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.

A monoclonal antibody reacting specifically with ganglioside GD1b in human brain.

A mouse monoclonal antibody directed against one of the major human brain gangliosides, GD1b, has been produced. The antibody binds specifically to the carbohydrate structure of GD1b as it does not react with structurally related gangliosides like GM1, GD2, GT1b or Fuc-GM1, or any other ganglioside of human brain. The results further indicate that terminal galactose as well as the disialosyl group linked to the inner galactose moiety are involved in binding to the antibody.

Large-scale production of monoclonal antibodies in dialysis tubing.

A simple procedure for the large-scale production of monoclonal antibodies has been developed. Hybridomas were grown in dialysis tubing containing medium with a low concentration of proteins. The dialysis tubing was inserted into a flask with medium containing 10% or 30% foetal calf serum. The flask was placed on a roller and medium was changed every other day. Monoclonal antibodies were harvested after about 10 days in culture. Immunoglobulin concentrations up to 5.4 mg/ml and cell yields of 85 X 10(6) cells/ml have been obtained. The low concentration of contaminating low molecular weight proteins in the supernatant from cells grown in dialysis tubing facilitated purification of the monoclonal antibodies.

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.

Amplified ELISPOT assay for the detection of HIV-specific antibody-secreting cells in subhuman primates.

A novel immunoenzyme amplification technique has been evaluated in an ELISPOT assay for the detection of antigen-specific antibody-secreting cells (ASC) in monkeys. In this assay, mononuclear cells containing putative ASC are incubated for a few hours in antigen-coated wells. Following removal of the cells, zones of solid phase bound antibodies secreted by individual ASC are visualized in four consecutive steps. First, a primary biotinylated anti-immunoglobulin (Ig) reagent is added followed by enzyme-labelled avidin. The amplification procedure comprises the addition of biotinylated anti-enzyme antibodies in the third stage, followed by enzyme-conjugated avidin and substrate. When evaluated in a modified ELISPOT assay for the detection of simian B cells secreting antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1), this amplification procedure proved to be suitable even when using anti-human Ig antisera as primary antibody reagents. This development should be useful for other ELISPOT assays where species specific anti-Ig reagents are not always available and, most importantly, for enumerating cells producing immunoreactive substances in such minute amounts that they may escape detection by conventional ELISPOT assays. Furthermore, a functional simian HIV-specific ELISPOT assay could prove valuable for assessing the humoral immunogenicity of future candidate vaccines against the acquired immunodeficiency syndrome (AIDS).

Evaluation of solubilized herpes simplex virus membrane antigens in diffusion in gel-enzyme-linked immunosorbent assay (DIG-ELISA).

Cell membranes were prepared from herpes simplex virus (HSV) type 1-infected cells and solubilized with a low concentration of sodium deoxycholate. The supernatant after ultracentrifugation was used as antigen in a newly developed solid phase assay, diffusion in gel-enzyme-linked immunosorbent assay (DIG-ELISA). Antigen solubilization was almost complete, all HSV glycoproteins were represented, the yield of antigen in the solubilization process was high, and the presence of a detergent, sodium deoxycholate, did not interfere with the adsorption of antigen to the solid phase. DIG-ELISA was compared with the neutralization test for the determination of HSC antibodies and zone diameters showed a good correlation with the titre obtained by the neutralization test.

Separation of herpes simplex virus virions and nucleocapsids on Percoll gradients.

Herpes simplex virus (HSV) virions and nucleocapsids were separated and purified by centrifugation in density gradient of polyvinylpyrrolidone-coated colloidal silica (Percoll). Virions and nucleocapsids banded at densities of 1.07 and 1.03 g/ml, respectively. The distribution in the gradient of virions and nucleocapsids suggested that particles were discriminated according to difference in size rather than in density. The reduction of cell proteins in preparations of purified virions was 1300--2100 times. The recovery of infective virus was approximately 30%.

Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2).

The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.

Long-term persistence of intrathecal virus-specific antibody responses after herpes simplex virus encephalitis.

Paired sera and cerebrospinal fluids (CSF) from nine surviving patients were collected 4.5 to 8 years after acute herpes simplex (HS) virus encephalitis. Oligoclonal bands of IgG were detected in the CSF of all, and seven patients had an elevated CSF IgG index. Antibodies to HS, varicella-zoster (VZ), measles, and cytomegalo viruses were analysed by enzyme-linked immunosorbent assay (ELISA) and by imprint immunofixation (IIF) of specimens separated by electrophoresis and by thin-layer electrofocusing. Intrathecal synthesis of HS and VZ IgG antibodies was demonstrated in all and of measles IgG antibodies in one patient by both methods. Intrathecal synthesis of HS IgA antibodies was demonstrated by ELISA in three and by IIF in seven patients; the latter method also disclosed intrathecal synthesis of VZ IgA antibodies in two. No patient had intrathecal synthesis of viral IgM antibodies. The intrathecally synthesized antibodies demonstrated by IIF displayed oligoclonal characteristics. The IIF analyses as well as virus absorption tests indicated that the intrathecally synthesized VZ IgG and IgA antibodies could be explained as HS antibodies cross-reacting with VZV. The results indicate that a long-term persistence of intrathecal antibody responses to HS virus is a common feature after acute HS encephalitis. The intrathecal production of measles IgG antibodies in one case may reflect a similar persistence of non-specific immune responses induced during the acute infection.

Prevalence of antibodies to herpes simplex virus types 1 and 2 in pregnant women, and estimated rates of infection.

There has been a recent increase in notifications of genital herpes but it is not known whether this has been reflected in the pregnant population. We have therefore carried out a study to determine the prevalence of herpes simplex antibodies in pregnant women and to estimate the incidence of primary infection. Sera were collected from 3533 women at antenatal clinics and tested for total antibodies to herpes simples virus (HSV), and if positive, for specific antibodies to HSV-2. Estimates of HSV-1 seroprevalence were derived from the HSV-2 seronegative population. HSV-1 seroprevalence was nearly 100% in black women born in Africa or the Caribbean and 60-80% in white, Asian and UK born black women. It was lower in women in non-manual employment. HSV-2 seroprevalence was related to age, rising from 0 at age 16 to 40% at age 35 in black women, and to about 10% in Asian and white women. The estimated incidence of primary HSV-2 infection during pregnancy, per 1000 pregnancies, was about 2.4 in Asian women, 5 in white women, and 20 in black women. Estimates of the incidence of neonatal infection were derived from these figures and compared to the nationally reported rates.

A 15-year surveillance study of antibodies to herpes simplex virus types 1 and 2 in a cohort of young girls.

A cohort of 839 young girls at the ages of 14 and 15 years was screened for total antibodies to herpes simplex virus (HSV) and, if positive, for specific antibodies to HSV-2, by means of a sensitive, enzyme-linked immunosorbent assay (ELISA). The cohort was followed from 1972-1987. Blood samples were obtained on six occasions during these 16 years. In total, 2270 blood samples were taken. The number of sero-converting girls was studied in relation to calendar time. Two methods were constructed for the statistical analyses. The first of these gave an estimate of the sero-prevalence at different points in time. This analysis showed that the sero-prevalence which was 23% against HSV-1 in 1972 had increased to 36% in 1976. At the end of the study in 1987, 50% of the cohort had sero-converted against HSV-1. The proportion of girls who had sero-converted against HSV-2 was 0.4% in the 14-15-year-olds and had reached 22% by the end of the study. The second statistical method used all the available information implicit in the observations so as to obtain a maximum-likelihood (ML) estimate of the prevalence. The ML estimates were slightly more precise, but the two estimates did not differ significantly. The observations were further analysed by the Mantel-Haenszel test in order to see if there was any dependence between positivity to HSV-1 and HSV-2 respectively but none was found.

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.

Production of human monoclonal antibodies in dialysis tubing.

Human x mouse hybridoma cells were grown in dialysis tubing (DT) to obtain large quantities of human monoclonal antibodies (MAb). Hybridoma cells were grown inside the DT, which was placed in a tissue culture flask containing medium. The medium inside the DT was supplemented with different additives which may be selected depending on the intended use of the MAb. About 10-50 times higher concentrations of immunoglobulin (Ig) were obtained after cultivation in DT compared to conventional tissue culture (CTC) for 2 days. The purity of the MAb was high which facilitated further antibody purification. Production of human MAb in DT proved to be excellent for evaluation studies in laboratory scale. It does not require expensive equipment and several hybridomas can be grown simultaneously.

Differentiation between herpes simplex virus type 1 and type 2 strains by immunoelectroosmophoresis.

A method has been elaborated to differentiate between herpes simplex type 1 and type 2 viruses by immunoelectroosmophoresis. With rabbit immune sera cross-absorbed with heterologous virus antigen, a distinct difference was shown between the two virus types. Herpes simplex type 1 virus tested against cross-absorbed type 1 antiserum gave two precipitin lines. Herpes simplex type 2 virus gave one precipitin line when tested against cross-absorbed homologous serum. When the viral antigens were tested against cross-absorbed heterologous immune sera, no or only very weak precipitin reactions were observed. The test is easy and rapid, requires relatively small quantities of antigen and antibody, and is suitable for typing of herpes simplex virus in diagnostic routine work.