bluuebunny/crossref_metadata_2025_split huggingface dataset:
Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists.
Authors: Mohler K M , Torrance D S , Smith C A , Goodwin R G , Stremler K E , Fung V P , Madani H , Widmer M B 
Published: December 2022

Abstract
Two forms (monomeric or dimeric) of the extracellular, ligand-binding portion of the human p80 cell-surface receptor for TNF were used to antagonize TNF activity in vitro and in vivo. The dimeric sTNFR:Fc molecule was a more potent inhibitor of TNF than the monomeric sTNFR (50 to 1000x), as assessed in vitro by inhibition of TNF binding or bioactivity and in vivo by protection of mice from an otherwise lethal injection of LPS. Surprisingly, the dimeric sTNFR:Fc construct demonstrated a beneficial effect even when administered 3 h after a lethal LPS injection (i.e., after serum TNF levels had peaked and receded). To study the mechanism by which the soluble TNFR functions in vivo, serum TNF levels were examined in mice given LPS in the presence or absence of soluble receptor. Administration of a mortality-reducing dose of sTNFR:Fc ablated the rise in serum TNF bioactivity that normally occurs in response to LPS. However, TNF bioactivity was revealed in these "TNF-negative" serum samples when the L929 bioassay was modified by inclusion of a mAb that blocks the binding of murine TNF to the human soluble TNFReceptor. These results indicate that the absence of direct cytolytic activity in the L929 assay was caused by neutralization of TNF, rather than to an absence of TNF in the serum. Moreover, administration of either monomeric sTNFR or low doses of dimeric sTNFR:Fc actually resulted in increased serum TNF levels compared to mice given LPS but no soluble receptor. However, these "agonistic" doses of soluble receptor did not lead to increased mortality when an LD60 dose of LPS was given. Thus, dimeric sTNFR are effective inhibitors of TNF and under some circumstances function simultaneously as both TNF "carriers" and antagonists of TNF biologic activity.
A hit-and-run system for introducing mutations into the Ig H chain locus of hybridoma cells by homologous recombination.
Authors: Bautista D , Shulman M J 
Published: December 2022

Abstract
The traditional method of site-specific mutagenesis is to introduce predetermined mutations into an expression vector, which is then transferred to cells so that the relevant gene product and the effects of the mutations can be measured. A problem with this approach is that the expression of the transferred genes varies from transformant to transformant, presumably because the number of vector copies and the site of chromosomal integration vary among transformants. While it should be possible to avoid this variability by mutagenizing the chromosomal gene itself, the labor involved in introducing predetermined mutations by homologous recombination with a mutagenized vector is usually so intense that this has not been the favored method. We describe here a system for introducing mutations into the IgH locus of hybridoma cells. This system greatly reduces the labor that would usually be required to identify and recover the rare recombinants. This is a two-step, so-called "hit-and-run," method, whereby mutations are first introduced into the chromosomal locus by targeted vector integration, after which the vector is excised so as to leave the mutation in the chromosomal target. The first step employs an enhancer trap vector bearing an enhancerless gpt gene; using this vector the frequency of randomly inserted transformants which grow in mycophenolic acid containing selective medium is so low that approximately 25% of the selected transformants have integrated the vector into the IgH locus by homologous recombination. Properly targeted transformants can then be used to derive secondary recombinants that have excised the vector and thus become gpt-. This second step which involves selection of gpt- cells by their resistance to 6-thioxanthine is also efficient, in that approximately 75% of the treated cells have excised the gpt gene by homologous recombination. Overall the labor involved in mutagenizing the chromosomal locus is not much more than is needed to produce the traditional transformants expressing a mutagenized transferred gene.
Molecular and kinetic analysis of an epitope-specific shift in the B cell memory response to a multideterminant antigen.
Authors: Press J L , Giorgetti C A 
Published: December 2022

Abstract
Our previous studies showed that the primary and memory B cell responses to the multideterminant antigen poly-(L-Tyr, L-Glu)-poly-D,L-Ala-poly-L-Lys ((T,G)-A-L), differ. The primary response is dominated by antibodies binding side-chain epitopes; there is little antibody response to epitopes on the poly-D,L-Ala-poly-L-Lys backbone of (T,G)-A-L. In contrast, B cells producing A-L+ antibodies constitute approximately a third of the memory response to (T,G)-A-L. To determine the basis of this epitope-specific repertoire shift, we have examined the kinetics of expression of A-L+ B cells and antibodies after in vivo antigen priming and identified VH and V kappa genes used by A-L+ hybridoma antibodies derived from primary vs memory B cells. Kinetic studies, using the splenic focus assay, showed that the clonal frequency of A-L+ B cells remains low (&lt;3% of (T,G)-A-L-specific B cells) 1 wk after Ag priming, increases (9%) by 2 wk, but does not reach the memory frequency (30%) until at least 3 wk after immunization. Molecular analyses showed that both the primary and memory A-L+ antibody responses are heterogeneous, using different VH and V kappa gene families as well as different germ-line genes within a VH gene family. Both H and L chain gene sequences showed somatic mutations in primary as well as memory antibodies. Analysis of antibody binding patterns and somatic mutations in a set of clonally related B cells that use a new germ-line VH gene in the VGAM3.8 family (VGK7, described here), showed a direct correlation between somatic mutation and change in antibody binding specificity. Our results demonstrate how somatic mutation and Ag selection play a role in the development of the memory response to a multideterminant Ag. The data are discussed in the context of the single vs dual lineage models for memory B cell generation.
Prostaglandin E2 limits arachidonic acid availability and inhibits leukotriene B4 synthesis in rat alveolar macrophages by a nonphospholipase A2 mechanism.
Authors: Christman B W , Christman J W , Dworski R , Blair I A , Prakash C 
Published: December 2022

Abstract
Prostaglandin E2 (PGE2), a potent mediator of inflammation released in large amounts by endotoxin-stimulated alveolar macrophages (AM), has been shown to inhibit leukotriene B4 (LTB4) release by activated neutrophils. We investigated the hypothesis that LTB4 synthesis by AM can be regulated by PGE2 and performed experiments to determine the biochemical site of regulation. AM obtained from Sprague-Dawley rats were preincubated with PGE2 before stimulation with the calcium ionophore A23187. LTB4, platelet-activating factor (PAF), lysoPAF, (AA), and 5-hydroxyeicosatetraenoic acid were isolated from AM cells and supernatants, then quantified by gas chromatography/electron capture negative ion mass spectrometry. Stimulated AM released 30.50 +/- 4.52 ng LTB4/10(6) cells and were inhibited by PGE2 in a dose dependent manner. PGE2 (1 microM) inhibited LTB4 synthesis by 37% and decreased release of both 5-hydroxyeicosatetraenoic acid and arachidonic acid by stimulated AM, but did not alter synthesis of PAF or lysoPAF. These mass measurements suggest that PGE2 does not affect the activity of phospholipase A2, PAF acetyltransferase, leukotriene A4 hydrolase. We conclude that PGE2 attenuates LTB4 production in alveolar macrophages by altering the activity of lipases other than phospholipase A2. PGE2-mediated inhibition of LTB4 synthesis by AM may regulate the initiation of lung inflammation.
Recombinant tetravalent group A streptococcal M protein vaccine.
Authors: Dale J B , Chiang E Y , Lederer J W 
Published: December 2022

Abstract
Previous studies have shown that the amino-terminal regions of group A streptococcal M proteins contain primarily protective (opsonic) epitopes and not tissue-cross-reactive epitopes. Limited primary structures from multiple serotypes of M protein containing only protective epitopes could potentially be linked together to form a broadly protective vaccine. The present studies were undertaken to determine the protective immunogenicity of a recombinant, multivalent hybrid molecule containing amino-terminal subunits of types 24, 5, 6, and 19 M proteins. Polymerase chain reaction primers were designed to amplify emm gene fragments ranging from 35 to 113 codons. The PCR products were ligated in tandem and inserted into pKK223-3. The tetravalent M protein that was purified from extracts of Escherichia coli migrated as a single band on SDS-PAGE with an apparent m.w. of 31 kDa. In immunoblot analyses, the hybrid protein reacted with serotype-specific antisera indicating that it contained all four M protein subunits. Rabbits immunized with the purified tetravalent M protein developed significant antibody levels against all four serotypes of native M proteins represented in the hybrid protein. None of the antisera cross-reacted with human tissues. The immune sera also opsonized all four serotypes of group A streptococci. Our data show that a hybrid protein containing subunits from multiple M proteins can evoke broadly protective immune responses without tissue-cross-reactive antibodies.
Induction of IFN-alpha by HIV-1 in monocyte-enriched PBMC requires gp120-CD4 interaction but not virus replication.
Authors: Francis M L , Meltzer M S 
Published: December 2022

Abstract
IFN-alpha is in plasma of HIV-1 infected patients during early and late-stage disease and may play a role in control of virus replication. The stimulus for IFN-alpha production, the cells that produce this cytokine, and the effectiveness of this IFN-alpha response for control of virus infection are not yet defined. Culture fluids from freshly isolated PBMC of HIV-1 seronegative donors contained high levels of IFN-alpha after exposure to 100 to 1000 infectious HIV-1 particles per culture. Levels of IFN-alpha induced by HIV-1 were directly dependent on the number of monocytes in cell preparations: No IFN-alpha was detected from T cell-enriched PBMC. In monocyte cultures, induction of IFN-alpha by HIV-1 was relatively specific: Levels of IL-1 beta, IL-6, IFN-gamma, and TNF-alpha remained at baseline. Capacity of HIV-1 virions to induce IFN-alpha was not dependent on virus replication. IFN-alpha was induced by (a) heat-inactivated HIV-1, (b) virions from 8E5 cells, a cell line that releases noninfectious HIV-1, (c) HIV-1-infected cells fixed in paraformaldehyde, and (d) T cell-tropic HIV-1 that binds to but does not infect monocytes. Capacity of HIV-1 virions and HIV-1 infected cells to induce IFN-alpha was completely inhibited by soluble rCD4 or mAb against CD4 or gp120. Antibodies against CD4, however, did not induce monocytes to produce IFN-alpha. HIV-1-induced IFN-alpha production was inhibited by antibodies against both V3 loop determinants and the CD4 binding site of gp120. Further, sera and purified immunoglobulin from HIV-1 infected patients also inhibited HIV-1-induced IFN-alpha production. These observations suggest that potentially protective antiviral responses associated with IFN-alpha production in HIV-1 infected patients are inhibited by the development of antibodies against gp120.
Trypanosoma cruzi: immunity-induced in mice and rats by trypomastigote excretory-secretory antigens and identification of a peptide sequence containing a T cell epitope with protective activity.
Authors: Taibi A , Plumas-Marty B , Guevara-Espinoza A , Schöneck R , Pessoa H , Loyens M , Piras R , Aguirre T , Gras-Masse H , Bossus M 
Published: December 2022

Abstract
In the present study, we investigate the immunoprotective properties of trypomastigote excretory-secretory Ag (ESA) in experimental models. In the case of BALB/c mice, the immunization with ESA resulted in the reduction of parasitemia during acute infection and a significant level of protection in terms of mortality with more than 60% survival, whereas none of the mice in the control groups survived after 39 days postinfection. The same experiments performed in Fischer rats showed a high degree of protection against acute lethal infection with 100% survival, whereas 20 to 40% of rats in the control groups survived the acute phase of T. cruzi infection. Mouse and rat immune sera presented trypanolytic activity against Trypanosoma cruzi infective forms, and recognized two major parasite components of 85 and 24 kDa. The analysis of specific isotype profiles showed a predominance of IgG1, IgG2a, and IgG2b antibody responses. Rat antisera to ESA were then used to screen a trypomastigote cDNA library. Several clones were identified, all of which encoded for the 24-kDa protein. Using a mAb (Tcr7) produced against the native protein, the 31-kDa recombinant fusion protein was purified by affinity chromatography. The antisera to the recombinant protein used in IFA and immunoelectron microscopy showed that the localization of the 24-kDa protein differs among T. cruzi developmental stages. Protection experiments were performed in BALB/c mice using two synthetic peptides (20-40 and 109-124) derived from the primary sequence of the 24-kDa polypeptide. The results obtained clearly indicated that the peptide 109 to 124 containing a putative T cell epitope represents the most protective epitope, which induced 30 to 50% of protection against mortality during acute infection, whereas the percent survival in the control groups (OVA and 20-40 OVA peptide-immunized mice) was around 16%. Moreover, analysis of T cell proliferation in response to OVA-coupled peptides clearly indicated that only the 109 to 124 peptide had the capacity to induce the proliferation of T cells from peptide-immunized mice. Interestingly, only the 109 to 124-coupled peptide induced the proliferation of T cells from T. cruzi-infected mice.
The role of CD4+ T cells in immunity to malaria sporozoites.
Authors: Weiss W R , Sedegah M , Berzofsky J A , Hoffman S L 
Published: December 2022

Abstract
BALB/c mice are strongly protected against malaria after immunization with Plasmodium yoelii (PY) sporozoites, and this is an important model for malaria vaccine development in humans. This paper explores the role of CD4+ cells in the induction of the antimalarial immune response. The method used has been to treat animals with anti-CD4 mAb before immunization, resulting in a profound depletion of CD4+ T cells. The primary finding is that mice are not protected by sporozoite immunization after depletion of CD4+ cells. Such mice make little antibody to malaria sporozoite Ag, showing the strong T-cell dependence of these humoral responses. However, infusion of hyperimmune serum does not restore immunity to anti-CD4 treated animals. Neither does injection of exogenous IL-2 compensate for the absence of CD4+ cells. However, regrowth of CD4+ cells does allow successful immunization of animals, showing that long-term suppression against malaria Ag has not been induced by immunization in the absence of CD4+ cells. It is thought that infiltrating CD8+ T cells are critical effector cells against PY parasites in the pre-erythrocytic stages. Mice immunized while depleted of CD4+ cells have normal numbers of CD8+ T cells infiltrating their livers. In addition, they have normal numbers of splenic CD8+ CTL directed at the PY circumsporozoite protein. Thus, it appears that although CD8+ cells have been activated in the absence of CD4+ cells, they cannot protect mice against malaria. We conclude that a successful vaccine against the pre-erythrocytic stages of malaria must activate both CD4+ and CD8+ T cells.
Protein tyrosine kinase inhibitors decrease induction of nitric oxide synthase activity in lipopolysaccharide-responsive and lipopolysaccharide-nonresponsive murine macrophages.
Authors: Dong Z , Qi X , Xie K , Fidler I J 
Published: December 2022

Abstract
We investigated tumoricidal activation and induction of nitric oxide synthase (NOS) activity in macrophages from LPS-responsive (C3H/HeN) and -unresponsive mice (C3H/HeJ). Macrophages were incubated in vitro with a synthetic lipopeptide or with LPS and IFN-gamma. LPS and IFN-gamma activated C3H/HeN but not C3H/HeJ macrophages to lyse B16 melanoma cells. In contrast, lipopeptide and IFN-gamma activated macrophages from both strains of mice. Genistein, a specific inhibitor of protein tyrosine kinase, significantly blocked tumoricidal activation of macrophages from both strains of mice. Genistein did not affect tumor cell binding but significantly inhibited the production of nitric oxide. Genistein, herbimycin A, and tyrphostin inhibited the induction of NOS activity in macrophages from both strains of mice. These data suggest that protein tyrosine kinase activity is involved in the signal transduction pathway of LPS and other synthetic bacterial-related immunomodulators at a point preceding triggering of macrophage tumoricidal activation and expression of inducible NOS activity.
Transcriptional down-regulation of tumor necrosis factor-alpha gene expression by a synthetic peptide homologous to retroviral envelope protein.
Authors: Haraguchi S , Good R A , Cianciolo G J , James-Yarish M , Day N K 
Published: December 2022

Abstract
We have previously shown that a synthetic peptide (CKS-17) homologous to retroviral envelope protein suppresses the accumulation of superantigen staphylococcal enterotoxin-induced TNF-alpha mRNA in human PBMC and in highly purified human monocytes. The present study was designed to examine the underlying mechanism(s) by which CKS-17 down-regulates the TNF-alpha mRNA expression using a human acute monocytic leukemia cell line THP-1 stimulated with the superantigen staphylococcal enterotoxin E. A cyclooxygenase inhibitor indomethacin does not reverse the inhibition of TNF-alpha mRNA expression by CKS-17, suggesting that prostaglandins are not responsible for the suppressive action of CKS-17. The inhibitory effect of CKS-17 is, however, significantly blocked by a protein synthesis inhibitor cycloheximide, indicating that CKS-17 requires de novo protein synthesis to induce the suppressive activity. The mRNA stability assays using actinomycin D show that CKS-17 does not decrease the TNF-alpha mRNA stability. Nuclear run-on transcription assays further reveal that CKS-17 suppresses the TNF-alpha mRNA transcription rate. Taken together, these results suggest that the synthetic retroviral peptide CKS-17 down-regulates TNF-alpha mRNA expression through inhibition of transcriptional activation of the TNF-alpha gene, which requires de novo synthesis of a transcriptional repressor protein(s).
Autosomal codominant inheritance and Japanese incidence of deficiency of OKT4 epitope with lack of reactivity resulting from conformational change.
Authors: Takenaka T , Kuribayashi K , Nakamine H , Yoshikawa F , Maeda J , Kishi S , Nakauchi H , Minatogawa Y , Kido R 
Published: December 2022

Abstract
A large Japanese family in which some members were homozygous or heterozygous for OKT4 epitope deficiency was studied. Homozygotes, heterozygotes, and normal individuals were identified by differences in the number of OKT4 epitopes on the surfaces of lymphocytes. This deficiency was transmitted as an autosomal codominant trait. The internalization of CD4 molecules and the production of IL-2 by lymphocytes of these subjects were examined. The OKT4 epitope was not needed for internalization of CD4 molecules, and IL-2 was produced in the same amounts by these different kinds of subjects. DNA from four clones lacking OKT4 established from four individuals of this family was sequenced. As reported elsewhere for different subjects, a single nucleotide substitution (CGG--&gt;TGG) was found in all four cell lines. The mutation results in arginine being replaced by tryptophan. Analysis showed different hydrophobicity at positions 239 and 240 from the control, probably giving rise to a conformational change in CD4 accounting for lack of reactivity with the OKT4 monoclonal antibody. The incidence of homozygotes in the Japanese population was found to be 0.47% by examination of 1478 random samples, and on the basis of this value, the incidence of heterozygotes was estimated to be 12.8%.
Binding and cytotoxicity characteristics of the bispecific murine monoclonal antibody 2B1.
Authors: Weiner L M , Holmes M , Richeson A , Godwin A , Adams G P , Hsieh-Ma S T , Ring D B , Alpaugh R K 
Published: December 2022

Abstract
Bispecific monoclonal antibodies (BsmAb) with specificity for tumor Ag and effector cell trigger molecules have been shown to redirect the cytotoxicity of several peripheral blood mononuclear cell populations against relevant tumor. The BsmAb, 2B1, binds to the extracellular domain of the c-erbB-2 gene product of the HER2/neu proto-oncogene and to CD16. In this report, the binding and cytotoxic characteristics of 2B1 are presented. Maximal saturation binding of 2B1 to PBL and c-erbB-2 expressing SK-OV-3 cells occurred in the 1 microgram/ml concentration range. However, substantial lysis potentiation was observed at 1000-fold lower BsmAb concentrations. Optimal tumor lysis was obtained when the BsmAb, PBL, and target cells were continuously coincubated. When PBL were franked with 2B1, washed, and added to labeled targets, substantially less lysis was observed. These results suggest that the best way to therapeutically exploit the cytotoxic attributes of 2B1 may be to obtain continuous BsmAb exposure to tumor. Approaches based on franking of this BsmAb to PBL may not be warranted.
Cellular and molecular mechanisms of activation of MHC nonrestricted cytotoxic cells by IL-12.
Authors: Cesano A , Visonneau S , Clark S C , Santoli D 
Published: December 2022

Abstract
Freshly separated PBL and two IL-2-dependent clonal T cell lines (TALL-103/2 [TCR-gamma delta+] and TALL-104 [TCR-alpha beta+]) were used to dissect, at the cellular and molecular levels, the mechanisms governing the enhancing effects of IL-12 on MHC nonrestricted cytotoxic activity. IL-12 induced cytotoxicity both in PBL and TALL cells, although less efficiently than IL-2, and the two cytokines displayed additive effects. Unlike IL-2, IL-12 failed to enhance the CD3 or CD2 redirected killing of the TALL cell lines. Analysis of the percentage of E/T conjugates induced by these cytokines showed that IL-12 could increase PBL, but not TALL cell, binding only when used in combination with IL-2. This and the failure of IL-12 to increase the expression of several adhesion molecules on PBL and TALL cells indicated no significant role for IL-12 in the binding step of the lytic interaction. We also excluded that production of TNF-alpha, IFN-gamma, or IL-2 by the IL-12-activated effectors might account for the cytotoxicity-inducing effects of this cytokine. By contrast, IL-12 was found to induce cytotoxic granule formation in PBL and TALL cells and to up-regulate the expression of their cytolytic components, including perforin, serine esterases, and TIA-1, both at the protein and mRNA levels. The costimulatory action of IL-2 and IL-12 on granulogenesis and induction of cytolytic proteins correlated well with the additive effects on cytotoxicity. Studies with cyclohexamide and actinomycin D demonstrated that TALL cells but not PBL require de novo protein synthesis for expression of serine esterases and perforin mRNA after IL-2 and/or IL-12 stimulation, and that both cytokines act directly or indirectly at the transcriptional level. Overall, these data indicate that IL-12 acts mostly at a postbinding level of the lytic interaction, involving enhanced expression of cytotoxic mediators, and that IL-2 and IL-12 use partially distinct pathways to activate the MHC nonrestricted killer function of mature T cells and resting lymphocytes.