bluuebunny/crossref_metadata_2025 huggingface dataset:
Human T cell leukemia/lymphoma virus type I infection of a CD4+ proliferative/cytotoxic T cell clone progresses in at least two distinct phases based on changes in function and phenotype of the infected cells.
Authors: Yssel H , de Waal Malefyt R , Duc Dodon M D , Blanchard D , Gazzolo L , de Vries J E , Spits H
Published: December 2022

The effect of human T cell leukemia/lymphoma virus type I (HTLV-I) infection on the function and the phenotype of a human proliferating/cytotoxic T cell clone, specific for tetanus toxin, was investigated. During the period after infection, two distinct phases were observed, based on growth properties, phenotype, and functional activity of the infected cells. Phase I HTLV-I infected cells (0 to about 150 days after infection) proliferated in an IL-2-dependent way, but without the requirement for repetitive antigenic stimulation. No differences in expression of the CD2, CD3, CD4, Tp103, and CD28 Ag between these cells and the parental cells could be demonstrated, with the exception of the expression of IL-R p55 and HLA-DR Ag, which were constitutively expressed on the phase I cells. The phase I HTLV-I-infected cells, as well as the parental 827 cells reacted with a mAb specific for an epitope on the variable part of the TCR beta-chain, indicating that the TCR was not altered after HTLV-I infection. Like the parental clone, the phase I cells proliferated in response to tetanus toxin, but the tetanus toxin-specific response of the phase I cells did not require the presence of APC. Results of experiments, in which the levels of intracellular Ca2+ were measured, indicated that HTLV-I cells can acquire the capability to process Ag and present that to themselves. Phase I HTLV-I-infected T cells had lost their cytotoxic activity which was likely to be due to an effect on the lytic machinery rather than on Ag recognition by the TCR, inasmuch as it was found that phase I HTLV-I-infected T cells did no longer contain N-alpha-benzyloxy-L-lysine thiobenzylester-serine esterase activity. Furthermore, it was found that phase I HTLV-I-infected T cells had a diminished capacity to form conjugates with target cells. From a period of about 200 days after HTLV-I infection, phase II cells emerged that proliferated strongly in the absence of IL-2 and that had lost all functional activity. These cells did not express the CD3/T cell receptor complex on their surface. Phase I as well as phase II HTLV-I-infected cells were targets for CTL raised in the autologous donor.
Characterization of a novel low affinity receptor for IFN-gamma on adherent human monocytes by radioligand binding studies and chemical cross-linking.
Authors: Finbloom D S , Wahl L M
Published: December 2022

Freshly isolated monocytes in suspension express 2000 to 4000 high affinity receptors for IFN-gamma. Because monocytes change phenotypically as they migrate out of the circulation and adhere to extracellular matrix, modulation of the expression of IFN-gamma receptors may occur. In order to determine if adherence alone modulates the receptor for IFN-gamma, we have studied receptor expression in adherent human peripheral blood monocytes. Elutriation-purified monocytes were allowed to adhere to polystyrene overnight at 37 degrees C. These cells now expressed 1 to 2 x 10(5) low affinity (Ka = 10(8) liters/M) receptors for [125I]rIFN-gamma. Binding to this receptor was specific and saturable. The expression of these receptors occurred rapidly (within 3 h) after adherence and was not inhibited by cycloheximide treatment. Binding to the receptor was abrogated by treating cells with trypsin, but was enhanced after treatment with alkaline protease or proteinase K. mAb against the high affinity receptor did not block binding to the low affinity receptor on adherent cells. The low affinity receptor transduced a signal to the cell as measured by the IFN-gamma-induced enhancement in FcR for human IgG1. The structure of the receptor on adherent cells was investigated by chemical cross-linking techniques. A receptor-[125I]rIFN-gamma complex was observed by SDS-PAGE to have a Mr of 180,000 to 200,000. Reduction of this complex with 2-ME resulted in the loss of the high Mr complex and the appearance of a doublet of lower Mr of 68,000 and 82,000. In contrast, cross-linking of monocytes in suspension yielded a complex of 110,000 to 120,000 Mr, which was unchanged upon reduction. Upon adherence, human monocytes express large numbers of a novel receptor for rIFN-gamma which is capable of stimulating the cell. This receptor appears to be composed of at least two components which are disulfide linked and structurally differs from the high affinity receptor on nonadherent monocytes.
Endotoxemia elicits increased circulating beta 2-IFN/IL-6 in man.
Authors: Fong Y , Moldawer L L , Marano M , Wei H , Tatter S B , Clarick R H , Santhanam U , Sherris D , May L T , Sehgal P B
Published: December 2022

beta 2-IFN/hepatocyte stimulating factor/IL-6 is a cytokine secreted by monocytes, fibroblasts, and endothelial cells in cell culture that possesses diverse biologic activity including the stimulation of acute phase plasma protein synthesis and immunomodulation. The circulating levels of this cytokine in man in response to bacterial LPS (endotoxin) were studied. A single i.v. bolus of endotoxin (20 U/kg) produced a monophasic rise in circulating immunoreactive IFN-beta 2/IL-6 and IFN-beta 2/IL-6 bioactivity (hepatocyte stimulation and B cell differentiation assays) peaking 2 to 4 h after the endotoxin challenge. Peak IFN-beta 2/IL-6 levels ranged from 4.1 to 27.5 ng/ml. Associated with this was a rise in circulating C-reactive protein levels detected 20 h after the endotoxin bolus. Thus, IFN-beta 2/IL-6 is likely one of the endogenous mediators which is triggered in man during bacterial infection and likely participates in the metabolic and immune responses of the infected host.
Net increase of pluripotential hematopoietic precursors in suspension culture in response to IL-1 and IL-3.
Authors: Iscove N N , Shaw A R , Keller G
Published: December 2022

A new short-term suspension culture system is described in which pluripotential hematopoietic precursors from mouse bone marrow increase 8 to 12 times in number over a 4-day period. The increase is shown to derive from myeloid precursors undergoing repeated cell divisions prior to definitive lineage restriction. The response, which occurs in the absence of any pre-formed feeder layer, depends on dual stimulation by both IL-1 and IL-3, and the maximum effect depends on the presence of both factors together from the initiation of the cultures. The observations extend the known range of targets of IL-1 and IL-3 to include primitive pluripotential precursors capable of some degree of self-renewal, and provide a specific and relatively simple assay both for the precursors and the soluble factors which regulate them.
Effect of complement on the lateral mobility of erythrocyte membrane proteins. Evidence for terminal complex interaction with cytoskeletal components.
Authors: Liu Z Y , Sanders M E , Hu V W
Published: December 2022

The lateral mobilities of erythrocyte membrane proteins and terminal complement complexes (TCC) were measured on C-treated erythrocyte ghosts by the technique of fluorescence redistribution after photobleaching. Results showed that the lateral diffusion coefficient of the bulk membrane proteins decreased with the assembly of TCC on the membrane at low C dose and was significantly reduced with assembly of the full membrane attack complex (C5b-9), even in the absence of cell lysis. At high serum doses, the mobility of the membrane proteins increased slightly above that of the control cells. The diffusion coefficients of the TCC on the erythrocyte membrane range from 1.18 to 4.37 x 10(-11) cm2/s, values characteristic of anchored membrane proteins. Spectrin-depletion of the C-lysed erythrocytes results in 25- and 45-fold increases in the diffusion coefficients of the membrane proteins and the C5b-9 complex, respectively. Conversely, oxidative cross-linking of spectrin by diamide reduced the diffusion coefficients of both membrane and C proteins. These studies indicate that the deposition of TCC on an erythrocyte can result in a substantial change in the physical and structural properties of the target membrane, aside from the creation of functional lesions. The low mobilities of the terminal complexes on the target membrane suggest possible interactions with cytoskeletal elements or with anchored membrane proteins.
Chemoattractant-induced cytoplasmic pH changes and cytoskeletal reorganization in human neutrophils. Relationship to the stimulated calcium transients and oxidative burst.
Authors: Naccache P H , Therrien S , Caon A C , Liao N , Gilbert C , McColl S R
Published: December 2022

The relationships between the chemotactic factor-stimulated mobilization of calcium, activation of the NADPH-oxidase, changes in cytosolic pH, and in the level of polymerized actin in human neutrophils have been examined. The approach taken was to use intracellular calcium chelators, and pharmacologic modulators (both positive and negative) of the NADPH-oxidase to measure the aforementioned responses under conditions where the calcium transients were abrogated and/or the generation of superoxide anions was either inhibited or augmented. The decrease in cytosolic pH induced by chemoattractants was inhibited by the calcium chelator BAPTA and by the diglyceride kinase inhibitor 6-[2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinylethyl ]-7-methyl-5H-thiazolo[3,2-alpha]pyriimidin-5-one (R59022) (this latter compound enhanced the oxidative response of the cells). Furthermore, a specific inhibitor of the NADPH-oxidase (diphenyleneiodonium) had no significant effect on the cytosolic acidification induced by FMLP or leukotriene B4. These results indicate that the initiation of the cytosolic acidification induced by chemotactic factors is a calcium-dependent event that is not directly linked to the activation of the NADPH-oxidase. In contrast, the stimulated polymerization of actin was insensitive to BAPTA, R59022, and diphenyleneiodonium. Thus, neither the calcium transients nor the oxidative burst play a signaling role in the initiation of actin polymerization elicited by chemoattractants. These data indicate that additional investigations are needed to uncover the biochemical basis of the signals initiated in human neutrophils by chemotactic factors that lead to the polymerization of actin and to the cytosolic acidification.
Effect of oligosaccharide chain length, exposed terminal group, and hapten loading on the antibody response of human adults and infants to vaccines consisting of Haemophilus influenzae type b capsular antigen unterminally coupled to the diphtheria protein CRM197.
Authors: Anderson P W , Pichichero M E , Stein E C , Porcelli S , Betts R F , Connuck D M , Korones D , Insel R A , Zahradnik J M , Eby R
Published: December 2022

Vaccines consisting of oligosaccharide (OS) derived from Haemophilus influenzae type b capsular polysaccharide and conjugated to carrier proteins had been shown capable of eliciting memory-type capsular polysaccharide of H. influenza type b antibody responses in human infants, but the structural variables governing immunogenicity were not defined. Here a series of conjugates were made with the diphtheria protein CRM197 and with uniterminally coupled OS haptens that varied in chain length, exposed terminal residue, or multiplicity of loading as defined by ribose/protein ratio. Adults were given a single injection, 1-yr-old infants were given a two-injection sequence, and capsular polysaccharide of H. influenzae type b antibody responses were assessed by radioantigen binding. Vaccines C-4r, C-6r, and C-12r, in which ribitol-ended OS of mean length 4, 6, or 12 repeat units were coupled at low hapten loading, were about equally immunogenic (geometric means 2 to 5 micrograms/ml in infants, 5 to 9 micrograms/ml in adults). Vaccine C7p was made with a higher loading of OS having mean length 7 repeat units and having mainly phosphate monoester at the exposed termini Vaccine C-7R was made from a portion of C-7p by enzymatic removal of most of the terminal phosphates. Compared to the C-4r, C-6r, and C-12r series, vaccines C-7p and C-7R induced geometric means about 10-fold higher in adults and 20-fold higher in infants. Thus OS chain length (in the range studied) and exposed terminus are less critical variables in this system than the extent of hapten loading.
Tropomyosin shares immunologic epitopes with group A streptococcal M proteins.
Authors: Fenderson P G , Fischetti V A , Cunningham M W
Published: December 2022

Tropomyosin is an alpha-helical coiled-coil protein with structural similarities to the streptococcal M protein. In order to show serologic cross-reactivity between streptococcal M proteins and tropomyosin, we selected from a panel of murine mAb those which reacted with M proteins and tropomyosins in the ELISA. Western blots were used to study the reactions of each mAb with human and rabbit cardiac and rabbit skeletal tropomyosins. The antibodies were further characterized for their reactions with the additional autoantigens myosin, actin, keratin, and DNA. Five mAb were found which reacted with either PepM5 or ColiM6 protein and tropomyosin in Western blots or ELISA. Two of the tropomyosin positive mAb were also antinuclear antibodies and were inhibited with DNA. In Western blots of cardiac tropomyosins, the mAb reacted with either the 70-kDa dimer of tropomyosin, the 35-kDa monomer, or both. Some differences were observed in the reactions of the mAb with the different tropomyosins in Western blots. The heart cross-reactive epitopes shared between M proteins and tropomyosin were in most instances shared with cardiac myosin. Differences were observed among the reactions of the mAb with the different tropomyosins. This report constitutes the first evidence of serologic cross-reactivity between streptococcal M proteins and tropomyosins.
Functional heterogeneity of macrophage precursor cells from spleen of Leishmania donovani-infected and untreated mice.
Authors: Hockertz S , Baccarini M , Lohmann-Matthes M L
Published: December 2022

We recently described the bone marrow-derived macrophage precursor, which is able to spontaneously and extracellularly kill protozoa of the genus Leishmania. These nonadherent, nonphagocytic macrophage precursor cells are present in the spleen of healthy mice only in a small quantity. However, high numbers of proliferating macrophage precursors are isolated from the spleen of Leishmania donovani-infected mice. Macrophage precursors from spleens of diseased animals are able to kill spontaneously the promastigote as well as the amastigote form of L. donovani. The mechanism of the spontaneous leishmanicidal activity of macrophage precursor cells derived from spleens of L. donovani-infected mice was investigated. This effector function could be defined in part as an antibody-dependent cellular cytotoxicity. In addition we assessed the role of CSF-1-containing L cell-conditioned supernatant at the leishmanicidal activity of these immature cells of the macrophage lineage. For that purpose, nonadherent spleen cells from healthy mice were cocultivated with this CSF-1-containing medium for 4 days. These in vitro proliferated macrophage precursor cells from untreated mice showed an increased leishmanicidal activity. Thereby we established a further activation mechanism for proliferating splenic macrophage precursor cells responsible for the observed killing of L. donovani pro- and amastigotes. The spontaneous cytotoxicity of macrophage precursors from spleens of L. donovani-diseased animals is thus defined as a cooperative effect of antibody-dependent cellular cytotoxicity and Macrophage-CSF activation.
Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type B polysaccharide. Demonstration of three types of V regions and their association with H and L chain isotypes.
Authors: Tarrand J J , Scott M G , Takes P A , Nahm M H
Published: December 2022

The antibody response to Haemophilus influenzae type b polysaccharide (Hib-PS) is pauciclonal but can vary between different individuals. To estimate the size of this antibody repertoire we examined the constant and V regions of human IgG anti-Hib antibodies from 14 individuals at the clonal level using various serologic and IEF methods. Examination of H chains showed that 11 of 14 individuals produced both IgG1 and IgG2 antibodies, two individuals produced only IgG2 and one individual produced only IgG1 antibody. All 14 individuals produced kappa-containing antibody clones and three persons also produced significant lambda-containing antibody clones. V region heterogeneity was examined by comparing cross-reactivity of anti-Hib-PS antibodies to the capsular polysaccharide of Escherichia coli K100 carbohydrate (K100 CHO). These studies showed that clones of IgG anti-Hib-PS antibodies cross-reactive with K100 CHO were present in 5 of 14 (36%) individuals and also revealed at least three types of V regions among these antibodies. The first type has no cross-reaction with K100 CHO and was found in 13 of the 14 individuals. The second type, found in three of 14 individuals, cross-reacts with K100 CHO and uses a lambda L chain V region. The third type, found in 2 of 14 individuals, cross-reacts with K100 CHO and uses a kappa L chain V region. Although the lambda type V region was found only in association with IgG2, the other two V region types associate with both IgG1 and IgG2. Thus, five IgG antibody clones are serologically discernable. An individual generally responds to Hib-PS by expressing several clones selected from these discernable antibody clones. Indeed, we can observe six individual response patterns among these 14 individuals and conclude that considerable variability in individual responses to Hib-PS can be achieved with very few V regions.
Isolation and characterization of the mouse CD8 beta-chain (Ly-3) genes. Absence of an intervening sequence between V- and J-like gene segments.
Authors: Nakayama K , Shinkai Y I , Okumura K , Nakauchi H
Published: December 2022

We have isolated and determined the nucleotide sequence and genomic organization of the genes encoding Ly-3.1 and Ly-3.2. These genes span approximately 14 kb on chromosome 6 and consist of six exons and five introns. The exons correlate roughly with the putative functional domains, namely, a leader exon, a variable and joining region-like exon, a hinge region-like exon, a transmembrane exon, and two intracytoplasmic exons. There is no intervening sequence between V- and J-like gene segments, indicating that rearrangement is not necessary for the expression of the Ly-3 gene. In the 5'-flanking region there is no "TATA" box nor "CAAT" box; however, three "GC" boxes are located upstream of the ATG initiator codon. There are short stretches of sequence homologous to 5'-flanking sequences of the Ly-2 gene. In addition, the sequences CTCTGTGGCA at -748 exhibits homology to the enhancer core sequence of the human Ig H chain and TCR genes. Comparison of the nucleotide sequence corresponding to the extracellular portion between Ly-3.1 and Ly-3.2 revealed a single base difference which results in an amino acid substitution. Therefore it is likely that this amino acid difference is responsible for the previously defined Ly-3 allotypes.
Structure of the gene encoding the human B lymphocyte differentiation antigen CD20 (B1).
Authors: Tedder T F , Klejman G , Schlossman S F , Saito H
Published: December 2022

The CD20 (B1) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In this report genomic DNA clones containing the human CD20 gene were isolated and the structure of the CD20 gene determined. Southern blot analysis revealed that CD20 mRNA was transcribed from a single-copy gene. The CD20 gene was 16 kb long and was composed of eight exons. The first exon marked the major transcription initiation site as determined by primer extension and S1 nuclease analysis. The translation initiation codon was located within the third exon. Exon VIII encoded the COOH terminus of the CD20 protein and the long 3' untranslated region. Three forms of CD20 mRNA were identified that all encode an identical protein product. The dominant form of 2.8 kb results from usage of exons I through VIII, whereas a second form that is 263 bp shorter had exon I spliced into an internal 3' splice site within exon III thereby skipping exon II. A minor 3.4-kb mRNA species most likely results from an uncharacterized upstream exon(s) splicing into an internal 3' slice site located in exon I. Nucleotide sequences of cDNA clones representative of each of these RNA forms are presented. The 5' splice site following exon V was found to be divergent from the consensus splice sequence. A relationship between the individual peptides encoded by the six exons and structurally distinct regions of the CD20 protein is likely.
Induction of antigen-specific antibody responses in primed and unprimed B cells. Functional heterogeneity among Th1 and Th2 T cell clones.
Authors: DeKruyff R H , Ju S T , Hunt A J , Mosmann T R , Umetsu D T
Published: December 2022

CD4+ T cells have been recently divided into two subsets. The functions of these subsets are thought to be distinct: one subset (Th1) is responsible for delayed type hypersensitivity responses and another (Th2) is primarily responsible for induction of antibody synthesis. To more precisely define the roles of both subsets in humoral immune responses, we examined the ability of a panel of nominal antigen specific Th1 and Th2 clones to induce anti-TNP specific antibody synthesis in TNP-primed or unprimed B cells. Four of nine Th1 clones induced little or no antibody synthesis with TNP-primed B cells. However, five other Th1 clones were very effective at inducing IgG anti-TNP plaque-forming cell (PFC) responses in primed B cells. One of these Th1 clones was analysed in detail and found to also provide helper function for unprimed B cells. Cognate B-T cell interaction was required for induction of both primary and secondary responses with this clone, indicating that a Th1 clone could function as a "classical" Th cell. The seven IL-4 producing Th2 clones examined were also heterogeneous in their ability to induce antibody secretion by TNP-primed B cells. Although four of the Th2 clones induced IgG and IgM anti-TNP PFC responses, two Th2 clones induced only IgM and no IgG antibody, and another clone failed to induce any anti-TNP PFC. All Th2 clones failed to induce any anti-TNP PFC. All Th2 clones produced high levels of IL-4, but "helper" Th2 clones produced significantly greater amounts of IL-5 than "non-helper" Th2 clones. These studies indicate that some IL-2- and some IL-4-producing T cell clones can induce TNP-specific antibody in cell clones can induce TNP-specific antibody in primed and unprimed B cells, and that Th1 and Th2 clones are heterogeneous in their ability to induce Ig synthesis. Therefore, although T cell clones can be classified as Th1 or Th2 types according to patterns of IL-2, IFN-gamma, or IL-4 synthesis, the functional capacity to induce antibody synthesis cannot be predicted solely by their ability to secrete these lymphokines.
Mechanisms of immune memory. T cell activation and CD3 phosphorylation correlates with Ta1 (CDw26) expression.
Authors: Hafler D A , Chofflon M , Benjamin D , Dang N H , Breitmeyer J
Published: December 2022

The Ta1 (CDw26) Ag distinguishes a subset of circulating T lymphocytes that is the major population proliferating to recall Ag challenge. Unlike receptors for growth factors such as IL-2 and transferrin, the Ta1 Ag is present on T cell lines and clones irrespective of cell cycle. The appearance of Ta1 on T cells that respond to recall Ag allowed us to investigate activation requirements that may be associated with T cell immune memory. Ta1+ peripheral blood T cells were induced to proliferate by mAb recognizing either the invariant chains of the TCR, or by pairs of mitogenic antibodies directed to the CD2 molecule. In contrast, Ta1- cells were not stimulated by these antibodies. In addition, Ta1-cells did not proliferate maximally after addition of the phorbol ester PMA in combination with the calcium ionophore Ionomycin, suggesting that the intracellular targets of these agents may not be fully active. Anti-CD3-induced elevation of intracellular calcium levels was equivalent in the two subpopulations, suggesting that calcium mobilization mechanisms were intact. In contrast, PMA-induced phosphorylation of TCR CD3 chains was significantly greater in Ta1+ cells as compared to Ta1- T cells. Taken together, our results indicate that Ta1 expression, which is associated with T cell activation and memory, may be causally related to TCR and CD2-mediated activation mechanisms. The PMA inducible TCR phosphorylation in Ta1+ memory cells associated with their increased ability to proliferate after CD3/TCR or CD2 stimulation suggests that intracellular phosphorylation events may be causally associated with T cell immune memory.
Responsiveness of fetal and adult CD4-, CD8- thymocytes to T cell activation.
Authors: Takashi T , Steinberg A D , June C H , Gause W C
Published: December 2022

Day-14 fetal CD4-, CD8- thymocytes showed a greater proliferative response to PMA + IL-4 than did adult double-negative thymocytes. In contrast, adult double-negative thymocytes were more responsive to PMA + IL-1 + IL-2 or to IL-1 + IL-2 alone. The adult double-negative thymocytes showed significantly greater proliferation than fetal thymocytes after stimulation via anti-CD3 or anti-Thy-1 in the presence or absence of interleukins (IL-1 + IL-2 or IL-4). Adult CD4-, CD8- thymocytes also exhibited greater calcium mobilization following anti-CD3 stimulation IL-2-dependent activation with anti-Thy-1 or IL-1 + IL-2 in the absence of PMA resulted in marked expansion of CD 3+, F23.1+, CD4-, CD8- thymocytes, a population absent in fetal thymocytes but constituting 4% of pre-cultured CD4-, CD8- adult thymocytes. IL-4 + PMA failed to expand this CD 3+ population. It is hypothesized that before expression of functional TCR, T cell development may be more dependent on activation pathways not using IL-2; after TCR expression, IL-2-dependent pathways, including Thy-1-mediated stimulation, become functional.
Calcium-independent pathway of tumor necrosis factor-mediated lysis of target cells.
Authors: Hasegawa Y , Bonavida B
Published: December 2022

The role of Ca2+ in cell-mediated cytotoxicity has been the subject of many investigations and both Ca2+-dependent and -independent pathways have been reported. TNF was suggested to play a role in NK and macrophage cell-mediated cytotoxicity. We assumed that its role in target cell lysis might take place by a Ca2+-independent mechanism. This hypothesis was investigated in assays of rTNF-mediated lysis of tumor target cells. Extracellular Ca2+ depletion by the calcium chelator EGTA (2 mM and 5 mM) and blocking of intracellular Ca2+ mobilization by 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride did not inhibit TNF-mediated tumor cell lysis. Furthermore, blocking of Ca2+ influx in the presence of the Ca2+ channel blocker Verapamil did not inhibit TNF-mediated tumor cell lysis. Previous reports showed that lysis of sensitive tumor cells by TNF is preceded by binding of TNF to TNF receptors, internalization, and DNA degradation. These events were tested in the absence of Ca2+. Treatment with Ca2+ inhibitors did not affect binding of 125I-TNF to target cells. Also TNF induced the fragmentation of cellular DNA in target cells without extracellular or intracellular Ca2+. These findings demonstrate that the mechanism of TNF-mediated tumor cell lysis does not depend on intracellular or extracellular Ca2+ and that events associated with target cell lysis can also function in the absence of Ca2+. Thus, our findings support the contention of a Ca2+-independent lytic pathway in which secreted or membrane-bound TNF may interact with the target cells and ultimately result in DNA degradation and target cell lysis.
Characterization of the inflammatory response in the central nervous system of mice susceptible or resistant to demyelination by Theiler's virus.
Authors: Lindsley M D , Rodriguez M
Published: December 2022

Intracerebral inoculation of mice with Theiler's murine encephalomyelitis virus results in an intense inflammatory response of mononuclear leukocytes which infiltrate into the central nervous system. Resistant strains of mice have the ability to clear virus whereas susceptible strains become infected persistently and are associated with chronic demyelination which is proposed to be immune-mediated. In an attempt to better understand the role of the immune response during demyelination, mononuclear leukocytes were isolated from the central nervous system of infected mice and stained by an immunoperoxidase technique with anti-Thy-1.2, anti-L3T4, anti-Lyt-2 and anti-MAC-1 mAb. Infection of susceptible SJL/J mice resulted in a biphasic immune response which peaked on days 7 and 27 post-infection. In contrast, a single peak (day 7) was observed in resistant C57BL/10SNJ mice. The presence of Thy-1.2, L3T4, and MAC-1+ cells was similar between the two strains. However, although the number of Lyt-2+ cells peaked on day 7 in C57BL/10SNJ mice, they were not detected in SJL/J mice until 14 days post-infection and gradually increased in number over the course of infection. To further study the role of T cells in demyelination, serial frozen sections of brain and spinal cord were stained for the presence of Lyt-2 and L3T4+ cells in the lesions of chronically infected SJL/J mice. L3T4+ cells were observed predominantly in perivascular regions while Lyt-2+ cells were observed infiltrating the parenchyma. These results provide further evidence that Lyt-2+ lymphocytes are important in the mechanism of susceptibility/resistance to Theiler's murine encephalomyelitis virus-induced demyelination.
Correlation between the biologic functions and the generation of lymphokines in T cell clones.
Authors: Tartakovsky B , Axelrod O , Blankenstein T , Mozes E
Published: December 2022

We have recently reported that various murine T cell clones produce IL-1. Based on this observation we have analyzed in the present study the correlation between the biological functions and the generation of different lymphokines in (T,G)-A--L specific CD4+ clones. One subset of clones--the "helper clones"--were found to provide help to primed B cells, in vitro. These cells could be shown to produce IL-1, IL-2, and B cell stimulatory factor 1 (IL-4) activities and to express mRNA encoding for these three cytokines. The second subset of clones, termed "proliferative clones", were unable to help B cells in vitro but expressed vigorous Ag-dependent proliferations. These cells did not express IL-1, IL-2, or IL-4 activities. They produced another lymphokine(s) which may be granulocyte-macrophage-CSF, or some other factor recognized by the HT2 cell line. This study further substantiates the link between T cell activities and lymphokine repertoire with a special emphasis on the potential role(s) of T cell-derived IL-1.
Probing the C4-binding site on C1s with monoclonal antibodies. Evidence for a C4/C4b-binding site on the gamma-domain.
Authors: Matsumoto M , Nagaki K , Kitamura H , Kuramitsu S , Nagasawa S , Seya T
Published: December 2022

The catalytic site for C4 of C1s has been presumed to consist of a C4-binding domain and a proteolytic domain. A mAb to C1s, M81, blocked C4 activation and C4 binding to C1s. M81 recognized the H chain of C1s. Using M81 as a probe, we tried to define C4-binding site on C1s. Plasmin digestion of C1s generated four products of Mr 58,000 (P1), 48,000 (P2), 37,000 (P3), and 27,000 (P4). These products, except for P2, all possessed a 26,000-Da H chain fragment (26k-HF) connected to variable-sized L chain pieces. 26k-HF alone had an ability to interact with M81. Amino-terminal amino acid analysis of 26k-HF mapped the epitope for M81 to domain IV and/or V of gamma-domain of C1s. The gamma-domain therefore contains the C4-binding site. The confirm and further elucidate the role of the C4-binding site for C4, we used a substrate-blotting technique in which labeled C4 was incubated with nitrocellulose membrane-fixed C1s and its fragments. C4 was successfully blotted onto C1s and P1, but not P2-P4; i.e., further degradation of the L chain led to the loss of C4-binding. During the incubation, most of the added C4 was converted to C4b. The binding was augmented, if the proteolytic activity of C1s and P1 was blocked, so that the added C4 remained intact. Although C4b also bound to C1s and P1, its binding was less effective and abolished by the addition of cold C4. Based on these results, the gamma-domain and the L chain constitute the catalytic site of C1s to activate C4 to C4b. Moreover, the generated C4b, although it still has weak affinity for C1s, can be replaced by newly coming C4.
CDw17: a neutrophil glycolipid antigen regulated by activation.
Authors: Symington F W
Published: December 2022

The plasma membrane and intracellular granules of human polymorphonuclear neutrophils (PMN) contain large amounts of the glycolipid, lactosylceramide (LacCer; Gal beta 1----4Glc beta 1----1Cer). Despite its abundance, novel subcellular distribution, and lineage-restricted expression, nothing of PMN LacCer function is known. We examined the relationship between LacCer and PMN activation by assessing binding of anti-LacCer mAb (T5A7; anti-CDw17) to PMN during and after cell stimulation. CDw17 expression markedly decreased after treatment with PMA, dioctanoylglycerol, calcium ionophore, FMLP (with or without cytochalasin B or added Ca2+), TNF-alpha, or lymphotoxin. Depending on the stimulus, CDw17 declined to levels ranging from 70% (TNF, lymphotoxin) to less than 5% (phorbol ester, dioctanoylglycerol) of levels detected on untreated PMN. Loss of CDw17 from PMA-treated PMN followed dose- and temperature-dependent kinetics, with loss being detected after PMA treatment for 1 min. Membrane internalization explained PMA-induced loss of CDw17, as cell-associated 125I-anti-CDw17 became inaccessible to fluorescent anti-Ig after PMA treatment. CDw17 on PMN cytoplasts or retinoic acid-induced HL-60 cells was only slightly affected by stimulation, suggesting that down-regulation of the epitope is associated with granule exocytosis rather than superoxide production. Results with PMN from a patient with chronic granulomatous disease confirmed that normal superoxide production is not required for CDw17 loss induced by PMA or FMLP treatment. The data collectively demonstrate that reduced levels of cell-surface CDw17 are associated with granule exocytosis after PMN activation.
Characterization of a CD3-like rat T cell surface antigen recognized by a monoclonal antibody.
Authors: Tanaka T , Masuko T , Yagita H , Tamura T , Hashimoto Y
Published: December 2022

A mouse mAb, which recognized a rat T cell surface Ag responsible for the T cell activation, was produced by a regular hybridoma method using F344 rat T cells stimulated with PMA and a calcium ionophore, as the Ag. The mAb termed 1F4 (kappa-IgM) was reactive with rat T cells but not with B cells and immunohistochemically it stained rat thymus tissues strongly at medulla and weakly rat cortex. Addition of 1F4 mAb to a culture of T cells resulted in the proliferation of T cells by a help of PMA or a solid support. 1F4 mAb also caused the modulation of the corresponding Ag but not other T cell markers such as CD5, CD2, and OX-52-defined Ag. The 1F4 mAb immunoprecipitated a cell surface component having an apparent m.w. of 25,000 from rat T cells which could be associated with a disulfide-linked heterodimer (m.w. 92,000) consists of subunits having m.w. of about 52,000 and 43,000. These results strongly suggest that the 1F4 mAb recognizes a rat T cell Ag homologous to the human and mouse CD3.
Characterization of Mycobacterium leprae cell wall-associated proteins with the use of T lymphocyte clones.
Authors: Mehra V , Bloom B R , Torigian V K , Mandich D , Reichel M , Young S M , Salgame P , Convit J , Hunter S W , McNeil M
Published: December 2022

Development of a vaccine against leprosy depends on the identification of Ag that stimulate cell-mediated immune responses. We have previously demonstrated that cell wall proteins of Mycobacterium leprae are highly immunogenic. By using human cell wall-specific T cell clones we have begun to characterize soluble proteins that integrate into the cell wall skeleton. T cells from leprosy lesions were expanded with IL-2 in vitro yet retained specificity to Ag of the insoluble cell wall core (CWC) in vitro, indicating that T cells had been activated by CWC Ag in vivo. A cell wall protein-peptidoglycan complex and cell wall protein preparations lacking carbohydrates and lipids from CWC retained T cell reactivity. To identify immunogenic protein component(s) of cell wall protein, T cell lines were established to cell walls and tested against M. leprae proteins separated by SDS-PAGE and transferred to nitrocellulose. Greatest T cell reactivity was observed to proteins of Mr 7 kDa, 16 kDa, and 28 kDa. T cell clones reactive with 7-kDa and 16-kDa Ag from gels failed to respond to proteins of other Mr separated under either reducing or nonreducing conditions, indicating that these molecules are not subunits of larger proteins and may represent monomeric units polymerized into cell walls. The approaches described herein for characterization of immunodominant T cell Ag of M. leprae may be useful for study of T cell Ag in cell walls of bacterial pathogens of man.
Delta-chains of dendritic epidermal T cell receptors are diverse but pair with gamma-chains in a restricted manner.
Authors: McConnell T J , Yokoyama W M , Kikuchi G E , Einhorn G P , Stingl G , Shevach E M , Coligan J E
Published: December 2022

To assess the diversity of TCR delta-gene expression in dendritic epidermal T cells (DETC), we characterized the delta-gene used by a panel of cell lines that express the gamma delta TCR on the cell surface. Northern hybridization analyses with a panel of V delta probes representing each of the six known V delta families showed that each of these lines expressed either V delta 1 or V delta 6 containing transcripts. Southern hybridization analysis with the V delta probes gave rearrangement patterns consistent with Northern hybridization results. The correlation of V delta expression with V gamma expression revealed that not only may V delta usage be restricted in DETC cells, but that the pairing of gamma- and delta-chains may not be random. In the DETC cells examined, V delta 1 chains paired exclusively with V gamma 3C gamma 1 chains and V delta 6 chains paired with C gamma 2 and C gamma 4 chains. Sequence analysis of delta-cDNA clones corresponding to the expressed delta-chains from one of the V delta 1 expressing cell lines and two of the V delta 6 expressing cell lines revealed that, although the V delta 1 gene segment sequence and one of the V delta 6 gene segment sequences were identical to previously published V delta sequences, considerable variable region diversity was generated by complex V-D-J rearrangement patterns that utilized various combinations of N-additions, D delta 2 and possibly D delta 1 segments, and J delta 1 or J delta 2 segments. These complex rearrangement patterns distinguish these DETC lines from early thymocytes and suggest that, if DETC are thymic derived, they originate from relatively mature thymic cells.
A family of cell-surface glycoproteins defined by a putative anti-endothelial cell receptor antibody in man.
Authors: Kansas G S , Wood G S , Dailey M O
Published: December 2022

An efficient immune response depends directly on the ability of lymphocytes and other blood borne leukocytes to leave the blood and enter lymphoid organs and other sites of immune response or inflammation. A new murine mAb termed 515 recognizes a 85- to 90-kDa structure involved in lymphocyte binding to specialized endothelium in postcapillary venules of lymphoid organs (high endothelial venules). In crossed immunoprecipitations followed by SDS-PAGE analysis, 515 appears to be identical to the antilymphocyte homing receptor mAb, Hermes-1. We show here that 515 recognizes peripheral blood leukocytes of all classes examined, but, like Hermes-1, fails to recognize germinal center cells or nearly all cortical (but not medullary) thymocytes. In addition, we demonstrate that 515 recognizes both fibroblasts and epidermal keratinocytes, both in situ and in cultured cell lines. Immunoprecipitation and SDS-PAGE analysis reveals that 515-defined Ag from T lymphocytes, neutrophils, and the epidermal keratinocyte cell line HaCaT are clearly distinct, both before and after treatment with peptide N-glycosidase F, which removes all asparagine-linked oligosaccharides. In contrast, the Mr of 515-defined Ag from T cells and fibroblasts appear indistinguishable. Thus, the 515 mAb recognizes a broadly distributed family of glycoproteins which may be involved in a variety of adhesive functions.
Expression of adhesion structures during B cell development in man.
Authors: Kansas G S , Dailey M O
Published: December 2022

We have used three-color flow cytometry to study the expression of adhesion structures during B cell development in man. The results indicate that the cell-surface molecule(s) recognized by 515, a mAb which defines a broadly expressed family of cell-surface glycoproteins that includes lymphocyte homing receptors, exhibit a clear bimodal distribution (515lo and 515hi); 515hi cells were found exclusively on more mature B cells which already expressed high levels of CD20. Earlier, less mature B cells, identified by their expression of CD10, were uniformly 515lo. In contrast, the CD11a/LFA-1 Ag was acquired gradually over the course of B cell development. B cells which expressed high levels of CD20 expressed three to six times as much CD11a/LFA-1 as cells which expressed CD10. Interestingly, expression of the 515hi phenotype was tightly correlated with that of Leu-8, a marker previously shown to define maturational and functions subsets of B cells. These data document the coordinated regulation of multiple cell surface structures during B cell ontogeny, and demonstrate that adhesion structures necessary for proper B cell function are precisely up-regulated during B cell differentiation in man.
Platelet-activating factor production in human neutrophils by sequential stimulation with granulocyte-macrophage colony-stimulating factor and the chemotactic factors C5A or formyl-methionyl-leucyl-phenylalanine.
Authors: Wirthmueller U , De Weck A L , Dahinden C A
Published: December 2022

Besides its function as a growth factor, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) "primes" polymorphonuclear leukocytes (PMN) for enhanced biologic responses to a number of secondary stimuli. We examined the effect of priming PMN with GM-CSF on the production of [3H] platelet-activating factor (PAF) from [3H]acetate upon stimulation with the chemotactic factors FMLP and C5a. In PMN stimulated with the individual peptide mediators alone [3H]PAF levels were close to controls, whereas considerable amounts of [3H]PAF are formed after stimulation of PMN which have been preexposed to GM-CSF. The priming effect was concentration and time dependent. It was optimal after a preincubation period of 2 h. A maximum of [3H]PAF accumulation is reached within 2.5 min (C5a) and 5.0 min (FMLP) after activation of GM-CSF-primed PMN. In addition, we show that PAF isolated from PMN preincubated with GM-CSF and triggered with chemotactic factors is able to enhance the respiratory burst in PMN. PAF formed by sequentially activated PMN could contribute to the enhanced oxygen radical production and cytotoxicity in effector cells and play a role in modulating and amplifying inflammatory reactions.
Tachykinin production in granulomas of murine schistosomiasis mansoni.
Authors: Weinstock J V , Blum A M
Published: December 2022

Preprotachykinins, the products of one gene, are the precursor molecules of three mammalian tachykinins called substance P (SP), substance K (SK), and neuropeptide K. An additional mammalian tachykinin, neurokinin B, has also been described. SP and possibly other tachykinins may modulate immunologic responses. Granulomas that form around parasite ova in murine schistosomiasis were examined for tachykinins. Tachykinins were extracted from granulomas by boiling or with detergent. Extracts examined by RIA and HPLC contained only immunoreactive SP. Granulomas were dispersed with collagenase and cultured in vitro for up to 4 h. Only immunoreactive SP appeared in the culture medium. SP immunoreactivity localized solely to granuloma eosinophils as demonstrated by a sensitive immunohistochemical technique. An antiserum that recognized SK, neuropeptide K, and neurokinin B, but which possessed low reactivity to SP, also stained these cells. Only prior absorption of each antiserum with the appropriate synthetic neuropeptide would abrogate the immunostaining. This suggested that tachykinins other than SP were present within these cells. However, results of in situ hybridization experiments intimated that eosinophils produced predominantly preprotachykinin mRNAs which encode SP but are devoid of the SK/neuropeptide K sequence. It is concluded that granuloma eosinophils make predominantly SP in deference to other tachykinins, and that tachykinins other than SP are unlikely to be important in the regulation of the early granulomatous response of murine schistosomiasis.
Inhibition of the growth of IL-3-dependent mast cells from murine bone marrow by recombinant granulocyte macrophage-colony-stimulating factor.
Authors: Bressler R B , Thompson H L , Keffer J M , Metcalfe D D
Published: December 2022

The mouse mast cell line PT-18 demonstrates [3H] thymidine uptake in the presence of either mouse IL-3 or mouse recombinant granulocyte-macrophage CSF (rGM-CSF). Experiments were thus undertaken to determine whether rGM-CSF would affect IL-3-dependent growth of mast cells from mouse bone marrow cells (BMC). BMC placed in liquid culture containing 50 U/ml of IL-3 gave rise to cultures containing up to 95% mast cells by 2 to 3 wk. The rise in percentage of mast cells was accompanied by an increase in total cell-associated histamine. In contrast, BMC grown in the presence of 50 U/ml of rGM-CSF gave rise to cultures containing primarily macrophages and granulocytes with less than 1% mast cells. The addition of increasing amounts of rGM-CSF to BMC cultures grown in the presence of IL-3 resulted in a decrease in the number of mast cells present in culture at 2 to 3 wk. Cells other than mast cells in these cultures consisted principally of granulocytes and macrophages. The rGM-CSF-related inhibition of mast cell growth was not abrogated by the addition of indomethacin to cultures. Granulocyte-macrophage cell populations added to IL-3-containing cultures did not inhibit mast cell growth. The suppressive effect of rGM-CSF on IL-3-dependent mast cell growth may indicate an important role for GM-CSF in the down-regulation of mast cell proliferation in tissues.