bluuebunny/crossref_metadata_2025 huggingface dataset:
Protease-modulation of neutrophil superoxide response.
Authors: Kusner D J , King C H
Published: December 2022

Although prior studies with mAb have defined an endogenous chymotrypsin-like protease in the neutrophil (polymorphonuclear leukocyte (PMN)) membrane that is associated with initiation of superoxide response to inflammatory stimuli, it is not known whether extracellular proteases (in the inflammatory milieu) can also influence PMN activation. This study examined the ability of four neutral proteases: cathepsin G, elastase, chymotrypsin, and trypsin, to modify PMN superoxide response to FMLP, PMA, and arachidonate. In response to 1 microM FMLP, PMN treated with cathepsin G, chymotrypsin, or elastase showed 64%, 60%, and 32% increases, respectively, in superoxide generation when compared with control, untreated cells (p less than 0.05 for each). These increments were dependent on intact enzymatic function of the proteases, were greatest when enzyme and stimulus were added concurrently, and persisted after PMN were washed free of enzyme. Enhancement of superoxide response was not stimulus specific; in response to 10 ng/ml PMA, cells treated with cathepsin G showed a 84%, and elastase a 57%, increase in superoxide generation (p less than 0.05 for both) with a marked reduction in the time required for onset of this response. For cell activation with 80 microM arachidonate, treatment with elastase produced a 180% increase in superoxide production (p less than 0.025). Neutrophils incubated with trypsin demonstrated significant decreases in superoxide response to PMA (-34%, p less than 0.05) and arachidonate (-39%, p less than 0.01). The enzymes themselves were not stimuli for superoxide production nor were they scavengers for superoxide in cellfree system. We conclude that local release of the PMN primary-granule neutral proteases, cathepsin G, and elastase within inflammatory sites can augment neutrophil effector function by up-regulating oxidative response to defined inflammatory stimuli. This autocrine/paracrine function may provide a significant increase in antimicrobial activity, but may also enhance the potential for host tissue injury.
Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro.
Authors: Seguin M C , Ballou W R , Nacy C A
Published: December 2022

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.
Origin and fate of IgE-bearing lymphocytes. II. Modulation of IgE isotype expression on Peyer's patch cells by feeding with certain bacteria and bacterial cell wall components or by thymectomy.
Authors: Durkin H G , Chice S M , Gaetjens E , Bazin H , Tarcsay L , Dukor P
Published: December 2022

Mechanisms regulating the appearance of sIgE+ B lymphocytes appear to be lacking in adult germfree (GF) rats in that their Peyer's patches (PP) contain high numbers of cells with sIgE (approximately 15% of total cells), one-half of which simultaneously express sIgA, whereas sIgE+ cells are absent from PP of conventional rats (less than 1%). GF rat PP also contain elevated numbers of sIgA+ cells and decreased numbers of sIgM+ cells, with elevated numbers of sThy-1+ RT 7.1+ Ig- T cells, and reduced numbers of sThy-1- RT 7.1+ Ig- T cells. The cellular composition of PP of GF rats was converted to that resembling a conventional rat within 18 h after either 1) use of standard (unautoclaved) food; 2) feeding with certain bacteria (Clostridium difficile, Corynebacterium pseudodiphtheriticum, Mycobacterium tuberculosis, and Klebsiella pneumoniae), in either live or heat-killed, but not autoclaved form; or with certain bacterial cell wall components: murein (peptidoglycan), and its synthetic derivatives, muramyltripeptide phosphatidylethanolamine and desmethyl-muramyldipeptide, but not with LPS, core lipid A or lipoprotein; there was no effect if any bacterial cell wall component was injected i.v.; or 3) thymectomy. Each procedure resulted in elimination of sIgE+ B cells and normalization of the other surface isotypes, and loss of sThy-1+ RT 7.1+ Ig- T cells and normalization of sThy-1- RT 7.1+ Ig- T cells. Irrespective of treatment, no sIgE+ cells were detected in bone marrow, thymus, other lymphoid organs or blood, excluding the possibility that the elimination of these cells from PP was associated with their redistribution to other sites. Thus, exposure to gut flora and bacterial peptidoglycan components may have resulted in IgE isotype switching, either directly or through the mediation of accessory and/or sThy-1+ RT 7.1+ regulatory T cells. The sites in which sIgE+ B cells are down-regulated appear to be PP.
Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters.
Authors: Kizaki H , Tadakuma T , Odaka C , Muramatsu J , Ishimura Y
Published: December 2022

Calcium ionophore, A23187, is known to be a comitogen, but it activates a suicide process characterized by DNA fragmentation at linker regions in mouse immature thymocytes. It did not induce DNA fragmentation in T lymphocytes prepared from lymph node and spleen cells. Induction of DNA fragmentation by A23187 depends on protein phosphorylation and synthesis of mRNA and protein, because an inhibitor of protein kinase, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7), actinomycin D, and cycloheximide, respectively, inhibits the DNA fragmentation and cell death. Studies adding the inhibitors at various times show that protein phosphorylation and mRNA synthesis occur within a few hours after incubation with A23187 followed by the protein synthesis responsible for inducing DNA fragmentation. Phorbol esters, 12-O-tetradecanoyl 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBD), which are capable of activating protein kinase C, also induced similar DNA fragmentation in immature thymocytes, followed by cell death. PBD committed the suicide process after 6 h of incubation, because the DNA fragmentation above the control level was not induced when PDB was removed from the medium before 6 h of incubation. A23187 or a phorbol ester alone induced DNA fragmentation followed by cell death, whereas the addition of TPA at low concentration inhibited the DNA fragmentation induced by A23187 accompanied with an increase in DNA synthesis. The result suggests that TPA switched a suicide process induced by A23187 to an opposite process: stimulation of DNA synthesis. Physiologic factors and mechanisms which regulate cell proliferation and death in the thymus are not known at present, but the signals by protein kinases and calcium ions may regulate both cell proliferation and death, independently, synergistically or antagonistically.
Two better cell lines for making hybridomas expressing specific T cell receptors.
Authors: White J , Blackman M , Bill J , Kappler J , Marrack P , Gold D P , Born W
Published: December 2022

Two variants of the AKR thymoma BW5147 have been isolated which can no longer express functional TCR alpha- and beta-chains. By generating hybridomas with these variant fusion lines, TCR of any normal T lymphocyte, including TCR-gamma/delta, can be studied at a clonal level, without interference of the BW5147-derived receptor chains. In this study one of the variants has been useful in identifying the reactivity to allogeneic MHC Ag of BW5147 itself.
Inhibition of erythroid progenitor growth is mediated by cytotoxic lymphocytes and not by natural killer cells or IFN-gamma.
Authors: Torok-Storb B , Graf L , Shankar P
Published: December 2022

Alloantigen primed T cells (PTC) were recovered from MLR at day 6 and 12, then added to cultures of erythroid progenitors, erythroid burst-forming units, BFU-E. The PBMC source of BFU-E was prepared either to retain or deplete APC by treatment with appropriate mAb and C. BFU-E grown in cocultures were counted at day 14 and replicate cultures assayed for IFN-gamma production on days 1 to 7. Analysis of MLR cells indicated that large, rapidly cycling cells recovered from MLR at day 6 have significant NK activity, whereas CTL activity is minimal, and production of IFN-gamma requires reexposure to APC. The smaller, noncycling cells recovered from MLR at day 12 have comparable NK activity, also require reexposure to APC for IFN-gamma production, but in addition have significant CTL activity. The addition of day 12 MLR cells to BFU-E cultures results in MHC restricted inhibition of BFU-E growth, suggesting that the CTL activity and not the NK activity contained within this population of cells is responsible for BFU-E inhibition. Studies using enriched population of BFU-E indicated that appropriate APC are needed to trigger both IFN-gamma production and BFU-E inhibition by the PTC. By using various APC-BFU-E combinations it was determined that after reexposure of PTC to appropriate APC, the inhibition of BFU-E was still target-specific indicating a direct effect between the PTC and BFU-E.
An IL-1 inhibitor from human monocytes. Production and characterization of biologic properties.
Authors: Arend W P , Joslin F G , Thompson R C , Hannum C H
Published: December 2022

Previous studies have described a 22 kD IL-1 inhibitor in the supernatant of human monocytes cultured on adherent immune complexes (J. Immunol. 134:3868, 1985). The studies reported herein further detail the conditions of production and biological properties of this IL-1 inhibitor. The inhibitor was produced by human monocytes cultured on adherent human IgG with maximal production between 8 and 24 hr. The IL-1 inhibitor was not performed in the cells but required transcription and new protein synthesis. The inhibitor blocked IL-1 augmentation of PHA-induced murine thymocyte proliferation but not IL-2-induced stimulation of CTLL or HT-2 cell lines. In addition, the inhibitor blocked IL-1-stimulated collagenase production from rabbit articular chondrocytes and IL-1-induced PGE2 production from human fibroblasts and synovial cells. The IL-1 inhibitor was not transforming growth factor beta (TGF beta) as determined by: the failure of anti-TGF beta antibodies to reduce IL-1 inhibitory activity, the separation of TGF beta from the IL-1 inhibitor by ion exchange chromatography, and the failure of TGF beta to inhibit IL-1-induced PGE2 production from synovial cells. IL-1 and the inhibitor showed no immunological cross-reactivity by Western blot analysis. The inhibitor specifically blocked binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1. These results indicate that a specific inhibitor of IL-1-induced immune and inflammatory cell responses is produced by monocytes cultured on adherent immune complexes or adherent IgG. This IL-1 inhibitor may be of importance in modulating the effects of IL-1 in the monocyte microenvironment.
Transcellular lipoxygenase metabolism between monocytes and platelets.
Authors: Bigby T D , Meslier N
Published: December 2022

We have examined the effects of co-culture and in vitro co-stimulation on lipoxygenase metabolism in monocytes and platelets. Monocytes were obtained from the peripheral blood of normal volunteers by discontinuous gradient centrifugation and adherence to tissue culture plastic. Platelets were obtained from the platelet-rich plasma of the same donor. When 10(9) platelets and 2.5 x 10(6) monocytes were co-stimulated with 1 microM A23187, these preparations released greater quantities of 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid, 5(S),12-(S)dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, and leukotriene C4, 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic (LTC4) when compared with monocytes alone. Release of arachidonic acid, 5-HETE, delta 6-trans-LTB4, and delta 6-trans-12-epi-LTB4 from monocytes was decreased in the presence of platelets. A dose-response curve was constructed and revealed that the above changes became evident when the platelet number exceeded 10(7). Dual radiolabeling experiments with 3H- and 14C-arachidonic acid revealed that monocytes provided arachidonic acid, 5-HETE, and LTA4 for further metabolism by the platelet. Monocytes did not metabolize platelet intermediates detectably. In addition, as much as 1.2 microM 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid and 12(S)-hydroperoxy-10-trans-5,8,14-cis-eicosatetraenoic acid had no effect on monocyte lipoxygenase metabolism. Platelets were capable of converting LTA4 to LTC4, but conversion of LTA4 to LTB4 was not detected. We conclude that the monocyte and platelet lipoxygenase pathways undergo a transcellular lipoxygenase interaction that differs from the interaction of the neutrophil and platelet lipoxygenase pathways. In this interaction monocytes provide intermediate substrates for further metabolic conversion by platelets in an unidirectional manner.
Quantitative analysis of idiotypic mimicry and allelic exclusion in mice with a mu Ig transgene.
Authors: Rath S , Durdik J , Gerstein R M , Selsing E , Nisonoff A
Published: December 2022

Analysis of C57BL/6 mice (IgM allotype, Igh-6b or mu b) that carry an Ig H chain transgene of a different allotype (mu a) shows that IgM molecules of mixed allotype (mu a mu b) are present among serum antibodies. The finding was extended to hybridomas prepared from nonimmune transgenic mice, many of which also failed to exhibit allelic exclusion. The proportions of mu a and mu b secreted by individual hybridomas varied markedly, and the product of an individual hybridoma was found to be heterogeneous with respect to the allotype content of individual molecules. The ratio of mu a:mu b chains secreted by individual hybridomas was found to correlate with the number of transgene copies remaining in each hybridoma, and several hybridomas that secrete only mu b-positive molecules had apparently lost all but one copy of the transgene. An idiotype characteristic of the transgene was found to be present only in association with the transgenic (mu a) allotype, and indirect evidence strongly suggests that the idiotype was present only on mu a polypeptide chains. Thus, there is no evidence in this system for the induction of idiotypically cross-reactive endogenous molecules.
Characterization of anti-CD8-resistant cytolytic T lymphocytes induced by multivalent cross-linking of CD8 on precursor cells.
Authors: McCarthy S A , Kaldjian E , Singer A
Published: December 2022

The lytic activity of most CD8+ MHC class I allospecific CTL generated in vitro can be inhibited by anti-CD8 antibodies. Such inhibition has led to hypotheses that CD8/class I interactions normally contribute to the triggering of CTL with low or moderate avidity Ag-specific TCR by providing those CTL with auxiliary binding avidity. However, CD8 has also been proposed to play an active signaling role in T cell activation. We have recently reported that multivalent cross-linking of CD8 on CTL precursors in MLC does appear to mediate activation signals, and induces the generation of CD8+ MHC class I allospecific CTL whose lytic activity cannot be blocked by anti-CD8 antibodies. In our present study, we have further characterized such anti-CD8 uninhibitable effector cells. These CTL are resistant to blocking of their lytic function by anti-Lyt-3 mAb as well as anti-Lyt-2 mAb, but remain sensitive to blocking by anti-LFA-1 mAb, indicating that they do use non-CD8 cell adhesion molecules during target cell recognition and lysis. As a consequence of mAb-induced multivalent CD8 cross-linking during their generation, anti-CD8 uninhibitable CTL significantly reduce their cell surface expression of CD8, which permits their identification and facilitates their purification from heterogeneous MLC populations. Such anti-CD8 uninhibitable effector cells can be maintained as stable CTL lines, in the absence of anti-CD8 mAb after the initial induction period. The in vitro generation of anti-CD8 uninhibitable CTL, which may be highly enriched for cells bearing high affinity TCR, could represent a new experimental approach to studies of TCR gene usage and repertoire, as well as a potentially important strategy for the deliberate generation of high affinity effector cells for adoptive immunotherapy.
Expression and function of the UM4D4 antigen in human thymus.
Authors: Fox D A , Chan L S , Kan L , Baadsgaard O , Cooper K D
Published: December 2022

UM4D4 is a newly identified T cell surface molecule, distinct from the Ag receptor and CD2, which is expressed on 25% of peripheral blood T cells, resting or activated. Monoclonal anti-UM4D4 is mitogenic for T cells and T cell clones. Since alternative activation pathways independent of Ag/MHC recognition may be important in thymic differentiation, the expression and function of UM4D4 was examined in human thymus. UM4D4 was found on the surface of 6% of thymocytes. All thymocyte subsets contained UM4D4+ cells but expression was greatest on thymocytes that were CD1- (12%), CD3+ (11%) and especially CD4-CD8- (18%). CD3+CD4- CD8- cells, most of which bear the gamma delta-receptor, were greater than or equal to 50% + for UM4D4. Moreover, anti-UM4D4 was comitogenic for thymocytes together with PMA or IL-2. Anti-UM4D4 also reacted strongly with a subset of thymic epithelial cells in both cortex and medulla. Dual color fluorescence microscopy, with anti-UM4D4 and antibodies to other thymic epithelial Ag, showed UM4D4 expression on neuroendocrine thymic epithelium but not on thymic fibrous stroma. Thus, UM4D4 is expressed on, and represents an activation pathway for, a subset of thymic T cells. In addition, this determinant, initially identified as a novel T cell activating molecule, is broadly expressed by neuroendocrine thymic epithelium. Although the function of UM4D4 on the thymic epithelial cells is not yet clear, it is possible that UM4D4 represents a pathway for the functional activation of a subset of the thymic epithelium as well as a subset of thymocytes, thus playing a dual role in T cell differentiation.
Induction of multinucleated giant cell formation from human blood-derived monocytes by phorbol myristate acetate in in vitro culture.
Authors: Hassan N F , Kamani N , Meszaros M M , Douglas S D
Published: December 2022

Multinucleated giant cells (MGC) of mononuclear phagocyte origin occur in different tissues in various inflammatory states and pathological conditions. Although MGC are believed to be derived from monocyte-derived macrophages by fusion, their mechanism of formation is not known. In this study, we investigated the role of PMA, a protein kinase C activator, in the induction and formation of MGC from blood monocyte-derived macrophages in in vitro culture. The addition of PMA (1 x 10(-9) to 8 x 10(-8) M) to 3-wk-old cultures of blood-derived monocytes induces cell fusion with a 30% to 80% fusion rate. Moreover, IFN-gamma-treated blood-derived monocyte cultures showed an additional enhancement of fusion rate with the addition of PMA. 1(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a protein kinase inhibitor, inhibited the formation of macrophage-derived giant cells when added before phorbol ester and IFN-gamma. These findings suggest that protein kinase C may play an important role in the formation of macrophage-derived MGC.
Cell-mediated suppression of HIV-specific cytotoxic T lymphocytes.
Authors: Joly P , Guillon J M , Mayaud C , Plata F , Theodorou I , Denis M , Debre P , Autran B
Published: December 2022

CTL specific for HIV have been described in lungs of infected patients at early stages of HIV disease. In order to characterize the evolution over time of HIV-specific CTL, we have analyzed the cytotoxic function and the cell surface phenotype of the alveolar lymphocytes from 41 patients at various stages of HIV disease. We demonstrated a progressive decline of alveolar anti-HIV CTL activity and detected Ts cells from the lungs of patients with advanced HIV disease. These alveolar T cells strongly suppressed the effector phase of anti-HIV CTL lysis. They lacked a marked specificity of function because they also block anti-HLA CTL response and were not restricted by the HLA-class-I transplantation Ag. They displayed the CD3, CD8, and HNK1 markers, were CD4 and CD16 negative, and lacked NK activity. The presence of Ts cells at late stages of HIV disease could thus partly explain the inefficiency of host defenses against HIV.
Effective production of monoclonal antibodies against phosphatidylserine: stereo-specific recognition of phosphatidylserine by monoclonal antibody.
Authors: Umeda M , Igarashi K , Nam K S , Inoue K
Published: December 2022

A system based on the direct immunization of phospholipid Ag into mouse spleen has been used to produce mAb against phosphatidylserine (PS). mAb that bind to PS but not to phosphatidylcholine were selected. Remarkable frequency of the production of mAb against PS was observed with the immunization protocol. The mAb exhibited three distinct reactivity profiles ranging from highly specific to broadly cross-reactive. Among 61 hybridomas, 15 mAb were established for further analysis. The reactivities of three typical mAb, designated PS4A7, PS3A, and PSC8, are described. PS4A7 is highly specific to PS and no cross-reaction with other acidic phospholipids was observed. In the experiments using PS derivatives with a modified polar head group, PS4A7 was shown to bind to 1,2-diacyl-sn-glycero-3-phospho-L-serine (PS) but not to 1,2-diacyl-sn-glycero-3-phospho-D-serine or 1,2-diacyl-sn-glycero-3-phospho-L-homoserine, indicating that the antibody recognizes the stereo-specific configuration of serine residue in PS. PS3A binds to both PS and phosphatidylethanolamine, whereas no cross-reaction with other acidic phospholipids was observed. The analysis using the derivatives of PS and phosphatidylethanolamine shows that the antibody recognizes the amino group of the phospholipid Ag and cannot distinguish the conformational structure of serine residue in PS. PSC8 represents the family of mAb that cross-react considerably with other acidic phospholipids.
Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes.
Authors: Eager K B , Hackett C J , Gerhard W U , Bennink J , Eisenlohr L C , Yewdell J , Ricciardi R P
Published: December 2022

A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and used to infect murine cell lines of fibroblast, mastocytoma and B cell lineages which are able to present antigens to MHC-restricted T cells. Stable cell lines were selected in which the retrovirus vector integrated as a single copy in almost all of the individual cell clones examined. The HA mRNA was shown to be of the expected length by Northern blot analysis, but the levels varied among the cell clones. Although the HA transcript was difficult to detect in any of the retrovirus-infected cell clones derived from fibroblasts, HA Ag was easily detected on the cell surface by cytofluorographic analysis. Significantly, retrovirus-infected clones derived from each cell type were recognized by HA-specific class I and class II MHC-restricted T lymphocytes. HA produced in these cells was able to be acquired, processed, and presented to class II-restricted T cells by additional, non-HA-expressing APC. This indicates that HA endogenously synthesized within these cell lines is available for Ag processing by an exogenous route.
Integration of the transposon-like element ETn upstream of V lambda 2 in the cell line P3X63Ag8.
Authors: Weiss S , Johansson B
Published: December 2022

The cell line P3X63Ag8 and hybridomas obtained with it contain two rearranged V lambda 2 EcoRI fragments--6.8 kb and 7.4 kb in size. Southern blot analysis, molecular cloning, and mapping revealed that the rearrangements are related to one another and are not due to normal VJ joining. Sequence analysis indicated that the rearrangement was due to the integration of the transposon like element ETn upstream of V lambda 2. Because a normal embryonic V lambda 2 is still retained in this cell line one of the rearranged fragments must be due to a secondary event after the aneuploidization of the plasmacytoma. RNA dot blot analysis showed that the transposon is strongly expressed not only in embryonic cells but also in plasmacytomas, B lymphomas and T cell lines.
Rejection of skin allografts by CD4+ T cells is antigen-specific and requires expression of target alloantigen on Ia- epidermal cells.
Authors: Rosenberg A S , Katz S I , Singer A
Published: December 2022

The effector mechanism of skin allograft rejection has been characterized as Ag specific, rejecting cells that express the target alloantigen but sparing those that do not. However, the rejection of MHC class II disparate skin grafts, in which very few cells (Langerhans cells) actually express the target Ia Ag could conceivably proceed by either one of two distinct rejection mechanisms. One possibility is that Ia- cells are destroyed by a sequence of events in which CD4+ T cells, activated by Ia+ LC, elaborate soluble factors that are either directly cytolytic or that recruit and activate non-specific effector cells. The alternative possibility is that activated CD4+ T cells elaborate soluble factors which induce Ia expression on Ia- cell populations, and that these Ia+ cells are subsequently destroyed by effector cells specific for the induced Ia alloantigens. We found that rejection of Ia+ LC was not of itself sufficient to cause rejection of skin grafts, indicating that skin allograft rejection is contingent on the destruction not only of LC but of other graft cell populations as well. We then investigated whether CD4+ T cells rejected allogeneic skin grafts in an antigen specific fashion. To do so, we engrafted immunoincompetent H-2b nude mice with trunk skin grafts from B6----A/J allophenic mice because such skin is composed of mutually exclusive cell populations expressing either H-2a or H-2b histocompatibility Ag, but not both. The engrafted mice were subsequently reconstituted with H-2b CD4+ T cells. The CD4+ T cells destroyed keratinocytes of A/J origin but spared keratinocytes of B6 origin, even though neither cell population constitutively expresses target IAk alloantigen. The targeted rejection of A/J keratinocytes but not of B6 keratinocytes indicates that the target Ia alloantigen must have been induced on Ia- A/J keratinocytes, rendering them susceptible to destruction by anti-Iak-specific CD4+ effector cells. These data demonstrate that CD4+ T cell rejection of skin allografts is mediated by Ag-specific CD4+ cytolytic T cells and hence, requires the induction of target Ia alloantigens on epidermal cells within the graft.
Properties and heterogeneity of human fetal pre-B cells transformed by EBV.
Authors: Hui M F , Lam P , Dosch H M
Published: December 2022

A series of 75 EBV-transformed pre-B and B-cell lines from fetal bone marrow at 14 to 18 wk of gestation was cloned for phenotypic, functional and molecular genetic studies. B-cell type volume regulation in response to hypotonic stress, low level CD9 (BA2), and, perhaps biased by our use of EBV, functional EBV receptors with expression of CD21 (B2) determinants characterized the most immature cells detected. These "B-progenitors" contained EBV genomes, maintained Ig H and L chain (as well as TCR) constant region genes in germ-line configuration and did not express other B, T, or myeloid lineage-associated surface markers including CD20 and MHC class II determinants. Such cells may represent B progenitor cells preceding classical pre-B-lymphocytes in pathways of B cell differentiation. Reminiscent of Abelson virus-induced transformation of immature murine B cells, EBV transformability together with the above properties may be the earliest markers of B lineage commitment in man. The expression of MHC class II Ag, CD20, C mu-H and then L chain rearrangements and expression followed in less immature pre-B lymphocytes and permitted a classification of lines into discrete subgroups of increasing maturity. The physical organization of the H chain locus in a given line was a stable characteristic. However, fetal pre-B cell lines showed considerable intraclonal heterogeneity with respect to H chain gene expression and that of differentiation markers such as CD20. Subcloning experiments indicated that this heterogeneity reflected clonally stable expression patterns distributed among subclones in an all-or-none fashion. The induction, by IL-6, of L chain expression in some but not all of these clones was linked to the presence of C mu transcription products, consistent with a possible regulatory role of mu protein in L chain rearrangement and expression. Although pre-B cell differentiation likely follows inherent programming, external signals seem able to hasten development along prescribed, hierarchical differentiation pathways.
In vitro responses to a 65-kilodalton mycobacterial protein by synovial T cells from inflammatory arthritis patients.
Authors: Gaston J S , Life P F , Bailey L C , Bacon P A
Published: December 2022

Bacterial Ag, especially those of mycobacteria, have been implicated in the pathogenesis of experimental inflammatory arthritis in rodents, while in man, reactive arthritis has a clear temporal relationship to infection with particular bacteria. To investigate the role of immune responses to bacterial Ag in inflammatory arthritis, we have examined the proliferative responses of paired synovial fluid and PBMC when stimulated with 1) suspensions of irradiated or heat-killed bacteria associated with reactive arthritis (ReA), 2) purified protein derivative, 3) a recombinant 65-kDa heat shock protein of Mycobacterium leprae. The 65-kDa Ag was stimulatory to synovial fluid mononuclear cells, but not PBMC, from patients with different arthropathies, including most of those with ReA, but also some with rheumatoid arthritis. Furthermore, the magnitude of these responses correlated more closely with responses to ReA-associated bacteria (such as Salmonella), than with responses to the mycobacterial Ag represented in purified protein derivative. These results suggest that the 65-kDa molecule, which is common to a wide range of bacteria, may be an important immunogen for the T cell-mediated immune responses within the joint in different clinically defined inflammatory arthropathies.
Prevention of murine cryoglobulinemia and associated pathology by monoclonal anti-idiotypic antibody.
Authors: Spertini F , Donati Y , Welle I , Izui S , Lambert P H
Published: December 2022

A murine IgG3 mAb, clone 6-19, derived from non-manipulated autoimmune MRL/MpJ-lpr/lpr mice is a rheumatoid factor specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intraperitoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces, within 18 h, remarkable pathology characterized by skin vasculitis and acute glomerulonephritis associated with cryoglobulinemia. In order to evaluate the possibility of modulating the development of tissue lesions by an anti-Id antibody, we have raised an IgG2b anti-Id mAb specific to the 6-19 mAb. The cryoprecipitation of 6-19 mAb was completely inhibited in the presence of excess amounts of anti-Id mAb in vitro. In vivo, pretreatment of BALB/c mice with anti-6-19 anti-Id mAb inhibited development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was markedly diminished due to enhanced elimination of the 6-19 mAb from the circulation. In contrast, pretreatment with an IgM anti-IgG3 rheumatoid factor mAb neither protected nor aggravated the development of tissue lesions. These results suggest possible implications in the anti-Id treatment of similar vascular diseases in man.
The invariant chain is a phosphorylated subunit of class II molecules.
Authors: Spiro R C , Quaranta V
Published: December 2022

The phosphorylation of the MHC, class II-associated invariant chain (gamma) is demonstrated in human B-lymphoblastoid, melanoma, and histiocytic lymphoma cell lines. Two-dimensional nonequilibrium gel electrophoresis of invariant chain and class II Ag immunoprecipitates isolated from [32P]orthophosphate-labeled cells demonstrates labeling of both free and class II-associated gamma, gamma s, and p41 forms of the invariant chain. The gamma 2/gamma 3 form of the invariant chain is not phosphorylated. Phosphoamino amino acid analysis of isolated invariant chain shows phosphorylation of serine residues. The isolation of invariant chain from 32P-labeled microsome preparations digested with proteinase K demonstrates that the phosphorylation occurs in the cytoplasmic tail. Limited proteolysis of [32P]orthophosphate-, [35S]cysteine-, and [35S]methionine-labeled invariant chain also indicates that the 32P-label is incorporated into the cytoplasmic domain. These results pinpoint serine residues at positions 9, 26, and 29 in the N-terminal cytoplasmic tail as potential sites for the phosphorylation of the invariant chain. Phosphorylation may be another mechanism by which the functions of invariant chain in class II-dependent immune responses are regulated.
A protective monoclonal antibody recognizes an epitope in the carboxyl-terminal cysteine-rich domain in the precursor of the major merozoite surface antigen of the rodent malarial parasite, Plasmodium yoelii.
Authors: Burns J M , Majarian W R , Young J F , Daly T M , Long C A
Published: December 2022

The 195-kDa major merozoite surface antigen of Plasmodium falciparum (Pf PMMSA) is a potential candidate for the development of a blood-stage malarial vaccine. We have focused on an analogous 230-kDa Ag of the rodent malarial parasite, Plasmodium yoelii, in an effort to study this protein in an experimental model system. Previously we reported the cloning and sequencing of a 2.1-kb portion of the gene encoding the carboxyl-terminal 77 kDa of the Py PMMSA. This region contained the B cell epitope recognized by mAb 302, a mAb shown to protect mice passively against P. yoelii challenge infection. To localize this B cell epitope, we have inserted various restriction fragments of the cloned Py PMMSA sequence into the bacterial expression vector pMG27NSTerm. Recombinant peptides of 74, 40, 34, 17, and 10 kDa have been produced which bear the epitope recognized by mAb 302. The results demonstrate that this B cell epitope is located within the most carboxyl-region of the Py PMMSA which contains a series of ten cysteine residues, also found in the PMMSA of P. falciparum. Further analysis showed that the reduction of disulfide bonds as well as the deletion of CYS-607 of the cloned sequence, resulted in the loss of the expression of this epitope. It is of interest that this epitope does not appear to be a dominant B cell determinant of the Py PMMSA molecule during infection. Inasmuch as this cysteine-rich domain of Py PMMSA displays considerable homology with that of Pf PMMSA, our data suggest that this region of the Pf PMMSA should be considered for inclusion in the development of a blood-stage vaccine.
Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes.
Authors: Gómez-Márquez J , Dosil M , Segade F , Bustelo X R , Pichel J G , Dominguez F , Freire M
Published: December 2022

A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.
Expression of B cell surface receptors. II. IL-4 can accelerate the developmental expression of the murine B cell IgE Fc receptor.
Authors: Waldschmidt T J , Conrad D H , Lynch R G
Published: December 2022

The ability of IL-4 to influence the developmental expression of the murine B cell IgE Fc receptor (Fc epsilon R) was examined. Spleen cells from neonatal mice of increasing age were incubated overnight with IL-4 and subsequently examined with multicolor flow cytometry. The results demonstrate that IL-4 can significantly increase the number of maturing B cells which express the Fc epsilon R. This effect was only seen however, on those neonatal B cells which already displayed surface IgD. Splenic B cells which were IgM+, IgD- failed to express the Fc epsilon R when treated with IL-4, even though they responded by increasing their level of class II Ag expression. Further experiments showed that the inability of IgD- immature B cells to express the Fc epsilon R could not be entirely explained by their assignment to the Ly-1 lineage. Taken together, these results indicate that IL-4 can accelerate the developmental expression of the B cell Fc epsilon R, but only on those B cells that are mature enough to express IgD.
Down-modulation of MHC-I in a CD4+ T cell line, CEM-E5, after HIV-1 infection.
Authors: Scheppler J A , Nicholson J K , Swan D C , Ahmed-Ansari A , McDougal J S
Published: December 2022

HIV-1 is capable of infecting many different cell types that express the CD4 molecule. In vivo and in vitro this infection is associated with profound immunologic defects. We have examined the effect of HIV-1 infection on the expression of MHC class I (MHC-I) molecules to explore the possibility that this important immune system molecule is perturbed after HIV-1 infection. Our data show that in vitro, HIV-1 infection of CD4+ PBL, and the CD4+ cell lines, CEM-E5, HT, and U937, results in decreased expression of MHC-I molecules on the cell surface. This down-modulation is transient, occurring 18 h after HIV-1 infection of CD4+ PBL and returning to normal expression by 24 h. In CEM-E5, MHC-I down-modulation occurs over the course of days, reaching its greatest decrease (40%) about the time the cells are producing the most virus. Reversal of MHC-I expression to normal levels occurs as viral production decreases. Down-regulation during the time periods examined appear to be specific for MHC-I and does not occur with other cell-surface Ag nor is it caused by selection of a preexisting cell population with low MHC-I expression. Radioimmunoprecipitation of MHC-I protein from CEM-E5 indicated that the decrease of surface MHC-I is caused by decreased total protein secondary to a decrease in the level of mRNA for MHC-I. These decreased levels of MHC-I are biologically relevant because HIV-1 infected CEM-E5 cells are less susceptible to CTL lysis determined by the use of MHC-I cytolytic T cell clones and with the use of cold target-inhibition assay.
Mechanism of action of anti-IL-2R monoclonal antibodies. ART-18 prolongs cardiac allograft survival in rats by elimination of IL-2R+ mononuclear cells.
Authors: Tanaka K , Hancock W W , Osawa H , Stunkel K G , Alberghini T V , Diamantstein T , Tilney N L , Kupiec-Weglinski J W
Published: December 2022

ART-18, a mouse IgG1 mAb recognizing the IL-2 binding domain of the rat p55 subunit IL-2R molecule, prevents graft rejection in various experimental models, although its mechanism of action in vivo, like that of anti-IL-2R mAb generally, remains elusive. These studies were designed to define whether IL-2R+ T effector cells were actually eliminated or their function merely inhibited by comparing directly the in vitro and in vivo efficacy of intact ART-18 and its F(ab)/F(ab')2 fragments. Addition of each mAb preparation profoundly suppressed MLR set up between naive LEW responders and x-radiated BN stimulators, suggesting that mAb fragments retained Ag binding functions in vitro. However, both ART-18 F(ab) and F(ab')2 were ineffectual in vivo as judged by their inability to affect acute (8 days) rejection of (LEW X BN)F1 cardiac allografts in LEW recipients (graft survival ca. 11 and 9 days, respectively, compared to ca. 21 days after therapy with intact ART-18, p less than 0.001). The sera levels of ART-18 and ART-18 F(ab')2 were 4 to 5 micrograms/ml, but only less than 0.5 micrograms/ml of F(ab) could be detected. The therapeutic failure of ART-18 fragments was unrelated to potential host sensitization, as rat antimouse F(ab) or F(ab')2 serum IgG titers remained in the same range as those against intact ART-18. The role of the Fc portion of Ig in the mode of action of ART-18 was then tested further by flow microfluorimetry analysis of host mononuclear spleen cells and immunoperoxidase stains of the graft infiltrate. IL-2R+ cells were abundant in rats treated with ART-18 fragments, comparable to acutely rejecting controls. In contrast, IL-2R expression was abolished in animals undergoing ART-18 therapy. The elimination of IL-2R+ cells is required to prolong cardiac allograft survival in rats after IL-2R targeted treatment with ART-18 mAb.
IL-7 promotes thymocyte proliferation and maintains immunocompetent thymocytes bearing alpha beta or gamma delta T-cell receptors in vitro: synergism with IL-2.
Authors: Okazaki H , Ito M , Sudo T , Hattori M , Kano S , Katsura Y , Minato N
Published: December 2022

IL-7 induced the proliferation of normal thymocytes and the effect was synergistically potentiated by a small dose of IL-2, which by itself hardly affected thymocyte proliferation. No synergism was observed between IL-7 and any one of the other lymphokines including IL-1, IL-3, and IL-4. The thymocyte culture stimulated with IL-7 and IL-2 consisted of single positive (CD4+CD8- and CD4-CD8+) and double negative (CD4-CD8-) populations, and double positive (CD4+CD8+) cells were completely deleted. Both single positive and double negative thymocytes expressed CD3, but only the former exhibited V beta 8 and V beta 6 in an expected proportion (approximately 30% in BALB/c mice) and the latter none at all. Immunoprecipitation of the cultured thymocytes by anti-TCR gamma antibody, on the other hand, revealed the presence of a TCR gamma chain. Taken together, these results indicated that the thymocyte cultured with IL-7 and IL-2 consisted of mature T cells bearing alpha beta or gamma delta TCR. Experiments using preselected thymocyte subpopulations indicated that double negative cells responded to both IL-7 and IL-2 with positive synergism when combined, while thymocytes enriched for single positive cells preferentially responded to IL-7 with little response to IL-2 and no detectable synergism. Double positive thymocytes showed no proliferation in response to IL-7 and IL-2. In contrast to single positive thymocytes, splenic T cells hardly responded to IL-7, although significant proliferation was induced in the presence of a low dose of IL-2. Thymocytes cultured with IL-7 and IL-2 showed little nonspecific cytotoxic activity, but responded to Con A or alloantigen, whereas those stimulated with a high dose of IL-2 alone exhibited potent cytotoxic activity. These results indicated that IL-7 was involved in the generation of immunocompetent T cells in the thymus in concert with IL-2.
Treatment of experimental disseminated Mycobacterium avium complex infection in mice with recombinant IL-2 and tumor necrosis factor.
Authors: Bermudez L E , Stevens P , Kolonoski P , Wu M , Young L S
Published: December 2022

Mycobacterium avium complex (MAC) is the most common bloodstream pathogen isolated from patients with AIDS. We have previously shown that TNF alone or in combination with IL-2 can activate human and murine macrophages in vitro to kill MAC strains isolated from disseminated infections. To determine whether treatment with TNF and IL-2 could effect the course of disseminated MAC infections in a murine model of disseminated MAC infection, we infected C57BL mice with 3 x 10(8) bacteria i.v. and 1 wk later administered: 1) IL-2, 100 micrograms/kg; 2) TNF, 25 micrograms/kg; 3) IL-2, 50 micrograms/kg, and TNF, 12.5 micrograms/kg; and 4) saline. IL-2 was injected i.p. daily with TNF being administered in cycles of 3 out of 4 consecutive days. Fourteen days after starting therapy, blood was cultured and mice were sacrificed for quantitative cultures of liver and spleen homogenates. IL-2, TNF, and IL-2/TNF treated groups showed an 87 +/- 5%, 57 +/- 9%, 88 +/- 6% decrease in bacteremia (p = 0.05 for TNF-treated animals and less than 0.04 for the other two groups, compared with control). The combination IL-2/TNF was the only treatment that showed a trend toward an absolute decrease in the number of bacteria in the blood. Reduction in colony counts of liver and spleen were 77 +/- 4% and 87 +/- 6%, respectively, for treatment with IL-2, 58 +/- 7% and 87 +/- 5% for TNF, and 60 +/- 10% and 82 +/- 6% for IL-2/TNF, respectively. These results suggest that both cytokines may play a role in the control of Mycobacterium avium infection and that the combination of a half-dose of IL-2 and TNF, despite not showing any greater efficacy, can be less toxic than TNF or IL-2 alone and might be useful for the therapy of disseminated infection.