Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity.

BACKGROUND: Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80-90% of peanut allergic patients. METHODS: The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients. RESULTS: The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP--Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct. DISCUSSION: The reduced IgE binding to the MBP--Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP--Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy.

Cockroach allergens: function, structure and allergenicity.

Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.

Effect of coinfection with GB virus C on survival among patients with HIV infection.

BACKGROUND: Previous studies have suggested that people with human immunodeficiency virus (HIV) infection who are coinfected with GB virus C (GBV-C, or hepatitis G virus) have delayed progression of HIV disease. GBV-C is related to hepatitis C virus but does not appear to cause liver disease. METHODS: We examined the effect of coinfection with GBV-C on the survival of patients with HIV infection. We also evaluated cultures of peripheral-blood mononuclear cells infected with both viruses to determine whether GBV-C infection alters replication in vitro. RESULTS: Of 362 HIV-infected patients, 144 (39.8 percent) had GBV-C viremia in two tests. Forty-one of the patients with GBV-C viremia (28.5 percent) died during the follow-up period, as compared with 123 of the 218 patients who tested negative for GBV-C RNA (56.4 percent; P<0.001). The mean duration of follow-up for the entire cohort was 4.1 years. In a Cox regression analysis adjusted for HIV treatment, baseline CD4+ T-cell count, age, sex, race, and mode of transmission of HIV, the mortality rate among the 218 HIV-infected patients without GBV-C coinfection was significantly higher than that among the 144 patients with GBV-C coinfection (relative risk, 3.7; 95 percent confidence interval, 2.5 to 5.4). HIV replication, as measured by the detection of p24 antigen in culture supernatants, was reproducibly inhibited in cultures of peripheral-blood mononuclear cells by GBV-C coinfection. Coinfection did not alter the surface expression of HIV cellular receptors on peripheral-blood mononuclear cells, as determined by flow cytometry. CONCLUSIONS: GBV-C infection is common in people with HIV infection and is associated with significantly improved survival.

Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor.

Hepatitis C virus (HCV) or HCV-low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm(3)), intermediate-density particles (1. 12 to 1.18 g/cm(3)), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4 degrees C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of alpha-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of alpha-sarcin, indicating viral entry. The amount of viral entry also correlated with LDLr expression and was independent of the CD81 expression. Using a solid-phase immunoassay, recombinant E2 protein did not interact with LDL. Our data indicate that E2 binds CD81; however, virus particles utilize LDLr for binding and entry. The specific mechanism by which HCV particles interact with LDL or the LDLr remains unclear.

Full-length GB virus C (Hepatitis G virus) RNA transcripts are infectious in primary CD4-positive T cells.

GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro.

Fluorescence-based quantitative methods for detecting human immunodeficiency virus type 1-induced syncytia.

Cell fusion induced by human immunodeficiency virus type 1 (HIV-1) is usually assessed by counting multinucleated giant cells (syncytia) visualized by light microscopy. Currently used methods do not allow quantification of syncytia, nor do they estimate the number of cells involved in cell fusion. We describe two fluorescence-based methods for the detection and quantification of HIV-1-induced in vitro syncytium formation. The lymphoblastoid cell lines MT-2 and SupT1 were infected with syncytium-inducing (SI) HIV-1 isolates. Syncytia were detected by DNA staining with propidium iodide using flow cytometry to determine cell size or by two-color cytoplasmic staining of infected cell populations by using fluorescence microscopy. Both methods were able to detect and quantify HIV-induced syncytia. The methods could distinguish between SI and non-SI HIV isolates and could be used with at least two separate types of CD4(+) T-cell lines. Small syncytia can be readily identified by the two-color cytoplasmic staining method. Both methods were also shown to be useful for evaluating antiretroviral compounds, as demonstrated by the accurate assessment of HIV inhibition by azidothymidine (zidovudine), dideoxycytidine (zalcytibine), and hydroxyurea. These fluorescence-based assays allow a rapid and practical method for measuring HIV replication and anti-HIV activity of potential inhibitory compounds.

[Pathological-anatomical findings after serologic and molecular biologic evidence of Hantaan virus infection]. / Pathologisch-anatomische Befunde nach serologisch und molekularbiologisch nachgewiesener Hantaan-virus-Infektion.

Post-mortem findings in a girl aged 2 years and 4 months who had been infected with Hantaan virus are reported. The child fell ill with acute renal failure in her 8th month of life and had to be subjected to chronic dialysis. There was a progressive cardiac insufficiency. Specific IgG and IgM antibodies to Hantaan virus were demonstrated by serology from the 18th month of life. Autopsy revealed endomyocardial fibrosis and cardiomegaly. Hantaan RNA could be demonstrated in formalin-fixed, paraffin-embedded specimens of lung, liver and spleen tissue by means of RT-PCR followed by DNA sequencing. A possible association between the Hantaan virus infection and the pathological findings in the heart are discussed.