Prenatal fever and autism risk.

Some studies suggest that prenatal infection increases risk of autism spectrum disorders (ASDs). This study was undertaken in a prospective cohort in Norway to examine whether we could find evidence to support an association of the prenatal occurrence of fever, a common manifestation of infection, with ASD risk. Prospective questionnaires provided maternal exposure data; case status was established from clinical assessments and registry linkages. In a large, prospectively ascertained cohort of pregnant mothers and their offspring, we examined infants born ⩾32 weeks for associations between fever exposure in each trimester and ASD risk using logistic regression. Maternal exposure to second-trimester fever was associated with increased ASD risk, adjusting for presence of fever in other trimesters and confounders (adjusted odds ratio (aOR), 1.40; 95% confidence interval, 1.09-1.79), with a similar, but nonsignificant, point estimate in the first trimester. Risk increased markedly with exposure to three or more fever episodes after 12 weeks' gestation (aOR, 3.12; 1.28-7.63). ASD risk appears to increase with maternal fever, particularly in the second trimester. Risk magnified dose dependently with exposure to multiple fevers after 12 weeks' gestation. Our findings support a role for gestational maternal infection and innate immune responses to infection in the pathogenesis of at least some cases of ASD.

Susceptibility and immune responses following experimental infection of MHC compatible Atlantic salmon (Salmo salar L.) with different infectious salmon anaemia virus isolates.

Infectious salmon anaemia virus (ISAV) is an aquatic orthomyxovirus causing a multisystemic disease in farmed Atlantic salmon (Salmo salar) where disease development, clinical signs, and histopathology vary to a large extent. Here, an experimental trial was designed to determine the effect of variation in viral genes on virus-host interactions, as measured by disease susceptibility and immune responses. The fish were infected using cohabitant transmission, representing a natural route of infection. Variation caused by host factors was minimized using MHC compatible A. salmon half-siblings as experimental fish. Virus isolates were selected according to HE genotype, as European ISAV isolates can be genotyped according to deletion patterns in their hemagglutinin-esterase (HE) surface glycoprotein, and the course of disease they typically induce, classified as acute versus protracted. The different ISAV isolates induced large variations in death prevalence, ranging from 0-47% in the test-group and 3-75% in the cohabitant fish. The use of MHC compatible experimental fish made it possible to determine the relative contribution of humoral versus cellular response in protection against ISA. Ability to induce a strong proliferative response correlated with survival and virus clearance, while induction of a humoral response was less protective.

Expression, antigenicity and studies on cell receptor binding of the hemagglutinin of infectious salmon anemia virus.

Infectious salmon anemia (ISA) virus belongs to the proposed genus Isavirus of Orthomyxoviridae and is an emerging pathogen in Atlantic salmon (Salmo salar) farming. The hemagglutinin-esterase (HE) of ISA virus share several characteristics with the influenza virus hemagglutinin. This study reports recombinant expression of different ISA virus HE mutants in fish cell lines. Some introduced mutations, representing minimal differences in single amino acid residues, resulted in remarkable effects on expression efficiency in general and on surface expression specifically. Receptor binding assays showed that amino acid residues in the N-terminal half part are important in receptor binding, either being directly involved in the binding, or by influencing the structure of the binding site. Deletion of the putative N-glycosylation sites of the ISA virus HE, located near the transmembrane region, did not influence expression, receptor binding properties or staining by either a neutralising MAb, or salmon convalescent sera. The humoral immune response of Atlantic salmon after ISA virus infection showed weak neutralising activity and the results indicated that it was directed against HE.

Characterization of the infectious salmon anemia virus genomic segment that encodes the putative hemagglutinin.

The genomic segment encoding the putative hemagglutinin of infectious salmon anemia virus (ISAV) is described. Expression of the putative hemagglutinin in a salmon cell line demonstrated hemadsorptive properties of the protein for salmon erythrocytes. The polypeptide was recognized by an ISAV-specific monoclonal antibody. Nucleotide sequencing indicated the occurrence of a variable region in the hemagglutinin gene.

Detection of infectious salmon anaemia virus (ISAV) by RT-PCR after cohabitant exposure in Atlantic salmon Salmo salar.

A reverse transcription polymerase chain reaction (RT-PCR) was used to study the early phase of infectious salmon anaemia virus (ISAV) infection in Atlantic salmon Salmo salar L. The detection threshold for the RT-PCR was estimated to be 0.01 to 0.1 TCID50. A protocol that closely mimics the conditions in populations of farmed salmon was used. The major port of ISAV entry was most likely the gills, but oral entry could not be excluded. The gills and heart were RT-PCR positive 5 d post infection and there was a rapid viraemic spread of the virus after entry. Ten or more days post infection, most organs yielded RT-PCR positive samples. The viral load of the fish followed a 2-phase curve with the first maximum at approximately 15 d and a minimum around 25 d. After 25 d, there was a steady increase in viral load until all sampled organs eventually became positive. In an experiment in which the transportation of material from field to diagnostic laboratory was simulated, the transportation of whole fish was found to be optimal for the performance of RT-PCR.

The viral RNA 3'- and 5'-end structure and mRNA transcription of infectious salmon anaemia virus resemble those of influenza viruses.

The nucleotide sequences of the termini of two of the genomic segments of the negative strand RNA virus infectious salmon anaemia virus (ISAV) were determined. The sequence of the terminal 9 nucleotides at both ends of the viral RNAs was identical, and showed distinctive sequence homology with the conserved terminal sequences found in the orthomyxoviruses. For both ISAV genomic segments a computer-based secondary structure modelling indicated that the terminal 21-24 nucleotides were able to form self-complementary panhandle structures. Comparison with ISAV-derived mRNA sequences showed that ISAV mRNAs have heterogeneous 5'-ends, and are polyadenylated from a signal sequence 13-14 nucleotides downstream of the 5'-end terminus of the vRNA. Furthermore, the in vitro replication of ISAV was hindered by the RNA polymerase II inhibitor alpha-amanitin. These findings indicate that the mechanisms for replication of ISAV are similar to those of the orthomyxoviruses, and add to the previously reported structural similarities between ISAV and the orthomyxoviruses.

Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.).

Infectious salmon anemia (ISA) virus is the cause of infectious salmon anemia in farmed Atlantic salmon. The virus has been shown to contain RNA with structural characteristics similar to those of accepted members of the Orthomyxoviridae. Further biochemical, physiochemical, and morphological characterization of ISA virus was undertaken to clarify its taxonomic position. The virus was found to be sensitive to chloroform, heat, and low pH and agglutinated erythrocytes from fish. Erythrocytes from mammals or birds were not agglutinated. Receptor-destroying enzyme activity was detected, and the nature of this enzyme was suggested to be an acetylesterase. The buoyant density of the virus was 1.18 g/ml in sucrose and CsCl gradients. The maximum rate of virus replication was observed at 15 degrees C, while no virus was produced at 25 degrees C. Actinomycin D inhibited viral replication, and viral antigen was detected in nuclei by immunofluorescence. The addition of trypsin to the culture medium during virus replication had a beneficial effect on virus replication. ISA virus contains four major polypeptides with estimated molecular sizes of 71, 53, 43, and 24 kDa. Electron microscopy revealed structures closely resembling the nucleocapsids of influenza virus. Mushroom-shaped surface projections were a distinctive morphological feature, which differed from the rod-shaped hemagglutinin projections of the influenza viruses. The data reported here support the relationship of ISA virus to the Orthomyxoviridae, although ISA virus differs from influenza viruses in some morphological characteristics and in showing restricted hemagglutination, in different specificity of the receptor-destroying enzyme, in different polypeptide profile, in being unable to replicate at temperatures above 25 degrees C, and in host range.

Genomic characterization of the virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L.): an orthomyxo-like virus in a teleost.

The genome of infectious salmon anemia virus (ISAV), which infects farmed Atlantic salmon (Salmo salar L.), is characterized here. The virus has an RNA genome, as shown by using specific DNA virus metabolic inhibitors and radioactive in vivo labeling of ISAV nucleic acid. Electrophoresis of [14C]uridine-labeled ISAV RNA revealed that the ISAV genome is segmented. The genome consists of eight segments that range from 1.0 to 2.3 kb, with a total molecular size of approximately 14.5 kb. One ISAV-specific molecular clone, corresponding to the smallest genome segment, was obtained by cDNA cloning of mRNA from an ISAV-infected cell culture. This clone gave a positive hybridization signal on Northern blots of pelleted ISAV. Pretreatment of the ISAV pellet with RNase A resulted in the disappearance of the positive hybridization signal, demonstrating that the genome is single stranded. Reverse transcriptase PCR with primers corresponding to sequences from the molecular clone and target RNA from ISAV-infected and noninfected fish tissues gave specific positive reactions. Alignments of the nucleotide sequence of the molecular clone did not reveal significant homology with any other available sequence in databases. However, the data presented here, together with morphological and replicational properties previously described, indicate that ISAV has a strong resemblance to members of the Orthomyxoviridae family. This is the first thoroughly characterized orthomyxo-like virus isolated from a teleost.

Dendritic leucocytes pulsed with monoclonal antibody-hapten conjugates elicit vigorous primary humoral responses in vivo.

Purified dendritic leucocytes (DL) were pulsed briefly in vitro with haptenated monoclonal antibodies (MoAb) to MHC class II and immediately injected i.v. into syngeneic recipients. Strong anti-hapten humoral responses were observed even though only a few picomoles of specific MoAb-hapten conjugates were injected with the DL. In contrast, DL pulsed with control conjugates, i.e. haptenated non-binding MoAbs, gave only weak responses. DL thus, can take up, process and present protein antigens even after brief exposure in vitro, and their immunogenicity is enhanced by pulsing with antigen conjugated to specific MoAbs. Furthermore, in the presence of fetal calf serum (FCS), but not normal rat serum, the control MoAb W6/32 (against human MHC class I) bound to DL. The vigorous primary humoral response achieved following this pulsing indicates that it is the binding and the corresponding increased uptake that enhances the immunogenicity of the DL.

Comparing macrophages and dendritic leukocytes as antigen-presenting cells for humoral responses in vivo by antigen targeting.

Immunotargeting is a novel technique whereby antigen is directed against antigen-presenting cells (APC) by conjugation to specific monoclonal antibodies (mAb). In this study we have employed the technique to investigate the efficiency of macrophages as APC compared with constitutively major histocompatibility complex (MHC) class II-positive cells, i.e. dendritic leukocytes and B cells, in vivo. We first studied the organ retention of the radiolabeled conjugates by gamma counting, and their distribution within the draining lymph nodes by autoradiography. We could confirm that the conjugates reached the cells at which they were aimed. We then measured primary and secondary humoral responses. The results confirmed previous findings that targeting with mAb against MHC class II, i.e. to dendritic leukocytes, strongly enhanced the primary humoral response. In contrast, anti-IgD conjugates, directed against B cells gave only weak primary responses. Although conjugates directed against macrophages were retained for a longer time than the other conjugates, the primary humoral response was virtually abolished. The secondary responses, however, were at least as strong as those obtained in animals primed with control conjugates, whereas animals primed with anti-MHC class II conjugates showed little if any amplification of the secondary response. The discrepancies between the various conjugates could not be ascribed to TH1 versus TH2 responses as IgG1, IgG2a, IgG2b and IgE titers all co-varied in single animals. A possible explanation for the observed results is that macrophages fail to induce cytokine production for terminal differentiation of B cells to plasma cells, whereas conversely, upon presentation by dendritic leukocytes most stimulated B cells mature to plasma cells, leaving less progeny for immunological memory.

Targeting antigens to antigen presenting cells.

Antigen may be targeted to antigen presenting cells (APC) by conjugating the antigen to monoclonal antibodies directed against surface molecules on APC. By now several laboratories have shown that immunotargeting enhances humoral responses, depending upon the targeted ligand or cell type, with low doses of antigen and without the use of adjuvants. There is also preliminary evidence that the method may be used to bias immune responses in desired directions, possibly also to induce tolerance. In addition to its use as an experimental tool for exploring immune reactions the method could in the future also be clinically important, e.g. in vaccination. In this article we give a brief account on work so far published with this novel method and discuss possible mechanisms behind its immunopotentiating effects.

Antigen targetting with monoclonal antibodies as vectors--II. Further evidence that conjugation of antigen to specific monoclonal antibodies enhances uptake by antigen presenting cells.

Immunization of rats with haptenized monoclonal antibodies (mAbs) against accessory cells enhances anti-hapten antibody responses. To see whether the mAb-conjugates really targetted the antigen (hapten) to the antigen presenting cells, we have investigated the lymph node distribution of locally injected radiolabelled conjugates. Compared with control conjugates, i.e. haptenized non-binding mAbs, a much larger proportion of the specific conjugates were retained in the draining lymph nodes. Whereas control conjugates were rapidly phagocytosed and degraded by macrophages, the specific conjugates were associated with the targetted accessory cells, which were radiolabelled for extended periods. Haptenated MRC OX6 (anti-MHC class II) gave strong labelling of interdigitating cells (IDC) in the paracortex with 70% of IDC still labelled by 4 days and 15% by 16 days following injection. By Western blots intact OX6 conjugates were still detected in the draining lymph node as long as 3 days after injections, whereas control conjugates were hardly detectable even by 24 h. The findings substantiate the idea that mAbs can be exploited for vectorial transport of antigens to accessory cells.

Modulation of immune responses with monoclonal antibodies. I. Effects on regional lymph node morphology and on anti-hapten responses to haptenized monoclonal antibodies.

Repeated injections of monoclonal antibody (mAb) culture supernatants into rat footpads increased the weights of the draining lymph nodes. Immunostained freeze sections showed that injection of MRC OX2, a mAb reacting with rat follicular dendritic cells and MRC OX7 (anti-Thy-1.1), led to gross hypertrophy primarily of the follicular areas, whereas MRC OX6 (anti-rat major histocompatibility complex class II molecules) resulted in selective stimulation of the paracortex. These findings indicate that mAb, when conjugated to certain antigens, would modulate the immune response to these antigens. Consequently, the mAb were conjugated with fluorescein isothiocyanate (FITC) and the humoral response against the hapten measured. The primary anti-FITC antibody response was tenfold stronger than after stimulation with FITC conjugated to a conventional carrier such as ovalbumin, and had some characteristics of a secondary response: a fast increase of IgG level to very high titers and a long duration without further amplification at later antigen challenges.

The localization of antigen in lymph node follicles of congenitally athymic nude rats.

We have examined the postulated dependence on T cells of follicular retention of antigen by studying antigen retention in the draining lymph nodes of congenitally athymic, nude rats after local injections of horseradish peroxidase (HRP). The lymphoid tissues of these rats contained germinal centres and follicular dendritic cells (FDC) that were ultrastructurally identical to those seen in euthymic rats and expressed the differentiation antigen MRC OX2. Nude rat FDC captured and retained locally injected antigen on their surfaces, but as with euthymic rats, only in the presence of previously injected anti-HRP antibody. This demonstrates that the FDC mature both morphologically and functionally in the absence of a thymus or T cells. However, in contrast to euthymic rats, there was no detectable antigen retention in nude rats that had been actively immunized by repeated intraperitoneal injections with HRP for 3 months. The lower number of germinal centres observed in athymic animals compared with their euthymic littermates could thus be explained by deficient production of specific antibody of the isotype necessary for follicular localization of environmental antigens.