T and B cells that recognize the same antigen do not transcribe similar heavy chain variable region gene segments.

We have attempted to determine whether T cells and B cells that have the same antigenic specificity and whose receptors share idiotypic determinants in fact express similar VH gene segments. To do this, we have obtained and characterized a cDNA clone containing the entire coding sequence for the VH gene from a glutamic acid60/alanine30/tyrosine10 (GAT)-binding immunoglobulin that carries the CGAT idiotype. The GAT-VH clone was hybridized to Northern blots of GAT-specific T cell RNAs; there was no evidence of a T cell transcript that hybridized to the GAT-VH probe. The T cells analyzed included: (a) 10 GAT-binding suppressor T cell hybridomas, 6 of which secreted factors with CGAT idiotypic determinants, (b) one GAT-specific helper T cell hybridoma, and (c) two GAT-specific helper T cell lines grown in the absence of feeder cells. The detection limit of the Northern blot analysis was 1-2 copies of a particular mRNA species per cell for the hybridomas and 5-10 copies per cell for the T cell lines. Therefore, we conclude that T and B lymphocytes responding to GAT do not utilize similar VH gene segments. Furthermore, the presence of idiotypic determinants on T lymphocytes does not necessarily imply close structural similarity between T and B cell antigen receptors.

Differences in clot lysis among patients demonstrated in vitro with three thrombolytic agents (tissue-type plasminogen activator, streptokinase and urokinase).

This study compares the ability of 3 thrombolytic drugs to promote clot lysis using a new in vitro testing procedure. Whole blood samples from 132 patients were tested using 5 different concentrations of tissue-type plasminogen activator (t-PA), streptokinase (SK) and urokinase. A mixture of blood and thrombolytic drug was placed on a dry-reagent test card containing reptilase, buffers and paramagnetic particles where clot formation occurred. Analysis of the motion of the clot-embedded paramagnetic particles caused by an oscillating magnetic field was used to define the lysis onset time. The slope of the linear regression plot of lysis onset time versus 1/[drug concentration] defined the kinetic rate constant (k) for each drug in each patient. Higher values of k indicated greater resistance to in vitro clot lysis. In the patients studied, there was a large range of k values for t-PA and SK (coefficient of variation 143 and 137%, respectively) but a smaller range of k for urokinase (coefficient of variation 32%). The coefficients of variation for t-PA and SK observed in the study group were five- to 10-fold greater than the coefficients of variation determined for replicate test measurements. Resistance to all SK concentrations tested was found in 9% of the patients. In vitro sensitivity to thrombolysis was compared among the drugs by correlating the derived k values. These comparisons indicated no relation for any of the drugs; many patients had a relatively low k value for 1 drug, while having a relatively high k value for a different drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen presentation to T cell lines and clones by peritoneal macrophages, peritoneal B cells and antigen-specific B cell hybridomas.

The antigen presenting cell (APC) activity of uninduced, resident peritoneal macrophages and B cells was compared to that of antigen-specific B cell hybridomas by measuring proliferative responses of antigen-specific, MHC-restricted T cell clones. The results demonstrate that peritoneal macrophages and B cells are much more efficient APC than irradiated splenic filler cells, and that unirradiated B cells were as good as, if not better than, macrophages. Both B cells and macrophages can be pulsed with antigen, although pulsed B cells were always found to be more efficient than pulsed macrophages. However, the APC activity of B cells was exquisitely sensitive to irradiation. The relative contribution of macrophages and B cells to the APC activity of mixed populations was easily distinguished by complement dependent lysis with monoclonal antibodies specific for unique differentiation antigens expressed by these cells. Normal peritoneal macrophages and B cells present the synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) to GAT-specific T cells clones and beef insulin to insulin-specific T cell lines nonspecifically. The APC activity of antigen-specific B cells was also examined by using novel GAT-specific, nonsecretor B cell hybridomas produced by fusing GAT-primed spleen cells to the HAT sensitive Balb/c lymphoma, M12.4.5. The hybridomas selected for these studies were GAT-specific, sIg+, Ia+ cells. These hybridomas presented GAT to GAT-specific T cells more efficiently than heterogeneous B cells suggesting that interaction with surface Ig receptors facilitated the uptake and/or processing of antigen. GAT-specific B cell hybridomas, like normal B cells, presented soluble beef insulin to an insulin-specific T cell clone nonspecifically. However, after pulsing with antigen overnight, the GAT-specific B cell hybridoma could activate only GAT-specific T cells.

Influence of recombinant human interleukin-2 administration on lymphocyte and neutrophil function in clinically normal and dexamethasone-treated cattle.

Recombinant human interleukin-2 (rhIL-2) was evaluated for its influence on total and differential WBC counts, lymphocyte blastogenic responsiveness to mitogens, and several measurements of neutrophil function in clinically normal and in dexamethasone-treated cattle. A single dose of rhIL-2 (2.5 X 10(7) U) given SC had no influence on the total or differential WBC count; however, it did cause an inhibition of neutrophil random migration. The other measurements of neutrophil function (Staphylococcus aureus ingestion, cytochrome C reduction, iodination, and antibody-dependent and antibody-independent cell-mediated cytotoxicity) evaluated were not significantly altered. The rhIL-2 treatment was associated with a significant (P less than 0.01) decrease in uptake of [3H]thymidine in unstimulated lymphocytes and a tendency toward enhanced blastogenesis of lymphocytes stimulated with phytohemagglutinin. This enhancement was significant (P less than 0.05) only when the results were expressed as a stimulation index. Lymphocyte responsiveness to concanavalin A and pokeweed mitogen was not significantly influenced by rhIL-2 administration. Dexamethasone (0.04 mg/kg) administered every 24 hours for 3 consecutive days altered the WBC count and several measurements of lymphocyte and neutrophil function. The administration of a single dose of rhIL-2 (2.5 X 10(7) U) 8 hours after the first dose of dexamethasone did not alter the influence of dexamethasone on any of the measurements. These results indicated that rhIL-2 has some biologic activity in cattle, but when used as administered here, did not overcome the influence of dexamethasone on the in vitro measurements of lymphocyte and neutrophil function that were evaluated.

Cloned bovine cytolytic T cells recognize bovine herpes virus-1 in a genetically restricted, antigen-specific manner.

The inability to demonstrate bovine herpes virus-1 (BHV-1)-specific lymphocyte responses from BHV-1-infected cattle has been a major difficulty in confirming the importance of cellular effector mechanisms during BHV-1 infection. We have examined the capacity of bovine cytolytic T-cell clones to lyse BHV-1-infected, concanavalin A-stimulated blast cells. Cytolysis was as high as 58% at an effector to target (E:T) ratio of 1:1. All cytolytic T-cell clones produced were genetically restricted in killing cells of the autologous genotype. Cytotoxic T-lymphocyte (CTL) clones were specific for BHV-1 but not related herpes viruses, i.e. BHV-2 and pseudorabies virus. These results provide evidence that cytolytic T lymphocytes have an antigen-specific role in the immune response of cattle against BHV-1, and that CTL may serve as effector cells in the identification of glycoproteins useful in recombinant vaccine preparation since only determinants of BHV-1 were recognized.

Antigen presentation by the BCL1 murine B cell line: in vitro stimulation by LPS.

We examined the antigen-presenting capacity of BCL1 tumor cells, which are capable of differentiating in vitro with respect to immunoglobulin synthesis/secretion under the influence of LPS. In vivo passaged BCL1 cells depleted of host cell contamination either by positive selection employing panning with anti-lambda reagents, or by elimination of latex-ingesting adherent cells, are capable of MHC-restricted antigen presentation to a GAT-immune T cell line. The BCL1 cells act as antigen-presenting cells when freshly explanted, but gradual loss of this function occurs, and cells cultured for 3.5 days cannot present antigen unless LPS is included during the culture period. BCL1 cells are equivalently Ia+ after the culture period with or without LPS stimulation. Other B cell lines capable of antigen presentation appear to express this trait constitutively, and the in vivo passaged BCL1 line is therefore unique among B cell lines in having antigen-presenting cell function that can be modulated. The data suggest that freshly explanted or LPS-cultured BCL1 cells are heterogeneous with respect to antigen-presenting capacity, and the basis for this heterogeneity is being sought. BCL1 offers an opportunity to study requirements for antigen presentation by B cells.

Dry reagent technology for rapid, convenient measurements of blood coagulation and fibrinolysis.

Rapid coagulation and fibrinolysis assays suitable for use with an imprecisely measured sample volume (either whole blood or plasma) have been developed, utilizing a technology based on paramagnetic iron oxide particles (PIOP) that move in response to an oscillating magnetic field. PIOP are combined with appropriate test reagents for clotting and thrombolysis assays and formulated as dry reagents within a capillary test chamber. The minima and maxima of the PIOP oscillations define a two-sided waveform that provides kinetic information on fibrin polymerization and lysis. Subject to the chemistry of the dry reagent formulation, the resulting waveform can be used to define clotting time, lysis onset time, or fibrinogen variables. Applications to one-stage prothrombin time and one-stage activated partial thromboplastin time tests have yielded assays with consistently good correlations with other test methods. Applications to fibrinolysis studies have yielded global assays of thrombolytic activity, in that the assay results reflect the interactions of multiple factors associated with the effectiveness of thrombolytic therapy. Depending on the components utilized in a particular reagent formulation, one can derive information about the activity of such factors as fibrinogen, plasminogen, and related inhibitors, as well as the lytic agent being administered. Use of these assays in a clinical setting should provide a rapid, convenient alternative to conventional testing of coagulation variables and a reliable method for monitoring thrombolytic therapy.

Helper T-cell clones that recognize autologous insulin are stimulated in nonresponder mice by pork insulin.

Murine antibody responses to various species of insulin are under major histocompatibility complex-linked Ir gene control. Beef insulin differs from pork insulin by only two amino acids in the A-chain loop, yet strain C57BL/10 (B10) mice produce insulin-specific antibodies after immunization with beef insulin and fail to produce antibody after stimulation with pork insulin. Nevertheless, pork insulin primes helper T cells in B10 mice that can be demonstrated if insulin-specific Lyt-1-, -2+ suppressor T cells are removed. Not only do the pork insulin-primed helper and suppressor T cells cross-react with autologous insulin, but also rat insulin (the amino acid sequence of which is identical to mouse insulin) elicits functionally identical helper and suppressor T cells. In this report, we demonstrate that in B10 mice the frequency of helper T cells stimulated by pork insulin is equivalent to that stimulated by beef insulin and that helper T-cell clones induced by beef and pork insulin are major histocompatibility complex-restricted T cells that proliferate, produce lymphokines, and provide helper activity after activation. These helper T-cell clones exhibit different antigenic fine specificities: beef insulin-induced clones respond to beef insulin but not pork or autologous insulin, whereas pork insulin-induced clones cross-react with all species of insulin tested, including rat insulin. In addition, the helper activity of cloned pork insulin-specific T cells is abrogated by pork insulin-primed suppressor T cells. These data support the hypotheses that Ir gene control of antibody responses to certain antigens involves mechanisms used for maintenance of self-tolerance.