adenovirus in longitudinally obtained faecal samples from infants is sufficiently high to enable studies of their association with chronic diseases. The present protocol for evaluating exposure to these viruses is well suited for large-scale efforts aimed at assessing possible long-term consequences, particularly in relation to type 1 diabetes.

Hepatitis C in the general adult population of Oslo: prevalence and clinical spectrum.

BACKGROUND: The prevalence of hepatitis C (HCV) in Northern Europe has not been well described. This study aimed to estimate the prevalence and spectrum of hepatitis C infection in the general adult population of Oslo, Norway. METHODS: The study was part of the Oslo Health Study 2000-2001 and included a random selection of individuals older than 30 years living in Oslo County. Sera from 11,456 participants were screened for anti-HCV (EIA-3), positive samples were confirmed (RIBA-3) and examined for HCV RNA (PCR). All anti-HCV positive patients were offered clinical evaluation. Routine biochemical liver tests were performed. Candidates for HCV treatment were asked to undergo a percutanous liver biopsy. RESULTS: Among 11,456 participants HCV RNA was detected in 62 (0.5%) and HCV RNA with raised serum alanine aminotransferase (ALT) in 46 (0.4%). Anti-HCV was detected in 78 (0.7%) with a peak prevalence of 1.5% among subjects 40 and 45 years old. Being anti-HCV positive was associated with being unmarried, unemployed and having low education. Anti-HCV prevalence was higher among subjects with alcohol-related problems compared to those without (4.4% versus 0.6%, P < 0.001). It was also higher among smokers compared to non-smokers (2.0% versus 0.2%, P < 0.001). In 33 liver biopsies, bridging fibrosis was seen in 8 (24%) and cirrhosis in 1 (3%). The route of transmission was injecting drug use in 67%, transfusion in 6% and unknown in 27%. CONCLUSION: In this population-based survey the prevalence of chronic hepatitis C was 0.5% and ALT was raised in 80% of those with chronic infection.

Is preeclampsia an infectious disease?

BACKGROUND: Studies have suggested a strong paternal factor in the etiology of preeclampsia. If preeclampsia is caused by an infectious agent transmitted by the woman's partner, seronegative women who may experience primary infection in pregnancy should be at increased risk of preeclampsia as compared to previously infected women. The aim of this study was to assess the impact of being seronegative for some viruses transmitted by close contact on the risk of developing preeclampsia. METHODS: Nine hundred and seventy-eight women were randomly drawn from a basic study population of 35,940 pregnant women in Norway. A serum sample drawn at the first antenatal visit was analyzed for specific IgG antibodies against herpes simplex virus type-2, cytomegalovirus and Epstein-Barr virus. For comparison, antibody status against Toxoplasma gondii was also assessed. Information on preeclampsia in pregnancy was obtained through linkage to the Medical Birth Registry of Norway. RESULTS: Thirty-three (3%) women developed preeclampsia. The risk of developing preeclampsia seemed to be increased for women who were seronegative for the viruses studied. Seronegativity for Toxoplasma gondii did not show such a pattern. INTERPRETATION: Women who are seronegative for antibodies against viral agents transmitted through close contact seem more likely to develop preeclampsia. This finding indicates that women who are seronegative to such agents may acquire primary infection in pregnancy, and subsequently be at increased risk of preeclampsia. This hypothesis could represent a new approach to the causes of preeclampsia, and encourage search for yet unidentified microbes as a possible causal factor.

T-cell responses to viral, bacterial and protozoan antigens in rheumatoid inflammation. Selective migration of T cells to synovial tissue.

OBJECTIVE: To identify any preferential or selective migration of T-cell specificities to inflamed tissues of rheumatoid arthritis (RA) patients. METHODS: Lymphocytes from peripheral blood (PB) and synovial tissue (ST) were isolated from RA patients and stimulated with a panel of crude antigen preparations from 18 bacterial, protozoan and viral sources. Proliferative responses of the T lymphocytes to each antigen and group of antigens were compared in PB and ST. Antigen-specific T-cell clones were developed and their migratory capacities towards synovial chemokines were compared. RESULTS: ST-derived T cells showed a small but significantly higher stimulation index (SI) to the group of intestinal bacteria compared with PB T cells. Conversely, responses of ST-derived T cells to Acanthamoeba polyphaga (AP) were both profoundly and significantly lower compared with PB-derived T cells. The viral antigens as a whole gave comparable reactivities in blood and ST. The migratory capacity of AP-specific T-cell clones towards chemokines produced by ST was profoundly poorer compared with Campylobacter jejuni- and herpes simplex virus-specific T-cell clones. CONCLUSIONS: The results indicate a selective migration of T cells of given specificities to the inflamed rheumatoid synovium.

Acanthamoeba polyphaga in rheumatoid arthritis: possibility for a chronic infection.

Acanthamoeba polyphaga (AP) is ubiquitous in nature and frequently infects humans. AP has some features, such as persistence, which makes it an attractive candidate in studies of a possible infectious aetiology in rheumatoid arthritis (RA). In this study the occurrence of AP-specific antibodies was compared between RA patients and matched controls.

[Hepatitis C virus among pregnant women in Norway--occurrence of antibodies and pregnancy outcome]. / Hepatitt C-virus blant gravide kvinner i Norge--forekomst av antistoffer og svangerskapsutfall.

BACKGROUND: Transmission of hepatitis C virus from mother to child is well documented. The prevalence of antibodies against hepatitis C virus among pregnant women in Norway is however, not known. The aim of this study was to estimate the maternal prevalence of antibodies against hepatitis C virus and to study the association between presence of antibodies and fetal death. MATERIAL AND METHODS: From a study of 35,940 pregnant women, a random sample of 970 women and all women with fetal death after 16 weeks of gestation (n = 283), were tested for antibodies against hepatitis C virus. RESULTS: 7 out of 970 women in the random sample (0.7%; 0.2-1.3%, 95% confidence interval) had antibodies against hepatitis C virus. The same prevalence (0.7%, 2 out of 283) was found among women with fetal death. INTERPRETATION: The prevalence of antibodies against hepatitis C virus among Norwegian women was unexpectedly high. Further research is necessary to understand the causes and implications of this observation.

Herpes simplex virus type-2 antibodies in pregnant women: the impact of the stage of pregnancy.

In this study the impact of pregnancy duration on the measured level of HSV-2 antibodies was assessed. The study population comprised 35,940 pregnant women in Norway, in 1992-4, followed during pregnancy. A random sample of 960 women was selected. A mean of 2.6 serum samples from each woman were analysed for HSV-2 specific IgG antibodies at different times in pregnancy. Crude and adjusted odds ratios were estimated in logistic regression models taking all observations per women into account. Twenty-seven percent of the pregnant women had antibodies against HSV-2 in the first trimester. The adjusted odds ratio of being HSV-2 antibody positive decreased during the pregnancy and was 0.5 (0.2-0.9, 95% confidence interval) in the 40th as compared to the 10th week of pregnancy. About 50% of initially HSV-2 positive women did not have detecable antibodies by the end of the pregnancy. This may be explained by haemodilution during pregnancy. Our findings have diagnostic implications and should encourage further studies.

Identification of type-specific domains within glycoprotein G of herpes simplex virus type 2 (HSV-2) recognized by the majority of patients infected with HSV-2, but not by those infected with HSV-1.

A combination of phage peptide display library mapping and pepscanning, with both murine monoclonal antibodies and a panel of well-characterized human sera, have been used in order to define type-specific epitopes of glycoprotein G of herpes simplex virus type 2 (HSV-2) (gG2). Both techniques revealed an immunodominant region of gG2, centred around amino acids 525-587 of the uncleaved gG2 molecule. A soluble peptide, equivalent to amino acids 551-570, when used as antigen in an ELISA format was recognized by three out of five murine MAbs and by 20/26 (77%) Western blot anti-HSV-2-positive human sera, but by only 1/63 Western blot anti-HSV-2-negative sera (specificity, 98%). The sensitivity of detection of human anti-HSV-2 antibodies was increased to 90% using a peptide derived from this region, presented on a nitrocellulose membrane. This highly antigenic and type-specific domain of gG2 is located at the junction between the 'unique' region of gG2 and its C-terminal end, which has approximately 50% identity with gG1. A second antigenic region of gG2, amino acids 351-427, which lies within the 'unique' part of gG2, was also identified by both techniques employed in this study and is recognized by a proportion of anti-HSV-2-positive sera. These findings demonstrate the feasibility of developing a peptide-based type-specific assay for the detection of anti-HSV-2 antibody in human sera based on type-specific epitopes of gG2 and have implications for the understanding of the three-dimensional topography of gG2.

[Antibodies against Herpes simplex virus type 2 among pregnant women in Norway]. / Herpes simplex-virus type 2-antistoffer hos gravide i Norge.

The prevalence of antibodies against herpes simplex virus type 2 (anti-HSV-2) among pregnant women in Norway is not known. To study the prevalence of anti-HSV-2, a random sample of 961 women was drawn from a study population of 35,940 pregnant women in Norway during 1992-94. 27% (256/961) had anti-HSV-2. The prevalence increased with age. 17% of the 20-24-year-olds and 34% of the 35 year-old or older had anti-HSV-2. The presence of antibodies also varied geographically, from 18% in the south to 39% in the north of Norway. Among women with repeated anti-HSV-2 tests during pregnancy, 2.6% of the seronegative women seroconverted (16/623). HSV-2 infection is common among pregnant women in Norway. The public health implications of this infection need to be clarified.

Localization of type-specific epitopes of herpes simplex virus type 2 glycoprotein G recognized by human and mouse antibodies.

Glycoprotein G is a major target for the humoral immune response against herpes simplex virus (HSV) and a prototype antigen for type-specific serodiagnosis discriminating HSV-1 and HSV-2 infections. The mature part of HSV-2 glycoprotein G-2 (gG-2) contains a unique stretch suspected to mediate type specificity, and in addition a region homologous to HSV-1 glycoprotein G-1 (gG-1). Antigenic determinants of the mature gG-2 were mapped by testing the reactivity of mouse anti-gG-2 monoclonal antibodies (MAbs) and purified human anti-gG-2 antibodies with synthetic peptides coupled to cellulose membranes. The anti-gG-2 MAbs bound to four epitopes localized in a narrow cluster within a gG-2 segment delimited by amino acids (aa) 552 and 611. This cluster was located between the predicted O-glycan-rich region and the transmembrane anchor sequence. The epitopes of the human anti-gG-2 antibodies were localized within three stretches of amino acids, two of which were overlapping with those recognized by anti-gG-2 MAbs. One of these stretches, delimited by aa 552 and 574, showed reactivity to all human HSV-2 sera tested, but not to HSV-1 sera or to purified anti-gG-1 antibodies. Neither the anti-gG-2 MAbs nor the purified human anti-gG-2 antibodies were cross-reactive to gG-1 peptides or HSV-1 antigen, although most of the epitopes were localized within the part of gG-2 which was homologous to gG-1. The findings concerning HSV-2 type-specific human antibody response to a defined stretch within gG-2 may be of importance for the further development of type-discriminating serodiagnosis.

Relationship of secretion pattern and MPB70 homology with osteoblast-specific factor 2 to osteitis following Mycobacterium bovis BCG vaccination.

Significant homology was found between MPB70 and each of four repeat domains of osteoblast-specific factor 2 (OSF-2). Two internal homology regions within each repeat domain of OSF-2 presumed to be related to the active site(s) of this bone adhesion molecule showed the highest homology. A literature search concerning osteitis after Mycobacterium bovis BCG vaccination in neonates revealed that MPB70-high-producer substrains were associated with an increased incidence of osteitis following vaccination. These observations indicate that the function of MPB70 is related to the interaction between bacilli and the host following vaccination or infection with mycobacteria.

An evaluation of a hemagglutination-inhibition test for the detection of antibodies to herpes simplex virus type 1.

BACKGROUND: We have recently demonstrated the ability of herpes simplex virus type 1 (HSV-1) to agglutinate mouse red blood cells, and identified glycoprotein C (gC-1) as a major virus hemagglutinin. Based on this a classical hemagglutination-inhibition (HI) assay was developed. OBJECTIVES: Regarding significant structural differences between HSV-1 gC-1 and its herpes simplex virus type 2 (HSV-2) counterpart, gC-2, the possibility of application of a classical HI assay for the detection of HSV-1-specific antibodies was explored. STUDY DESIGN: HI antibody titers were compared with those of gC-1-specific enzyme-linked immunoassay (ELISA), and with the results of the standard gG-1- and gG-2-specific immunodot enzymatic assays for the detection of type-specific antibodies to HSV-1 and HSV-2 respectively. RESULTS: The sensitivity of HI test was 89% and 97% of that gC-1-ELISA and gG-1-immunodot respectively. Approximately 21% of serum specimens, defined as containing antibodies specific for only HSV-2, showed low HI titers. Heterotypic reactivity with purified gC-1 antigen was also observed in both ELISA and immunoblot assays. CONCLUSION: Antibodies detectable in HI assay were mainly HSV-1-specific; however, a limited degree of serologic reactivity between HSV-2-specific sera and HSV-1 hemagglutinin also occurred. Thus, our results confirmed prevalent opinion about the presence of a limited number of antigenic determinants shared by HSV-1 gC-1 and HSV-2 gC-2.

Infection of vaginal and colonic epithelial cells by the human immunodeficiency virus type 1 is neutralized by antibodies raised against conserved epitopes in the envelope glycoprotein gp120.

The rectal and genital tract mucosae are considered to be major sites of entry for the human immunodeficiency virus (HIV) during sexual contact. We now demonstrate that vaginal epithelial cells can be infected by HIV type 1 (HIV-1) via a mechanism similar to that described for neuroglial cells and, more recently, for colorectal epithelial cells, involving initial interaction of the HIV-1 envelope glycoprotein gp120 with a cell-surface glycosphingolipid (sulfated lactosylceramide). A hyperimmune serum against gp120 was able to neutralize HIV-1 infection of vaginal epithelial cells. Site-directed immunization was employed to identify sites on gp120 recognized by antibodies neutralizing HIV-1 infection of vaginal and colonic epithelial cells. Hyperimmune sera were raised in monkeys against a series of 40 overlapping synthetic peptides covering the entire sequence of HIV-1 (HTLV-IIIB) gp120. Antisera raised against five synthetic peptides, corresponding to three relatively conserved regions and to the hypervariable region (V3 loop), efficiently neutralized HIV-1 infection of human vaginal epithelial cells in vitro. Similar results were obtained with the colonic cells. Hyperimmune sera to all five peptides have been shown earlier to neutralize HIV-1 infectivity in CD4+ T cells. These results have obvious implications for the design of mucosal subunit vaccines against sexually transmitted HIV-1 infections.

Herpes simplex virus type 2 infection of heterosexual men attending a sexual health centre.

OBJECTIVE: To identify risk factors, particularly circumcision status, associated with serological evidence of herpes simplex virus type 2 (HSV-2) infection of heterosexual men. DESIGN: A cross-sectional case-control study employing an anonymous delinked interviewer-administered questionnaire, clinical examination, and a type-specific serological test for HSV-2. PARTICIPANTS AND SETTING: Three hundred consecutive heterosexual male patients at a public sexually transmissible diseases (STD) clinic in Sydney, Australia. MAIN OUTCOME MEASURES: Associations between serological evidence of HSV-2 infection and history of genital herpes or contact with genital herpes, history of other common STDs, and demographic and behavioural factors such as age, education level, number of sexual partners and lack of circumcision. RESULTS: One hundred and ninety-four patients (64.7%) had antibodies to HSV-2 but only 24% of these gave a history of genital herpes. A history of genital herpes or sexual contact with genital herpes, reported total lifetime number of sexual partners, failure to complete high school and a history of non-gonococcal urethritis or genital warts were associated with serological evidence of HSV-2 infection at the univariate level. Neither increasing age nor lack of circumcision was associated with HSV-2 infection. Following multivariate analysis only the lifetime number of partners and failure to finish high school were significantly strong predictors of HSV-2 infection. CONCLUSION: This is the highest prevalence of HSV-2 infection ever detected in an Australian population and one of the highest recorded globally. As younger men were as commonly infected as older men, and an earlier (1985) study involving the same clinic yielded a lower prevalence, it appears that a high level of ongoing HSV-2 transmission is occurring among Sydney heterosexuals. Increased awareness of this fact could enhance safer sex campaigns.

Localization of a functional site on herpes simplex virus type 1 glycoprotein C involved in binding to cell surface heparan sulphate.

The amino acid residues critical for interaction between herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) and cell surface heparan sulphate (HS) were localized to two separate regions within antigenic site II of this glycoprotein. These amino acids were Arg-143, Arg-145, Arg-147 and Thr-150 in one region and Gly-247 in the other. This conclusion is based on the following observations. (i) Monoclonal antibodies defining gC-1 antigenic site II, and not those reactive with antigenic site I, inhibited HSV-1-induced haemagglutination and virus binding to susceptible cells. (ii) A number of HSV-1 mar mutants, altered at these critical residues, were impaired in attachment to cells. (iii) Synthetic peptides, corresponding to these two regions inhibited virus attachment to cells and infectivity. In addition these peptides were found to agglutinate red blood cells. This agglutination was inhibited by soluble HS, and was prevented by the pretreatment of red blood cells with heparitinase suggesting that cell surface HS was a site of peptide binding. The same was observed with the polycationic substances neomycin and poly-L-lysine. In conclusion, we propose that the regions of gC-1 represented by the HS-binding peptides may form a functional site of a polycationic nature, active in attachment to the polyanionic glycosaminoglycan chain of cell surface HS.

Prevalence of antibodies to herpes simplex virus in pregnant women in Stockholm in 1969, 1983 and 1989: implications for STD epidemiology.

Prevalence of antibody to herpes simplex virus types 1 and 2 was assessed in consecutive serum samples from a total of 3700 women pregnant in 1969, 1983, or 1989 from the same catchment area in Stockholm. There was little change in seroprevalence of antibody to herpes simplex type 1 in the 3 groups, but age-adjusted herpes simplex virus type 2 antibody prevalence was 19, 33, and 33% respectively. Increase in type 2 seropositivity with age was slight and similar in 1969 and 1989, but steep in 1983, indicating a shift in sexual behaviour. However, rising prevalence in women will be mirrored by rising prevalence in their male partners. The increase from 1969 to 1989 will thus partly be due to higher risk of infection per partner, and cannot be taken as direct evidence of increased rate of partner change during this 20-year period.

The intrabronchial microbial flora in chronic bronchitis patients: a target for N-acetylcysteine therapy?

Chronic bronchitis is common among smokers, often together with recurrent infectious exacerbations. Streptococcus pneumoniae and Haemophilus influenzae are the pathogens traditionally considered most important. N-acetylcysteine (NAC) treatment has been shown to reduce the number of infectious exacerbations in patients with chronic bronchitis. The mechanism behind this is unknown. We attempted to characterize the intrabronchial bacterial flora in patients with chronic bronchitis in an infection-free interval, and to determine whether pharmacological and immunological factors effected the bacterial occurrence. Twenty two smokers with non-obstructive chronic bronchitis, 19 smokers with chronic bronchitis and chronic obstructive pulmonary disease (COPD) and 14 healthy nonsmokers underwent bronchoscopy. To obtain uncontaminated intrabronchial samples, a protected specimen brush was used. Quantitative bacterial cultures and virus isolations were performed. Significantly positive bacterial cultures (> 1,000 colony-forming units (cfu).ml-1) were found only in the patients. S. pneumoniae and H. influenzae were found in five patients, and only in the patients without NAC treatment. The most common bacterium was alpha-haemolytic streptococcus. Negative cultures were more common in the healthy controls. Of the various factors examined, only NAC medication had an influence on bacterial numbers. Significantly fewer patients with NAC medication had positive cultures (3 out of 16) than in the group of patients without NAC therapy (15 out of 21). Our results confirm that chronic bronchitis in smokers leads to increased intrabronchial bacterial colonization. We could also confirm that 1,000 cfu.ml-1 is an adequate cut-off level for significant bacterial growth when using the protected specimen brush. NAC medication was associated with low bacterial numbers.

Production of large amounts of recombinant interleukins by cDNA transfected mouse myeloma cells cultured in dialysis tubing.

Studies of interleukin function often require large quantities of these highly expensive substances. The available interleukins are generally recombinant proteins produced in bacteria or yeast and, less commonly, interleukins produced by mammalian cells, which provide appropriate glycosylation and other post-translational modifications. Due to differences in biosynthesis, difficulties in production and purification the quality of the interleukin preparations may vary. We have taken advantage of the recently developed constitutively interleukin-secreting mouse myeloma cell lines and the dialysis tubing culture technique, which permit cells to be grown at high densities, in order to establish a method for the production of large amounts of recombinant murine IL-2 and IL-4. We show that these interleukins can be produced at low cost and in concentrations 20-30-fold higher than in conventional culture flasks. A single dialysis tubing culture will produce more than 10(6) U of interleukin which may be compared with the available commercial preparations containing between 10- and a 100-fold less per vial. The IL-2 and IL-4 produced in this manner are biologically active molecules as demonstrated by the strong proliferative response of clonal T cells and the isotype-switching effect in LPS-stimulated splenic B cell cultures. The dialysis tubing culture technique is a simple and highly cost-effective means of generating large quantities of biologically active interleukins and is especially suitable for research laboratories interested in functional studies of these proteins.

Anti-idiotypic antibodies to poliovirus antibodies in commercial immunoglobulin preparations, human serum, and milk.

Our previous studies have suggested that fetal antibody production can be induced by maternal antiidiotypic antibodies transferred to the fetus via the placenta. We tested commercial Ig, sera, and milk for the presence of anti-idiotypic antibodies to poliovirus type 1, using affinity chromatography combined with ELISA systems and virus neutralization techniques. Our results indicate that commercial Ig, serum, and milk samples contain antibodies recognizing idiotypic determinants on antibodies to poliovirus. Several lines of evidence support this conclusion. Thus, in an ELISA with poliovirus as a solid phase, binding of specific antibodies could be inhibited by addition of an eluate from the Ig preparation containing anti-idiotypic antibodies against poliovirus type 1. Also, antiidiotypic antibodies from pooled human Ig, serum, and colostrum samples against poliovirus bound directly to solid-phase-attached MAb against poliovirus type 1. In addition, in a competitive inhibition ELISA, where antiidiotypic antibodies isolated from the Ig preparation competed with the poliovirus antigen for binding to monoclonal or polyclonal idiotypic antibodies on the solid phase, inhibition of antigen binding was seen at low antigen concentrations. When single-donor serum or milk was used, this inhibition was even more pronounced and could be demonstrated at almost all antigen concentrations. The finding that anti-idiotypes are present in maternal serum and milk imply, in agreement with our previous studies, that anti-idiotypes may actively induce a specific immune response in the fetus without previous exposure to the antigen by being transferred across the placenta or by being passively transferred to the newborn via mother's milk.