bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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 > B*2703 > B*2702 > B*2701 > B*2704 > 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

A subset of resting T cells expressing low levels CD45RA and CD45RO molecules (< 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 (< 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, > 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

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

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

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

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

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

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

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

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

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.
Neonatal VH, D, and JH gene usage in rabbit B lineage cells.
Authors: Friedman M L , Tunyaplin C , Zhai S K , Knight K L
Published: December 2022

The neonatal antibody repertoire in both mouse and humans differs from that of the adult repertoire in that the neonatal repertoire uses a limited set of JH-proximal VH genes but the adult repertoires use many different VH genes. Rabbits are unusual in that adults use only three or four VH genes, with approximately 80% of the B cells using VH1, the 3'-most VH gene. To investigate whether the repertoire of neonatal rabbits differs from that of adults, we analyzed VH, D, and JH gene usage in B cells of neonatal rabbits. A total of 68 rearranged VDJ genes was cloned from mRNA and genomic DNA isolated from lymphoid tissues of newborn to 10-day-old rabbits. We found that 74% of the VDJ gene rearrangements utilized VH1 and 15% utilized the genes that we designated VHx or VHy. From the remaining VDJ genes we identified seven novel VH genes, one, VHz, which was found in mRNA. We conclude that the repertoire of utilized VH genes in neonates is limited and is similar to that of adult rabbits. We also found the D1, D2a, D2b, and JH4 gene segments preferentially rearranged. We suggest that the preferential usage of VH, D, and JH gene segments in VDJ genes is caused by preferential rearrangement rather than by selective expansion of B cells that utilize the gene segments.
Molecular analysis of mercury-induced antinucleolar antibodies in H-2S mice.
Authors: Monestier M , Losman M J , Novick K E , Aris J P
Published: December 2022

In H-2S mice, the administration of mercuric chloride results in the development of antinucleolar autoantibodies. These mice represent a valuable model to study the role of environmental factors in the development of systemic autoimmunity. We have obtained seven antinucleolar mAb from mercury-injected A.SW mice and characterized their specificities and V genes. All mAb immunoprecipitate the U3 and U8 ribonucleoprotein particles (RNP) and some (but not all) react with fibrillarin, the only currently characterized protein component of mammalian nucleolar RNP. Several VH and V kappa genes are recurrently used by these antinucleolar RNP mAb and their H chain CDR3 segments contain several acidic residues that may be important for binding to the cationic proteins composing the nucleolar RNP. Our results support the concept that in H-2S mice administration of mercury induces a specific loss of tolerance to nucleolar RNP.
Differential recognition of murine tumor-associated oncofetal transplantation antigen and individually specific tumor transplantation antigens by syngeneic cloned BALB/c and RFM mouse T cells.
Authors: Rohrer J W , Rohrer S D , Barsoum A , Coggin J H
Published: December 2022

We have previously demonstrated in several species that sarcomas, lymphomas, and carcinomas express a common Ag that cross-reacts with midgestation fetal cells. We also produced a mAb to that protein and characterized it as a 44-kDa glycoprotein. The cross-reactive immunity induced by immunization with tumor or fetal cells expressing the oncofetal Ag (OFA) can be adoptively transferred with cell populations containing T lymphocytes. The experiments discussed within this paper describe the establishment and characterization of two types of T lymphocytes induced by immunization with syngeneic tumor cells in two mouse strains. We find that five of the eight cloned T cells derived from spleens of BALB/c mice that had been immunized with MCA1315 fibrosarcoma cells are specific for an Ag shared by MCA1315 and MCA1321 cells. The other three clones are specific for an Ag present on MCA1315 but not on MCA1321. Also, none of the clones were reactive with the BALB/c plasmacytoma MOPC-315, which does not express OFA. We also find that 75% of the RFM T cell clones from spleens of RFM mice immune to the RFM thymoma 5T show a 5T-specific proliferative response. One of the four clones, however, responds to both 4T and 5T RFM thymoma cells. The BALB/c and RFM cross-reactive clones specifically respond to purified 44-kDa OFA derived from MCA1315 fibrosarcoma cells in the presence of syngeneic irradiated spleen cells and IL-2. All of the clones from both strains of mice, be they tumor-specific transplantation Ag specific or OFA specific, are CD4+, CD3+, alpha beta TCR+ T cells that secrete IFN-gamma on Ag stimulation.
Transforming growth factor-beta 1 inhibits inflammatory cytokine-induced C3 gene expression in astrocytes.
Authors: Barnum S R , Jones J L
Published: December 2022

In this report, we show that transforming growth factor-beta (TGF-beta) can significantly inhibit the capacity of IFN-gamma, IL-1 beta, and TNF-alpha to augment expression of the central component of complement C3 in the human astroglioma cell line D54-MG. Treatment of D54-MG cells with TGF-beta alone had no dose- or time-dependent effect on basal C3 protein or mRNA levels. However, TGF-beta suppressed induction of C3 expression at both the protein and mRNA level in D54-MG cells treated with inflammatory cytokines. The extent of TGF-beta-mediated suppression was cytokine-specific, and suppression of protein production did not necessarily correspond to reductions in steady-state mRNA levels for each cytokine. Similar findings were obtained at the mRNA level using primary rat astrocytes, indicating that TGF-beta can modulate C3 gene expression in nontransformed astrocytic cells. Kinetic studies demonstrated that TGF-beta mediates its suppressive effect for at least 72 h, and that pretreatment of cells with TGF-beta for as little as 2 h significantly reduced the ability of all three inflammatory cytokines to enhance C3 expression. Our results suggest that TGF-beta may play an important role in modulating the endogenous synthesis of complement by astrocytes under inflammatory conditions.
Leukotrienes in renal transplant rejection in rats. Distinct roles for leukotriene B4 and peptidoleukotrienes in the pathogenesis of allograft injury.
Authors: Spurney R F , Ibrahim S , Butterly D , Klotman P E , Sanfilippo F , Coffman T M
Published: December 2022

To investigate the role of leukotrienes in renal allograft rejection, we studied the effects of specific leukotriene inhibitors in a rat kidney transplant model. The enhanced renal production of leukotrienes observed in allograft recipients was reduced in a dose-dependent manner by the specific 5-lipoxygenase inhibitor MK886. This suppression of leukotriene production caused a substantial improvement in renal function. Inhibition of 5-lipoxygenase also reduced the severity of vascular inflammation and endothelial injury in allografts, and profoundly inhibited expression of donor MHC class II Ag on kidney cells. Survival of renal allograft recipients was prolonged from 10 +/- 1 days in controls to 16 +/- 1 days in animals that received a 6-day course of MK886 (p < 0.05). To investigate the relative roles of LTB4 compared to peptidoleukotrienes in these processes, we treated a separate group of animals with the specific peptidoleukotriene receptor antagonist SKF106203. This agent inhibits the interaction of peptidoleukotrienes with their receptor(s) but does not affect the biologic actions of LTB4. In these studies, SKF106203 caused a modest improvement in renal allograft function that was of lesser magnitude than that seen with the 5-lipoxygenase inhibitor. SKF106203 also reduced vascular inflammation in allografts, but had no effect on expression of MHC class II Ag. We conclude that leukotrienes play a key role in the pathogenesis of renal allograft rejection. Furthermore, the detrimental effects of leukotrienes in rejection are mediated by distinct actions of LTB4 and peptidoleukotrienes.
Effects of IL-12 on in vivo cytokine gene expression and Ig isotype selection.
Authors: Morris S C , Madden K B , Adamovicz J J , Gause W C , Hubbard B R , Gately M K , Finkelman F D
Published: December 2022

The effects of murine rIL-12 on cytokine gene expression and Ig secretion were studied in vivo. In untreated mice IL-12 enhanced IFN-gamma and IL-10 gene expression and protein secretion, reduced base line IL-3 and IL-4 gene expression, and increased serum IgG2a concentration. In mice that had been injected with goat anti-mouse IgD antibody (G alpha M delta) to induce increases in IL-3, IL-4, and IL-10 gene expression and serum IgE, IgG1, IgG2a, and IgG3 concentrations, the simultaneous injection of IL-12 enhanced IFN-gamma and IL-10 gene expression and suppressed IL-3 and IL-4 gene expression and serum IgG and IgE responses. Anti-IFN-gamma mAb neutralized most, but not all, IFN-gamma produced by mice treated with G alpha M delta and IL-12. Anti-IFN-gamma mAb enhanced IL-3 and IL-4 gene expression, did not affect IL-10 or IFN-gamma gene expression, and increased serum IgG1, IgG2a, and IgG3 levels, but had relatively little effect on serum IgE in these mice. In contrast to its effects in G alpha M delta-treated mice, IL-12 failed to inhibit the IgE response to G alpha M epsilon antibody, which stimulates mIgE+ B cells to secrete IgE. These observations demonstrate that: 1) IL-12 may limit its own effects by inducing the production of a cytokine (IL-10) that down-regulates both IL-12 production and IL-12-induced IFN-gamma production; 2) IL-12 inhibits the production of at least one cytokine, IL-3, that is not generally regarded to be strictly Th1- or Th2-associated; 3) IL-12 inhibits switching to IgE secretion to a greater extent than it inhibits switching to other Ig isotypes; and 4) the in vivo effects of IL-12 are, to a large extent, IFN-gamma-dependent.
Peptide binding to surface class II molecules is the major pathway of formation of immunogenic class II-peptide complexes for viable antigen presenting cells.
Authors: Nygard N R , Giacoletto K S , Bono C , Gorka J , Kompelli S , Schwartz B D
Published: December 2022

Although studies on fixed APCs have demonstrated that peptide can bind to cell surface class II molecules, the mechanisms by which peptide-class II complexes are formed in viable cells is largely unexplored. To explore the possibility that peptide loading of class II molecules was occurring after endocytosis of peptides as well as by surface binding, we utilized an immunogenic hemagglutinin peptide (HAP 128-145) from the influenza strain A/Japan/57, and studied the appearance of surface complexes of HAP 128-145 bound to HLA-DRw11 molecules on human B-lymphoblastoid cells (BLCLs). Detection of the bound peptide was made possible by a rabbit anti-serum (alpha HAP) raised against HAP 128-145, which recognizes both the free peptide as well as peptide bound to DRw11 on living APCs. Pretreatment of the BLCLs with a variety of inhibitors of protein synthesis and intracellular trafficking failed to decrease the levels of HAP 128-145/DRw11 surface complexes. However, significant inhibition in the appearance of these complexes was caused by a decrease in the temperature at which the cells were incubated with peptide. Temperature-specific inhibition was also observed for fixed DRw11-positive APCs and purified DRw11 molecules indicating that the effect of temperature was directly on the class II molecules. We conclude that surface binding of peptide by class II molecules on human B cells is a major pathway of formation of immunogenic class II-peptide complexes for at least some soluble antigenic peptides, and that endocytosis of soluble peptides with subsequent binding of peptide by intracellular class II molecules plays little if any role in the formation of such complexes. Moreover, class II molecules have evolved to stably bind peptide optimally at physiologic temperatures, independent of cell metabolism.
Immunization with soluble hepatitis B virus surface protein elicits murine H-2 class I-restricted CD8+ cytotoxic T lymphocyte responses in vivo.
Authors: Schirmbeck R , Melber K , Kuhröber A , Janowicz Z A , Reimann J
Published: December 2022

Immunization with soluble proteins only rarely induces a specific response of CD8+ CTL. We describe experiments that demonstrate the efficient and specific in vivo priming of CTL in BALB/c mice immunized with soluble hepatitis B virus (HBV)-derived surface (S) protein. A single (s.c., i.p. or i.v.) injection of a low dose (30 ng to 3 micrograms per mouse) of recombinant S protein particles without adjuvants induced a CTL response. This specific cytotoxic response was read out against a panel of different S protein-expressing transfected mouse cell lines. Effector cells of this response were Ld-restricted, CD3+ CD4- CD8+ CTL. H-2d/Ld+ (BALB/c, C.B-17) mice were responders; H-2d/Ld- (dm2) mutant mice and H-2b (C57BL/6) mice were nonresponders. Injections of various dosages of a S protein-derived, immunogenic, synthetic peptide into BALB/c mice by various routes did not prime CTL. After incorporation of S protein particles into IFA or aluminum hydroxide, these protein Ag lost their ability to specifically stimulate CTL in vivo. After priming of mice with S protein emulsified in IFA or adsorbed to aluminum hydroxide boost injections with native S protein particles were inefficient in stimulating a specific CTL response. These findings are of relevance for the design of synthetic subunit vaccines for which specific stimulation of CD8+ T effector functions is desired.
A monoclonal antibody that recognizes HLA-B27 in the context of peptides.
Authors: Wang J , Yu D T , Fukazawa T , Kellner H , Wen J , Cheng X K , Roth G , Williams K M , Raybourne R B
Published: December 2022

The T2 mutant cell line is unable to load peptides into the MHC class I Ags inside the cells. These "empty" MHC class I Ags are not expressed on the cell surface unless the cells are cultured at low temperatures. Expression will occur at 37 degrees C only in the presence of peptides that bind to and stabilize the class I Ags. T2 cells transfected with the B*2705 gene were tested with a panel of anti-HLA-B27 mAb. Two of the antibodies, ME1 and KS3, reacted with the "empty" HLA-B27 expressed at low culture temperatures. Three antibodies, B27.M1, B27.M2, and Ye-2, were unreactive with these "empty" HLA-B27. The cells were then incubated with a panel of HLA-B27-binding peptides. One of the antibodies, Ye-2, became reactive when the cells were incubated with a peptide derived from HIV gp120 and to a less degree with a peptide derived from histone H3.3. Mouse L cells transfected with the B*2705 and the human beta 2m genes also reacted very poorly with B27.M1, B27.M2, and Ye-2. Those two peptides were also able to induce high increase in Ye-2 reactivity. Alternately, increase in Ye-2 reactivity was also observed when the L cells were incubated with IFN-gamma or TNF-alpha. These experiments indicate that the Ye-2 anti-HLA-B27 mAb recognizes HLA-B27 in the context of certain residing peptides either added exogenously or expressed endogenously. The B27.M1 and B27.M2 antibodies might share similar characteristics.
Retinoic acid inhibition of IL-1-induced IL-6 production by human lung fibroblasts.
Authors: Zitnik R J , Kotloff R M , Latifpour J , Zheng T , Whiting N L , Schwalb J , Elias J A
Published: December 2022

IL-6 is a multi-functional cytokine that plays an important role in normal biologic homeostasis and disease pathogenesis. Retinoids are vitamin A analogs that regulate the function of a wide variety of inflammatory and structural cells. To further understand the biology of retinoids and IL-6 we determined whether all-trans-retinoic acid (RA) and other retinoids regulate lung fibroblast IL-6 production. RA did not stimulate fibroblast IL-6 production. Instead, it inhibited the production of IL-6 by IL-1-stimulated cells. This effect was dose-dependent with an IC50 of 10(-7) M RA and significant inhibition being noted with doses of RA as low as 10(-8) M. These inhibitory effects could not be explained by cytotoxicity or a shift in the kinetics of IL-6 production. They also did not appear to involve alterations in the early events in IL-1-induced IL-6 production, because RA inhibited IL-6 production even when added 6 h after IL-1 and RA did not inhibit IL-1 binding to cell surface IL-1 receptors. RA inhibition of IL-6 protein production was associated with a comparable decrease in IL-6 mRNA accumulation and gene transcription. 13-cis-retinoic acid, retinol, retinaldehyde, all-trans etretin, Ro 13-6298, and 9-cis retinoic acid also inhibited IL-1-induced IL-6 production. However, 4-hydroxyphenyl retinamide and etretinate did not share this property. The inhibitory effects of these analogues may be mediated by nuclear retinoic acid receptors as mRNA encoding RAR-alpha, RAR-gamma, and RXR-alpha were present, and RAR-beta was induced by RA in human lung fibroblasts. These studies demonstrate that RA and other retinoid analogs inhibit IL-1-induced IL-6 production and that this effect is analog-specific and, at least partially, transcriptionally mediated.
VH and V kappa segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus. Evidence for somatic selection.
Authors: Ikematsu H , Ichiyoshi Y , Schettino E W , Nakamura M , Casali P
Published: December 2022

In some patients with insulin-dependent (type I) diabetes mellitus (IDDM), autoantibodies to insulin are present at diagnosis. After initiation of the treatment with not only animal but also human insulin, anti-insulin, mainly IgG, autoantibodies become a major component of the autoimmune response in virtually all IDDM patients. Their structure, however, is still relatively unknown. We analyzed the structure of the VH and V kappa segments of three human IgG mAb derived from three IDDM patients. The sequences of VH genes of two IgG, mAb13 and mAb48, were 98.3 and 96.6% identical with those of the H11 and 1.9III genes (VHIII family), respectively. The sequence of the VH gene of the third IgG, mAb49, was 98.6% identical with that of the 51p1 gene (VHI family). All three IgG mAb used V kappa III segments. The V kappa III gene sequences of mAb13 and mAb49 were 97.9 and 98.9% identical, respectively, to that of the kv3g gene; the mAb48 V kappa gene sequence was 96.5% identical to that of the kv328 gene. The VH and/or V kappa segments of these anti-insulin IgG mAb are similar to Ig V genes expressed in the fetal, and adult normal and autoimmune B cell repertoires. The nucleotide differences displayed by the three anti-insulin IgG mAb VH gene sequences, when compared with those of the closest reported germ-line genes, were concentrated in the CDR (6.2 x 10(-2) and 0.8 x 10(-2) difference/base in CDR and FR, respectively; p < 0.01, chi 2 test), and yielded a significantly higher putative replacement (R) to silent (S) mutation ratio in the CDR (12.0) than in the framework (0.2). The concentration of nucleotide differences in the CDR and their high R:S putative mutation ratios were consistent with the hypothesis that these expressed VH genes underwent a process of somatic mutation and Ag-driven clonal selection. That such differences constituted somatic point-mutations was formally proved in IgG mAb13, by differentially targeted PCR amplification and Southern blot hybridization of the mAb13-producing cell line DNA. The putative germ-line gene that gave rise to the expressed VH segment was cloned using genomic DNA from PMN of the same patient whose B cells were used for the generation of this mAb. Overall, in the anti-insulin IgG mAb VH and V kappa III genes, the (putative and verified) somatic point-mutations yielded 27 amino acid replacements, of which 14 nonconserved. Four of these resulted in positively charged residues, three Arg and one His.(ABSTRACT TRUNCATED AT 400 WORDS)
Up-regulation of the MHC class II molecules on B cells by peptide ligands.
Authors: Agrawal B , Fraga E , Kane K , Singh B
Published: December 2022

MHC class I and class II molecules present peptide Ag to T lymphocytes. Peptides are critical in class I heavy chain folding and/or stable association with beta 2m. A recent study suggests the role of peptide Ag binding for MHC class II alpha- and beta-chain heterodimers to enter into a compact state and allow their transport to the cell surface. We have investigated the effect of peptide ligands on the expression of MHC class II I-A(d) molecules on the B cell hybridoma, TA3. These cells, when cultured in vitro, gradually lost the surface expression of I-A(d) molecules. Incubation with peptides, having high affinity for binding to intact I-A(d) molecules, significantly increased the surface expression of I-A(d) in less than 24 h. The ability of peptides to induce increased expression of I-A(d) correlated with the affinity of peptide to intact I-A(d), and an I-Ak-restricted peptide did not have an effect on I-A(d) expression. The effect could be reversed after the removal of the peptide Ag. Based on our studies with inhibitors of protein synthesis and intracellular transport, the mechanism for up-regulation of I-A(d) expression by peptides seems to involve intracellular pathway but appears not to require new protein synthesis or transport from ER. Our results suggest that the decrease in surface expression of I-A(d) on TA3 cells may result from their failure to generate or to be saturated with naturally processed peptide ligands. Thus peptide ligands are evidently important in regulating surface expression of MHC class II molecules and their recognition by T lymphocytes.
IL-6-induced calmodulin-dependent protein phosphorylation in B9 hybridoma cells.
Authors: Choi I , Na W J , Kang H S , Pyun K H
Published: December 2022

The addition of IL-6 to the IL-6-dependent B9-55 hybridoma cells starved for IL-6 for 16 h induced an increased phosphorylation of a 25-kda protein (p25). Phosphorylation of p25 was IL-6 dependent and maximal at 5 min after IL-6 stimulation. Phosphorylation of p25 was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), an inhibitor of calmodulin-dependent protein kinase, but not by tyrphostin nor ionomycin. When enolase was used as an exogenous substrate, similar results were obtained. Furthermore, calmodulin stimulated p25 phosphorylation in vitro. IL-6-starved B9 hybridoma cells synthesized DNA at 9 to 12 h after addition of IL-6. Preincubation of cells with W-7 before IL-6 stimulation blocked DNA synthesis. Similar effects were observed when cells were preincubated with tyrphostin or ionomycin but to a lesser extent. The concentrations of W-7 required for blocking p25 phosphorylation and DNA synthesis correlated relatively well. These results suggest that an IL-6-inducible calmodulin-dependent protein kinase is involved in IL-6 signal transduction, including p25 phosphorylation and B9 hybridoma proliferation.
Heat shock enhances antigen processing and accelerates the formation of compact class II alpha beta dimers.
Authors: Cristau B , Schafer P H , Pierce S K
Published: December 2022

The heat shock response is a universal and highly conserved cellular response to stress. Here we describe the effect of heat shock induced by elevated temperatures on the processing and presentation of an exogenous Ag, cytochrome c, to an Ag-specific class II-restricted T cell hybrid. Heat shock markedly enhances processing of Ag entering the B cell either through fluid phase pinocytosis or through receptor-mediated endocytosis. B cells undergoing a stress response require less time to process and present Ag and achieve higher levels of T cell activation as compared with control cells. Augmented processing and presentation requires that the Ag be present during the stress response. Heat shock has no effect on the presenting ability of B cells that had already processed Ag; and heat shock in the absence of Ag has little effect on subsequent processing or presentation of Ag. Heat shock has no measurable effect on the cell surface expression of class II as measured by flow cytometry but markedly accelerates the formation of compact alpha beta dimers in B cells. The class II purified from heat shocked cells is more active in Ag presentation assays in vitro as compared with class II purified from cells grown at 37 degrees C, indicating that class II formed during a stress response is not identical to that formed under normal conditions. The effect of heat shock on B cell Ag processing reported here is likely to be relevant to processing in vivo, which may often proceed under conditions that induce the heat shock response, such as during viral or bacterial infections, inflammation, and fever.
Cellular basis of differential responsiveness of lymph nodes and spleen to 23-valent Pnu-Imune vaccine.
Authors: Garg M , Kaplan A M , Bondada S
Published: December 2022

Subcutaneous administration with 23-valent pneumococcal polysaccharide vaccine (Pnu-Imune) elicited a good antipneumococcal capsular polysaccharide response from the spleen but not from peripheral lymph nodes (LN) (cervical, brachial, axillary, popliteal, inguinal) of BALB/c mice. To evaluate the cellular basis of the unresponsiveness of LN to this vaccine and to identify the cell types in spleen that are necessary for induction of polysaccharide Ab responses, a reliable in vitro culture system was developed to obtain plaque-forming cell responses to the Pnu-Imune vaccine. The in vitro antipneumococcal polysaccharide response of mouse spleen cells was found to be relatively T cell independent. Unlike spleen cells, LN cells did not respond to the Pnu-Imune vaccine in vitro. However, LN cells responded to Pnu-Imune vaccine when co-cultured with irradiated spleen cells or splenic dendritic cells but not with accessory cell-depleted spleen cells. LN cells were found to contain the necessary accessory cells that can support the polysaccharide response but in reduced numbers. The LN response to the vaccine was also reconstituted by IL-1 or Th2-associated cytokine IL-5. In contrast, IFN-gamma, a Th1 cell-derived lymphokine was able to suppress the in vitro splenic response to the vaccine suggesting that differential activation of Th1 and Th2 types of Th cells might be one mechanism by which T cells regulate Ab responses to pneumococcal polysaccharides.
Resistance to natural killer cell lysis conferred by TAP1/2 genes in human antigen-processing mutant cells.
Authors: Salcedo M , Momburg F , Hämmerling G J , Ljunggren H G
Published: December 2022

The human Ag-processing-defective cell line 721.174, and its derivative T2, have a large homozygous deletion in the MHC class II region encompassing the TAP1 and TAP2 as well as the LMP2 and LMP7 genes. A similar, but smaller defect encompassing the TAP1 gene is observed in the 721.134 mutant cell line. With a few recent exceptions, these lines are unable to present endogenous Ag to class I-restricted CD8+ CTL. However, restoration of Ag processing and presentation to CD8+ cytotoxic T cells can be achieved by transfecting TAP1 and TAP2 genes into the T2 and .174 cells, or the TAP1 gene into .134 cells. In contrast to their resistance to MHC class I-restricted CTL-mediated lysis, T2 and .174 as well as .134 cells are highly sensitive to lysis by human NK cells. In the present study, we demonstrate the reversal of the NK-sensitive phenotype of these mutant cell lines upon transfection of TAP genes. The induced NK cell-resistant phenotype, studied in detail with TAP1/2-transfected T2 cells, appeared independent of the MHC class I haplotype of the NK cell donors and was also observed with xenogeneic NK cells. The resistance to NK cell lysis induced by re-expression of TAP1/2 seemed to be independent of the origin of the TAP genes; both human and rat transporters efficiently conferred NK cell resistance. Transfection of single TAP genes to T2 or .174 cells, whether TAP1 or TAP2, did not markedly affect NK cell susceptibility. This indicates that an intact TAP1/2 dimer, and thus efficient peptide translocation into the ER, is necessary for rendering T2 and .174, as well as .134 cells, resistant to NK cell-mediated lysis. Proper translocation of peptides across the ER membrane and loading to class I molecules may represent critical events in rendering an NK-resistant phenotype.