The sensitivity to Zn2+ discriminates between typical and atypical mast cells.

The effects of zinc gluconate have been studied on rat peritoneal mast cells and rat basophilic leukemia cells (RBL 2H3) stimulated by various secretagogues. The IC50's of zinc gluconate on peritoneal cells were (microM): 1.6, 1.9, 5.4 and 18 for ionophore A23187, phorbol 12-myristate 13-acetate, substance P and immunoglobulin E-antigen, respectively. Higher concentrations of zinc gluconate were required to inhibit histamine secretion from RBL 2H3 cells, i.e. 12 microM (ionophore A23187) and 140 microM (immunoglobulin E-antigen). Zinc gluconate (10(-4) to 10(-3) M) also inhibited the IgE-dependent contraction of guinea pig trachea but was unable to affect that induced by exogenous histamine. These results suggest that zinc gluconate acts intracellularly and is selective of "typical" or "connective tissue" mast cells.     

Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency

Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed ‘foldon’ (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10⁹ M⁻¹ and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10⁷ M⁻¹). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy.     

Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate: a novel in vitro tool to estimate rabies viral glycoprotein antigen in vaccine manufacture

The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to qualify the whole reagent preparation procedure, to be used to quantify rabies viral antigen preparation in a simple and rapid format for potency control of rabies vaccines. 50AD1 is a neutralizing monoclonal antibody directed against the rabies virus glycoprotein that binds to native conformational antigenic site III. In the present study, the DNA fragments encoding the variable domains of 50AD1 were inserted into a prokaryotic expression vector so as to produce a single-chain Fv antibody fragment (scFv) genetically fused to the bacterial alkaline phosphatase (AP). The recombinant fusion protein preserved both the AP enzymatic activity and the antigen-binding activity against the rabies virus glycoprotein nearly identical to the parental antibody, and was used successfully in different assays including ELISA, dot-blot and cell culture tests. The present study shows that the genetic fusion protein provides a new tool for one-step rabies virus immunodetection, which can be produced in homogeneous bifunctional reagent, easily, quickly and reproducibly. In addition, this recombinant immunoconjugate is a promising alternative reagent for applications involving immunodetection, it presents a similar sensitivity and specificity to that obtained with classical reagents.     

Role of phospholipase A2 and pertussis toxin-sensitive G proteins in histamine secretion induced by substance P

Incubation of mast cells with substance P or ionophore A23187 resulted in histamine release and arachidonic acid liberation from purified rat peritoneal mast cells. The treatment of mast cells with 100 ng/ml of pertussis toxin for 2h inhibited the effect of substance P on both histamine release and arachidonic acid liberation, but the response to the ionophore A23187 was not affected. Para-bromophenacyl bromide, a selective inhibitor of phospholipase A₂, inhibited similarly histamine and arachidonate release induced by substance P but not that evoked by ionophore A23187. These results suggest that phospholipase A₂ plays a key role in the histamine secretion induced by substance P.

     

ICBP90 belongs to a new family of proteins with an expression that is deregulated in cancer cells

ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa) is a recently identified nuclear protein that binds to one of the inverted CCAAT boxes of the topoisomerase IIalpha (TopoIIalpha) gene promoter. Here, we show that ICBP90 shares structural homology with several other proteins, including Np95, the human and mouse NIRF, suggesting the emergence of a new family of nuclear proteins. Towards elucidating the functions of this family, we analysed the expression of ICBP90 in various cancer or noncancer cell lines and in normal or breast carcinoma tissues. We found that cancer cell lines express higher levels of ICBP90 and TopoIIalpha than noncancer cell lines. By using cell-cycle phase-blocking drugs, we show that in primary cultured human lung fibroblasts, ICBP90 expression peaks at late G1 and during G2/M phases. In contrast, cancer cell lines such as HeLa, Jurkat and A549 show constant ICBP90 expression throughout the entire cell cycle. The effect of overexpression of E2F-1 is more efficient on ICBP90 and TopoIIalpha expression in noncancer cells (IMR90, WI38) than in cancer cells (U2OS, SaOs). Together, these results show that ICBP90 expression is altered in cancer cell lines and is upregulated by E2F-1 overexpression with an efficiency depending on the cancer status of the cell line.     

Evaluation of an enzyme-linked immunosorbent assay based on crude leishmania histone proteins for serodiagnosis of human infantile visceral leishmaniasis

Human visceral leishmaniasis (VL) is routinely diagnosed by detecting IgG that specifically binds to Leishmania antigens. The enzyme-linked immunosorbent assay (ELISA) remains a widely used method. However, the biggest challenge remains the choice of antigen with the highest specificity and sensitivity. This study is aimed at assessing the diagnostic performances of crude Leishmania histone (CLH) protein-based ELISAs in Mediterranean VL patients. The CLH proteins were biochemically purified from promastigote nuclear extracts. Their reactivities were analyzed by Western blotting (WB) using rabbit polyclonal antibodies against Leishmania recombinant histones and sera from VL patients, respectively. Then, the diagnostic potential of CLH proteins was validated by the CLH-based ELISA using 42 infantile VL patients' sera and 70 control subjects. The CLH-based ELISA performance was compared to that of the soluble Leishmania antigen (SLA)- and the recombinant K39 (rK39)-based ELISAs. Analysis of the WB profile with the use of polyclonal antibodies confirmed the histone origin of low molecular mass proteins (12 to 16 kDa). All VL samples tested presented antibodies reacting against different antigen fractions; however, recognition patterns were different depending on the reactivity of each serum. CLH-based ELISA showed an excellent ability to discriminate between VL cases and healthy controls (97.6% sensitivity and 100% specificity). It had a diagnostic performance similar to that of rK39-based ELISA (97.6% sensitivity and 97.1% specificity, P = 0.5) and a better serodiagnosis accuracy than the SLA-based ELISA (85.7% sensitivity and 90% specificity, P < 0.05). Therefore, crude Leishmania histone extract could be a valuable antigen for clinical use.     

A pertussis toxin-sensitive G protein is required to induce histamine release from rat peritoneal mast cells by bradykinin.

Bradykinin, kallidin (Lys-bradykinin) and [Thi 5,8, D-Phe7]-bradykinin, a functional B2 antagonist, induce histamine release from rat peritoneal mast cells. The histamine release is dependent upon added calcium when mast cells are placed in calcium-free medium 30 min before being triggered with the kinins. Histamine release was dose-dependently inhibited by pertussis toxin (1-100 ng/ml) and by benzalkonium chloride (0.1-3 micrograms/ml). The efficiency of ionophore A23187 on histamine release was affected neither by pertussis toxin nor by benzalkonium chloride. The parallel response of rat peritoneal mast cells to kinins and to substance P suggest that these peptides have the same mechanisms of action i.e. activation of a pertussis toxin-sensitive G protein and of phospholipase C defining a peptidergic triggering pathway of mast cells.     

Evidence for the interaction of mast cell-degranulating peptide with pertussis toxin-sensitive G proteins in mast cells.

K(+)-channel blocker properties have been reported for mast cell-degranulating peptide (MCD) in the central nervous system, but its action mechanism in mast cells remains unknown. We studied the effect of MCD on the membrane potential of rat peritoneal mast cells using the fluorescent probe bis-oxonol. Unexpectedly, MCD induced a decrease in bis-oxonol fluorescence, in a rapid and then a slower phase, suggesting hyperpolarization of mast cells. Other K(+)-channel blockers, tetraethylammonium and 4-aminopyridine, did not significantly modify the bis-oxonol fluorescence and did not alter the effect of MCD. The late phase of bis-oxonol fluorescence decrease was inhibited by ouabain and by potassium deprivation, whereas histamine release was not affected. The first phase of putative hyperpolarization induced by MCD coincided with histamine release and with the generation of inositol polyphosphates. Prior treatment of the cells with pertussis toxin inhibited these effects of MCD. MCD stimulated the GTPase activity of purified G proteins (G0/Gi) in a concentration-dependent manner. These results indicate that the effect of MCD on mast cells is unrelated to K+ channels but that it is relevant to the activation of pertussis toxin-sensitive G proteins leading to the activation of phospholipase C. A direct interaction of MCD with G proteins is proposed, which, unlike mastoparan, does not require positive cooperativity.