Hepatitis B virus Dane particles bind to human plasma apolipoprotein H.

Human apolipoprotein H (apo H) was found to bind specifically to hepatitis B surface antigen (HBsAg) from hepatitis B virus (HBV)-infected individuals. We used recombinant HBsAg proteins to analyze HBV domains recognized by apo H. We showed that the myristylated pre-S1 domain of HBsAg strongly interacted with apo H. This binding involved phospholipid components of the HBV envelope because their removal by detergent prevented apo H-HBsAg interaction. The opposite effects of iron and zinc metal ions on binding suggest that the oxidation of phospholipids also affects apo H-HBsAg interaction. After fractionation of viral particles on a sucrose gradient, and their addition to microtiter plates coated with apo H or anti-HBsAg, we observed that the maximal anti-HBsAg capture activity corresponded to a sucrose concentration of 36%, whereas the maximal apo H capture activity corresponded to a concentration of 39%. Electron microscopy and polymerase chain reaction (PCR) Southern blot studies of these fractions showed that the fraction with maximal apo H binding predominantly contained full Dane particles. Finally, we studied apo H-HBsAg binding relative to the presence of hepatitis B virus markers and observed that apo H binding activity for HBsAg was higher in sera from patients in the active virus replication phase.

HEED, the product of the human homolog of the murine eed gene, binds to the matrix protein of HIV-1.

heed, the human homolog of mouse eed and Drosophila esc, two members of the trithorax (trx) and Polycomb group (Pc-G) of genes, was isolated by screening an activated lymphocyte cDNA library versus the immunodeficiency virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system in yeast. The human EED protein (HEED) had 99. 5% identity with the mouse EED protein and contained seven WD repeats. Two heed gene transcripts were identified, with a putative 407-nucleotide-long intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively. The shorter HEED isoform, originated from the unspliced message, lacked the seventh WD repeat. HEED was found to bind to MA protein in vitro, as efficiently as in vivo in yeast cells. Site-directed mutagenesis and phage biopanning suggested that the interaction between HEED and MA involved the N-terminal region of the MA protein, including the first polybasic signal, in a MA conformation-dependent manner. In the HEED protein, however, two discrete linear MA-binding motifs were identified within residues 388-403, overlapping the origin of the fifth WD repeat. Deletion of the C-terminal 41 residues of HEED, spanning the seventh WD repeat, as in the 494-residue HEED protein, was detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a conformational role of the HEED C-terminal domain in the MA-HEED interaction. MA and HEED proteins co-localized within the nucleus of co-transfected human cells and of recombinant baculovirus co-infected insect cells. This and the failure of HEED to bind to uncleaved GAG precursor suggested a role of HEED at the early stages of virus infection, rather than late in the virus life cycle.

Structural and functional determinants in adenovirus type 2 penton base recombinant protein.

Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle.

Inhibition of T cell apoptosis in the rheumatoid synovium.

Synovial T cells in rheumatoid arthritis are highly differentiated and express a phenotype suggesting susceptibility to apoptosis (CD45RB dull, CD45RO bright, Bcl-2 low, Bax high, Fas high). However, no evidence of T cell apoptosis was found in synovial fluid from any of 28 patients studied. In contrast, synovial fluid from 10 patients with crystal arthritis showed substantial levels of T cell apoptosis. The failre of apoptosis was not an intrinsic property of rheumatoid synovial T cells, as they showed rapid spontaneous apoptosis on removal from the joint. Synovial T cells from rheumatoid arthritis and gout patients could be rescued from spontaneous apoptosis in vitro either by IL-2R gamma chain signaling cytokines (which upregulate Bcl-2 and Bcl-XL) or by interaction with synovial fibroblasts (which upregulates Bcl-xL but not Bcl-2). The phenotype of rheumatoid synovial T cells ex vivo (Bcl-2 low, Bcl-xL high) suggested a fibroblast-mediated mechanism in vivo. This was confirmed by in vitro culture of synovial T cells with fibroblasts which maintained the Bcl-xL high Bcl-2 low phenotype. Synovial T cells from gout patients were Bcl-2 low Bcl-xL low and showed clear evidence of apoptosis in vivo. Inhibition experiments suggested that an integrin-ligand interaction incorporating the Arg-Gly-Asp motif is involved in fibroblast-mediated synovial T cell survival. We propose that environmental blockade of cell death resulting from interaction with stromal cells is a major factor in the persistent T cell infiltration of chronically inflamed rheumatoid synovium.

T cell receptor distribution in rheumatoid synovial follicles.

OBJECTIVE: In rheumatoid arthritis (RA) joint erosion is accompanied by T cell infiltration of the synovial tissue. The resulting T cell receptor (TCR) repertoire is a combination of antigen driven shaping, nonspecific selection by endothelial cell-T cell interactions, and cytokine mediated chemoattraction. Considering the conflicting results of molecular biology studies of the TCR repertoire in RA, we attempted to obtain new data to clarify the situation through microscopic distribution analysis of TCR beta chain diversity in synovium follicular CD4/CD8 subsets. METHODS: We used dual color fluorescence confocal microscopy with CD4/CD8 monoclonal antibodies (Mab) and a panel of new anti-V beta Mab. The analysis focussed on lymphocyte rich, follicle-like areas of synovial tissue from 4 patients with RA. RESULTS: The expression of individual TCR beta families varied between areas within the T cell follicles, and between patients. Normal absolute levels of some beta chains can be completely skewed towards one subset, indicating that overall TCR evaluation is insufficient. CONCLUSION: Confocal microscopy analysis of localized TCR diversity in RA synovium offers novel insight into overall V beta gene usage, which appears to be the accumulation of several localized microexpansions.

Jacalin, a lectin that inhibits in vitro HIV-1 infection, induces intracellular calcium increase via CD4 in cells lacking the CD3/TcR complex.

The lectin jacalin interacts with the CD4 cell surface antigen; this lectin inhibits in vitro infection by human immunodeficiency virus type 1 without preventing virus binding on the host cell. The infection process is known to involve cellular events triggered by the binding of the viral external glycoprotein gp120 to CD4. Herein we demonstrate that jacalin induces cell signaling directly through the CD4 antigen and that independently of the CD3/TcR complex. The capacity of jacalin to trigger cell signals through the CD4 molecule is discussed in relation to its ability to inhibit HIV infection.

Human mucosyl lymphocyte marker expression in synovial fluid lymphocytes of patient with rheumatoid arthritis.

OBJECTIVE: Human mucosyl lymphocyte marker (HML-1) antigen is an activation antigen and adhesion molecule of the beta 7 integrin family, which is generally restricted to T cells found in the intestinal epithelium. Expression of the membrane antigen as defined by the monoclonal antibody HML-1 was studied on peripheral blood (PB) lymphocytes and synovial fluid (SF) lymphocytes in 10 patients with rheumatoid arthritis (RA) and in a control group of patients with osteoarthritis (OA). METHODS: Double fluorescence activated flow cytometry was used to assess HML-1 expression with T cell subtype antigens (CD3, CD4, CD8) or activation markers interleukin 2 receptor (IL-2R) (CD25), HLA-DR, and lymphocyte function associated antigen (LFA-1) (CD11a) were assessed by flow cytometry. RESULTS: HML-1 antigen expression in PB lymphocytes of patients with RA (7.3%) was found to be comparable to the control group (6.4%). In contrast, 25.4% (range 14-43%) of SF lymphocytes expressed HML-1 antigen, compared to 13.6% of SF lymphocytes in patients with OA (p < 0.001). In RA, 62% of HML-1 positive cells from SF lymphocytes were the CD8 subtype, compared to 10.6% of PB lymphocytes (p < 0.003), and 18% of control SF lymphocytes (p < 0.05). Furthermore, HML-1 antigen and HLA-DR antigen were coexpressed in 75% of RA SF lymphocytes compared to 29.6% of control SF lymphocytes (p < 0.01). In contrast, coexpression of LFA-1 and the Il-2R did not differ from that of control. CONCLUSION: We describe overexpression of the adhesion molecule HML-1 in SF lymphocytes of patients with RA, preferentially in the CD8 subset. These results suggest a similarity between the expression of activation antigens in SF lymphocytes of patients with RA and T lymphocytes present in the intestinal epithelium.

CD57+ T lymphocytes are derived from CD57- precursors by differentiation occurring in late immune responses.

CD3+ T cells expressing the 110-kDa CD57 antigen are found in survivors of renal, cardiac and bone marrow transplants, in patients with acquired immune deficiency syndrome and in patients with rheumatoid arthritis. They are also present in normal individuals and expand upon ageing. They do not grow in culture and their role in the immune response is poorly understood. The expression of the various isoforms of the leukocyte common antigen (CD45) identifies a spectrum of differentiation in CD4+ and CD8+ T cells ranging from naive (CD45RA+CD45RBbrightCD45RO-) through early primed cells (CD45RA-RBbrightROdull) to highly differentiated memory cells which are CD45RA-RBdullRObright. CD45 isoforms expressed by CD57+ T cells showed distinct differences between CD4+ and CD8+ populations, but in each case indicated an advanced state of differentiation. The expression of T cell receptor V beta families was highly variable between individuals, but both CD57+ and CD57- cells show a full range of the specificities tested. V beta expression was more closely related within either the CD4+ or the CD8+ subsets, irrespective of CD57 expression, than between these subsets, suggesting a relationship between CD57+ and CD57- cells within the same T cell pool. This possibility was supported by experiments showing that CD3+CD57+ lymphocytes were similar to CD3+CD57- T cells in terms of the production of basic T cell cytokines [interleukin (IL)-2, IL-4, and interferon-gamma]. Furthermore, in vitro stimulation of CD3+CD57- T cells in secondary mixed leukocyte reaction or by co-culture with IL-2 and IL-4 induced the appearance of CD3+CD57+ cells with phenotypic and functional similarities to in vivo CD3+CD57+ cells. These data strongly suggest that the expression of CD57 is a differentiation event which occurs on CD57- T cells late in the immune response.

Rheumatoid pericarditis: new immunopathological aspects.

Rheumatoid pericarditis (RP) is a well known extraarticular manifestation of rheumatoid arthritis (RA). It is not frequently diagnosed despite its high reported prevalence in post-mortem studies. There have been no immunohistological studies of its presence in pericardial membranes. Here we report a complete immunohistological study of two RA patients with RP complications, using a panel of monoclonal antibodies (mAbs) for the recognition of B, T, and NK cells. Both cases showed strong and almost exclusive pericardial membrane infiltration of CD8+ T-cells which was correlated with a higher than expected similar increase in the subset of peripheral blood lymphocytes (PBL). These findings suggest an important role for CD8+ T-cells in chronic RA, especially in this extraarticular manifestation of the disease.

T cell receptor gamma variable gene locus polymorphism in patients with rheumatoid arthritis.

OBJECTIVE: We sought new susceptibility markers for rheumatoid arthritis (RA) among the T cell receptor gamma (TCR gamma) genes. METHODS: We analyzed restriction fragment length polymorphisms (RFLP) of the first variable subgroup of TCR gamma genes in a group of French control subjects and a group of French RA patients. RESULTS: No significant difference in Eco RI RFLP was found between the 2 study populations: Allele frequencies were virtually identical. There was no polymorphism using Hind III. CONCLUSION: These results exclude TCRV gamma I polymorphism as a disease susceptibility marker in RA.

Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin-1 and phorbol ester activation pathways in the EL4-6.1 thymoma cell line.

EL 4-6.1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL-1) or phorbol 12-myristate-13-acetate (PMA), or PMA+IL-1 to secrete interleukin 2 (IL-2) and interleukin 4 (IL-4) and to express the IL-2 receptor (IL-2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL-2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20- to 30-fold) the production of IL-2 or IL-4, when EL 4-6.1 cells were induced by IL-1 alpha (or IL-1 beta) alone. By contrast, at identical concentrations, STAR dose-dependently inhibited the production of IL-2 and IL-4 resulting from PMA or PMA+IL-1 cell treatment. STAR also negatively affected the expression of IL-2R, which was dependent on PMA-sensitive PKC with either IL-1, PMA, or PMA+IL-1 stimulation. The changes in interleukin production and IL-2R expression in EL 4-6.1 activated cells were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretranslational effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL-1-triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL-1 receptor (IL-1R) expression in EL 4-6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL-1 and PMA stimulatory pathways. In addition, they suggest that the IL-1 stimulatory pathway involves PKC(s) [or other undefined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA.

Transforming growth factor type beta 1 (TGF-beta 1) down-regulates interleukin-2 production and up-regulates interleukin-2 receptor expression in a thymoma cell line.

Transforming growth factor type beta 1 (TGB-beta 1) belongs to a family of polypeptides with regulatory effects on growth and differentiation of a variety of cell types. TGB-beta 1 plays an important role in regulation of immune response by acting as a negative control signal for T cell proliferation through still unknown mechanisms. In this study we have analysed the effects of TGB-beta 1 on EL 4-6.1, a variant of the murine EL 4 thymoma, which can be induced by phorbol 12-myristate 13-acetate (PMA) and/or interleukin 1 (IL-1) to secrete interleukin 2 (IL-2) and express IL-2 receptors (IL-2R). Using this defined model system, we show that TGB-beta 1 simultaneously down-regulates IL-2 expression and up-regulates the number of both high and low affinity IL-2R. These changes correlate with changes at the mRNA level, suggesting an effect at the pre-translational level. The specificity of both TGF-beta 1 effects was demonstrated using a neutralizing antiserum to TGF-beta 1. Our data also suggest that TGF-beta 1 does not interfere with early activation signals of PMA and/or IL-1. This model might be useful for elucidating the complex role of TGF-beta 1 in the regulation of T cell responses.

Interleukin 2 (IL 2) inhibitor in rheumatoid synovial fluid: correlation with prognosis and soluble IL 2 receptor levels.

A soluble activity inhibiting over 50% of the CTLL-2 cell line response to recombinant human interleukin 2 (IL 2) was found in 17 of 29 (59%) rheumatoid synovial fluids. To study the prognosis value of this activity, 16 rheumatoid synovial fluids were collected before a radiation synovectomy of the knee with 7 mCi of 90Yt. Patients with a good clinical result after the synovectomy had a lower IL 2 inhibitory activity than those with a bad or incomplete result (P less than 0.01). Levels of inhibitory activity and of soluble IL 2 receptors were correlated with each other and with the response of the synovitis to the radiation synovectomy. These results extend the clinical usefulness of soluble IL 2 receptor measurements and indicate a correlation between the immune activation of the rheumatoid synovitis and its clinical activity.

Hemoglobin chains during foetal development in mice.

Hemoglobin components present in C3H mice during foetal development were investigated. Two major embryonic hemoglobins were found, one composed of alpha and non-alpha globin chains and the other of non-alpha chains only. No foetal hemoglobin different from the adult ane embryonic hemoglobins was detected. The preparation of adult C3H alpha chain is a mixture of two alpha chains: one comprising 70 per cent of the whole amount is identical with the alpha-chain of the C57B1 strain of mice while the other 30 per cent is identical with the alpha chain of NB strain. The synthesis of these two chains is not activated at the same time during foetal development. No C57B1 alpha chain is synthesized by foetal erythrocytes on the 13th day of gestation whereas on the 18th day the synthesis of C57B1 chain is the same in the foetus and in the adult C3H mouse.