Production of TNF-alpha and TNF-beta by staphylococcal enterotoxin A activated human T cells

Staphylococcal enterotoxin at concentrations of less than 1 pg/ml induces significant TNF activity in human peripheral blood T cells and monocytes. Maximal TNF activity is routinely detected after 48 to 72 h of culture. IL-2 and IL-4 were both growth promoting for human T cells but only IL-2 could efficiently induce TNF production. The production of TNF-alpha and TNF-beta differed greatly in kinetics. An early intracytoplasmatic production of TNF-alpha after 6 h was detected in both monocytes and T cells whereas a late production of TNF-beta (lymphotoxin) after 48 h, occurred in the T cell population. Induction of TNF-alpha and TNF-beta production by Staphylococcal enterotoxin requires the presence of both monocytes and T cells. The CD4+45R- but not CD4+45R+ and CD8+ cells supported TNF-alpha production in monocytes. The main lytic component from Staphylococcal enterotoxin-activated mononuclear cells is TNF-beta. CD4+ and CD8+ T cells produced about equal amounts of biologically active TNF into the culture supernatants but a fourfold higher frequency of TNF-beta producing cells was demonstrated among CD4+ vs CD8+ cells. The CD4+45R- T cell subset was an efficient producer of TNF-beta and IFN-gamma whereas the CD4+45R+ T cell subset produced significant amounts of TNF-beta but only marginal amounts of IFN-gamma.     

Lectin binding to cells of Schistosoma mansoni sporocysts and surrounding Biomphalaria glabrata tissue

The binding of different lectins to the surface of mother and daughter sporocysts of Schistosoma mansoni (Trematoda) and to cells of its intermediate host Biomphalaria glabrata (Gastropoda) was investigated. The test system consisted of several biotin-labeled lectins, an avidin-biotin-peroxidase complex and 3,3'-diaminobenzidine. The fixatives used were Formalin, Bouin's and Zenker's solutions; unfixed material was also studied. Most lectins reacted equally with host tissue and parasite tissue. However, receptors for Ulex europaeus I (most probably fucose) were only demonstrated on daughter sporocysts. Thus, a method was found to specifically mark Schistosoma mansoni daughter sporocysts in the digestive gland tissue of its intermediate host. Mother sporocysts and surrounding host tissue differed in their distribution of galactosyl groups. Both lack fucose and N-acetyl-galactosamine. The differences in lectin binding of galactosyl determinants were also observed during the in vitro development of mother to daughter sporocysts.     

Identification of metabolites from peroxisomal beta-oxidation of prostaglandins

We have recently shown that isolated rat liver peroxisomes can chain-shorten prostaglandin F2 alpha and prostaglandin E2 to tetranor-metabolites. In the present report dinor-metabolites of these two prostaglandins were also identified, suggesting that the peroxisomal chain-shortening reaction of prostaglandins is a beta-oxidation reaction. Furthermore, an intermediate containing an extra double bond was isolated from incubates of prostaglandin F2 alpha with peroxisomes. This intermediate was tentatively assigned the structure 2,3-dehydroprostaglandin F2 alpha. Prostaglandin E1 and a major circulating prostaglandin F2 alpha metabolite were also metabolized to chain-shortened products by peroxisomes. The accumulation of the 2,3-dehydro-metabolite and the dinor-metabolites suggest that the peroxisomal beta-oxidation sequence is not tightly coupled, in contrast to mitochondrial fatty acid oxidation.     

[3H]1-Methyl-4-Phenyl-2,3-Dihydropyridinium Ion Binding Sites in Mouse Brain: Pharmacological and Biological Characterization

Because 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPP+) appears to damage the dopaminergic neuron and cause neuronal death, we characterized [3H]MPP+ binding sites in mouse brain membranes. Among several compounds tested, debrisoquin [3,4-dihydro-2(1H)-isoquinolinecarboxamidine] and some analogues were able to antagonize [3H]MPP+ binding. Debrisoquin is able to block adrenergic transmission and inhibit the activity of monoamine oxidase A (MAO-A). We found a certain correlation between the ability of these agents to displace [3H]MPP+ from its binding sites and their capacity to inhibit MAO-A activity. These data and the finding of a higher number of [3H]MPP+ binding sites in human placenta compared to mouse brain suggest that these sites may correspond to MAO-A enzymes. Recently it has been demonstrated in human brain that neurons in regions rich in catecholamines are positive for MAO-A. Accordingly, we suggest MAO-A as a possible accumulation site of MPP+ within the dopaminergic neuron. We also indicate the chemical structural requirement associated with the best binding of debrisoquin analogues with [3H]MPP+ sites. It would be reasonable to test the effects of debrisoquin-like drugs able to pass the blood-brain barrier on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity.     

Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production.

We have investigated the roles of cytokines in the modulation of human immunodeficiency virus (HIV) production in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3. HIV-infected U937 cells exhibited markedly lower levels of CD4 and HLA-DR antigens than uninfected cells did. Vitamin D3 induced a time-dependent macrophagelike differentiation, as determined by monitoring the expression of some surface antigens by means of the monoclonal antibodies OKM1, OKM5, OKM13, OKM14, OKT4, anti-HLA-DR, TecMG2, TecMG3, LeuM3, LeuM1, anti-HLA-DP, and anti-HLA-DQ. Treatment with hydroxyvitamin D3 resulted in a marked increase in HIV production compared with control cultures. Interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were detected in the culture media, whereas interferon (IFN) was not generally found. Using the polymerase chain reaction technique, we found HIV-infected U937 cells to express detectable levels of mRNAs for alpha interferon (IFN-alpha), IFN-beta, TNF-alpha, and IL-1 beta. The addition of TNF resulted in a marked increase of HIV production, whereas IL-1 beta was ineffective. In contrast, both IFN-alpha and IFN-beta exerted some inhibitory effect on HIV production, which was more marked in vitamin D3-treated cultures than in untreated cultures. HIV production was significantly increased by antibodies to IFN-alpha in both untreated and vitamin D3-treated cultures. Anti-IFN-beta antibody increased HIV production only in vitamin D3-treated cells. In contrast, anti-TNF-alpha antibodies markedly decreased HIV production in both control and differentiating U937 cells. Vitamin D3 treatment resulted in a higher expression of TNF receptors in differentiating cells than in control HIV-infected cells. These data demonstrate a strong correlation between HIV production and macrophagelike differentiation in chronically infected U937 cells and suggest that endogenous IFN and TNF exert opposite effects in the regulation of virus production in both undifferentiated and vitamin D3-treated cell cultures.     

Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23.

A set of B-cell activation molecules, including the Epstein-Barr virus (EBV) receptor CR2 (CD21) and the B-cell activation antigen CD23 (Blast2/Fc epsilon RII), is turned on by infecting EBV-negative B-lymphoma cell lines with immortalizing strains of the viruslike B95-8 (BL/B95 cells). This up regulation may represent one of the mechanisms involved in EBV-mediated B-cell immortalization. The P3HR1 nonimmortalizing strain of the virus, which is deleted for the entire Epstein-Barr nuclear antigen 2 (EBNA2) protein open reading frame, is incapable of inducing the expression of CR2 and CD23, suggesting a crucial role for EBNA2 in the activation of these molecules. In addition, lymphoma cells containing the P3HR1 genome (BL/P3HR1 cells) do not express the viral latent membrane protein (LMP), which is regularly expressed in cells infected with immortalizing viral strains. Using electroporation, we have transfected the EBNA2 gene cloned in an episomal vector into BL/P3HR1 cells and have obtained cell clones that stably express the EBNA2 protein. In these clones, EBNA2 expression was associated with an increased amount of CR2 and CD23 steady-state RNAs. Of the three species of CD23 mRNAs described, the Fc epsilon RIIa species was preferentially expressed in these EBNA2-expressing clones. An increased cell surface expression of CR2 but not of CD23 was observed, and the soluble form of CD23 molecule (SCD23) was released. We were, however, not able to detect any expression of LMP in these cell clones. These data demonstrate that EBNA2 gene is able to complement P3HR1 virus latent functions to induce the activation of CR2 and CD23 expression, and they emphasize the role of EBNA2 protein in the modulation of cellular gene implicated in B-cell proliferation and hence in EBV-mediated B-cell immortalization. Nevertheless, EBNA2 expression in BL/P3HR1 cells is not able to restore the level of CR2 and CD23 expression observed in BL/B95 cells, suggesting that other cellular or viral proteins may also have an important role in the activation of these molecules: the viral LMP seems to be a good candidate.     

Identification of individual human immunodeficiency virus type 1 gp120 amino acids important for CD4 receptor binding.

The binding of the CD4 receptor by the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein is important for virus entry and cytopathic effect. To investigate the CD4-binding region of the gp120 glycoprotein, we altered gp120 amino acids, excluding cysteines, that are conserved among the primate immunodeficiency viruses utilizing the CD4 receptor. Changes in two hydrophobic regions (Thr-257 in conserved region 2 and Trp-427 in conserved region 4) and two hydrophilic regions (Asp-368 and Glu-370 in conserved region 3 and Asp-457 in conserved region 4) resulted in significant reductions in CD4 binding. For most of the mutations affecting these residues, the observed effects on CD4 binding did not apparently result from global conformational disruption of the gp120 molecule, as assessed by measurements of precursor processing, subunit association, and monoclonal antibody recognition. The two hydrophilic regions exhibit a strong propensity for beta-turn formation, are predicted to act as efficient B-cell epitopes, and are located adjacent to hypervariable, glycosylated regions. This study defines a small number of gp120 residues important for CD4 binding, some of which might constitute attractive targets for immunologic intervention.     

Processing of pseudorabies virus glycoprotein gII.

The glycoprotein complex gII of pseudorabies virus was isolated by immunoprecipitation with the monoclonal antibody M5, which was covalently linked to protein A-Sepharose. After sodium dodecyl sulfate-polyarylamide gel electrophoresis under reducing conditions and blotting onto poly(vinylidene difluoride) membrane, its subunits, gIIa, gIIb, and gIIc, were subjected to N-terminal sequencing. gIIa and gIIb start at position 59 and gIIc starts at position 503 according to the amino acid sequence deduced from the gene, indicating that there is one major protein (gIIa) which is cleaved into the two protein fragments gIIb and gIIc. Protein labeling with 14C-amino acids gave no indication that the three proteins (gIIa, gIIb, and gIIc) of the complex are present in equimolar ratios. It seems that gIIa is only a minor component of the complex, whereas gIIb and gIIc are contained in equimolar amounts.