Properties of a collagenolytic enzyme from Bipalium kewense.

A collagenolytic enzyme from the land planarian Bipalium kewense has been purified by preparative isoelectric focusing. The enzyme has a molecular weight of 47,000 +/- 2,000 and appears to be dimeric. It has an isoelectric point of 4.6 +/- 0.1 and a high content of acidic amino acids. The amino acid composition of the Bipalium collagenase is similar to that of human skin fibroblast collagenases but clearly different from previously reported collagenolytic proteases from other invertebrates, Uca pugilator and Hypoderma lineatum. In its action on guinea-pig collagen, the enzyme produces distinct products, at low incubation temperatures, different from those produced by vertebrate and other invertebrate collagenolytic enzymes. These products have glycine as their N-terminal amino acids. As determined by viscosity measurements, the Bipalium collagenase is more active on invertebrate, earthworm, collagen than it is on the vertebrate, Type I guinea-pig skin, collagen. The Bipalium collagenase differs from both bacterial and vertebrate collagenases as well as from invertebrate, collagenolytic serine proteases.

Binding of human IgG myeloma proteins to autologous T-cell receptor determinants.

IgG myeloma proteins (MPs) produced by monoclonal plasma cells derived from B2 lymphocytes have been reported to bind to various autoantigens but the binding generally has been of low affinity. Moreover, T cells from some multiple myeloma patients can respond specifically to idiotypes of their own paraproteins. We analyzed the capacity of more than 20 human IgG MP to bind, a recombinant single-chain molecule containing complete V beta 8.1 and V alpha 1 structures, sets of synthetic peptide epitopes corresponding to a complete TCR beta chain, and a set of CDR1 epitopes corresponding to 24 human V beta gene products, and intact monoclonal T cells. Two of 20 MPs bound strongly to the recombinant TCR. Five of the same set, including these, bound to a synthetic epitope corresponding to the CDR1 segment. On a mass basis, the binding was approximately 1000-fold greater than that of pooled polyclonal IgG. The binding activity was confined to the Fab fragment and was specifically inhibitable by appropriate peptide determinants. Spectrotypic analysis using a set of CDR1 epitopes indicated that individual proteins showed characteristic binding patterns ranging from highly specific to relatively promiscuous. Highly reactive MPs also bound to TCR on intact cells in immunocytofluorescence by flow cytometry. These results are consistent with the relatively frequent occurrence of autoantibodies to TCR determinants and indicate that MPs can be derived from this autoantibody subset.

Characterization of autoantibodies directed against T cell receptors.

Recently it has been observed that administration of intravenous immunoglobulin (IVIG) can have profound effects on a wide variety of diseases related to the dysregulation of the immune system. The mechanisms which explain these activities are poorly understood. Human IVIG and various Cohn plasma fractions contain autoantibodies directed against T cell receptors (Tcr). Previous studies have shown that IVIG contains autoantibodies against T cell receptor peptides. In order to further our understanding of autoantibody specificities, a single chain Tcr (scTcr) was constructed by recombinant DNA techniques from the variable alpha and variable beta chains of the Jurkat cell line. Anti-Tcr autoantibodies were isolated from IVIG and Cohn fractions I + III using a scTcr affinity column. This scTcr affinity purified material reacted with the surfaces of T cells at 10 micrograms/ml whereas non-purified IVIG did not. Sera from patients with rheumatoid arthritis (RA) as well as serum from patients with systemic lupus erythematosus (SLE) reacted with the scTcr at levels above that of normals.

NAD+-malic enzyme. Regulatory properties of the enzyme from Ascaris suum.

The regulatory properties of the NAD-dependent malic enzyme from the mitochondria of Ascaris suum have been studied. The malate saturation curve exhibits sigmoidicity and the degree of this sigmoidicity increases as the pH is increased. Fumarate was the only compound tested that stimulated the enzyme activity, whereas oxalacetate was the most powerful inhibitor. Activation by low levels of fumarate was found to be competitive with malate. It is proposed that this stimulation has physiological significance in controlling the dismutation reaction in the parasite. The branched-chain volatile fatty acid excretion products, tiglate, 2-methylbutanoate, and 2-methylpentanoate, inhibited the enzyme activity and this inhibition was competitive with malate. The Ki values for these compounds are in the physiological range of their concentrations; therefore, it is suggested that they may aid in controlling the malic enzyme activity in vivo. Oxalacetate inhibition of malic enzyme activity was competitive with malate, and the Ki values decreased with an increase in pH. Two alternatives are proposed which could account for the lack of oxalacetate decarboxylation by the ascarid malic enzyme.

Characterization of monoclonal antibodies to Pseudomonas aeruginosa type A flagellar antigen.

Flagellar murine MAbs (53B and 29) to strain a-type 170018 (a0,a3,a4, 45 kDa) and a human a-type MAb were studied in ELISA, Immunoblot, colony blot, agglutination and motility assays to evaluate the degree of cross-reactivity within the dominant a0 epitope. No specific effect of possible subtypes (a1, a2, a3, a4) was observed. An association of cross-reactivity and molecular weight was observed for 53B. A broader cross-reactivity as seen with MAbs 29 and A522 including high molecular weight flagellins (49-52 kDa), and particularly in the motility assay, may predict protective potential. Moderate reactivity with strain 5939 (a0 a3) was only seen with A522 MAb. These data indicate the presence of several cross-reactive sites associated with the common a0 antigen.

Purification and properties of a proteolytic enzyme from the cercariae of the human trematode parasite Schistosoma mansoni.

Skin penetration by the cercarial stage of the human trematode parasite Schistosoma mansoni is mediated by the secretion of proteolytic enzymes able to digest components of mammalian connective tissues. In the present study the purification of these proteinases from cercarial homogenates is reported. The major proteinase species has a mol. wt. of approx. 25 000 and exists in monomeric form as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This proteinase has an isoelectric point of 6.0. Studies presented here, with a variety of substrates and inhibitors, confirm previous claims that these proteinases belong to the serine class, and, in addition, suggest that they resemble the vertebrate chymotrypsins rather than trypsins or elastases. However, the amino acid composition of the cercarial proteinase differs significantly from bovine chymotrypsin and from the human leucocyte chymotrypsin-like cathepsin G. The amino-acid-composition differences between these proteinases are consistent with their differences in isoelectric point. In order to obtain an insight into the role of the proteinase in skin penetration, its activity on cartilage proteoglycan monomers and on the isolated peptide backbone of proteoglycan was studied. The results of the present study indicate that the cercarial enzyme catalyses a limited specific digestion of the peptide core.

Physical and epitope analysis of a recombinant human T-cell receptor V alpha/V beta construct support the similarity to immunoglobulin.

The genetic organization and protein structure of T-cell receptors (TCR) and immunoglobulins (Ig) are remarkably similar. Through recombinant, physical, and peptide-based immunological studies we demonstrated that rabbit antisera generated against a recombinant single-chain TCR (scTCR) react with defined peptide epitopes of their constituent TCR alpha and beta chains. These antisera cross-react with the lambda light-chain Mcg as well as with peptides duplicating its covalent structure. Conversely, rabbit antisera generated to human lambda light chains cross-reacted with the recombinant scTCR. Rabbit anti-lambda antibodies purified on an scTCR affinity column bound to T-cell lines and to T and B lymphocytes from peripheral blood. Circular dichroism analysis demonstrated plots characteristic of beta-sheets for both Mcg and recombinant scTCR. Antisera directed against TCR alpha-chain synthetic peptides reacted with scTCR, Mcg lambda light-chain protein, synthetic peptides from regions of sequence homology in beta-chains, and Mcg. Based upon this homology and the serological cross-reactions which reflect conformational determinants, we suggest that the V alpha/V beta antigen-binding domain of this particular monoclonal scTCR construct is substantially similar to the conformational structure of lambda light chains.

Recognition of defined epitopes by affinity-purified anti-immunoglobulin fab autoantibodies isolated from HIV-infected humans.

Infection of humans with HIV-1 has previously been independently shown to result in the generation of autoantibodies (AAbs) reactive with immunoglobulin Fab fragments (Heidelberg), and with autoantibodies to T-cell receptors (TCRs) (Tucson). Here, we carry out epitope mapping studies of affinity-purified AAbs to Fab fragments prepared from individual HIV-positive patients for their capacity to bind recombinant constructs and peptide-defined epitopes modeling TCR and Ig light chains. Some affinity-purified autoantibodies reacted strongly with TCRs expressed by intact T-cells, and recombinant Valpha/Vbeta constructs as well as with certain synthetic peptide epitopes. The binding reactions of affinity-purified AAbs of individual patients were distinct, and the AAb preparations consisted of populations of polyclonal lgs as reflected in specificity and isotype. AAb pools from individual patients all bound particular regions of TCR and Ig chains defined by comprehensive peptide synthesis including the CDR1 and Fr3 segments of the variable domains and the joining segment/switch peptide. In addition, other reactivities to restricted regions of alpha, beta and lambda light chains were documented. These results substantiate the cross-reactivity of TCR and Ig-Fab determinants, and are consistent with the hypothesis that autoantibodies arising as a consequence of HIV infection can have an immunomodulatory role.

Autoantibodies to T-cell receptor beta chains in human heart transplantation: epitope and spectrotype analyses and kinetics of response.

Autoantibodies against T-cell receptors have been found in two alloimmunization situations in humans: renal transplantation and pregnancy. We carried out longitudinal studies of human heart transplant recipients monitoring their autoantibody production to a recombinant single chain T-cell receptor V alpha/V beta construct, a set of nested, overlapping peptides duplicating the complete covalent structure of an individual T-cell receptor beta chain and a set of peptides duplicating the first complementarity determining segments of 24 distinct human V beta gene products in order to define the time course, epitope specificity and recognition heterogeneity of the response. Autoantibodies against intact and peptide-defined V beta and C beta determinants were generated following human heart allotransplantation. The responses generally show an increase following transplantation that subsequently decreases with time, a result which is consistent with a single immunization. However, some patients showed elevated responses as long as 12 months following the transplant. Autoantibody anti-CDR1 spectrotype analyses detected individual differences among patients, but 5 of 8 patients characterized in detail showed elevated IgG binding to CDR1 peptide epitopes of V beta 6.1, 21.1 and 22.1 gene products. Autoantibodies to CDR1 epitopes of V beta 7.1 and 8.1 were high pretransplant and remained high, although the relative increases with respect to the pretransplant values were not as impressive as those for the above CDR1 epitopes and others usually present in low quantity, e.g. anti-V beta 2.1, 3.1 and 24.1. Although there was great disparity between the MHC haplotypes of donors and recipients, and individual differences among patients, the degree of restriction in the autoantibody response was surprising and suggests a common step in recognition and regulation of the response to allografts.

Analysis of autoantibodies to T-cell receptors among HIV-infected individuals: epitope analysis and time course.

Individuals seropositive for human immunodeficiency virus type 1 (HIV) express elevated levels of autoantibodies (AAbs) directed against recombinant T-cell receptors (TCRs) and synthetic peptide epitopes duplicating beta chain markers. We performed longitudinal studies of anti-TCR AAbs in HIV-1-infected individuals, making comparisons with uninfected sera and sera from other individuals infected with a nonviral agent. We determined levels of autoantibodies by titration using enzyme-linked immunosorbent assay (ELISA) and developed a means for characterizing "autoantibody CDR recognition spectrotypes" for individual sera. Antibody levels against certain defined synthetic epitopes were substantially elevated in HIV-infected subjects relative to reactivities by control groups. Individual sera showed relatively high AAb levels to a subset of CDR1 peptide epitopes. Two patients who subsequently developed AIDS showed particular reactivity to Vbeta2.1, 8.1, 10.1, and 22.1 epitopes. Our results show that production AAbs to TCR Vbeta epitopes is a general consequence of HIV infection. The response is individual but shows some restriction and shifts in AAb subpopulations often occur with time.

Inhibition of bacterial motility with human antiflagellar monoclonal antibodies attenuates Pseudomonas aeruginosa-induced pneumonia in the immunocompetent rat.

Two human monoclonal antibodies, directed against the type a and type b flagellar proteins of Pseudomonas aeruginosa, inhibited bacterial motility in vitro specifically and in a concentration-dependent manner. In order to determine if this decreased bacterial motility was associated with a decreased pathogenicity, the ability of these human antiflagellar monoclonal antibodies to attenuate P. aeruginosa-induced pneumonia in the rat was assessed. Incubation of P. aeruginosa with a 1:1 mixture of the human antiflagellar monoclonal antibodies prior to pulmonary instillation significantly (P < 0.05) ameliorated the bacterium-induced decrease in arterial blood oxygen pressure, blunted the increase in respiratory rate, and markedly reduced the area of pulmonary inflammation. Similarly, intravenous administration of the human antiflagellar monoclonal antibodies 1 h after pulmonary instillation of the bacteria also reduced the in vivo pathogenicity of P. aeruginosa. Therefore, human antiflagellar monoclonal antibodies can decrease the in vitro motility of P. aeruginosa and can reduce its in vivo pathogenicity when administered either before or after bacterial challenge.