Insulin-stimulated translocation of GLUT4 to the plasma membrane in rat hippocampus is PI3-kinase dependent.

In the central nervous system (CNS) insulin mediates a variety of effects including feeding, metabolism and cognition. The cognitive enhancing effects of insulin are proposed to be mediated through activation of insulin receptors in the hippocampus, an important integration center for learning and memory in the mammalian brain. Since less is known regarding insulin signaling events in the hippocampus, the aim of the current study was to determine whether insulin stimulates similar signaling cascades and GLUT4 translocation in the rat hippocampus as has been described in peripheral tissues. Intracerebroventricular administration of insulin increases hippocampal insulin levels and also stimulates the phosphorylation of Akt in a time-dependent manner. Insulin also stimulates the translocation of GLUT4 to hippocampal plasma membranes in a time course that mirrors the increases in glucose uptake observed during the performance of hippocampal-dependent tasks. Insulin stimulated phosphorylation of Akt and translocation of GLUT4 were blocked by pretreatment with the PI3-kinase inhibitor LY294002. Confocal immunofluorescence determined that insulin stimulated phosphorylation of Akt was localized to neurons and colocalized with the insulin receptor and GLUT4 in the rat hippocampus, thereby identifying the functional anatomical substrates of insulin signaling in the hippocampus. These results demonstrate that insulin-stimulated translocation of GLUT4 to the plasma membrane in the rat hippocampus occurs via similar mechanisms as described in peripheral tissues and suggests that insulin-mediated translocation of GLUT4 may provide a mechanism through which hippocampal neurons rapidly increase glucose utilization during increases in neuronal activity associated with hippocampal-dependent learning.

Serotyping HIV-1 with V3 peptides: detection of high avidity antibodies presenting clade-specific reactivity

The main objective of the present study was to assess the specificity and sensitivity of a modified assay using short synthetic peptides of the V3 region of HIV-1 gp120, which is the main target for neutralizing antibodies. Results from an enzyme immunoassay (EIA) employing a panel of synthetic peptides of HIV-1 subtypes and using urea washes to detect high avidity antibodies (AAV3) were compared with those obtained by the heteroduplex mobility assay and DNA sequencing. The EIA correctly typed 100 percent of subtype B (sensitivity = 1.0; specificity = 0.95), 100 percent of HIV-1 E samples (sensitivity = 1.0; specificity = 1.0), and 95 percent of subtype C specimens (sensitivity = 0.95; specificity = 0.94). In contrast, only 50 percent of subtype A (sensitivity = 0.5; specificity = 0.95), 60 percent of subtype D (sensitivity = 0.6; specificity = 1.0), and 28 percent of subtype F samples (sensitivity = 0.28; specificity = 0.95) were correctly identified. This approach was also able to discriminate in a few samples antibodies from patients infected with B variants circulating in Brazil and Thailand that reacted specifically. The assays described in this study are relatively rapid and simple to perform compared to molecular approaches and can be used to screen large numbers of serum or plasma samples. Moreover, the classification in subtypes (genotypes) may overestimate HIV-1 diversity and a classification into serotypes, based on antigenic V3 diversity or another principal neutralization domain, may be more helpful for vaccine development and identification of variants

Adenovirus type 3 viremia in an adult with toxic shock-like syndrome.

Surveillance by the Unexplained Deaths and Critical Illnesses Project (UNEX) uncovered a novel presentation of adenovirus type 3 infection that satisfied the criteria for toxic shock-like syndrome in a 28-year-old immunocompetent man. Adenovirus may be a cause of toxic shock syndrome; surveillance systems such as UNEX may uncover additional causes of this and other clinically defined infectious syndromes.

Identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein G.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A-->T198) and B (183K-->K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence 184A/183K-->198T/K197 for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.

Neutralization of primary HIV-1 isolates by anti-envelope monoclonal antibodies.

OBJECTIVE: To evaluate human monoclonal antibodies (MAb) for neutralizing activity against primary HIV-1 isolates in peripheral blood mononuclear cells. DESIGN: Neutralization activity data were obtained from 11 laboratories on a coded panel consisting of six human MAb to HIV envelope V3, CD4-binding region or gp41. Hyperimmune globulin against HIV-1 and normal human immunoglobulin G were supplied as controls. Each laboratory received pre-titered virus for use in the studies. METHODS: Each laboratory measured neutralization of the MAb against laboratory strain HIVMN, genomic clone HIVJR-CSF, two subtype B and one subtype D primary isolates. RESULTS: The titers of the centrally supplied virus stocks as determined by re-titration or back-titration varied among laboratories and were generally 10-100-fold less than provided. The neutralizing activity of each MAb varied by as much as a 1000-fold among laboratories. These differences may result from varying sensitivity in neutralization assay protocols and the differing susceptibility of primary cells to infection with HIV-1. CONCLUSIONS: To consolidate the data from multiple laboratories, the neutralization titers were compared by classifying antibodies as neutralizing if the antibody concentration for 50% virus inhibition was < or = 10 micrograms/ml. By this criterion, the CD4-binding region and gp41 MAb neutralized all four subtype B viruses and the subtype D isolate in a few of the laboratories. The V3 MAb neutralized only HIVMN and the closely related HIVJR-CSF viruses.

Evaluation of monoclonal antibodies to HIV-1 envelope by neutralization and binding assays: an international collaboration.

OBJECTIVE: To characterize a purified panel of monoclonal antibodies (MAb) to epitopes in HIV-1 envelope V3, CD4-binding region, C4 and gp41. DESIGN: Neutralization and/or binding activity data were obtained from 21 laboratories on a coded panel consisting of seven human MAb, seven mouse MAb, recombinant human CD4 immunoadhesin [CD4-immunoglobulin G (IgG)], normal human and normal murine Ig. METHODS: Laboratories performed a variety of neutralization assays and antigen binding assays with HIVIIIB, HIVMN and other laboratory strains of HIV-1. RESULTS: For a single MAb, there was up to a 10(3) range of neutralizing antibody titers between laboratories. The range in titers appeared to depend on the sensitivity of the neutralization assay. Two methods were used to consolidate the data from all laboratories, the geometric mean titer (GMT) and the median neutralizing titer (MNT). The panel of MAb were also analyzed by a variety of assays that measure binding activity to native or denatured epitopes. The relative binding activity of the MAb did not appear to correlate with neutralizing activity. CONCLUSION: Neutralization results from any single laboratory did not correlate with the collective data. The relative potency (rank order) of the MAb in the panel were equivalent when determined by GMT or MNT. These values may be useful to individual laboratories for estimating the sensitivity of their neutralization assays. The study also identified potential reference reagents with which neutralizing activity could be compared.

High prevalence of antibodies to the gp120 V3 region principal neutralizing determinant of HIV-1MN in sera from Africa and the Americas.

Neutralizing antibodies (NA) against HIV-1MN and HIV-1IIIB, and antibodies binding to synthetic peptides (BA) derived from the gp120 envelope V3 region principal neutralizing determinants (PND) of the HIV-1MN, HIV-1IIIB, and HIV-1Z3 virus strains were assayed in HIV-1 antibody-positive sera from the United States, Haiti, Brazil, Zaire, and Zimbabwe. The ability of soluble PND peptide to block neutralization of the corresponding virus by representative sera was also tested. In each country, NA and BA titers were highest against the HIV-1MN strain, and compared with other countries, NA and BA titers against HIV-1MN were higher in sera from the United States and Haiti. When NA titers were compared with BA titers against either HIV-1MN or HIV-1IIIB, no correlation was found for the HIV-1IIIB strain, but there was a significant correlation for HIV-1MN. Addition of the HIV-1MN strain peptide to a neutralization assay for HIV-1MN resulted in a four- to tenfold reduction in NA titers in sera from the United States, Zaire, and Brazil. The results suggest that HIV-1MN and closely related variants are prevalent in many parts of the world, and that antibodies directed against the PND account for most of the neutralizing activity in sera of infected individuals.

PIP: Virologists assessed the extent of neutralizing antibody cross-reactivity to multiple virus strains in sera from 112 HIV-1 infected individuals from the US, Brazil, Haiti, Zaire, and Zimbabwe. They also looked at the association between virus neutralization and the level of antibody binding to synthetic peptides representing the HIV-1 gp120 V3 region principal neutralizing determinant (PND) sequences. The 3 strains observed included HIV-1 MN, HIV-1 Z3, and HIV-1 IIIB. Neutralizing antibodies (NA) and antibodies binding to synthetic peptides (BA) titers ranked highest against the PND sequence HIV-1 MN in all countries (p.01). These titers were higher in sera from the US and Haiti than sera from Brazil and Africa (p.05). A significant correlation existed between the NA and BA titers for HIV-1 MN (p.01), but not for HIV-1 IIIB. When the virologists added HIV-1 MN strain peptide to a neutralization assay for HIV-1 MN, NA titers in sera from the US, Zaire, and Brazil fell 4-10 fold. These findings intimated that HIV-1 MN and closely related variants are commonplace in several locations around the world, and that antibodies directed against HIV--1 gp120 V3 region PND sequences make up most of the neutralizing activity in sera of infected individuals. In conclusion, virologists need to conduct more studies that examine the true extent of strain variation worldwide. These studies could lay the groundwork for the development of an effective HIV-1 vaccine.

Multicenter study of strains of respiratory syncytial virus.

Two major groups of respiratory syncytial virus (RSV) strains, A and B, have been identified and their patterns of isolation determined in different communities but not simultaneously in multiple communities. In this study, we tested 483 RSV isolates from 14 university laboratories in the United States and Canada for the 1984/1985 and 1985/1986 RSV seasons; 303 (63%) isolates were group A, 114 (24%) were group B, and 66 (14%) could not be grouped. Isolates were subdivided into six subgroups within group A and three within group B; up to six and often four or more different subgroups were isolated in the same laboratory during the same RSV season. The pattern of group and subgroup isolations varied among laboratories during the same year and between years for the same laboratory. These differences suggest that RSV outbreaks are community, possibly regional, but not national phenomena. The ability to identify group and subgroup differences in isolates is a powerful tool for epidemiologic studies of RSV.

Possible beneficial effects of neutralizing antibodies and antibody-dependent, cell-mediated cytotoxicity in human immunodeficiency virus infection.

We studied the relationship between early human immunodeficiency virus type 1 (HIV-1) specific immune responses and pathogenesis of infection in participants enrolled in the multicenter AIDS cohort study (MACS). Sera collected at 6-month intervals for 2 years (visit 1-5) from 39 persons who seroconverted by enzyme-linked immunosorbent assay (ELISA) 6 months (visit 2) after enrollment were examined for isotype-specific Western blot reactivity, neutralizing antibodies (NA) against two divergent strains of HIV-1 (HIV-1IIIB and HIV-1RF), and for antibodies capable of participating in antibody-dependent, cell-mediated cytotoxicity (ADCC). These results were compared with changes in CD4+ cell number and episodes of lymphadenopathy. Twenty-five subjects had antibodies of at least one isotype reactive to at least one HIV-1 protein by Western blot at visit 1, before they became ELISA positive. NA reactive with HIV-1IIIB were detected before those reactive with HIV-1RF. NA were first observed in 11 sera at visit 2, in 22 sera at visit 3, and in 3 sera at visit 4; sera from three patients remained nonneutralizing through visit 5. In most cases, NA were detected after a decline in CD4+ cell numbers. The data are consistent with the interpretation that NA develop after about 16 to 18 months of declining CD4+ cell numbers, following which the rate of decline in CD4+ cell numbers slows. In contrast, HIV-1 envelope antigen-specific ADCC responses were first observed in 11 subjects at visit 1 when all 39 were NA and ELISA negative, in 12 subjects at visit 2, in 13 subjects at visit 3, and 1 subject at visit 4. Early ADCC responses were associated with high mean % CD4+ cell numbers and absence of lymphadenopathy throughout the 2-year observation period. Not all subjects who developed ADCC developed NA. In some subjects, ADCC and NA were detectable for the first time at the same visit, for others ADCC was detectable prior to NA, and for a few NA was detectable prior to ADCC. These findings suggest that ADCC and neutralization are mediated by different antibody populations, that they may partially inhibit the progress of HIV-1 infection, and that the late appearance of NA may relate to the failure of immunity to effect recovery from this infection.

Clinical and laboratory characteristics of HIV-1 infection in Zimbabwe.

To define the impact of human immunodeficiency virus (HIV) infection in Africa, clinical and laboratory investigations were conducted on 265 HIV-seropositive outpatients in Zimbabwe. Twenty-four of the study subjects were asymptomatic (ASX), 124 had persistent generalized lymphadenopathy (PGL), and 117 had AIDS-related complex (ARC). HIV infection was assessed by commercial ELISA, Western blots, synthetic peptide ELISA, and measurement of p24 antigen. Serum immunoglobulins, lymphocyte mitogen responses, and CD4+ cell numbers were obtained in 54 sequential patients. Compared to seronegative subjects, mean CD4+ cell numbers were decreased and serum immunoglobulins, particularly IgM and IgG, were increased in all groups of seropositive subjects. Lymphocyte proliferative responses to phytohemagglutinin and concanavalin A decreased progressively in ASX, PGL, and ARC patients and were significantly lower in PGL and ARC patients compared to seronegative controls. Generalized lymphadenopathy was present in 234/265 (88%) of patients. Lymph node biopsies in 100 patients demonstrated follicular hyperplasia in 97 and Mycobacterium tuberculosis in 3. Of 165 patients followed for a median of 6 months, 5 developed the acquired immune deficiency syndrome (AIDS). Symptoms of ARC, low CD4+ cell number, and p24 antigen were predictive of the development of AIDS in Zimbabwe.

Characterization of the serologic profile of children with human immunodeficiency virus infection: correlation with clinical status.

Serum samples from 28 children with symptomatic human immunodeficiency virus (HIV) infection were studied for the presence of HIV antigen. Their humoral immune response profile, including anti-HIV specific isotypic responses and neutralizing titers, was characterized. Additionally, serum specimens from 12 of these children were tested for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) against HIV envelope antigens. Analysis of our results showed that children with acquired immunodeficiency syndrome (AIDS) were much more likely to have serum antigenemia and an absence of anti-p24 antibodies than those with AIDS-related complex (ARC). A significant association was also noted between a more stable clinical status and a strong anti-p24 antibody response with detectable antibodies to other HIV antigens in multiple antibody subclasses. This suggests that the longitudinal evaluation of antigen/antibody profiles may aid in the assessment of prognosis for children with HIV infection. Sera from 6/6 patients with ARC and 4/6 patients with AIDS were able to mediate ADCC. No correlation was found between clinical status and the titers of neutralizing antibodies.

Quantification of respiratory syncytial virus polypeptides in nasal secretions by monoclonal antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibody as solid-phase immunosorbent was developed to measure specific polypeptides of respiratory syncytial virus (RSV). The assay was used to examine 43 nasopharyngeal (NP) aspirates from RSV-positive infants that had been examined previously for RSV by culture, direct immunofluorescence, and polyclonal antibody ELISA. Frozen NP aspirates were serially diluted and examined for the 66K mol. wt. fusion glycoprotein (F), the 84K large surface glycoprotein (G) and the 41K nucleoprotein (N) by monoclonal capture ELISA. F protein was detected in all 43 specimens, G protein was detectable in 20 (47%) and N protein in 22 (51%) of 43 NP aspirates. In specimens with detectable G and N proteins, F was detected by endpoint titration at approximately tenfold greater dilutions than either G or N. In 19 sequential NP aspirates from five patients with RSV infection, F was present in higher titre throughout infection. In 20 cases, matching cell culture isolates were examined by immunofluorescence with strain-specific monoclonal antibodies. Three of 20 isolates showed strain-specific differences by their lack of reaction with anti-G monoclonal antibody. Titration of the 20 cell culture isolates by monoclonal antibody capture ELISA showed the relative amount of F and N proteins to be equal in all cases, whereas levels of G protein tended to be slightly lower. Reconstruction experiments with NP aspirates demonstrated that degradation of F and N proteins did not occur in NP aspirates, but that G protein antigenicity appeared to be affected by nasal secretions. When compared with cell culture-grown material, nasal secretions contained abundant F protein but a surprisingly low concentration of N protein.

Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies.

To study the antigenic characteristics of respiratory syncytial virus (RSV), we developed and evaluated monoclonal antibodies (MAbs) to three strains of RSV: 11 to Long, 4 to 18537, and 9 to A2. Six of these MAbs immunoprecipitated the nucleoprotein, six the large glycoprotein, and 11 the fusion protein. By the pattern of the reactions of these MAbs to 16 strains of RSV in an indirect immunofluorescence assay or enzyme-linked immunosorbent assay, we were able to distinguish three subgroups. With a panel of 10 of these 24 MAbs, we tested 26 strains isolated between 1979 and 1982 in Boston and found that 22 belonged to group 1, 4 to group 2, and none to group 3. The pattern of the reactions of the MAbs against representative strains from the three groups identified nine epitopes by indirect immunofluorescence assay: three of each on the nucleoprotein, the large glycoprotein, and the fusion protein. These results, along with those of previous studies, suggest that groups 1 and 3 are antigenically similar and group 2 is antigenically more distinct.

Comparison of enzyme immunoassay with radioimmunoassay for the detection of hepatitis B surface antigen.

A direct solid-phase enzyme immunoassay (Auszyme I) and a direct solid-phase radioimmunoassay (Austria II) for detection of hepatitis B surface antigen (HBsAg) were compared in tests with a panel of 347 human sera. Compared with RIA, EIA showed a sensitivity of 98% with 153 HBsAg-positive sera and a specificity of 99% with 194 HBsAg-negative sera. Sera that gave false negative and false positive results by EIA were re-examined by both RIA and EIA to confirm the initial result. Use of less than the recommended volume of serum for EIA produced results inconsistent with RIA in four of 27 sera examined. Quantitative correlation between RIA and EIA was low (r = 0.691). Positive controls used for EIA showed considerable variation from day to day, although intra-assay variation was much less. The sensitivity of the EIA method examined compares favourably with previously published EIA studies and with the RIA used in this study. Auszyme I EIA is a sensitive and specific third generation test for HBsAg that offers several advantages over currently used RIA techniques.

Detection and identification of influenza virus antigens by nylon-coupled enzyme-linked immunoassay.

A direct solid-phase enzyme-linked immunoassay for rapid detection and typing of influenza virus was developed utilizing antibodies immobilized by covalent linkage to nylon beads. Covalent linkage of antibody to nylon was accomplished by treatment of partially hydrolyzed nylon with glutaraldehyde. For comparison to conventional enzyme-linked immunosorbent assays (ELISA), IgG fractions were adsorbed to polystyrene beads. Influenza type-specific immunoglobulins coupled to nylon beads were used in an enzyme-linked immunoassay to identify influenza A/USSR/77(H1N1), and A/Texas/75 (H3N2). In titrations of viral antigen, antibody coupled to nylon beads detected 1.9 X 10(4) plaque-forming units (PFU) per assay, whereas 2.2 X 10(5) PFU were required in assays utilizing antibody adsorbed to polystyrene beads. Use of fluorogenic or radioactive substrates for alkaline phosphatase-labeled antibodies increased the sensitivity for virus detection 10-fold with this enzyme, but were only slightly more sensitive than chromogenic substrates with peroxidase-labeled antibody.

Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: development and description.

An indirect enzyme-linked immunosorbent assay for detection of respiratory syncytial virus (RSV) antigens was developed, using commercially available antisera. Horse anti-RSV and calf antiserum to bovine RSV were used as capture and detector antibodies, respectively. The assay could detect as few as 50 PFU of unpurified RSV per ml in infected cell culture supernatant fluids and as little as 10 ng of affinity-purified RSV antigen per ml. No cross-reactions were observed with heterologous virus types. Freeze-thaw treatment had no effect on RSV enzyme-linked immunosorbent assay titers, but viral transport medium inhibited RSV enzyme-linked immunosorbent assay titers from 10- to 100-fold. The assay can be easily performed in 24 h and is a sensitive and specific method for the detection of RSV antigens.