bluuebunny/crossref_metadata_2025_split huggingface dataset:
Probing the C1q-binding site on human C-reactive protein by site-directed mutagenesis.
Authors: Agrawal A , Volanakis J E 
Published: December 2022

Abstract
We have used oligonucleotide-directed site-specific mutagenesis to investigate structural determinants of the C1q-binding site of C-reactive protein (CRP). Eleven mutant rCRP cDNAs, D112N; D112A; D112K; D112E; K114T; K114A; K114E; K114R; R116L; D112N, K114T; and D112N, R116L were constructed and expressed in COS cells. Wild-type (wt) and all mutant rCRPs bound to phosphocholine-substituted BSA and also to pneumococcal C-polysaccharide with apparent avidities similar to native CRP, except for the R116L mutant which bound slightly less avidly. Substitution of Asn, Ala, or Lys for Asp-112 resulted in decreased avidity of ligand-bound CRP for C1q and also in decreased C-activating efficiency as estimated from a C3-fragment deposition assay. However, complexes of the D112E mutant reproducibly bound better to C1q and activated the classical pathway more efficiently than wt rCRP. Substitution of Thr, Ala, or Glu for Lys-114 increased the avidity for C1q by 2- to 3-fold and the efficiency of classical pathway activation by 20- to 30-fold compared with wt CRP. In contrast, the K114R mutant was only slightly different from wt CRP. Substitution of Leu for Arg-116 did not significantly affect C1q-binding but resulted in increased C-activating efficiency. The data indicate that the negative charge of residue Asp-112 plays a major role in the formation of the C1q-binding site of CRP and that the positively-charged residue Lys-114 and to a lesser extent also Arg-116 play important but indirect roles in C1q-binding and activation of C by CRP complexes.
IL-6 production by rat peritoneal mast cells is not necessarily preceded by histamine release and can be induced by bacterial lipopolysaccharide.
Authors: Leal-Berumen I , Conlon P , Marshall J S 
Published: December 2022

Abstract
Mast cells produce a number of cytokines including IL-6. In view of the large amounts of de novo synthesis induced by the activation of rat peritoneal mast cells and previous observations of expression of this cytokine by human lung mast cells, we have studied the regulation of IL-6 production. We examined the hypothesis that mast cell IL-6 production is not related to previous histamine release. Highly purified rat peritoneal mast cells were activated with anti-IgE, calcium ionophore A23187, or LPS. Histamine was used as a marker of preformed mediator release and IL-6 production was assessed by using the B9 hybridoma growth factor bioassay. Anti-IgE activation of rat peritoneal mast cells induced IL-6 production and histamine release. In contrast, LPS activation induced substantial, serum-dependent, IL-6 production without a significant level of histamine release. No preformed IL-6 was detected in the cells. Calcium ionophore induced histamine release from mast cells to a greater extent than did anti-IgE, but no A23187-induced IL-6 production was observed. A23187-treated cells retained high viability and produced a significant amount of TNF-alpha. To further examine the concordance of IL-6 production and histamine release we used mast cell stabilizing drugs. Dexamethasone and nedocromil significantly inhibited IL-6 production in response to anti-IgE. Our results demonstrate that there is not a direct relationship between mast cell degranulation and IL-6 production. Our observations are important for understanding the role of mast cells in inflammation and for developing strategies to modulate mast cell function in disease.
Pattern of anti-small nuclear ribonucleoprotein antibodies in MRL/Mp-lpr/lpr mice suggests that the intact U1 snRNP particle is their autoimmunogenic target.
Authors: Fatenejad S , Brooks W , Schwartz A , Craft J 
Published: December 2022

Abstract
MRL/Mp-lpr/lpr (MRL/lpr) mice develop anti-Sm Abs that bind the B and D proteins of the U1 and other U small nuclear ribonucleoproteins (snRNPs). Humans with SLE also develop anti-Sm, but in contrast to MRL/lpr mice, anti-Sm Abs in humans are virtually always accompanied by anti-snRNP Abs that bind the A and 70 kDa proteins of the U1 snRNP. In this study, we identified anti-U1 snRNP Abs in 60% of MRL/lpr mice (40 of 66 animals), with the earliest and most frequent response directed against the A protein. This response was either accompanied or closely followed by Abs to other snRNP proteins, including the 70-kDa protein of the U1 particle and the B and D proteins that represent the anti-Sm response. Other U snRNPs were rarely bound by these sera, analogous to previous observations in human SLE. Using in vitro translated 5' and 3' deletion mutants, we found that these early Abs principally bound an epitope on the COOH-terminal of the murine A protein. As shown previously, human sera with anti-U1 snRNP reactivity bind a similar epitope. In summary, we have shown that anti-snRNP Abs in MRL/lpr mice are composed of a linked group of specificities against proteins of the U1 snRNP particle, similar to human SLE. These observations, in conjunction with our previous findings that intact snRNPs are necessary for diversification of Abs in normal mice immunized with snRNPs, strongly suggests that intact U1 particles may be targets of the immune response in this disease.
Clonal analysis of alloreactive T cell responses against the closely related B*2705 and B*2703 subtypes. Implications for HLA-B27 association to spondyloarthropathy.
Authors: López D , García-Hoyo R , López de Castro J A 
Published: December 2022

Abstract
Alloreactive responses against closely related HLA-B27 subtypes were compared at the clonal level by fine specificity analysis of anti-B*2705 and anti-B*2703 CTL clones from unrelated HLA-B27- individuals with target cells expressing B*2701 to B*2706, and other HLA Ags. T cell epitope sharing between B*2705 and other subtypes was B*2705 &gt; B*2703 &gt; B*2702 &gt; B*2701 &gt; B*2704 &gt; B*2706, and correlated with their amino acid differences. This suggests that identical or similar peptides, or peptide motifs, can be presented by multiple HLA-B27 subtypes to T cells, a feature that may be critical for the similar linkage of several subtypes to spondyloarthropathies. Other cross-reactions were predominantly with HLA-B61 and HLA-B60. Marked differences were observed in the nature and frequency of clonal reaction patterns among individuals. They correlated with structural features of the HLA-B Ags from each donor, suggesting that anti-HLA-B27 T cell responses are partially determined by the HLA-B phenotype of the responder. Ability to respond to particular HLA-B27-associated epitopes may determine differences in disease susceptibility among HLA-B27+ individuals. The anti-B*2703 and anti-B*2705 responses in the same individual were different. A major feature of anti-B*2703 CTL was that a large majority cross-reacted with B*2705. This can be explained by the effect of the single amino acid change in B*2703 on peptide binding and suggests that the B*2703-bound peptide repertoire is mainly a subset of that bound to B*2705, with few peptides being specifically presented by B*2703 to T cells.
Immature CD45RA(low)RO(low) T cells in the human cord blood. I. Antecedents of CD45RA+ unprimed T cells.
Authors: Bofill M , Akbar A N , Salmon M , Robinson M , Burford G , Janossy G 
Published: December 2022

Abstract
A subset of resting T cells expressing low levels CD45RA and CD45RO molecules (&lt; 1 x 10(3)/cell; CD45RA(low)CD45RO(low)) but high levels of CD45RB and CD38 (5 to 25 x 10(3)/cell) were identified in human cord blood. When these CD45RA(low)RO(low) cells were isolated, they failed to survive in culture (&lt; 10% viability at day 3) unless they were co-cultured on fibroblast monolayers. During the co-culture with fibroblasts, these lymphocytes acquired high levels of cytoplasmic and then membrane CD45RA by day 3 without signs of activation. When stimulated with PHA in the absence of fibroblasts the CD45RA(low)RO(low) cells required monocytes or IL-1 to respond; they rapidly perished if neither were present. On optimal mitogenic stimulation for 48 h in the presence of monocytes, &gt; 90% of CD45RA(low)RO(low) T cells showed only transient CD45RA expression and rapidly acquired CD45RO reactivity. After activation for 48 h the stimulated CD45RA(low)RO(low) subset synthesized high levels of IL-2, comparable to mature peripheral T cells. No IL-4 was detected in these stimulated cultures of cord blood T cells. These data taken together suggest that CD45RA(low)RO(low) T cells in the cord blood are the relatively immature antecedents of CD45RA+RO T cells that require stromal factors for survival in a resting state. The same cells need monocytes or IL-1 for their activation to develop after a short CD45RA+ stage into activated CD45RO+RA(low) T cells with potent IL-2 biosynthetic capacity. An additional study of these cells is warranted to confirm that they are in fact the recent emigrants from the thymus, as suggested by similar observations in animal models.
Inhibition of ceramide pathway does not affect ability of TNF-alpha to activate nuclear factor-kappa B.
Authors: Johns L D , Sarr T , Ranges G E 
Published: December 2022

Abstract
TNF-alpha is a multifunctional cytokine that has been shown to activate a number of intracellular second messenger pathways. Recent studies demonstrate that the sphingomyelinase-ceramide pathway plays a potential role in the activation of nuclear factor-kappa B (NF-kappa B) by TNF-alpha. The following experiments both confirm that the addition of ceramide to cells can activate NF-kappa B and demonstrate that a 48-h pretreatment with phorbol 12-myristate (PMA) results in the loss of the ceramide-induced NF-kappa B response. In parallel experiments, in which SW480 cells were pretreated with PMA, TNF-alpha provided a signal resulting in the nuclear translocation of NF-kappa B that was similar to untreated cells. These data combined suggest that additional pathways exist that TNF-alpha can use for the activation of NF-kappa B. Supplementary data demonstrates that TNF-alpha, ceramide, and PMA activate a human cytomegalovirus-(HCMV) beta gal construct (promoter is responsive to NF-kappa B) that was stably transfected into the TNF receptor-bearing tumor cell line, SW480. PMA pretreatment of these cells resulted in a significant decrease in both the PMA and ceramide generated responses, 6% and 0% of controls, respectively. However, the response generated by TNF-alpha was not inhibited significantly (96% of control cells). This data suggests that although ceramide and 1,2-diacylglycerol (DAG) pathways may contribute to TNF-alpha activation of NF-kappa B, impedance of these pathways does not block TNF-alpha from activating NF-kappa B nor induction of the functional activation of the NF-kappa B responsive reporter construct, HCMV.
CD31 (PECAM-1), CDw32 (Fc gamma RII), and anti-HLA class I monoclonal antibodies recognize phosphotyrosine-containing proteins on the surface of human neutrophils.
Authors: Skubitz K M , Goueli S A 
Published: December 2022

Abstract
Although most studies of protein phosphorylation have focused on intracellular reactions, studies have provided evidence for the existence of ectoprotein kinase activity on the surface of some cells including human neutrophils. The identification and characterization of physiologic substrates of ectoprotein kinase activity should aid the understanding of the role of this enzyme activity in cell function. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [gamma-32P]ATP under conditions initially designed to detect ectoprotein kinase activity revealed that CD31, CDw32, and anti-HLA class I mAbs specifically recognize phosphoproteins on the surface of human neutrophils. Phosphorylation of these proteins was inhibited by pretreatment of cells with an impermeant sulfhydryl reagent before radiolabeling. Phosphoamino acid analysis of the proteins revealed that they contained predominantly phosphotyrosine. However, controlled proteolysis of intact cells and purified HLA class I revealed that the HLA class I heavy chain was phosphorylated on the cytoplasmic domain. These results suggest that the molecules recognized by CD31 (PECAM-1) and CDw32 (Fc gamma RII) Abs may also be phosphorylated on cytoplasmic domains under conditions originally designed to detect ectoprotein kinase activity. Phosphorylation of CD31 (PECAM-1), Fc gamma RII, and HLA class I heavy chain on tyrosine may play a role in regulating their function. These results emphasize that the demonstration that a membrane protein is an ectokinase substrate is complex and requires the definitive localization of the phosphorylated residue to the extracellular domain of the protein.
Signal transduction pathway in human polymorphonuclear leukocytes for chemotaxis induced by a chemotactic factor. Distinct from the pathway for superoxide anion production.
Authors: Yasui K , Yamazaki M , Miyabayashi M , Tsuno T , Komiyama A 
Published: December 2022

Abstract
The tyrosine kinase inhibitors erbstatin and herbimycin A inhibited the chemotactic response to FMLP (2 x 10(-7) M) and the superoxide anion (O2-) production stimulated by FMLP (1 x 10(-6) M) in human polymorphonuclear leukocytes (PMN) in similar manners. These compounds also inhibited phospholipase D (PLD)-catalyzed breakdown of phosphatidyl choline, suggesting a possible link between tyrosine kinase and PLD. In the presence of propranolol (phosphatidic acid (PA) phosphohydrolase inhibitor), or ethanol, the activation of PLD results in the modulation of PA and/or diglyceride (DG) generation, producing an irregularity in O2- production. However, PMN motility was not affected in these conditions. These results suggest that PLD is a downstream effector of FMLP-induced tyrosine kinase activation that leads to activation of the PMN superoxide release but not to chemotactic migration. In contrast, the tyrosine kinase inhibitors did not inhibit inositol 1,4,5-triphosphate generation and increase of intracellular concentration of free calcium. Furthermore, a protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), did not affect the migration of PMN and the activation of PLD induced by FMLP at concentrations of less than 50 microM. These results support the premise that there is a specific signaling pathway for chemoattractant-induced PMN locomotion.
Overexpression of zinc-finger transcription factor Z-225/Egr-1 in synoviocytes from rheumatoid arthritis patients.
Authors: Aicher W K , Heer A H , Trabandt A , Bridges S L , Schroeder H W , Stransky G , Gay R E , Eibel H , Peter H H , Siebenlist U 
Published: December 2022

Abstract
In rheumatoid arthritis (RA) the proliferation of synovial lining cells appears to be one of the initial pathologic changes that contributes to the destruction of articular joints. To understand the pathomechanisms involved in these functional changes, we analyzed the transcriptional regulation of the zinc-finger gene 225 (Z-225/Egr-1), a transcription factor expressed in the immediate early events of cellular activation. We found that Z-225 transcripts were significantly up-regulated in RA synoviocytes. In primary and long term culture Z-225 was spontaneously transcribed at elevated levels. In situ hybridization of zinc-finger probe showed characteristic Z-225 transcripts in RA synovial tissues. Identity of these signals to the Z-225 gene product were confirmed in freshly isolated synovial tissue by enzymatic amplification of cDNA by the PCR technique. Z-225 transcripts were also detected and characterized in a cDNA library established from a RA synovial explant. We therefore conclude that RA synoviocytes spontaneously produce Z-225 gene products at elevated levels. Because early growth response gene Z-225 is involved in the regulation of expression of other genes such as proto-oncogenes c-ras and c-sis, which are also up-regulated in the RA synovium, activation of Z-225 transcription in RA may represent a key event in articular joint destruction.
Characterization and function of the NKR-P1dim/T cell receptor-alpha beta+ subset of rat T cells.
Authors: Brissette-Storkus C , Kaufman C L , Pasewicz L , Worsey H M , Lakomy R , Ildstad S T , Chambers W H 
Published: December 2022

Abstract
MHC-unrestricted cytotoxicity is mediated primarily by NK cells. However, some subsets of TCR-alpha beta+ and TCR-gamma delta+ T cells also have the capacity to mediate MHC-unrestricted cytotoxicity, particularly after incubation in high concentrations of IL-2. Currently, it is not known what receptors on T cells are responsible for this activity, nor whether such receptors are the same as those on NK cells. We have recently described a type II integral membrane protein, termed NKR-P1, that is expressed at high levels on rat NK cells (NKR-P1bright). NKR-P1 contains a carbohydrate recognition domain characteristic of C-type (Ca(2+)-dependent) animal lectins and is a representative member of a distinct group of this superfamily. By a variety of criteria, NKR-P1 is linked to a signaling pathway that activates NK cell lytic function. Based on its structure and function, NKR-P1 has been implicated as a candidate molecule involved in or contributing to MHC-unrestricted cytotoxicity. We describe herein the expression of NKR-P1 at low levels on a small subset of rat T cells with an NKR-P1dim/TCR-alpha beta+ phenotype and on a small subset of cells with an NKR-P1dim/TCR-alpha beta- phenotype (presumably containing gamma delta+ T cells). Before incubation with IL-2, the NKR-P1dim subsets of cells lack MHC-unrestricted cytolytic capacity and lack the capacity for reverse antibody-dependent cellular cytotoxicity (rADCC) mediated via NKR-P1. However, culture of NKR-P1dim/TCR-alpha beta+ T cells in IL-2 led to the acquisition of both MHC-unrestricted cytotoxicity and the capacity for rADCC via NKR-P1. NK-like cytolytic function was not found among IL-2-activated NKR-P1-/TCR-alpha beta+ T cells. These data suggest that expression of functional NKR-P1 (i.e., ability to signal rADCC) correlates with and potentially contributes to MHC-unrestricted cytotoxicity.
Expression of tenascin in thymus and thymic nonlymphoid cells.
Authors: Hemesath T J , Stefansson K 
Published: December 2022

Abstract
Tenascin (TN) is an extracellular matrix glycoprotein that is widely expressed in fetal tissues and tumor matrices but is absent from most normal adult tissues. It is transiently expressed at sites of wound healing and has been shown to inhibit some types of T cell activation. We have examined the expression of TN in rat and human thymic tissue. Our results indicate that TN is expressed in both neonatal and adult rat thymus, and that in human thymus TN is present in a meshlike network at the corticomedullary junction. In addition, cultured human thymic non-lymphoid cells grown in serum-containing medium synthesize TN, whereas under serum-free conditions these cells secrete TN in response to transforming growth factor-beta.
Mechanism of virus-induced Ig subclass shifts.
Authors: Nguyen L , Knipe D M , Finberg R W 
Published: December 2022

Abstract
Infection of mice with live viruses leads to a dramatic increase in the amount of IgG2a Ig with a consequent shift in the ratio of IgG1/IgG2a. To examine the Ig subclass shift induced by viral infection, we challenged mice with live virus, inactivated virus, or replication-defective mutant viruses that were able to infect cells and produce some viral proteins but were not able to complete a replicative cycle. While killed (or inactivated) virus was capable of inducing HSV-specific antibody, it did not stimulate a shift in the subclass of the total Ig. Replication-defective mutant viruses that fail to express a functional ICP8 or ICP27 protein, but not a mutant expressing a defective ICP4 protein, were able to stimulate the shift. Thus, only a portion of the lytic cycle is sufficient to induce the shift. At least part of the effect is mediated by IFN-gamma.
Isolation and characterization of CD6- T cells from peripheral blood.
Authors: Rasmussen R A , Counts S L , Daley J F , Schlossman S F 
Published: December 2022

Abstract
Antibodies to the CD6 Ag have been described as having pan-T cell reactivity. We have recently demonstrated, however, that after treatment of PBL with an anti-CD6-blocked ricin-conjugated immunotoxin, clonal populations of CD3+, CD6- cells can be identified. Herein we show that through dual parameter staining of freshly isolated E-rosette+ cells, an average of 5 to 6% of either CD3+ or CD5+ cells express little or no CD6 on their surface. After negative selection by antibody-coated paramagnetic bead depletion, expanded CD6- T cells were shown to be CD1a-, CD2+, CD3+, CD5+, CD16-, CD56-, TCR-gamma/delta-, and consisted of both CD4+ and CD8+ cells. Furthermore, staining of digitonin permeabilized cells showed no cytoplasmic expression of the CD6 Ag and CD6 mRNA was not detected by Northern blot analysis. Identical staining patterns were observed for T cell clones isolated through bead depletion or immunotoxin treatment and expanded with either PHA or immobilized anti-CD3 mAb. It was also found that, relative to unfractionated T cells, the surface expression of CD5 was significantly diminished on CD6- T cells. Functionally, freshly isolated CD6- T cells showed substantially reduced alloreactivity in MLR compared with unfractionated E-rosette+ cells, yet both gave similar proliferative responses to either PHA or soluble tetanus toxin Ag. We conclude that there exists a minor subpopulation of mature T cells in peripheral blood that lack CD6. The diminished alloreactivity of these cells may help to explain the low incidence of graft-vs-host disease, despite high levels of engraftment, that has been reported in allogeneic bone marrow transplant patients receiving marrow treated with anti-CD6 (T12) mAb plus C'.
Helper-cytotoxic T lymphocyte (CTL) determinant linkage required for priming of anti-HIV CD8+ CTL in vivo with peptide vaccine constructs.
Authors: Shirai M , Pendleton C D , Ahlers J , Takeshita T , Newman M , Berzofsky J A 
Published: December 2022

Abstract
CTL are a critical component of protective immunity against viral infections, but requirements for in vivo priming of CTL are not completely understood. Covalent linkage of a helper determinant to a CTL determinant, analogous to that required for cognate help for antibody production, does not appear to be necessary in vitro, but its necessity has not been extensively explored in vivo, especially at a molecular level. We previously defined peptides encompassing multideterminant regions of HIV-1 gp160 (cluster peptides) recognized by Th from mice and humans of multiple MHC types. To investigate the requirement for Th in the development of CTL in vivo, in the context of developing a synthetic peptide vaccine for HIV active in multiple strains of mice, we immunized with compound peptides representing an immunodominant CTL epitope, P18, of gp160, co-linearly synthesized at the C-terminus of three cluster peptides. Spleen cells from compound-peptide-immunized mice of three MHC haplotypes sharing the Dd class I MHC molecule but with different class II molecules exhibited enhanced gp160-specific CD8+ CTL activity and CD4+ Th. In contrast, immunization with P18 alone or a mixture of cluster peptide and P18 elicited only marginal CTL activity. These results imply a requirement for determinant linkage in CTL induction in vivo similar to that already well recognized for cognate help for antibody induction. The results also define promising peptide HIV vaccine candidates for induction of CTL, as well as neutralizing antibodies, in diverse MHC types.