Fc gamma receptor-mediated interplatelet activation by a monoclonal antibody against beta 2 microglobulin.

Three different mAb directed against beta 2 microglobulin (two IgG1 and one IgG2a) were tested for their ability to activate human platelets. Although all three antibodies bound to platelets, only one of them, B2.62.2, of the IgG1 subclass, induced platelet activation. This activation is similar to the activation by SYB-1, a CD9 antibody of the same subclass previously described as activating platelets through platelet Fc gamma R. These similarities include serotonin secretion, a lag time preceding aggregation and the induction of a strong intracellular calcium mobilization from storage pools. As with CD9 antibodies, the F(ab')2 fragments of B2.62.2 did not induce activation but blocked the activation by the native antibody, by preventing the binding to beta 2 microglobulin. Also, this activation was inhibited by pretreating the platelet with IV-3, a mAb that blocks the Fc binding site of the FcR. Inasmuch as the same antibody does not prevent the binding of B2.62.2 on platelets, we conclude that the activation by B2.62.2 is mediated by the FcR. Nevertheless, there were differences with the activation by SYB-1. B2.62.2 activation was more dependent on thromboxane A2 formation and no cytoplasmic alkalinization was detected. Finally, contrary to SYB-1, B2.62.2 activation proved to be sensitive to platelet count, suggesting that it involves the formation of immune complexes consisting of antibodies and platelets, that activate nearby platelets.     

Restricted Ig H chain V gene usage in the human antibody response to Haemophilus influenzae type b capsular polysaccharide

The mechanisms that govern the content of the human antibody repertoire are poorly understood. To investigate the antibody response to a clinically relevant Ag, we have produced heterohybridomas secreting human antibodies directed against the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). Immune lymphocytes were harvested 7 days after immunization with either of two vaccine formulations, a plain polysaccharide vaccine (Hib PS) or a polysaccharide-protein conjugate of Hib PS and diphtheria toxoid (Hib PS-D). H chain V region gene nucleic acid sequences were determined for five stable hybridomas. All use members of the VHIII gene family and are 83% to 98% homologous to two candidate germ-line sequences. A variety of D and JH segments are used. Thus the Ig H chain repertoire appears to be restricted to a limited group of VHIII family members. The previously reported expression of homologous sequences in the human fetal repertoire suggests that the inability of young children to respond to this Ag is not caused by deficiencies of these important elements early in development. The restricted use of VHIII gene segments suggests that this gene family plays a pivotal role in the immune response to this important childhood pathogen.     

In vivo administration of anti-CD3 monoclonal antibodies or immunotoxins in murine recipients of allogeneic T cell-depleted marrow for the promotion of engraftment.

The role of host anti-donor cells in rejection of fully allogeneic donor T cell-depleted marrow was investigated by using mAb or immunotoxins directed against T cell or NK cell determinants. Immunotoxins consisting of mAb conjugated to a low oligosaccharide-containing fraction of purified ricin toxin A chain (RTA) facilitated in vivo-depletion of target cell populations. BALB/c and DBA/1 donors were selected based upon their expression (BALB/c) or lack of (DBA/1) hemopoietic histocompatibility (Hh1) Ag, which may serve as targets for donor rejection in C57BL/6 hosts. When studies directed toward eliminating CD3+ cells were performed in both systems, injections of intact anti-CD3 mAb or anti-CD3-RTA reproducibly produced the highest engraftment values. The fact that engraftment values obtained with anti-CD3 or anti-CD3-RTA therapy in allogeneic systems were substantially higher than in syngeneic controls suggested that engraftment stimulatory proteins were released upon TCR engagement. Elevated levels of cytokines and a high mortality rate in allogeneic recipients confirmed that this was the case. Nonstimulatory preparations of anti-CD3F(ab')2 fragments and anti-CD3F(ab')2-RTA promoted engraftment of both types of allogeneic marrow, as measured by short term 125I-IUdR assays, suggesting that stimulation was not a prerequisite for engraftment. Recipients of anti-CD3F(ab')2 or anti-CD3F(ab')2-RTA showed a marked reduction of host CD3+ cells as measured by immunofluorescence and flow cytometry. In long term chimerism studies, recipients of Hh1-disparate marrow and anti-CD3F(ab')2 had a dramatic increase in donor cell engraftment as compared to controls, indicating that positive effects on engraftment were long lived. Studies further showed that BALB/c donor cells exhibiting an Hh1 disparity were rejected by host cells expressing NK1.1 or Ly-1 (NK cells and T cells). In contrast, DBA/1 donor cells that were not Hh1-disparate were rejected by cells expressing Ly-1, but not NK1.1 (T cells only). These studies provide definitive data that CD3+ cells participate in the rejection of either Hh1+ or Hh1null T cell-depleted allografts and offer new strategies for alloengraftment using regimens containing nonmitogenic anti-CD3.     

Anti-idiotypic antibody response to monoclonal anti-CD4 preparations in nonhuman primate species.

A series of mouse monoclonal anti-CD4 preparations was characterized for the ability to recognize overlapping epitopes on CD4 and to inhibit HIV/simian immunodeficiency virus (SIV) syncytium formation. Based on this characterization, mAb able to recognize CD4 epitopes overlapping the HIV binding site were selected and used to immunize nonhuman primates to elicit the production of specific anti-Id antibodies. Five baboons and five rhesus monkeys were immunized with either individual or a cocktail consisting of several monoclonal anti-CD4 preparations. All the nonhuman primates produced specific anti-Id that recognized either private or cross-reactive Id depending on the monoclonal anti-CD4 used to generate the anti-Id response. Inhibition assays were performed to ascertain the ability of: 1) soluble CD4 to inhibit the Id-anti-Id reaction and 2) the various anti-Id to inhibit the CD4-monoclonal anti-CD4 reaction. These studies demonstrated that some of the anti-Id recognized a cross-reactive Id that was associated with the Ag-combining site. In addition, some of the anti-Id weakly recognized SIV gp120 by Western blot analysis. These studies may be useful in designing experiments that may lead to a better understanding of the CD4-HIV gp120 interaction and to the production of Id and/or anti-Id reagents that might be used to manipulate this virus-receptor interaction.     

Epitope mapping of apolipoprotein A-I using endoproteinase cleavage and monoclonal antibodies in an enzyme-linked immunosorbent assay.

The epitopes for two monoclonal antibodies (MAbs) directed towards human apolipoprotein A-I (apoA-I), designated AI-1 and AI-3, have been more precisely defined. Previous work in our laboratory demonstrated that AI-1 and AI-3 recognize antigenic determinants located within cyanogen bromide (CNBr) fragments 1 (CF1) and 3 (CF3), respectively. Using peptides generated from endoproteinase cleavage of CF1 and CF3, we now report that both MAbs are specific for two previously unreported epitopes along the apoA-I molecule. The ability of whole endoproteinase digest mixtures to bind the MAbs, as determined by means of a competitive enzyme-linked immunosorbent assay (ELISA), indicated regions of CF1 and CF3 that were likely to form the epitopes. Purified peptides derived from the digests were then used to localize the epitopes recognized by MAbs AI-1 and AI-3 to within residues 28-47 and 140-147 of apoA-I, respectively. We have previously reported that the epitopes for both MAbs are exposed on HDL2, HDL3, and free apoA-I. Thus, the precise mapping of the binding sites recognized by AI-1 and AI-3 has enabled the identification of regions along apoA-I that are exposed on the surface of lipoprotein particles.     

Involvement of beta-COP in membrane traffic through the Golgi complex

Non-clathrin-coated vesicles mediate membrane traffic through the Golgi complex. The proteins that constitute the coats of these vesicles have similar molecular weights to the clathrin coat proteins. A major component of the coat of non-clathrin-coated vesicles, beta-COP, has significant homology with the clathrin coat protein beta-adaptin, indicating that the coats of the two different classes of vesicles may be structurally and functionally homologous.     

A comparison of the intracellular distribution of mu-calpain, m-calpain, and calpastatin in proliferating human A431 cells.

Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.     

Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome.

The globular domain of the linker histone H5 has been expressed in Escherichia coli. The purified peptide is functional as it permits chromatosome protection during micrococcal nuclease digestion of chromatin reconstituted with the peptide, indicating that it binds correctly at the dyad axis of the nucleosomal core particle. The globular domain residue lysine 64 is highly conserved within the linker histone family, and site-directed mutagenesis has been used to assess the importance of this residue in the binding of the globular domain of linker histone H5 to the nucleosome. Recombinant peptides mutated at lysine 64 are unable to elicit chromatosome protection to the same degree as the wild-type peptide, and since they appear to be fully folded, these observations confirm a major role for this residue in determining the effective interaction between the globular domain of histone H5 and the nucleosome.     

Cytosolic Free Calcium and Gene Expression During Chemical Hypoxia

Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.