bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and characterization of antiserum to murine granulocyte-macrophage colony-stimulating factor.
Authors: Mochizuki D Y , Eisenman J R , Conlon P J , Park L S , Urdal D L
Published: December 2022

The expression in yeast of a cDNA clone encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has made possible the purification of large quantities of this recombinant protein. Rabbits immunized with pure recombinant GM-CSF generated antibodies that were shown to be specific for both recombinant GM-CSF and GM-CSF isolated from natural sources. Other lymphokines, including IL 1 beta, IL 2, IL 3, and recombinant human GM-CSF did not react with the antiserum. The antiserum, together with recombinant GM-CSF that had been radiolabeled with 125I to high specific activity, formed the foundation for a rapid, sensitive, and quantitative radioimmunoassay specific for murine GM-CSF. Furthermore, the antiserum was found to inhibit the biologic activities of GM-CSF as measured in both a bone marrow proliferation assay and a colony assay, and thus should prove to be a useful reagent for dissecting the complex growth factor activities involved in murine hematopoiesis.
Xenogeneic antibodies with apparent public idiotypic specificity for anti-Ia.7 antibodies are directed in part against V kappa 21D and E subgroup marker.
Authors: Devaux C A , Pierres M , Epstein S L , Sachs D H
Published: December 2022

Public idiotypes (IdX) expressed on monoclonal antibodies (mAb) against a monomorphic alpha-chain determinant of the I-E molecule (Ia.7 epitope cluster I) have been studied by using xenogeneic anti-Id reagents derived from pig, rabbit, and rat. IdX+ anti-Ia.7 mAb were recently demonstrated to be structurally related by a high frequency expression of the V kappa 21E light chain subgroup. This raised the question of whether V region determinants of the IdX were related to V kappa 21E sequences or whether they were unique to hypervariable regions of Ia.7 binding antibodies. To clarify this question, the possible association between the expression of the public Id (IdX(s)Ia.7) and the presence of V kappa sequences (V kappa 21E and/or J kappa segment) was examined. The reactivity of the anti-Id reagents with a random panel of 28 myeloma products (each containing a light chain from one of the different V kappa 21 subgroups) was studied by assaying the ability of these mAb to inhibit the binding between the anti-Id and anti-Ia.7 mAb. This analysis demonstrates that what has previously been defined as IdX Ia.7 includes determinants shared by V kappa 21E and V kappa 21D light chain V regions. The structures recognized are expressed irrespective of the J kappa segment. In addition, this study demonstrates interspecies variation in immune responses to such V kappa 21E antigenic determinants. Additional IdX components are found on anti-Ia.7 mAb but not on other V kappa 21E or D proteins. Thus V region subgroup considerations have crucial implications for Id characterization. In addition, this work describes the first division of the V kappa 21 subgroup into component parts by a mAb.
Biochemistry of HLA-DRw6: evidence for seven distinct haplotypes.
Authors: Haziot A , Lepage V , Freidel A C , Betuel H , Degos L , Charron D J
Published: December 2022

The DRw6 specificity, which has a frequency of 11% in the Caucasian population, cannot be positively defined, since no monospecific allo-antiserum is available. This particular status among DR specificities led us to study the DRw6 haplotypes at the molecular level. We performed 2D-PAGE analysis of HLA-DR molecules in 44 different DRw6 haplotypes. The data were obtained from six homozygous typing cells, eight families informative for the segregation of the DRw6 haplotype, and 15 unrelated donors. Five unique beta-chain electrophoretic patterns were detected, indicating the existence of five structurally distinct DRw6 beta-chains. Each haplotype expresses one or two beta-chains. The different combinations of the DR beta-chains present in a single haplotype allow to characterize seven unique DRw6 haplotypes. In contrast to what has been previously found for DR2 and DR4, there is no DR beta-chain common to all the DRw6 cells. Correlation of the biochemical data with the recent serologic (DRw13 vs DRw14) and cellular (Dw9, Dw18, Dw19) splits of the DRw6 specificity will be discussed.
Interaction of culture-derived macrophages with the fibroblast-binding domain of fibronectin is a necessary but inefficient signal for fibronectin enhancement of CR1-mediated phagocytosis.
Authors: Bohnsack J F , Takahashi T , Brown E J
Published: December 2022

Human plasma fibronectin (Fn) enhances ingestion of opsonized particles through its interaction with phagocytic cells. To better characterize the site or sites on Fn responsible for this effect, we subjected Fn to limited proteolytic cleavage by chymotrypsin and used affinity and gel filtration chromatography to isolate a 110,000 dalton cell-binding fragment, a 60,000 dalton fragment that bound both heparin and gelatin, and 50,000 and 45,000 dalton fragments that bound to gelatin but not heparin. The cell-binding fragment mediated adhesion and spreading of fibroblasts on glass slides, whereas the heparin-gelatin and gelatin-binding fragments failed to cause fibroblast spreading. At high concentrations, the cell-binding fragment doubled phagocytosis of C4b-coated sheep erythrocytes by human monocyte-derived macrophages, whereas equal concentrations of the other fragments had minimal enhancing effect on phagocytosis. Interestingly, the effect of the cell-binding fragment on CR1-mediated phagocytosis was always less than the effect of intact Fn, even when a 40-fold higher molar concentration of the cell-binding fragment was used. Fab of a monoclonal anti-Fn, HFn 7.1, which recognizes the 110,000 dalton cell-binding fragment of Fn and inhibits fibroblast binding, blocked enhancement of CR1-mediated phagocytosis by intact Fn. Fab of Fn 8, a monoclonal anti-Fn that binds the heparin-gelatin-binding fragment, failed to inhibit the Fn effect. These data suggest that interaction of the macrophage with the cell-binding domain of Fn is critical for the Fn effect on CR1-mediated phagocytosis. In addition, there may be other domains of the Fn molecule that have a role in augmenting the Fn-phagocyte interaction.
Monoclonal T cell responses to two epitopes on a single immunogen controlled by two distinct genes.
Authors: Lai C H , Babu U M , Turchin H A , Maurer P H
Published: December 2022

The fine specificities of immune T cells were studied in a system in which the response to the antigen can involve two immune response (Ir) genes and two epitopes on a single synthetic polypeptide immunogen. The (BALB/c X SJL)F1 (H-2d X H-2s) mice can respond to the random terpolymer poly(Glu55, Lys36, Phe9) (GLPhe) through the H-2d-linked Ir gene (Ir-d) or through the complementing Ir gene (Ir-dxs), which controls the immune response to poly(Glu, Phe), epitopes that are present in GLPhe. Nine groups of monoclonal T cells were obtained from (H-2d X H-2s)F1 mice immunized with GLPhe. These groups were delineated by the differences in major histocompatibility (MHC) restriction on antigen-presenting cells (APC) and the cross-reactions with GPhe or GLT. A unique T cell line was discovered that can react to the three polymers (GLPhe, GLT, GPhe) even though GLT and GPhe immune T cells do not normally show reciprocal cross-reactions. The monoclonal T cells retain helper activities in the Mishell-Dutton culture. Although the activation of T cells is antigen specific and MHC restricted, the subsequent B cell response is nonspecific.
Rat serosal mast cell degranulation mediated by chymase, an endogenous secretory granule protease: active site-dependent initiation at 1 degree C.
Authors: Schick B , Austen K F
Published: December 2022

Exposure at 37 degrees C of rat serosal mast cells (RSMC) to chymase, an endogenous secretory granule serine protease, results in exocytosis as determined by the release of another secretory granule enzyme, beta-hexosaminidase. Chymase-mediated RSMC degranulation does not occur at 1 degree C; however, exposure of RSMC to chymase at 1 degree C followed by the removal of buffer and the resuspension of the cells in buffer alone at 37 degrees C results in exocytosis equivalent to that obtained by direct exposure of RSMC to chymase at 37 degrees C. Maximal chymase-mediated RSMC degranulation at 37 degrees C is Ca2+-dependent and Mg2+-independent. The dose-dependent degranulation-inducing interaction of chymase and alpha-chymotrypsin with RSMC at 1 degree C is Ca2+-independent, whereas subsequent exocytosis at 37 degrees C in new buffer without added enzyme still requires Ca2+. Specific binding of 125I-labeled alpha-chymotrypsin to RSMC does not occur at 1 degree C, implying that the inducing action of chymase is not a simple ligand-receptor binding. The enzyme inhibitors diisopropyl fluorophosphate and lima bean trypsin inhibitor inhibit subsequent exocytosis at 37 degrees C only if they are added within the first 10 min of the interaction of RSMC and chymase at 1 degree C, implying that an active site-dependent inducing event occurs between RSMC and chymase at 1 degree C. Thus, chymase-induced coupled activation-secretion can be divided into a cation- and temperature-independent initiation phase, which is dependent on the active site of exogenously added chymase and a subsequent temperature-dependent and calcium-augmented cellular secretion phase.
Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation.
Authors: Ledbetter J A , June C H , Martin P J , Spooner C E , Hansen J A , Meier K E
Published: December 2022

We have studied the relationship of valency of CD3 stimulation and modulation of the CD3 receptor complex with biochemical and proliferative responses of T cells. Anti-CD3 Fab, as well as F(ab')2 and whole antibody caused rapid modulation of the CD3 antigen, whereas anti-CD3 conjugated to Sepharose did not. In the absence of monocytes, T cells stimulated with anti-CD3 Fab, F(ab')2, or F(ab')2-Sepharose showed differences in their ability to respond to second signals given by PMA, IL 1, IL 2, or antibodies to Tp67 and Tp44. None of the anti-CD3 signals alone caused resting T cells to produce IL 2, and only the Sepharose-bound anti-CD3 F(ab')2 caused T cells to express high levels of functional IL 2 receptors. Anti-CD3 F(ab')2-Sepharose-stimulated T cells produced IL 2 and proliferated in response to each of the second signals. Because anti-CD3-Sepharose did not cause modulation of the CD3 antigen, the ability of the Sepharose-bound antibody to induce T cells to express IL 2 receptors and to respond to individual second signals may be related to lack of modulation rather than valency of binding. Anti-CD3 Fab-stimulated T cells responded to PMA but required combinations of other second signals. T cells stimulated with unmodified anti-CD3 antibody or F(ab')2 fragments responded to PMA but did not respond to any other second signals alone or in combination. Stimulations that resulted in modulation (i.e., anti-CD3 whole antibody, anti-CD3 F(ab')2, or anti-CD3 Fab fragments) caused an increase in cytoplasmic calcium levels in resting T cells but blocked proliferation of T cells in response to mitogenic lectins or CD2 stimulation. Anti-CD3 F(ab')2 on Sepharose, however, did not block T cell proliferation. Whole bivalent anti-CD3 antibody or F(ab')2 fragments, but not monovalent Fab fragments, caused a rapid translation of protein kinase C activity from cytosol to membrane in the Jurkat T cell line. Because all of these modulate the receptor, these data indicate that the functional difference between monovalent and bivalent binding to CD3 is related to antibody valency and not to antigenic modulation. The use of Fab anti-CD3 stimulation that requires combinations of second signals for proliferation allowed an analysis of the functional relationships between IL 1, anti-Tp67, and anti-Tp44.
Isotype-specific immunoregulation; characterization and function of Fc receptors on T-T hybridomas which produce murine IgA-binding factor.
Authors: Kurita T , Kiyono H , Komiyama K , Grossi C E , Mestecky J , McGhee J R
Published: December 2022

Several methods have been used in the present study to characterize Fc receptors (FcR) expressed on T-T hybridomas derived from mouse Peyer's patch T helper (Th) cell clones that preferentially support IgA responses. These T hybridomas (designated Th HA cells) produce IgA-binding factor (IBF alpha) which regulates antigen-dependent IgA responses. The ultrastructure of Th HA cells and the distribution of Fc alpha R on these cell lines were determined by colloidal gold (CG) immunoelectron microscopy (IEM). When Th HA cells were incubated with purified mouse IgA followed by CG-labeled anti-IgA, an even pattern of CG was distributed on the cell membrane. To ensure that binding occurred through Fc alpha R, Th HA cells were mixed with MOPC 315 IgA anti-DNP, followed by staining with CG-labeled TNP-human serum albumin. This resulted in an identical pattern of gold particle distribution, confirming expression of Fc alpha R on Th HA cells. No Fc mu R or Fc gamma 1R were detectable on Th HA cells by IEM. Immunocytoadherence with TNP-conjugated erythrocytes confirmed that Th HA cells were Fc alpha R+; however, no IgM or IgG rosettes were seen. When these cell lines were analyzed by flow cytometry (FACS) using IgA, IgM, or IgG1 and FITC-labeled anti-H chain-specific antibodies, 55 to 65% of cultured Th HA cells expressed Fc alpha R, and 11 to 18% expressed Fc mu R; however, no Fc gamma 1R was detectable on Th HA cells. The use of ELISA with Th HA cells as antigen confirmed the expression of Fc alpha R and the presence of less Fc mu R on these two cell lines. Solubilized membrane fractions derived from Th HA cells were tested for the presence of FcR by ELISA and for biologic function for support of IgA responses in Peyer's patch B cell cultures. Both Fc alpha R and Fc mu R were detected in fractions derived from Th HA cells. Furthermore, these fractions supported in vitro IgA anti-sheep erythrocyte responses, comparable to those obtained with Th HA cell culture supernatants containing IBF alpha. These studies show that Th HA cells express Fc alpha R with less Fc mu R, and the solubilized form of Fc alpha R exhibits IBF alpha-like activity. The significance of FcR expression by Th cell clones and cell lines and the relationship of soluble Fc alpha R and IBF alpha for IgA response regulation are discussed.
An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes.
Authors: Gomard E , Hénin Y , Sterkers G , Masset M , Fauchet R , Lévy J P
Published: December 2022

The clone TA10 is a T3+ T4+ T8- proliferative and cytolytic human T cell clone. This clone has been shown to be specific for the hemagglutinin of influenza A Texas virus and restricted by an HLA class II molecule associated with the DRw8-Dw8.1 phenotype. Here we show that TA10 and all of its subclones can also react with eight HLA-DRw8 negative, Epstein-Barr virus (EBV)-transformed cell lines or phytohemagglutinin blasts in the absence of influenza antigens. All of these cell lines are HLA-DR2/DR4 with a classic DR2 long haplotype. The only nonreactive HLA-DR2/DR4 cell line observed bears a DR2 short haplotype. Only heterozygous HLA-DR2/DR4 but not parental DR2 or DR4 EBV-transformed cell lines can be recognized by TA10, indicating that the cross-reacting determinant is a transcomplementation product between HLA-DR2 and HLA-DR4 haplotypes. DR-specific, but not DQ- or DP-specific monoclonal antibodies, inhibit in the proliferation assay and in the chromium release test both the DRw8-Dw8.1-restricted and the anti-DR2/DR4 reactions. These results show that HLA-DR-restricted, anti-viral human T cell clone can evidence cross-reactivity for allospecific class II molecules of the major histocompatibility complex, and human CTL can recognize transcomplementation products of class II HLA genes. In addition, the results suggest that a beta-chain coded for by an HLA-DR gene and associated with an alpha-chain coded for by a still unidentified but possibly HLA-DQ gene constitute this functional transcomplementation product.
Polyclonal activation of rat B cells. I. A single mitogenic signal can stimulate proliferation, but three signals are required for differentiation.
Authors: Stunz L L , Feldbush T L
Published: December 2022

A water-soluble, proteinaceous preparation derived from the cell walls of Salmonella typhimurium Re mutants has recently been tested in our laboratory for its ability to act as a mitogen for rat lymphocytes. We have found this preparation (STM) to be a potent stimulator of B lymphocyte proliferation, as measured both by 3H-TdR incorporation and by cell cycle analysis performed with flow cytofluorometry. STM stimulates approximately 50% of rat B cells to enter cycle. Previous investigations by others have shown that at least two sets of signals are required for B cell differentiation; a) proliferation signals that may consist of both a stimulator of B cell conversion from G0 to G1 and growth factors, and b) differentiation signals that probably include at least two B cell differentiation factors (BCDF). When STM was tested in a differentiation system it did not drive purified B cells to differentiate to PFC, either alone or when supplemented with a supernatant from concanavalin A-stimulated spleen cells (CAS). However, when both CAS and dextran sulfate (DXS) were supplied to the STM-stimulated cells, a large number of PFC resulted. DXS does not act by stimulating an additional, CAS-responsive B cell subset, since it has only a marginal effect upon 3H-TdR uptake and does not increase the number of B cells in cycle when used together with STM. We postulate that the two agents may be acting sequentially: STM stimulates the B cells to proliferate, and DXS drives the proliferating cells to become responsive to CAS. This suggests that the signals for B cell differentiation must consist of at least three activities: a trigger to stimulate the cells to proliferate, a factor to drive the cells to a BCDF-responsive state, and a BCDF that can drive the cells to secrete antibody.
Cyclosporin A inhibits IL 2-driven proliferation of human alloactivated T cells.
Authors: Buurman W A , Ruers T J , Daemen I A , van der Linden C J , Groenewegen G
Published: December 2022

The influence of cyclosporin A (CsA) on the interleukin 2 (IL 2)-driven proliferation of allo-activated human T lymphocytes has been studied. CsA (50, 100, and 250 ng/ml) appeared to affect the IL 2-driven proliferation. The impaired proliferation could not be reversed with exogenous interferon-gamma. The doubling time of the cell populations appeared to increase with higher CsA doses. The cytotoxic capacity of the cells was also strongly inhibited by CsA. The cells regained proliferative and cytotoxic properties after CsA has been removed and cells were additionally cultured in normal medium. The data strongly suggest that CsA has an inhibitory influence on essential basic processes in T cells.
Monoclonal antibody analysis of MHC expression in human brain biopsies: tissue ranging from "histologically normal" to that showing different levels of glial tumor involvement.
Authors: Lampson L A , Hickey W F
Published: December 2022

The expression of class I and class II MHC products in human brain was studied. Radioimmunoassay confirmed weak expression of HLA-A,B,C and beta 2-microglobulin (beta 2-m) in brain extract. Quantitative inhibition assay showed brain had 1/70 as much activity as spleen, per microgram of extract protein. Immunoblot assay confirmed that HLA chains and beta 2-m were present in the brain extract. Class II was not detected. Microscopic analysis was performed on eight brain biopsies. The histologic appearance ranged from "apparently normal," to the presence of reactive astrocytes, to the presence of glial tumor. In every case, HLA-A,B,C and beta 2-m activity was concentrated at blood vessel walls. Small and medium-sized vessels were uniformly stained. Cell body staining was not seen in neurons, glia, oligodendrocytes, microglia, reactive astrocytes, or the majority of glial tumor cells. Class II activity was seen in occasional cell bodies in both grey matter and white matter in the microscopic assays. These cells had the morphologic appearance of microglia or reactive astrocytes. Occasional blood vessels also showed class II activity. Unlike the class I activity, the class II blood vessel stain was often discontinuous. More class II+ cell bodies were seen in tumor-associated tissue.
Phenotypic, functional, and molecular genetic comparisons of the abnormal lymphoid cells of C3H-lpr/lpr and C3H-gld/gld mice.
Authors: Davidson W F , Dumont F J , Bedigian H G , Fowlkes B J , Morse H C
Published: December 2022

Lymph node cells from C3H mice homozygous for lpr and gld were compared for expression of cell surface antigens, lectin-binding sites, functional characteristics, expression of ecotropic MuLV, and organization of Ig and T cell receptor (TcR) beta-chain genes. The abnormal cells (Ly-2-/L3T4-) populating nodes of both mutant strains were specifically purified by using plate separation techniques. The purified abnormal cells were shown to express the beta-chain of the TcR, to exhibit rearrangements of the beta-chain genes, and to express TcR beta and alpha gene mRNA, demonstrating the T cell origin of these populations. FMF analyses of the separated abnormal cells showed them to be Thy-1+, Ly-1+, Ly-2-, L3T4-, Ly-5(B220)+, Ly-6+, Ly-22+, Ly-24+, sIg-, ThB-, Ia-, HSA-/+, and PC.1+ and to bind at high levels lectins that normally bind preferentially to B cells. These cells did not proliferate or generate CTL in response to stimulation with alloantigens, and supernatants of cells stimulated with Con A were devoid of IL 2. These characteristics do not correspond to those of any known immature or mature population of normal T cells. The findings that the abnormal T cells of lpr and gld homozygotes are indistinguishable for each parameter examined support the suggestion that these mutations may affect different enzymes in a common metabolic pathway of major importance to T cell differentiation and function.
The murine interleukin 2 receptor. IV. Biochemical characterization.
Authors: Malek T R , Korty P E
Published: December 2022

The IL 2 receptor isolated from the IL 2-dependent CTL-L cell line was subjected to biochemical analysis. Pulse-chase and tunicamycin studies, as well as digestion with the endoglycosidases, Endo-F and Endo-H, of 35S-methionine-labeled IL 2 receptors suggested a single protein precursor of 32,000 (p32) daltons. The p32 precursor was rapidly processed by addition of high-mannose-containing core N-linked sugars to intracytoplasmic precursor intermediates of 38,000 (p38) and 40,000 (p40) daltons, which undergo further processing to yield a mature surface receptor with heterogeneous apparent m.w. of 52,000 to 65,000 (p58). Two-dimensional gel studies indicated that p58 exhibited broad charge heterogeneity between pH 4.6 and 6.3. Endo-F digestions of p58 shifted the isoelectric focus point to a more basic 5.5 to 7.4. This considerable charge heterogeneity is consistent with the possibility that other posttranslational modifications to the mouse IL 2 receptor occur besides addition of complex N-linked glycans. Immunoprecipitations of the IL 2 receptor from surface iodinated cells also revealed an additional band at 110,000 (p110) daltons. IEF vs SDS-PAGE two-dimensional gel studies demonstrated that p110 also had an isoelectric focus point identical to p58. Western blot studies with an anti-IL 2 receptor monoclonal antibody (7D4) demonstrated that p38, p40, p58, and p110 each expressed the epitope recognized by this antibody. Thus, it is likely that p110 is not a unique molecule that coprecipitates with the IL 2 receptor. Western blot analysis of mitogen-stimulated T and B lymphocytes also revealed bands similar to p58 and p110, although these bands had an average apparent m.w. 3000 to 6000 less than those seen for CTL-L cells.
T cell receptor triggering induces responsiveness to interleukin 1 and interleukin 2 but does not lead to T cell proliferation.
Authors: Meuer S C , Meyer zum Büschenfelde K H
Published: December 2022

The antigen-like activity of monoclonal antibodies directed at the T3-Ti antigen receptor complex of human T lymphocytes was employed to study activation requirements of resting T cells. Efficient antigen recognition (signal 1) by T lymphocytes requires multimeric antigen receptor triggering because under appropriate experimental conditions soluble ligands do not produce this initial signal for T cell activation. The latter leads to receptiveness for both interleukin 1 (IL 1) and interleukin 2 (IL 2). Importantly, induction of proliferation requires an additional signal (signal 2), namely IL 1, which appears to be required to enable optimal secretion of IL 2. In contrast, presensitized T lymphocytes do not require IL 1 for IL 2 production. In this case, antigen receptor oligomerization is in itself sufficient to induce IL 2 receptor expression, and IL 2 secretion as well.
Biochemical and antigenic analysis of the Epstein Barr virus/C3d receptor (CR2).
Authors: Siaw M F , Nemerow G R , Cooper N R
Published: December 2022

Four monoclonal antibodies (OKB7, HB-5, AB-1, and anti-B2) that recognize a 145-kDa B cell-specific membrane structure have markedly different abilities to 1) inhibit C3d and EBV binding to B cells, 2) immunoprecipitate a 145-kDa B cell protein, and 3) stimulate B cell proliferation and differentiation into Ig-secreting cells. This study was initiated to determine whether these four monoclonal antibodies (MoAb) react with the same protein; a related goal was to determine whether the structure(s) recognized by these antibodies constitutes an antigenically related family of structurally distinct molecules. In the studies presented here, the four MoAb were found to fully immunoprecipitate the purified 145-kDa B cell molecule isolated by immunoaffinity chromatography on either OKB7, HB-5, or AB-1 columns, findings that show conclusively that the antibodies all react with the same B cell protein. The variable ability to immunoprecipitate this B cell membrane protein was found to result from differences in exposure or accessibility of the relevant antigenic epitopes in the detergent extract. The 145-kDa molecule immunoprecipitated with the four MoAb was equivalently sensitive to endoglycosidase F and yielded the same banding pattern after digestion with endoglycosidase F and after partial digestion with either S. aureus V8 protease or with trypsin. Within the limits of the sensitivity of these techniques, therefore, there is no evidence for carbohydrate or protein differences in the EBV/C3d receptor (CR2) molecule recognized by the four MoAb. Additional studies showed that the four MoAb react with distinct and nonoverlapping antigenic epitopes on the 145-kDa molecule. The variable abilities of the four MoAb to inhibit CR2 function and EBV binding and to trigger B cell activation, together with the other findings noted above, indicates that the 145-kDa EBV/C3d receptor possesses discretely localized functional domains.
Location of the interchain disulfide bonds of the fourth component of human complement (C4): evidence based on the liberation of fragments secondary to thiol-disulfide interchange reactions.
Authors: Seya T , Nagasawa S , Atkinson J P
Published: December 2022

Treatment of human C4 with chemical denaturants and heat produces rapid, autolytic peptide bond cleavage of the alpha-chain. These alpha-chain fragments are linked to the parent C4 molecule through disulfide bonds. On more prolonged heating, however, there is liberation of several peptides, including the beta-chain, the gamma-chain, and a C-terminal alpha-chain fragment. This reaction is inhibited by iodoacetamide. By using a fluorescent thiol reagent and 14C-iodoacetamide, the thiol group present on each peptide was analyzed. The results suggest that the thiol residue exposed by cleavage of the thioester bond induces thiol-disulfide interchange reactions to liberate the peptides. Based on the identification of fragments liberated, the kinetics of their appearance, their sulfhydryl content, and the reported primary structure of human C4, a model of the interchain disulfide bonds is proposed in which the amino terminal portion of the alpha-chain is disulfide-linked to both the beta- and gamma-chains, whereas the carboxyl terminal portion of the alpha-chain is disulfide-linked to only the gamma-chain.
Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin.
Authors: Stanker L H , Branscomb E , Vanderlaan M , Jensen R H
Published: December 2022

Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.
Release of leukotrienes C4 and B4 and prostaglandin E2 from human monocytes stimulated with aggregated IgG, IgA, and IgE.
Authors: Ferreri N R , Howland W C , Spiegelberg H L
Published: December 2022

Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins of different classes or the calcium ionophore A23187 and the release of leukotrienes C4 and B4 (LTC4, LTB4), and prostaglandin E2 (PGE2) into the supernatant was determined. The ionophore induced release of 10 +/- 5 ng LTC4/10(6) cells and 25 +/- 8 ng LTB4/10(6) cells. Aggregated IgG, IgA, and IgE, but not IgM or monomeric immunoglobulins (Ig), induced release of LTC4 and LTB4 that was approximately 10 to 20% of that induced by ionophore. In addition, IgG, IgA, and IgE, but not IgM, induced release of PGE2 (range 0.015 to 0.22 ng/10(6) cells). Aggregated Ig induced LTC4, LTB4, and PGE2 release in a dose-dependent manner; maximal leukotriene (LT) release was observed by 30 min, in contrast to PG release, which continued to increase up to 2.5 hr. Both ionophore- and Ig-induced LTC4 and LTB4 release were completely inhibited by removal of calcium from the media and by preincubation of cells with nordihydroguaiaretic acid. Indomethacin inhibited Ig-induced PGE2 release by 80%. Phagocytosis of the Ig aggregates was not required for LT or PGE2 release, since release was not inhibited by cytochalasin B. Release of LTC4, LTB4, and PGE2 induced by IgG, IgA, and IgE, but not IgM, correlated with the presence or absence of monocyte Fc receptors (FcR) as determined by rosette assays. The data suggest that IgG, IgA, and IgE immune complexes mostly likely induce monocyte arachidonic acid metabolism via cross-linking of FcR. The ability of monocytes to release eicosanoids in the absence of phagocytosis suggests that interaction of monocytes with immobilized immune complexes, such as those deposited in blood vessel walls or glomerular basement membranes, could initiate metabolism of arachidonic acid by monocytes. Such a mechanism could contribute to inflammatory reactions characterized by mononuclear cell infiltrates.
Functional activity of different IgG subclass antibodies against type b capsular polysaccharide of Haemophilus influenzae.
Authors: Weinberg G A , Granoff D M , Nahm M H , Shackelford P G
Published: December 2022

The biologic activity of different human IgG subclass antibodies directed against the Haemophilus influenzae type b (Hib) capsular polysaccharide (PRP) was compared by using an in vitro complement-mediated bactericidal assay and an in vivo passive protection assay in infant rats. An IgG pool was made by Sephacryl S-300 chromatography of sera from adults immunized with PRP vaccine. An IgG2 subclass fraction was prepared by column immunoabsorption of the IgG pool with anti-IgG1 monoclonal antibody. An IgG1 subclass fraction was eluted from the affinity matrix. IgG1, IgG2, IgG3, and IgG4 concentrations in the fractions were measured by solid-phase competitive radioimmunoassays, and anti-PRP antibody was measured by a modified Farr assay. Each fraction was greater than 90% pure IgG2 or IgG1, respectively. There were no significant differences in the minimal anti-PRP antibody concentrations required to kill 50% of Hib cells in vitro (IgG, 0.22; IgG1, 0.21; and IgG2, 0.42 microgram/ml). Similarly, equivalent amounts of anti-PRP antibody of the IgG1 or IgG2 fractions protected against bacteremia (IgG1, 0.12; IgG2, 0.24 microgram per rat). IgG absorbed to remove anti-PRP antibody was neither bactericidal nor protective. Thus IgG1 and IgG2 anti-PRP antibody have equivalent functional activities against Hib as determined by these biologic assays.
Characterization of four constant region genes of rabbit immunoglobulin-lambda chains.
Authors: Duvoisin R M , Heidmann O , Jaton J C
Published: December 2022

A cDNA plasmid insert encoding the constant (C) region of a rabbit immunoglobulin-lambda light chain was used as a probe for screening a rabbit liver genomic DNA cosmid library. This allowed the isolation and identification of four distinct C lambda genes, designated C lambda 1, C lambda 2, C lambda 3, and C lambda 4, which were shown to be widely separated from each other along chromosomal DNA. Their nucleotide sequences have been determined. No in-frame termination codons were found within the coding regions. The C lambda 1, C lambda 2, and C lambda 3 sequences are quite similar to each other, but share less homology with the C lambda 4 gene or the cDNA-C lambda sequence used as a probe. The C lambda gene coding for the cDNA sequence was not isolated. Translation of the C lambda 1, C lambda 2, and C lambda 3 sequences predicts a Cys-Pro carboxy-terminal amino acid sequence, as found so far only for horse lambda-chains. Compared to the other rabbit C lambda genes, the C lambda 3 sequence exhibits two deletions, one of 9 bp, the other of 3 bp. The latter occurs at the same position as in the mouse C lambda 2 and C lambda 3 genes. These two deletions are located in the loops between anti-parallel beta-pleated sheets of the C lambda domain. When the C lambda nucleotide sequences from man, mouse, and rabbit are compared, there is less divergence within the same species than for interspecies comparisons. Possible genetic implications of this finding are discussed.
Differential expression of HLA-DR, -DQ, and -DP antigens on malignant B cells.
Authors: Pesando J M , Graf L
Published: December 2022

HLA class II antigens mediate interactions among cells involved in the immune response and play an important role in the process of self recognition. We made use of conventional alloantisera and six well-characterized monoclonal antibodies (MoAb) to study the HLA class II antigens on CALLA-positive malignant B cell populations and autologous normal B cell lines. Forty additional HLA class II-specific MoAb were also tested for their ability to bind to these cells. By using indirect immunofluorescence and immune precipitation assays, we find that malignant B cells often fail to express one or more of the three known types of HLA class II antigens. Cell lines with the following five phenotypes have been identified: HLA-DR+, -DQ+, -DP+; HLA-DR+, -DQ-, -DP+; HLA-DR-, -DQ+, -DP+; HLA-DR-, -DQ-, -DP+; and HLA-DR-, -DQ-, -DP-. These cell lines have been used to characterize the subregion specificity of MoAb that react with HLA class II antigens. This work confirms the existence of complicated patterns of serologic cross-reactivity between the three different types of HLA class II molecules. It also increases our understanding of the specificity of individual MoAb, thereby facilitating future investigation of the distribution and function of individual antigens. Our studies are consistent with the proposal that altered expression of HLA antigens on tumors might impair recognition of these cells by the immune system of the host, thereby contributing to the proliferation of a malignant clone.
Antigen presentation by Langerhans cells in vivo: donor-derived Ia+ Langerhans cells are required for induction of delayed-type hypersensitivity but not for cytotoxic T lymphocyte responses to alloantigens.
Authors: Peeler J S , Niederkorn J Y
Published: December 2022

T cell activation in response to allogeneic stimulation and hapten-specific delayed-contact hypersensitivity responses in vivo can be initiated by Ia-bearing epidermal Langerhans cells (LC). By using a murine heterotopic corneal allograft model, we have investigated the requirement for allogeneic LC as antigen-presenting cells (APC) in the in vivo induction of delayed-type hypersensitivity (DTH) and cytolytic T lymphocyte (CTL) responses to alloantigens in fully allogeneic and H-2 I region-disparate strain combinations. LC-deficient, avascular central corneal allografts from BALB/c donors failed to induce DTH responsiveness when grafted to a subdermal bed on C57BL/6 recipients (p greater than 0.05), yet antigen-specific primary CTL reactivity developed within 7 days after grafting. LC-containing corneal-limbus allografts or central corneal allografts containing a latex bead-induced infiltrate of LC resulted in intense DTH as well as CTL responsiveness when grafted in this same strain combination. Similarly, LC-containing but not LC-deficient corneal allografts from A.TL donors induced DTH responsiveness in I region-disparate A.TH hosts despite the fact that these grafts survived for prolonged duration (less than 28 days). By contrast, CTL induction in I region-disparate hosts was independent of the presence of allogeneic LC. Corneal epithelial cells of grafts removed from I region-disparate hosts 7 days posttransplantation were shown by immunohistology to express the Iak antigens of donor origin. The possibility that bone marrow-derived allogeneic LC were a sufficient requirement for DTH induction was confirmed in experiments performed with CB6F1----B6 bone marrow chimeras used as corneal allograft donors. Corneal-limbus grafts obtained from mice 90 days after chimerization were shown by immunohistology to contain Iad-bearing CB6F1 LC as a sole source of class II alloantigens. When grafted to C57BL/6 recipients, LC-containing chimeric corneas induced DTH responsiveness that was similar in magnitude to that observed in C57BL/6 mice grafted with chimeric skin, yet no DTH response to LC-deficient chimeric central corneal grafts was observed. Moreover, in all cases, the chimeric corneal and skin allografts survived for prolonged duration (greater than 28 days). These results demonstrate that donor-derived LC act as APC in the induction of DTH responsiveness to allogeneic tissue; however, there was no apparent requirement for allogeneic LC in the induction of CTL responses to class I or class II MHC alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of a subset of bovine T lymphocytes that express BoT4 by monoclonal antibodies and function: similarity to lymphocytes defined by human T4 and murine L3T4.
Authors: Baldwin C L , Teale A J , Naessens J G , Goddeeris B M , MacHugh N D , Morrison W I
Published: December 2022

We report the identification of a subset of bovine T cells by two monoclonal antibodies (mAb), IL-A11 and IL-A12, and some functional analyses of these cells. Both mAb precipitate two polypeptides, called BoT4, with apparent molecular mass of 52 and 55 kilodaltons. The epitopes recognized by these two mAb are different, however. BoT4 is found on approximately 70% of thymocytes and 30% of peripheral blood mononuclear cells (PBM), is not expressed by monocytes or B cells, and is found on cells in the T-dependent areas of lymph nodes. BoT4+ lymphocytes purified by a fluorescence-activated cell sorter proliferate in response to mitogenic and alloantigenic stimulation without addition of exogenous growth factors, whereas BoT4- lymphocytes do not. Monoclonal antibodies IL-A11 and IL-A12 have no effect on mitogen- (PHA and Con A) or alloantigen-induced proliferation of PBM. Monoclonal antibody IL-A12 has no inhibitory effect on the cytolytic activity of bulk populations of alloreactive T lymphocytes, and most of the cytolytic activity generated in mixed leukocyte culture is ascribable to the BoT4- population. Using cloned alloantigen-specific lymphocytes, we found that the ability of BoT4+ clones to proliferate to alloantigenic stimuli without exogenous growth factors is a characteristic of some clones, as is susceptibility to inhibition of proliferation by mAb IL-A12. These results implicate the BoT4 molecule in antigen recognition but indicate that the requirement for BoT4 is variable among clones. Our findings, together with those in the companion paper, which provides evidence that BoT4+ lymphocytes are class II restricted, indicate that BoT4+ lymphocytes are the bovine equivalent of Leu3/T4+ lymphocytes of humans and analogous lymphocytes of other species.
Full reconstitution of the immune deficiency in scid mice with normal stem cells requires low-dose irradiation of the recipients.
Authors: Fulop G M , Phillips R A
Published: December 2022

Mice homozygous for an autosomal recessive mutation for the scid gene exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Such mice can be cured of their lymphoid deficiency by grafts with normal bone marrow, although full reconstitution of lymphoid function is seldom obtained. Long-term bone marrow cultures (LTBMC) are devoid of all mature B and pre-B cells but contain lymphoid stem cells. We therefore reconstituted scid mice with LTBMC cells to study the kinetics of B lymphocyte reconstitution in normal and irradiated (4 Gy) scid recipients and in irradiated (9.5 Gy) co-isogenic C.B-17 mice. Detectable colony-forming B cells rapidly increased in the spleen and bone marrow of irradiated C.B-17 and irradiated scid recipients, reaching normal levels between 4 and 6 wk post-grafting. Unirradiated scid recipients showed limited reconstitution in spleen and very poor reconstitution in bone marrow. Unirradiated scid recipients also had relatively few surface Ig+ cells in spleen or bone marrow, whereas both groups of irradiated recipients had normal numbers between 4 and 6 wk post-reconstitution. Normal levels of cytotoxic T cell activity by 8 wk after reconstitution were observed only in the irradiated C.B-17 and irradiated scid recipients. Analysis of mice reconstituted with cells from LTBMC indicates that these cultures contain lymphoid stem cells with significant proliferative and self-renewal potential, and that full reconstitution of lymphoid function requires prior irradiation of the scid recipient.
Characterization of C1q receptor expression on human phagocytic cells: effects of PDBu and fMLP.
Authors: Bobak D A , Frank M M , Tenner A J
Published: December 2022

The receptor-mediated binding of C1q to human phagocytic cells was investigated in this study. By using a C1q binding assay, we determined that purified, elutriated monocytes expressed an average of 4.6 X 10(5) C1q receptors (C1qR) per cell, with an equilibrium binding constant (Keq) of 0.91 X 10(7) (M-1). When analyzed in an identical manner, the polymorphonuclear leukocytes (PMN) expressed an average of 4.2 X 10(5) C1qR per cell, with a Keq for C1q of 1 X 10(7) (M-1). Fluorescent flow cytometric analysis showed that C1q was bound by 98% of the monocytes studied. Further, the pattern formed by these cells was consistent with a normal log distribution, indicating that this was a homogeneous population of cells. When PMN were assayed with flow cytometry, however, we found that C1q was bound by an average of only 45% of the PMN analyzed. Further, these PMN were not dispersed in a normal log distribution, indicating some heterogeneity among the cells that bind C1q. We examined the abilities of the chemoattractant N-formylmethionylleucylphenylalanine (fMLP) and the phorbol ester phorbol dibutyrate (PDBu) to modulate expression of C1qR as compared to the receptor for C3b (CR1). Pretreatment of the monocytes and the PMN with either 10(-6)M fMLP or 10 ng/ml of PDBu significantly increased cell surface CR1 expression, as reported previously by other investigators. In contrast, no significant increase in the number of C1qR on the monocytes or the PMN was observed with any of the concentrations of fMLP or PDBu used during pretreatment. However, with certain pretreatment doses of these agents, some reduction was noted in the amount of 125I-C1q bound to the monocytes and the PMN. This study characterizes the binding of C1q to purified monocytes and confirms previously published values for PMN. The distribution of cells expressing C1qR is shown to be significantly different between identically treated populations of monocytes and PMN. Finally, the abilities of fMLP and PDBu to modulate the binding of C1qR are examined. Our results indicate that the control of C1qR expression differs markedly from that of CR1.