bluuebunny/crossref_metadata_2025_split huggingface dataset:
Patients with multidrug-resistant tuberculosis with low CD4+ T cell counts have impaired Th1 responses.
Authors: McDyer J F , Hackley M N , Walsh T E , Cook J L , Seder R A 
Published: December 2022

Abstract
Multidrug-resistant tuberculosis (MDRTB) has emerged as a challenging clinical problem in both HIV-infected and -uninfected individuals. In this study, immune responses from HIV-negative patients with MDRTB were compared with those of healthy purified protein derivative (PPD)-positive and PPD-negative individuals. These responses were characterized by measuring the proliferation and cytokine production from PBMCs stimulated in vitro with Mycobacterium tuberculosis, PPD, or mitogens. MDRTB patients with CD4 counts &gt;500/microl stimulated in vitro with M. tuberculosis had similar immune responses (proliferation, IFN-gamma, and IL-2 production) as the PPD-positive and -negative controls. By contrast, MDRTB patients with CD4 counts &lt;500/microl had markedly deficient immune responses to similar stimuli. In these patients, IFN-gamma production could be restored by adding IL-12 to the in vitro cultures. IL-12 also caused a striking increase in the amount of IFN-gamma produced from PBMCs of both PPD-positive and -negative controls. The role of endogenous IL-12 production was also studied. Addition of anti-IL-12 to cultures resulted in a two- to eightfold decrease in IFN-gamma production in response to PHA stimulation. Inhibition of IFN-gamma was also observed when cells were stimulated by M. tuberculosis and PPD. Using Staphylococcus aureus Cowan strain as a mitogenic stimulus, IL-12 p70 was produced in similar amounts in all groups tested. TNF-alpha production was also assessed from cells stimulated by M. tuberculosis. Addition of IL-12 to the cultures did not cause a significant enhancement of TNF-alpha production. Last, production of IL-10 and IL-4 in response to M. tuberculosis and PHA, respectively, was not significantly different among all groups tested. These results suggest that patients with MDRTB tuberculosis with CD4 T cell counts &lt;500/microl have impaired IFN-gamma and IL-2 responses and might benefit by adjunctive IL-12 therapy.
Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations.
Authors: Torres C A , Iwasaki A , Barber B H , Robinson H L 
Published: December 2022

Abstract
Surgical ablations revealed different dependencies on the injection site tissue for i.m. needle and epidermal gene gun DNA immunizations. Excision of an injected muscle bundle within 10 min of DNA inoculation did not affect the magnitude or longevity of Ag-specific Ab responses. By contrast, biopsy of the skin target site up to 24 h after gene gun bombardment completely abrogated the Ab response in the majority of mice. The form of a DNA-expressed Ag (membrane bound, secreted, or intracellular) did not affect the temporal requirements for the skin and muscle target sites. Skin cells, but not muscle cells, were also required for DNA-induced CTL. We conclude that transfected cells in gene gun-bombarded skin, but not needle-injected muscle, play a central role in DNA-initiated Ab and CTL responses.
In vivo migration of dendritic cells differentiated in vitro: a chimpanzee model.
Authors: Barratt-Boyes S M , Watkins S C , Finn O J 
Published: December 2022

Abstract
Dendritic cells with potent Ag-presenting function can be propagated from peripheral blood using recombinant cytokines, and these cells have potential usefulness as immunotherapeutic agents in the treatment of cancer and other disease states. However, it is not known if these in vitro differentiated dendritic cells have the capacity to migrate in vivo, especially to T cell areas of lymphoid tissue. We have used a fluorescent marker system to track the migration of dendritic cells, propagated in vitro from chimpanzee peripheral blood, following s.c. injection. We report that injected dendritic cells migrate spontaneously and rapidly to draining lymph nodes, where they remain for at least 5 days. The injected cells interdigitate with T cells in the parafollicular and paracortical zones and retain high level expression of CD86, CD40, and MHC class II molecules, reflecting a phenotype of potent APC. We conclude that dendritic cells differentiated in vitro from peripheral blood and administered s.c. behave in a manner very similar to endogenous Langerhans cells. These data provide strong experimental support, in a highly relevant large animal model, for the use of in vitro differentiated dendritic cells as vehicles for immunotherapy. More importantly, they show that the s.c. route of injection delivers these APC to sites of T cell activation, a prerequisite for the generation of an effective immune response.
P-4 and RNKP-7, new granzyme-like serine proteases expressed in activated rat lymphocytes.
Authors: Ewoldt G R , Smyth M J , Darcy P K , Harris J L , Craik C S , Horowitz B , Woodard S L , Powers J C , Hudig D 
Published: December 2022

Abstract
Serine proteases (granzymes) in killer lymphocytes are required for lymphocyte cytotoxic granules to lyse target cells. Herein we report the development of a 3-step PCR cloning technique to amplify novel granzyme genes and two new rat granzymes are described. Degenerate oligonucleotide primers were designed based on sequence motifs selectively expressed in granzymes. These motifs flank "delta" regions that are unique for each granzyme. Total RNA of RNK-16 cells or activated splenocytes was amplified by reverse transcriptase-PCR to obtain cDNA fragments of several new granzymes. Gene-specific primers based on these delta regions were then used for 3'-RACE to obtain clones with the 3' gene ends. Reverse (antisense) delta-based or active site serine primers were used with a granzyme 5'-UTR primer to obtain clones extending to the 5' ends. Using this technique, two new cDNAs, RNKP-4 and RNKP-7, which encode granzymes of 248 and 241 amino acids, respectively, were cloned from activated lymphocytes. RNKP-4 is likely the rat equivalent of mouse granzyme C. RNKP-7 is most closely related to granzymes F and G. Modeling of the predicted proteins suggests large/polar P1 (Gln/Asn) specificity for RNKP-4 and large/hydrophobic P1 (e.g., Phe) specificity for RNKP-7. These specific protease activities were found in cytotoxic RNK-16 lymphocyte granules indicating that the two new genes may be translated and stored as active granzymes.
Expression of the murine CD21 gene is regulated by promoter and intronic sequences.
Authors: Hu H , Martin B K , Weis J J , Weis J H 
Published: December 2022

Abstract
Murine CD21 gene products are expressed primarily on the surface of B lymphocytes and follicular dendritic cells. To identify the genetic elements that control the tissue-specific expression of the CD21 gene, we analyzed, via transient transfections, the 5' proximal promoter region of the CD21 gene (1272 bp 5' of the initiating ATG). This region possessed strong promoter activity, but it was not tissue specific, in that T cell expression was equivalent to that of B cells. These data suggested that the anticipated tissue-specific control element(s) lies 3' of the initiating ATG. Analysis of a novel minigene construct that possessed both the 5' promoter region and a large region (9 kb) of the CD21 gene 3' of the initiating ATG demonstrated the expected tissue-specific expression. Further analysis using the luciferase reporter system indicated that such control elements reside in the first intron (5.5 kb in size), which separates the exons encoding the signal sequence and the first extracellular short consensus repeat domain of the mature protein. Further dissection of intron 1 demonstrated that the sequences controlling the tissue-specific expression of the murine CD21 gene are contained in the 5' 1.6-kb region of this intron. This 1.6-kb fragment was fractionated into an 800-bp sequence at the 5' end that showed very significant inhibitory activity in both B and T cells and a 3' 800-bp sequence that demonstrated moderate repression in T cells, but enhancer activity in B cells. These data suggest this region of the CD21 gene possesses a number of functionally distinct sites that positively and negatively regulate CD21 gene transcription.
The pentanucleotide ATTGG, the "inverted CCAAT," is an essential element for HLA class I gene transcription.
Authors: Schoneich J , Lee J L , Mansky P , Sheffery M , Yang S Y 
Published: December 2022

Abstract
The HLA class I genes, HLA-A, -B, and -C, contain an inverted CCAAT sequence (ATTGG) located 20 bp upstream of the canonical CCAAT and approximately 70 bp upstream of the transcription initiation site. We have investigated the transcriptional function of the class I inverted CCAAT sequence using the HLA-normal cell line, HeLa. Deletion, mutation, or inversion of the inverted CCAAT sequence abrogated or reduced the activity of the class I promoter, as assessed by luciferase reporter gene assays in transient gene expression experiments. This activity coincided with occupancy of the inverted CCAAT motif, as tested by electrophoretic mobility shift assays using the wild-type sequence and mutated variants of the sequence. The ATTGG-binding protein was not CP2, NF-1, or other known CCAAT-binding proteins, but the complex may contain a CP1/NF-Y-like protein. Our results indicate that this inverted CCAAT sequence is an essential element for the expression of HLA class I genes and that its transcriptional activity depends upon the sequence, position, and orientation of the pentanucleotide.
Phosphorylation and subcellular redistribution of pleckstrin in human neutrophils.
Authors: Brumell J H , Craig K L , Ferguson D , Tyers M , Grinstein S 
Published: December 2022

Abstract
Pleckstrin, originally described as a major substrate of protein kinase C (PKC) in platelets, was found to be highly expressed in human neutrophils (intracellular concentration, approximately 15 microM). As PKC isoforms play an important role in mediating neutrophil antimicrobial responses, we studied the regulation of pleckstrin phosphorylation in response to inflammatory stimuli. Following treatment of neutrophils with FMLP, 12-O-tetradecanoylphorbol-13-acetate, or opsonized zymosan, pleckstrin was rapidly phosphorylated, which resulted in a shift in its electrophoretic mobility. Several lines of evidence suggest that pleckstrin is phosphorylated in part by a nonconventional PKC following stimulation by FMLP: 1) chelation of intracellular Ca2+ had only a partial inhibitory effect; 2) diacylglycerol kinase inhibitors shortened the duration of phosphorylation, while the phosphatidic acid phosphohydrolase antagonist propranolol extended it; and 3) wortmannin and erbstatin blocked the phosphorylation of pleckstrin. These results suggest that nonconventional PKC isoforms, possibly delta or zeta, mediate the phosphorylation of pleckstrin. Both PKCdelta and -zeta are expressed in human neutrophils. Increased association of pleckstrin with both microsomes and with the cytoskeleton was observed in stimulated cells. These findings suggest that phosphorylation by nonconventional PKC isoforms induces a conformational change in pleckstrin that promotes its interaction with membranes and/or with the cytoskeleton. Such a translocation may serve to target proteins or lipids recognized by pleckstrin homology domains to sites where they can contribute to the microbicidal response.
Leukotriene C4 secretion from normal murine mast cells by a probenecid-sensitive and multidrug resistance-associated protein-independent mechanism.
Authors: Nguyen T , Gupta S 
Published: December 2022

Abstract
P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) are members of the superfamily of ATP-binding cassette transporter proteins. Because the ATP-dependent export system has been implicated in the release of leukotriene C4 (LTC4), we examined the roles of P-gp and MRP in the release of LTC4 from normal murine mast cells (MC-9). We have previously shown that MC-9 cells express P-gp at the level of protein and mRNA. In the present study, MRP expression in MC-9 cells was examined at the protein level by anti-MRP Ab, using flow cytometry and at the level of mRNA by PCR and Northern blot analyses. MC-9 cells were stimulated with calcium ionophore A23187 for 15 min in the presence or the absence of various concentrations of cyclosporin A (CsA) and its nonimmunosuppressive analogue CsA-1, which are known to inhibit P-gp efflux function, or in the presence or the absence of probenecid, an organic ion transport inhibitor that appears to inhibit MRP-mediated transport function. Culture supernatants were collected, and LTC4 was measured by ELISA assay. CsA and CsA-1 had no effect on LTC4 secretion from MC-9 cells, suggesting that P-gp is not involved in LTC4 release from MC-9 cells. In contrast, probenecid, in a concentration-dependent manner, inhibited LTC4 secretion from MC-9 cells without inhibiting its synthesis. However, MC-9 lacked MRP at both the protein and mRNA levels. These data suggest that LTC4 is secreted by normal mast cells by a probenecid-sensitive mechanism that is independent of MRP.
Inhibition of prostaglandin endoperoxide synthase-2 expression in stimulated human monocytes by inhibitors of p38 mitogen-activated protein kinase.
Authors: Pouliot M , Baillargeon J , Lee J C , Cleland L G , James M J 
Published: December 2022

Abstract
Prostaglandin endoperoxide synthase (PGHS; cyclooxygenase), the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids, has two isoforms. PGHS-1 is constitutively expressed and involved in homeostasis, whereas PGHS-2 is inducible in monocytes in response to proinflammatory agents. Using freshly elutriated human monocytes, we examined the effect on PGHS-2 expression of certain cytokine-suppressive anti-inflammatory drugs such as SK&F 86002. Incubation with serum-treated zymosan (STZ) stimulated the expression of PGHS-2 in a time- and dose-dependent manner. SK&F 86002 dose-dependently inhibited this STZ-induced expression of PGHS-2 protein, which correlated with a decrease in prostaglandin E2 and thromboxane A2 production. However, suppression of PGHS-2 expression is not the result of suppressed cytokine production, because SK&F 86002 suppressed PGHS-2 expression initiated by IL-1beta and TNF-alpha, in addition to other stimuli. Moreover, this effect was selective in that the protein expression of two other important enzymes involved in the metabolism of arachidonic acid, cytosolic phospholipase A2 and 5-lipoxygenase, was not affected. Stimulation with STZ caused a time-dependent increase in levels of PGHS-2 mRNA; incubation with cytokine-suppressive agents caused a decrease of these levels, suggesting the involvement of transcription and/or mRNA stability events in the inhibition of PGHS-2. These results indicate a new and potentially important anti-inflammatory property of SK&F 86002, namely the specific suppression of PGHS-2 induction.
The balance between nitric oxide and superoxide determines apoptotic and necrotic death of rat mesangial cells.
Authors: Sandau K , Pfeilschifter J , Brüne B 
Published: December 2022

Abstract
Nitric oxide (NO.) and superoxide (O2-) are inflammatory mediators. Their formation seems to be associated with apoptotic and/or necrotic cell death in diseases such as mesangioproliferative glomerulonephritis in which the early phase of mesangiolysis is linked to significant NO. production. Notably, mesangial cells (MC) not only generate NO. but also O2- after cytokine stimulation. Here we investigated the interrelation between NO. and O2- in MC death by generating both radicals with the use of NO donors (S-nitrosoglutathione, spermine-NO) and O2(-)-generating systems (2,3-dimethoxy-1,4-naphtoquinone, hypoxanthine/xanthine oxidase). Exogenously supplied NO. or O2- in a concentration-dependent manner induced apoptosis and/or necrosis. Apoptosis is characterized by chromatin condensation and DNA fragmentation in contrast to necrotic cytoplasmatic membrane rupture. Noteworthy, coincubation of NO. and O2- was cross-protective. Maximum protection required the existence of a balanced NO./O2- ratio. Analysis in cytokine-stimulated MC suggests endogenous radical formation, which may participate in modulating apoptosis. Manipulation of the endogenous NO./O2- ratio by exogenous, sublethal S-nitrosoglutathione in addition to cytokines produced death, which was antagonized by inducible nitric oxide synthase (iNOS) inhibition. Moreover, pyrrolidine dithiocarbamate supplementation, which down-regulates iNOS expression and blocks superoxide dismutase activity, initiates apoptosis. Our results imply the participation of reactive nitrogen and oxygen species in determining life and death of MC.
Mitogen-activated protein kinase activation during IgG-dependent phagocytosis in human neutrophils: inhibition by ceramide.
Authors: Suchard S J , Mansfield P J , Boxer L A , Shayman J A 
Published: December 2022

Abstract
In FMLP-activated polymorphonuclear leukocytes (PMNs) challenged with IgG-opsonized erythrocytes (EIgG), the termination of phagocytosis correlates with an accumulation of ceramide, a product of sphingolipid metabolism. Furthermore, the exogenous addition of short chain ceramides inhibits EIgG-mediated phagocytosis. In the present study, we identified p42 and p44 mitogen-actived protein (MAP) kinases, referred to as extracellular signal-regulated kinases ERK2 and ERK1, respectively, as intracellular targets of ceramide action during Fc gammaR-mediated phagocytosis. The tyrosine phosphorylation of ERK1 and ERK2 increased within 30 s of addition of EIgG, with maximal phosphorylation by 1 to 5 min. By 30 min, ERK1 and ERK2 were almost completely dephosphorylated. The kinetics of ERK1 and ERK2 tyrosine phosphorylation indicated that MAP kinase activation preceded target ingestion. N-Acetylsphingosine (C2-ceramide) inhibited phagocytosis, reduced ERK1 and ERK2 phosphorylation to basal levels, and reduced ERK1 and ERK2 activity by 85 to 90% and 70 to 80%, respectively. In contrast, N-acetyldihydrosphingosine (dihydro-C2-ceramide) had no effect on either tyrosine phosphorylation or activity of ERK1 and ERK2. In the presence of the MAP kinase kinase (MEK) inhibitor, PD 098059, phagocytosis was reduced by approximately 50%, while ERK1 and ERK2 activity was reduced by 85 to 90%. Thus, engagement of Fc gammaRs led to ERK1 and ERK2 phosphorylation and activation, and the activation of these enzymes was critical for phagocytosis. Furthermore, the inhibition of phagocytosis by C2-ceramide correlated with the inhibition of tyrosine phosphorylation and activation of ERK1 and ERK2. These results suggest that ceramides generated during phagocytosis act on the MAP kinase signaling pathway, ultimately "turning off" the phagocytic response.
Antigen activation of mitogen-activated protein kinase in mast cells through protein kinase C-dependent and independent pathways.
Authors: Zhang C , Hirasawa N , Beaven M A 
Published: December 2022

Abstract
We demonstrate discrete pathways for activation of mitogen-activated protein (MAP) kinase in cultured RBL-2H3 mast cells through protein kinase C (PKC), cytosolic calcium, and a third pathway that provides sustained signals for activation in Ag-stimulated cells. Thus, p42 MAP kinase was activated by increasing intracellular free Ca2+ with thapsigargin or by stimulating PKC with PMA. The latter stimulation was selectively blocked by the protein kinase C inhibitor, Ro31-7549. Stimulation of p42 MAP kinase by Ag resulted in relatively sustained activation of MAP kinase which was only partially suppressed by Ro31-7549. Kinetic studies revealed two components of the MAP kinase response to Ag: a rapid but transient component that was Ro31-7549 sensitive and presumably PKC dependent; and a more sustained component that was Ro31-7549 resistant and presumably PKC independent. Similarly, Ro31-7549 inhibited the early but not late release of arachidonic acid, a finding that was consistent with the known regulation of phospholipase A2 by MAP kinase. Early tyrosine phosphorylation events which were thought to be essential for Ag-induced activation of p42 MAP kinase and release of arachidonic acid were unaffected by Ro31-7549. The findings suggested that release of arachidonic acid was regulated primarily through MAP kinase but that PKC may transiently influence this release, either directly or indirectly through MAP kinase.
Human anti-nuclear ribonucleoprotein antigen autoimmune sera contain a novel subset of autoantibodies that stabilizes the molecular interaction of U1RNP-C protein with the Sm core proteins.
Authors: Satoh M , Richards H B , Hamilton K J , Reeves W H 
Published: December 2022

Abstract
Anti-Sm Abs recognize Sm core proteins B'/B, D, E, F, and G, shared by U1, U2, U4-6, and U5 small nuclear ribonucleoproteins (snRNPs), while anti-nuclear ribonucleoprotein Ag (nRNP) Abs recognize the U1 RNP-specific 70K, A, and C proteins. However, although the autoimmune response to U1 snRNPs involves all components of the particle, not all are recognized equally. For example, all human anti-nRNP sera contain Abs against native U1-C, in contrast to their absence in MRL/lpr mice. In this study, autoantibody recognition of native U1 snRNPs was investigated by dissociating the particle into four components (U1-70K, U1-A, U1-C, and the Sm core particle) using 1 M MgCl2 or ribonuclease treatment. As expected, human anti-Sm and MRL/lpr sera immunoprecipitated only the Sm core proteins, and human anti-nRNP/Sm sera immunoprecipitated the Sm core proteins plus U1-C under both conditions. However, although human anti-nRNP sera immunoprecipitated U1-C when U1 snRNPs were dissociated before Ab binding, they unexpectedly immunoprecipitated the Sm core proteins when Abs were bound before dissociation. This apparent paradox was explained by the stabilizing effects of anti-nRNP sera on interactions of U1-C with the Sm core particle. All human anti-nRNP sera contained high levels of autoantibodies that prevent dissociation of U1-C from the U1 snRNP. These Abs were absent in MRL/lpr mice. Human autoimmune sera may prevent dissociation by recognizing the quaternary structure of the U1-C-Sm core protein complex or by altering its conformation. Stabilization of U1 snRNPs by autoantibodies could influence Ag processing and presentation, possibly with important effects on the development of autoimmunity to U1 snRNPs.
Abnormal T lymphocyte development induced by targeted overexpression of IkappaB alpha.
Authors: Esslinger C W , Wilson A , Sordat B , Beermann F , Jongeneel C V 
Published: December 2022

Abstract
A role in thymic maturation for factors of the NF-kappaB family has long been suspected, but not yet proven. Transgenic mice with a lymphocyte-specific defect in NF-kappaB activation were produced by targeted expression of human IkappaB alpha. The thymic cellularity of these mice was significantly decreased. The proportion of mature, TCRhigh thymocytes of the alphabeta lineage was reduced, and the remaining TCRhigh population contained an unusually high proportion of double-positive cells. This defect in maturation resulted in a transgene dose-dependent reduction in peripheral T lymphocytes, with the CD8 lineage being more severely affected. These data provide direct evidence for the involvement of NF-kappaB/Rel family proteins in late stages of T lymphocyte development, coincident with positive and negative selection.