Presence of Aedes albopictus in Lebanon and Syria.

The occurrence of Aedes albopictus in Lebanon and Syria is reported for the first time. Larvae were found in 4 localities in Lebanon, and 1 female was captured inside a house located in a coastal locality in Syria. The potential of the species to vector arboviral disease in the region is noted.

Immunomodulation of autoimmune and inflammatory diseases with intravenous immunoglobulin.

Intravenous immunoglobulin (IVIg) has been used in the treatment of primary and secondary antibody deficiencies for over two decades. Since the early 1980s, the therapeutic efficacy of IVIg has been established in idiopathic thrombocytopenic purpura, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, dermatomyositis and Kawasaki syndrome, and the prevention of graft versus host disease in recipients of allogeneic bone marrow transplants. Its use has also been reported in a large number of other autoimmune and systemic inflammatory conditions. In this review, we discuss the mechanisms by which IVIg exerts immunomodulatory effects in immune pathologies.

Protective effect of taurine against free radicals damage in the rat myocardium.

Free radicals are highly cytotoxic to the heart and are involved in ischemia/reperfusion injury. In this study, we tested the ability of taurine to neutralize the deleterious effects of free radicals generated ex vivo and in vitro. Taurine was added at a concentration of 0.1 mM to the drinking water of experimental rats during 6 months. The animal hearts were then isolated and submitted to regional ischemia and reperfusion; ventricular fibrillation was significantly reduced as compared to a control group of non-treated animals. Moreover, at a concentration of 1 mM, taurine provided significant cardio-protection against the deleterious effect of free radicals generated by the electrolysis of Krebs-Henseleit buffer. When isolated hearts were perfused with electrolysed buffer, extensive fiber necrosis occurred, as observed by staining with nitro blue tertrazolium, a soluble dye which yields a dark blue formazan stain in the presence of reducing agents This stain was barely detectable when taurine was added to the perfusing electrolysed buffer. To further understand the protecting mechanism of taurine, we used xanthine-xanthine-oxidase as a superoxide (O2-) generating system and monitored the O2- through yield O2--dependent cytochrome c reduction. We demonstrated that taurine did not affect this system, which indicated that it did not scavenge O2- directly. On the other hand, taurine inhibited the auto-oxidation of adrenaline to adrenochrome at pH 7.8 where this auto-oxidation is O2--independent and superoxide dismutase insensitive. We thus conclude that taurine acts as a potent, but non-specific, scavenger of free radicals that cause heart damage and protects against reperfusion-induced ventricular

In vitro IgE inhibition in B cells by anti-CD23 monoclonal antibodies is functionally dependent on the immunoglobulin Fc domain.

CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab')(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab')(2) of p5E8G1, the F(ab')(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.

Human monoclonal antibodies isolated from spontaneous Epstein-Barr virus-transformed tumors of Hu-SPL-SCID mice and specific for fusion protein display broad neutralizing activity toward respiratory syncytial virus.

Two human monoclonal antibodies, RF-1 and RF-2, specifically recognize the fusion protein of the human respiratory syncytial virus (RSV). These were isolated from spontaneous tumors in SCID mice reconstituted with human splenocytes and boosted with fusion protein. The tumors consisted of Epstein-Barr virus-transformed human B cells in animals with antigen-specific antibody titers>105. The binding affinity of RF-1 and RF-2 to the fusion protein is 1010 and 109 M-1, respectively. The antibodies bind specifically to a conformational epitope of the fusion protein on RSV-infected HEp-2 cells. Both antibodies display virus-neutralizing properties in vitro at concentrations varying between 8 and 1000 ng/mL. Virus neutralization applies to a broad variety of wild and laboratory-adapted virus strains belonging to both virus types A and B. These antibodies are potential candidates for passive immunotherapy of severe RSV infections.

Two neutralizing human anti-RSV antibodies: cloning, expression, and characterization.

BACKGROUND: Respiratory syncytial virus (RSV) infection is a major problem in the newborn and aging populations. Fully human monoclonal antibodies with the ability to neutralize RSV could have a major impact on the immunotherapy of the disease. The generation of human antibodies has been difficult because there exists no general way to activate B cells against an antigen of choice in vitro. MATERIALS AND METHODS: Human spleen cells from individuals exposed to RSV were used to repopulate SCID mice. Hu-SCID mice were boosted with RSV fusion (F)-protein and subsequently developed B cell tumors. The tumors were removed and cultured and subcloned in vitro, using a feeder layer of CD154-expressing T cells. Two of these tumors produced the antibodies designated RF-1 and RF-2. VL genes were isolated by standard PCR techniques, however, it was necessary to use high-temperature reverse transcriptase to clone the VH genes. RESULTS: RF-1 and RF-2 VH genes were both found to be closely related members of the VH2 family. Vk genes originated from the VK III family. RF-1 and RF-2 recombinant antibodies expressed in CHO cells (cRF-1 and cRF-2) were found to have affinities for RSV F-protein of 0.1 nM and 0.07 nM, respectively, and both were able to neutralize several A and B subtypes of RSV. CONCLUSION: The technique of immortalizing human B lymphocytes, by passage in SCID mice and expression as recombinant antibodies in CHO cells, provides a method by which high-affinity human antibodies can be developed for immunotherapy of viral diseases.

Antigen-specific IgG responses from naive human splenocytes: in vitro priming followed by antigen boost in the SCID mouse.

High titers of Ag-specific human IgG were consistently achieved in SCID mice reconstituted with human splenocytes that had been primed with Ag in vitro and then boosted with Ag after engraftment into SCID mice. Specific human IgG titers in the hu-SPL-SCID mice reached approximately 1:4 x 10(5) when the mice were immunized with a neo-antigen, whereas titers reached 1:2 x 10(6) when recall responses were induced. Booster immunizations with Ag 21 days after the initial in vivo boost further enhanced this response, and specific human IgG titers of 1:17 x 10(6) were achieved. This represented an essentially monospecific IgG population. These responses were CD4+ T cell dependent. In addition, affinity maturation of the human Ab responses was observed. Spleens of hu-SPL-SCID mice with Ag-specific titers < or = 1:1 x 10(6) were often significantly enlarged and often displayed visible tumors. Fourteen of sixteen B cell tumors removed from spleens of five such hu-SPL-SCID mice, produced Abs that were specific for the immunizing Ags. From such tumor, cloned cell lines were established. One such mAb, MLN-7 (gamma1, kappa), was raised to tetanus toxoid and had no identified cross-reactivity.

Two major groups of neutralizing anti-gp120 antibodies exist in HIV-infected individuals. Evidence for epitope diversity around the CD4 attachment site.

The aim of this study was to dissect neutralizing anti-gp120 antibody populations in seropositive asymptomatic individuals. Murine anti-Id mAb were raised against polyclonal affinity-purified human anti-gp120 antibodies. These anti-Id mAb were used to fractionate anti-gp120 antibodies from a pool of HIV-positive sera into idiotypically distinct anti-gp120 antibody (Id+Ab) preparations. Immunochemical and neutralization studies indicated that all Id+Ab that neutralized HIV-1 in vitro interacted with either the V3 loop or the CD4 attachment site of gp120. The V3-specific Id+Ab neutralized HIV-1 in a strain-restricted manner. Id+Ab specific for the CD4 attachment site exhibited different spectra of neutralizing activities against multiple strains of HIV-1. This finding indicates that multiple, antigenically diverse epitopes reside around the CD4 attachment site of gp120. Significantly, depletion of the Id+Ab from affinity-purified total anti-gp120 antibodies abrogated most of the neutralizing activities of these antibodies, suggesting that neutralizing anti-gp120 antibodies consist of two major specificities, either to the V3 region or to the CD4 attachment site. The understanding of specificities and neutralizing activities of different anti-gp120 antibodies in seropositive healthy individuals will be helpful for designing effective vaccines and immunotherapeutic strategies for AIDS.

Anti-idiotype monoclonal antibody elicits broadly neutralizing anti-gp120 antibodies in monkeys.

Murine monoclonal antibodies (mAbs) were raised against human, polyclonal, anti-gp120 antibodies (Ab1) and were selected for binding to broadly neutralizing anti-gp120 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gp120 antibodies directed against an epitope around the conserved CD4 attachment site of gp120. The 3C9 reactive human anti-gp120 antibodies (3C9+ Ab) neutralized MN, IIIB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gp120 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gp120SF and gp120IIIB. Furthermore, purified gp120-specific Ab3 neutralize MN, IIIB, and RF isolates. These results demonstrate that primates immunized with an anti-idiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the results of clonotypic B-cell stimulation.

Synthetic peptides from a conserved region of gp120 induce broadly reactive anti-HIV responses.

In our efforts to identify products that might be used for active immunotherapy in human immunodeficiency virus (HIV) infection, we have studied synthetic peptides derived from the CD4 attachment site of gp120. Two peptides have emerged with particularly interesting properties. The first (B138) is linear and spans the envelope residues 421-438; the second (1005/45) encompasses amino acids 418-445 and is cyclized by way of a disulphide bond joining its terminal cysteines. Both species have been shown to inhibit syncytial formation in a conventional bioassay, B138 being the most efficient. Both peptides elicit high titres of anti-peptide antibodies in immunized mice, rabbits and goats, with titres exceeding 1:10(5) in many cases. A substantial portion of this response is directed against gp120 as determined by enzyme-linked immunosorbent assay (ELISA). Analysis by flow cytometry has demonstrated that the antisera are broadly reactive with multiple diverse strains of HIV. The anti-gp120 activity of the anti-peptide antiserum was further confirmed by radioimmuno-precipitation (RIP) assays. Furthermore, RIP analysis and inhibition experiments in a GD4-gp120 binding assay have revealed that anti-peptide sera contain antibodies directed against the CD4 attachment site on gp120 and interfere with this receptor-ligand interaction.

Evidence for non-V3-specific neutralizing antibodies that interfere with gp120/CD4 binding in human immunodeficiency virus 1-infected humans.

Total anti-gp120 antibodies (total anti-gp120 Abs) were purified from a pool of four human immunodeficiency virus-positive (HIV+) sera by affinity chromatography on a gp120SF2-Sepharose column and exhibited both type- and group-specific neutralizing activities. To dissect the epitope specificity of the group-specific neutralizing antibodies, CD4 attachment site-specific antibodies (CD4-site Abs) were isolated from total anti-gp120 Abs by using a CD4-blocked gp120SF2-Sepharose column. The CD4-site Abs exhibited group-specific neutralizing activities. Another approach to dissecting type- and group-specific neutralizing activities of total anti-gp120 Abs was to separate the third variable region (V3)-specific antibodies (V3-region Abs) from non-V3-region-specific antibodies (non-V3 Abs). The results indicated that V3-region Abs exhibited type-specific neutralizing activities, whereas non-V3 Abs exhibited group-specific neutralizing activities. By comparing the neutralizing activities of V3-region Abs to those of non-V3 Abs, we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. Collectively, this study indicates that group-specific neutralizing anti-gp120 antibodies are specific for the CD4 attachment site.

The immune response towards beta-adrenergic ligands and their receptors--VII. Equilibrium and kinetic binding studies of l-alprenolol to a monoclonal anti-alprenolol antibody.

Binding of the catecholamine beta-adrenergic antagonist, l-alprenolol, by the IgGl anti-alprenolol monoclonal antibody 37A4 was examined using the radioligand 3H-dihydroalprenolol as an extrinsic signal and the increase in antibody fluorescence upon l-alprenolol binding as intrinsic signal. Equilibrium binding studies based on both signals indicated that the binding process was exothermic with a positive entropy change. The difference in the affinity constants obtained by radioligand binding studies and by fluorescence analysis could be ascribed to the higher affinity of the hydrogenated tritiated l-dihydroalprenolol compared to the unsaturated l-alprenolol. The association rate constants determined by both signals were 10(4)-10(5)/M/sec and showed a high activation enthalpy (8-10 kcal/mol), thus excluding a diffusion controlled reaction. At low temp (7 degrees C), the fluorescence stopped-flow studies showed non-linear pseudo first order kinetics, indicating the existence of a fast pre-equilibrium of low affinity, followed by a conformational change leading to the tight binding of the ligand. The dissociation rate constants determined using both signals were very similar. Thus, the differences in affinity between the hydrogenated and non-saturated l-alprenolol could be ascribed to the association rate constants. Affinity constants and thermodynamic parameters calculated from the kinetic data were in close agreement with those determined by equilibrium binding. The mechanisms of ligand binding are discussed in terms of the interactions of idiotypes and anti-idiotypes in the anti-catecholamine immune response.

The immune response towards beta-adrenergic ligands and their receptors. VI. Idiotypy of monoclonal anti-alprenolol antibodies.

Four murine monoclonal antibodies specific for alprenolol, a synthetic beta-adrenergic ligand, with different binding properties towards alprenolol and other beta-adrenergic antagonists and agonists (as described in a previous report) were used to induce anti-idiotypic responses in rabbits and mice. Three of the rabbit anti-idiotypes inhibited, and one increased the binding of tritiated dihydroalprenolol to the Ab1 against which they were induced. The syngeneic mouse anti-idiotypes all had an inhibitory effect on the ligand binding to their corresponding Ab1. Cross-reactivity tests of the xenogeneic and syngeneic anti-idiotypes were positive only for two monoclonal anti-alprenolol antibodies. Cross-reaction could be shown neither on a panel of 15 other monoclonals, nor on polyclonal anti-alprenolol antibodies of the BALB/c and the C57BL/10 mice. These results suggest that the immune response against alprenolol results in antibodies with mostly private idiotypic determinants. Moreover, the properties of the anti-idiotypic response against the same monoclonal antibody seem to be different according to the species used for anti-idiotypic induction.

Idiotypy of catecholamine-binding proteins.

The idiotypic and antiidiotypic response to alprenolol, a beta-adrenergic antagonist, was studied both in rabbits and in mice. Rabbit polyclonal anti-alprenolol antibodies showed binding properties for catecholamine analogs, agonists as well as antagonists, similar to those of the beta-adrenergic receptors. The variability of the anti-alprenolol response was studied by using mouse monoclonal antibodies specific for alprenolol. While the binding properties showed great variations in affinity, the response seemed restricted to the heavy chain classes gamma 1 and gamma 2a. N-terminal sequencing of the light and heavy chains and restriction maps of the corresponding genes suggest that the antibodies use particular subgroups infrequently found in antibodies specific for other antigens. The cyclical antiidiotypic response in rabbits immunized with polyclonal antibodies and in mice immunized with monoclonal antibodies were compared. The response of the latter was dependent on the choice of the monoclonal antibody used to elicit the antiidiotypic response. Finally, the agonist-like properties of a monoclonal antiidiotypic antibody directed against one of the monoclonal anti-alprenolol antibodies were studied extensively. The ability to recognize beta-adrenergic receptors was documented by Western blot and direct immunoprecipitation and visualized by immunofluorescence. The antiidiotypic antibody stimulated catecholamine-sensitive adenylate cyclase and this effect was blocked by the beta-adrenergic antagonist propranolol.

Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-beta-adrenergic ligand antibody.

A new method has been developed to raise monoclonal anti-idiotypic antibodies. Monoclonal anti-idiotypic antibodies were obtained by fusion of NS-1 myeloma cells with splenocytes of mice immunised by intravenous injections of fixed hybridoma cells bearing a monoclonal antibody specific for beta-adrenergic ligands. New screening tests were developed to analyse the resulting hybridoma supernatants for different anti-idiotypic properties. Among 23 hybridoma supernatants recognising the idiotype, 6 were found to inhibit hapten binding and 3 of these recognised beta-adrenergic receptors.

Monoclonal antibodies specific for beta-adrenergic ligands.

After somatic cell fusion between splenocytes of immunized BALB/c mice and NS-1 myeloma cells, eight clones were obtained secreting anti-alprenolol antibodies as characterized by means of an ELISA. Four of these were subcloned and were studied further. The association constant for alprenolol ranged from 1.9 X 10(6) M-1 to 24 to 10(6) M-1. Competitive inhibition of [3H]-l-dihydroalprenolol binding revealed cross-reactivity with beta-adrenergic ligands, with a higher avidity for antagonists than for agonists. Two of the antibodies had a higher affinity for the l-isomer than for the d-isomer. The most stereospecific of these antibodies showed only affinity for beta-adrenergic antagonists and for the agonist isoproterenol. The other recognized both beta-adrenergic antagonists and agonists; it also showed an increase in tryptophan fluorescence after ligand binding. This property was used for the physicochemical study of the hapten-antibody interaction.