Arg89Cys substitution results in very low membrane expression of the Duffy antigen/receptor for chemokines in Fy(x) individuals

The Duffy (FY) blood group antigens are carried by the DARC glycoprotein, a widely expressed chemokine receptor. The molecular basis of the Fya/Fyb and Fy(a-b-) polymorphisms has been clarified, but little is known about the Fyx antigen and the FY*X allele associated with weak expression of Fyb, Fy3, Fy5, and Fy6 antigens. We analyzed here the structure and expression of the FY gene in 4 Fy(a-bweak) individuals. As compared with Fy(a-b+) controls, the Fy(a-bweak) red blood cell membranes contained residual amount of DARC polypeptide and these cells were poorly bound by anti-Fy antibodies and chemokines. The FY gene from Fy(a-b+) and Fy(a-bweak) individuals differed by one substitution, C286T. The resulting Arg89Cys amino acid change reduced the binding of anti-Fy antibodies and chemokines to DARC transfectants. We concluded that the Fybweak donors carried the FY*X allele at the FY locus and that the Fyx antigen corresponds to highly reduced expression of a grossly normal Fyb polypeptide caused by the Arg89Cys substitution. Because FY is a single copy gene, this defect should also affect DARC expression in nonerythroid cells. Because the Fyx phenotype is not associated with apparent clinical consequences, we discussed these findings in the light of the putative roles of DARC in various tissues. Finally, we developed a Fyx DNA typing assay that should be useful for genetic studies and clinical transfusion medicine.     

HIV-1 Tat protein mimicry of chemokines

The HIV-1 Tat protein is a potent chemoattractant for monocytes. We observed that Tat shows conserved amino acids corresponding to critical sequences of the chemokines, a family of molecules known for their potent ability to attract monocytes. Synthetic Tat and a peptide (CysL24-51) encompassing the "chemokine-like" region of Tat induced a rapid and transient Ca2+ influx in monocytes and macrophages, analogous to beta-chemokines. Both monocyte migration and Ca2+ mobilization were pertussis toxin sensitive and cholera toxin insensitive. Cross-desensitization studies indicated that Tat shares receptors with MCP-1, MCP-3, and eotaxin. Tat was able to displace binding of beta-chemokines from the beta-chemokine receptors CCR2 and CCR3, but not CCR1, CCR4, and CCR5. Direct receptor binding experiments with the CysL24-51 peptide confirmed binding to cells transfected with CCR2 and CCR3. HIV-1 Tat appears to mimic beta-chemokine features, which may serve to locally recruit chemokine receptor-expressing monocytes/macrophages toward HIV producing cells and facilitate activation and infection.     

Close association of the first and fourth extracellular domains of the Duffy antigen/receptor for chemokines by a disulfide bond is required for ligand binding

It has been demonstrated that the promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines (DARC) is given by its extracellular NH2-terminal region. However, the relationship among the Fy6, Fya/b, and Fy3 epitopes, localized in the first and fourth extracellular domains of DARC, respectively, and the chemokine binding sites remained a matter of controversy. Here, we performed cross-displacement and cross-inhibition experiments indicating that all anti-Fy6, anti-Fya, and anti-Fy3 monoclonal antibodies and interleukin 8 are antagonists for binding to red cells. Biopanning of phage peptide libraries with an anti-Fy6 monoclonal antibody led to the identification of the motif Phe22-Glu23, the mutation of which altered the binding of both anti-Fy6 and chemokines (interleukin 8, MGSA, RANTES (regulated on activation normal T cell expressed)) to DARC transfectants. These results characterized the core of the Fy6 epitope and provided definitive proof of the tight relationship between Fy6 and the chemokine receptor site. Analysis of red cells treated by sulfhydryl group-modifying reagents suggested that the chemokine receptor function of DARC required the integrity of disulfide bond(s) but not that of free sulfhydryl group(s). Accordingly, mutation of cysteines 51 and 276 abolished chemokine binding to DARC transfectants. Altogether, our results suggested that the chemokine binding pocket of DARC included sequences located in the first and fourth extracellular domains which are brought into close vicinity by a disulfide bridge.     

Detection of human immunodeficiency virus type 1 (HIV-1) RNA in pools of sera negative for antibodies to HIV-1 and HIV-2

A total of 234 pools were prepared from 10,692 consecutive serum samples negative for antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 collected at five virological laboratories (average pool size, 45 serum samples). Pools were screened for the presence of HIV-1 RNA by a modified commercial assay (Amplicor HIV-1 Monitor test) which included an additional polyethylene glycol (PEG) precipitation step prior to purification of viral RNA (PEG Amplicor assay). The sensitivity of this assay for HIV-1 RNA detection in individual serum samples within pools matches that of standard commercial assays for individual serum samples, i.e., 500 HIV-1 RNA copies per ml. Five pools were identified as positive, and each one contained one antibody-negative, HIV-1 RNA-positive serum sample, corresponding to an average of 1 infected sample per 2,138 serum samples. Retrospective analysis revealed that the five HIV-1 RNA-positive specimens originated from individuals who had symptomatic primary HIV-1 infection at the time of sample collection and who were also positive for p24 antigenemia. We next assessed the possibility of performing the prepurification step by high-speed centrifugation (50,000 x g for 80 min) of 1.5-ml pools containing 25 microl of 60 individual serum samples, of which only 1 contained HIV-1 RNA (centrifugation Amplicor assay). The sensitivity of this assay also matches the sensitivities of standard commercial assays for HIV-1 RNA detection in individual serum samples. The results demonstrate that both assays with pooled sera can be applied to the screening of large numbers of serum samples in a time- and cost-efficient manner.     

Peptide antigen or superantigen-induced down-regulation of TCRs involves both stimulated and unstimulated receptors

T cell activation by peptide/MHC complexes, superantigens, or mAbs induces the down-regulation of cell surface TCRs. We addressed the question of whether TCR down-modulation affects only TCRs that had directly interacted with their ligand or whether down-modulation could also affect TCRs that had not interacted with their ligand. To this end, we generated T cells coexpressing equal levels of two different TCRs by transfecting the appropriate cDNAs into cells of the human T cell line, Jurkat. Each set of TCRs can be distinguished by means of anti-Vbeta mAbs and can be stimulated separately with peptide Ag, bacterial superantigens, or mAbs. We found that activation of these cells with each of these stimuli down-modulated not only directly stimulated TCR complexes but also unstimulated ones. Comodulation of stimulated and unstimulated receptors may reflect functional interactions between surface TCRs that could take place during Ag or superantigen recognition by T cells without the need for ligand cross-linking. Consistent with this idea, both stimulated and unstimulated receptors colocalized in patches on the cell surface after activation.     

The modulation by neurosteroids of the scopolamine-induced learning impairment in mice involves an interaction with sigma1 (sigma1) receptors

Pulmonary tuberculosis: primary tuberculosis, usually asymptomatic, represents the first infection and is shown by a parenchymal mostly mid-pulmonary focus and satellite lymphadenopathy. Postprimary pulmonary tuberculosis, mostly located in the upper fields may be caused by endogenous reinfection for reactivation of a hematogenous focus formed during primary infection or from exogenous reinfection. Extrapulmonary tuberculosis: it includes numerous forms mostly from hematogenous spread. Miliary tuberculosis may involve a number of organs and apparatus besides the lung. Tuberculous meningitis predominantly involves the base of the skull, the fluid is clear with hypoglycorrhachia and lymphocyte pleocytosis. Lymph node tuberculosis is generally unilateral and cervical. Tuberculous pleuritis is exudative or dry. Other forms of tuberculous serositis are pericarditis and peritonitis. Renal tuberculosis involves the medullaris and intestinal tuberculosis the ileocecum; tuberculous spondilitis (Pott's disease) involves the last dorsal vertebrae. Other forms are osteoarthritis, genital tract tuberculosis, pancreatitis, laryngitis, otitis.     

Detection of antibody IgG to HIV-1 in urine by ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 as antigen for diagnosis of HIV-1 infection

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 gag protein (p24) of HIV-1 as antigen and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate and recombinant p24-beta-D-galactosidase conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. This assay was at least 3,000-fold more sensitive than conventional methods. The lowest signal among 49 asymptomatic carriers was 3.1-fold higher than the highest nonspecific signal among 100 seronegative subjects. The sensitivity and specificity were both 100%. The positivity could be confirmed by preincubation of urine samples with excess of the antigen. Thus, this assay would be a powerful tool for detecting IgG antibody to HIV-1 in urine.     

Immune complex transfer enzyme immunoassay for antibody IgM to HIV-1 p17 antigen

The surgical management of valvular heart disease nowadays includes the use of various valvular substitutes and sophisticated reparative surgical techniques. Characteristics and indications of these different surgical options are discussed and the objective criteria that justify the use of a given technique in a given clinical situation are provided.     

Sensitive enzyme immunoassay of antibodies to HIV-1 p17 antigen using indirectly immobilized recombinant p17 for diagnosis of HIV-1 infection

Recombinant p17 (rp17) antigen of HIV-1 and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized onto polystyrene beads in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption; biotinyl-rp17 and biotinyl-MBP-rp17 were immobilized indirectly onto streptavidin-coated polystyrene beads; and 2,4-dinitrophenyl (DNP)-MBP-rp17 was immobilized indirectly onto (anti-DNP) IgG-coated polystyrene beads. These directly and indirectly immobilized antigens were incubated with urine samples containing antibody IgG to p17 antigen and subsequently with rp17-beta-D-galactosidase conjugate or (anti-human IgG gamma-chain) Fab'-beta-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene beads was assayed by fluorometry. When rp17-beta-D-galactosidase conjugate was used, signals (fluorescence intensities for bound beta-D-galactosidase activity) were much higher with the indirectly immobilized antigens than those with the directly immobilized antigens. By experiments using (anti-human IgG gamma-chain)Fab'-beta-D-galactosidase conjugate, the binding of rp17-beta-D-galactosidase conjugate to antibodies against p17 antigen bound to directly immobilized rp17 antigen was shown to be seriously limited as compared with that to antibodies against p17 antigen bound to indirectly immobilized DNP-MBP-rp17. When rp17-beta-D-galactosidase conjugate and serum samples were used, serum interference was much less with indirectly immobilized DNP-MBP-rp17 than with directly immobilized rp17 antigen, and the sensitivity of enzyme immunoassay for antibody IgG to p17 antigen using indirectly immobilized DNP-MBP-rp17 was 1,000- to 3,000-fold higher than that of enzyme immunoassay using directly immobilized rp17 antigen and Western blotting for p17 band. This sensitive enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as conventional methods for antibodies to HIV-1 and earlier than Western blotting for p17 band.     

Characterization of the MN gp120 HIV-1 vaccine: antigen binding to alum

PURPOSE: The characterization of recombinant MN gp120/alum vaccine requires the study of the gp120-alum interaction for the successful formulation of an alum-based HIV-1 vaccine. METHODS: Several observations suggest that the gp120-alum interaction is weak, wherein buffer counterions such as phosphate, sulfate, bicarbonate may cause the desorption of gp120 from alum. Comparison of gp120 with other proteins using particle mobility measurements shows that the weak binding of gp120 to alum is not an anomaly. Serum and plasma also cause desorption of gp120 from alum with a half-life of only a few minutes, wherein this half-life may be faster than the in-vivo recruitment of antigen presenting cells to the site of immunization. RESULTS: Immunization of guinea pigs, rabbits and baboons with gp120 formulated in alum or saline demonstrated that alum provides adjuvant activity for gp120, particularly after early immunizations, but the adjuvant effect is attenuated after several boosts. CONCLUSIONS: These observations indicate that both the antigen and the adjuvant require optimization together.     

Surface expression of CD63 antigen (AD1 antigen) in P815 mastocytoma cells by transfected IgE receptors

We have examined epidermal growth factor (EGF) signalling in a CHO cell line (CHO11) which expresses a human EGF receptor truncated at amino acid 990. Previous studies showed that EGF treatment of these cells failed to increase prostaglandin production or phospholipase A2 activity. In the current study EGF increased the tyrosine phosphorylation of the intracellular signalling protein Shc in CHO11 cells but did not activate either of the downstream signalling enzymes raf or mitogen activated protein kinase (MAPK). The uncoupling of Shc activation from distal signalling in CHO11 cells contrasts with other cells which express similar mutant EGF receptors. The failure of She to activate distal signalling may reflect qualitative differences in the way that this protein is activated or could result from the activation of an inhibitory signalling pathway.     

HIV-1 envelope gp120 inhibits the monocyte response to chemokines through CD4 signal-dependent chemokine receptor down-regulation

Since HIV-1 infection results in severe immunosuppression, and the envelope protein gp120 has been reported to interact with some of the chemokine receptors on human T lymphocytes, we postulated that gp120 may also affect monocyte activation by a variety of chemokines. This study shows that human peripheral blood monocytes when preincubated with gp120 either purified from laboratory-adapted strains or as recombinant proteins exhibited markedly reduced binding, calcium mobilization, and chemotactic response to chemokines. The gp-120-pretreated monocytes also showed a decreased response to FMLP. This broad inhibition of monocyte activation by chemoattractants required interaction of gp120 with CD4, since the effect of gp120 was only observed in CD4+ monocytes and in HEK 293 cells only if cotransfected with both chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Anti-CD4 mAbs mimicked the effect of gp120, and both anti-CD4 Ab and gp120 caused internalization of CXCR4 in HEK 293 cells provided they also expressed CD4. Staurosporine blocked the inhibitory effect of gp120 on monocytes, suggesting that cellular signaling was required for gp120 to inhibit the response of CD4+ cells to chemoattractants. Our study demonstrates a broad suppressive effect of gp120 on monocyte activation by chemoattractants through the down-regulation of cell surface receptors. Thus, gp120 may be used by HIV-1 to disarm the monocyte response to inflammatory stimulation.     

Binding of gamma-aminobutyric acidA receptors to tubulin

Tubulin cofractionated with gamma-aminobutyric acidA (GABAA) receptors upon affinity chromatography on Affigel 15 coupled to the benzodiazepine Ro 7-1986, and this cofractionation was not due to unspecific adsorption of tubulin to the column material. In addition, GABAA receptors not only bound to microtubules but also coassembled with added tubulin through three cycles of microtubule polymerization and depolymerization. These data indicate that GABAA receptors may be associated with the microtubule cytoskeleton in the brain. In an experiment designed to detect a protein that possibly could form a bridge between tubulin and the GABAA receptor, only a single protein band containing tubulin could be identified that was able to bind polymerized tubulin after sodium dodecyl sulfate-gel electrophoresis of affinity-purified GABAA receptors. These results are discussed with respect to a possible mechanism of association between GABAA receptors and microtubules.     

Human immunodeficiency virus 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors may suppress the replication of specific drug-resistant (E138K)RT HIV-1 mutants or select for highly resistant (Y181C-->C181I)RT HIV-1 mutants

Mutant HIV-1 that expresses a Glu138-->Lys substitution in its RT [(E138K)RT] is resistant to the HIV-1-specific RT inhibitor 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)pyrimidine (TSAO). However, cell cultures infected with this mutant were completely protected against virus-mediated destruction by micromolar concentrations of the HIV-1-specific RT inhibitors tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), nevirapine, and bis(heteroaryl)piperazine (BHAP). In contrast, cells infected with a virus mutant that expresses a Tyr181-->Cys substitution in its RT [(Y181C)RT] were not protected by nevirapine and TIBO and were only temporarily protected by BHAP. HIV-1 mutant that emerged under the latter conditions contained a Cys181-->Ile substitution in their RT [(LC181I)RT]. This mutant proved highly resistant to all HIV-1-specific RT inhibitors tested, except for several 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivatives. When recombinant (C181I)RT was evaluated for susceptibility to the HIV-1-specific RT inhibitors, it was resistant to all inhibitors except the HEPT compounds. Since a (Y181F)RT HIV mutant strain was isolated from cells infected with (Y181C)RT HIV-1 and treated with BHAP, we postulate that the Ile codon was derived from a Cys-->Phe transversion mutation (TGT-->TTT), followed by a Phe-->Ile transversion mutation (TTT-->ATT).     

Blocking of CD4 cell receptors for the human immunodeficiency virus type 1 (HIV-1) by chemically modified bovine milk proteins: potential for AIDS prophylaxis

An evaluation of the effects of the ABLE (Analyzing Behavior State and Learning Environments) model was presented. Eight teachers received training on observing behavior state and relevant environmental variables; analyzing potential effects of nutrition and medications; identifying strategies for individual students; and participating in a process to generate classroom-based intervention strategies based on their observations. Results showed that teachers were able to reliably implement the model, which produced significant increases in occurrences of the preferred alert and active states and significant decreases in nonpreferred states, such as sleep, drowse, daze, and crying/agitated. Additional findings indicated that the teachers were satisfied with the training procedures and found the model to be relevant and appropriate for students with profound mental retardation. Case studies were briefly presented.