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PubMed1992

Heat shock alters the composition of heteromeric steroid receptor complexes and enhances receptor activity in vivo.

Under normal cellular conditions, human progesterone receptors (PR), immune-isolated from cytosols of T47D breast cancer cells, associate with two heat shock proteins (hsps), hsp 90 and hsp 70. Receptors activated by hormone binding in vivo and extracted from nuclei with 0.5 M NaCl no longer associate with hsp 90 but retain association with hsp 70. We have examined the effect of heat shock treatment of cells on hsp-receptor interactions and on receptor function. Heat shock resulted in a partial reduction in cellular levels of PR, but receptors that remained were functional for both steroid and DNA binding activities. By steady-state [35S]methionine labeling prior to heat shock treatment, it was determined that heat shock did not affect the composition or maintenance of preexisting cytosolic PR.hsp 90.hsp 70 complexes. By contrast, immune isolation of PR complexes from cells pulse-labeled with [35S]methionine showed that heat shock altered the composition of newly synthesized hsps associated with PR. After heat shock, both the highly inducible form of hsp 70 (72K hsp) and a 100K hsp were bound to cytosol PR, and inducible 72K hsp remained bound with the nuclear-activated PR. Neither of these hsps were associated in detectable amounts with PR under normal cellular conditions. With respect to receptor function, heat shock treatment substantially enhanced the activity of PR in vivo as determined by measuring hormone-dependent PR-mediated transcription of a target reporter gene (MMTV-CAT) that was stably transfected into T47D cells. Heat shock treatment alone, in the absence of hormone, did not stimulate MMTV-CAT expression nor did it affect transcription from a control reporter gene, pSV2-CAT, suggesting that enhanced receptor activity was due to an effect on PR-mediated processes and not to a general effect on transcription. Induction of the heat shock response by a related chemical stress (sodium arsenite) also enhanced PR activity in vivo. Interestingly, sodium arsenite produced both a greater induction of hsp 90 and hsp 70 synthesis and a greater fold enhancement of PR-mediated gene transcription than did heat shock. This suggests that enhancement of PR activity is related not only to induction of hsp synthesis but also to the severity of the stress response. The present results provide an indication that in certain cells there may exist an interrelationship between the activation pathways by which cells respond to stress and to steroid hormones. Possible mechanisms responsible for heat shock effects on PR activity are discussed.

Arsenic↗
PubMed1988

Tumor necrosis factor and immune interferon act in concert to slow the lateral diffusion of proteins and lipids in human endothelial cell membranes.

Vascular endothelial surface-related activities may depend on the lateral mobility of specific cell surface macromolecules. Previous studies have shown that cytokines induce changes in the morphology and surface antigen composition of vascular endothelial cells in vitro and at sites of immune and inflammatory reactions in vivo. The effects of cytokines on membrane dynamic properties have not been examined. In the present study, we have used fluorescence photobleaching recovery (FPR) to quantify the effects of the cytokines tumor necrosis factor (TNF) and immune interferon (IFN-gamma) on the lateral mobilities of class I major histocompatibility complex protein, of an abundant 96,000 Mr mesenchymal cell surface glycoprotein (gp96), and of a phospholipid probe in cultured human endothelial cell (HEC) membranes. Class I protein and gp96 were directly labeled with fluorescein-conjugated monoclonal antibodies; plasma membrane lipid mobility was examined with the phospholipid analogue fluorescein phosphatidylethanolamine (Fl-PE). In untreated, confluent HEC monolayers, diffusion coefficients were 30 x 10(-10) cm2 s-1 for class I protein, 14 x 10(-10) cm2 s-1 for gp96, and 80 x 10(-10) cm2 s-1 for Fl-PE. Fractional mobilities were greater than 80% for each probe. Cultures treated at visual confluence for 3-4 d with either 100 U/ml TNF or 200 U/ml IFN-gamma did not exhibit significant changes in protein or lipid mobilities despite significant changes in cell morphology and membrane antigen composition. In HEC cultures treated concomitantly with TNF and IFN-gamma, however, diffusion coefficients decreased by 71-79% for class I protein, 29-55% for gp96, and 23-38% for Fl-PE. Fractional mobilities were unchanged. By immunoperoxidase transmission electron microscopy, plasma membranes of untreated and cytokine-treated HEC were flat and stained uniformly for class I antigen. "Line" FPR measurements on doubly treated HEC demonstrated isotropic diffusion of class I protein, gp96, and Fl-PE. Finally, although TNF and IFN-gamma retarded the growth of HEC cultures and disrupted the organization of cell monolayers, the slow diffusion rates of gp96 and Fl-PE in confluent doubly treated monolayers were not reproduced in sparse or subconfluent untreated monolayers. We conclude that the slowing of protein and lipid diffusion induced by the combination of TNF and IFN-gamma is not due to plasma membrane corrugations, to anisotropic diffusion barriers, or to decreased numbers of cell-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Membrane↗
PubMed1990

[Mechanisms in the modulation of the reactions of immune splenocytes by macroheterocyclic compounds].

The effect of macroheterocyclic compounds possessing immunostimulating properties in vivo on the reactions of the immune splenocytes in vitro has been studied. It has been shown that the cell activity is determined by the medium ionic composition. In the medium with the optimal content of the bivalent cations the azacrown ethers decrease the intensity of the immune splenocytes reactions depending on the dose. In the media of the modified ionic composition the studied compounds in concentration 10 microM activate all the reactions of the spleen cells, in concentration 100 microM they suppress or do not change them.

Animals↗
PubMed1979

Guinea pig lung lavage cells after intranasal BCG sensitization.

Recent studies have suggested that intranasal administration of antigen can induce local cell-mediated immunity in lung lavage cells. The present study was designed to examine the changes in composition of lung lavage cells and their capacity to produce the lymphokine migration inhibitory factor after intranasal immunization with BCG in guinea pigs. Results indicate that guinea pigs responded to respiratory tract BCG infection with an increase in immunocompetent cells in the bronchoalveolar tract and with production of migration inhibitory factor. After local pulmonary BCG administration, the total number of cells increased as compared with that of the uninfected animals, the increase being statistically significant within 2 weeks. This marked increase in the total cell population is due to a more than doubling of the number of macrophages in the lavage fluid. Animals also developed at this time positive delayed hypersensitivity to intradermally administered purified protein derivative. A significant increase in the total lymphoid cells and macrophage population was observed again at 6 weeks after sensitization, suggesting that the response is biphasic in nature. At 6 weeks, however, there was also a significant rise in total lymphocytes and T cell population in addition to macrophage numbers. This increase in T cells correlated with an increase in production of migration inhibitory factor in the presence of purified protein derivative. These data suggest that the immune response of the respiratory tract after BCG challenge involves increased recruitment of immunocompetent cells locally at the site of infection and that these cells are capable of producing effector molecules in terms of the elaboration of migration inhibitory factor.

Animals↗
PubMed1999

Aging and T-cell-mediated immunity.

Changes in the T-lymphocyte compartment represent the most critical component of immunological aging. Recent studies have demonstrated that the age-related decline in T-cell-mediated immunity is a multifactorial phenomenon affecting T-cell subset composition as well as several proximal events such as protein tyrosine phosphorylation, generation of second messengers, calcium mobilization and translocation of protein kinase C, and distal events such as lymphocyte proliferation and cytokine production of the T-cell activation pathway. Age-related T-cell immune deficiency is preceded by thymic involution and is influenced by several intrinsic as well as extrinsic factors. Further, the role of monocytes and macrophages in T-cell activation changes with advancing age. This brief review will summarize the current knowledge of the cellular as well as molecular aspects of immunodeficiency of T cells due to aging, some of the paradoxes of aging as related to T-cell-mediated immunity, and possible factors which contribute to this paradox. Finally, experimental approaches will be suggested that might resolve these controversies and that might provide insights into the diverse and complex mechanisms that contribute to immunodeficiency of T cells. Ultimately these studies may suggest possible therapeutic interventions to enhance immune function in the elderly.

Aging↗
PubMedDate not supplied

Escherichia coli as a probiotic?

The influence of oral treatment with a suspension of non-pathogenic Escherichia coli cells (commercially available as: Symbioflor II) on the morphological composition of the gut microflora and on the systemic humoral immune response (the IgG-, IgA- and IgM-isotype) against the bacterial cells in the Symbioflor II preparation was measured. After a pretreatment period of 21 days, ten healthy human volunteers ingested 1*10(8) cells of E. coli daily for 14 days. Thereafter a follow-up period of 28 days completed the study. The results of this study indicated that no effect of the treatment on the composition of the gut microflora could be observed. However, the immune-fluorescence measurements revealed a significant increase in circulating amounts of IgG directed against the administered E. coli cells. It is concluded that the treatment only resulted in a specific humoral immune response, while the gut microflora is not modulated.

Antibodies, Bacterial↗
PubMed2005

The Influence of long-chain polyunsaturated fatty acids on total lipid fatty acid composition of a canine mastocytoma cell line.

Cutaneous mast cells are considered as key immune effectors in the pathogenesis of canine atopic dermatitis (CAD). These cells release immediate-phase and late-phase mediators of inflammation. Dietary fatty acids are incorporated in cellular membranes and seem to influence mediator production and release. A dietary intervention with n6- and n3-fatty acids is thought to alleviate clinical symptoms in atopic dogs. The purpose of this study was to examine the effects of n6- and n3-fatty acids on the fatty acid composition of canine mastocytoma cells (C2) as a possible model for CAD. The C2 was cultured in a basic medium called Dulbecco's modified Eagle's medium (DEH) or with additional 14 mum linoleate (C18:2n6, DEH-LA), gamma-linolenate (C18:3n6, DEH-GLA), arachidonate (C20:4n6, DEH-AA), alpha-linolenate (C18:3n3, DEH-LnA), eicosapentaenoate (C20:5n3, DEH-EPA) or docosahexaenoate (C22:6n3, DEH-DHA). Cell growth was examined for 11 days in all media. Cell growth increased from days 1 to 8 and decreased thereafter in all media conditions. The fatty acids supplied did not influence cell growth. The cells were harvested after 8 days for fatty acid analysis. The fatty acid composition was determined by gas chromatography after extraction and trans-esterification of the lipids. The added fatty acids increased the concentration of these fatty acids in C2 differently (LA 4.9-fold, GLA 6.9-fold, AA 6-fold, LNA 9.3-fold, EPA 6.5-fold and DHA 8.4-fold). Furthermore, elongated and Delta6-desaturated products of the corresponding fatty acids were significantly elevated. However, Delta5-desaturated products were not measurable. These results let us assume that C2 has no measurable activity of the Delta5-desaturase. In case the low activity of Delta5-desaturase is one of the mechanisms underlying the pathogenesis of CAD, C2 seems to be an adequate model for investigations in CAD.

Animals↗
PubMed1995

FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen.

In establishing the memory B-cell population and maintaining self-tolerance during an immune response, apoptosis mediates the removal of early, low-affinity antibody-forming cells, unselected germinal center (GC) cells, and, potentially, self-reactive B cells. To address the role of the apoptosis-signaling cell surface molecule FAS in the B-cell response to antigen, we have examined the T-cell-dependent B-cell response to the carrier-conjugated hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) in lpr mice in which the fas gene is mutated. High levels of FAS were expressed on normal GC B cells but the absence of FAS did not perturb the progressive decline in numbers of either GC B cells or extrafollicular antibody-forming cells. Furthermore, the rate of formation and eventual size of the NP-specific memory B-cell population in lpr mice were normal. The accumulation of cells with affinity-enhancing mutations and the appearance of high-affinity anti-NP IgG1 antibody in the serum were also normal in lpr mice. Thus, although high levels of FAS are expressed on GC B cells, FAS is not required for GC selection or for regulation of the major antigen-specific B-cell compartments. The results suggest that the size and composition of B-cell compartments in the humoral immune response are regulated by mechanisms that do not require FAS.

Amino Acid Sequence↗
PubMed1998

Zoocin A immunity factor: a femA-like gene found in a group C streptococcus.

A 6.8-kb fragment of Streptococcus equi subsp. zooepidemicus 4881 DNA containing the zoocin A gene (zooA) was cloned in Escherichia coli and sequenced. We have identified a gene we call zoocin A immunity factor (zif), which protects the producer cell from the otherwise lethal action of its own product. Transformation of Streptococcus gordonii DL1 with zooA and zif changed its phenotypic character from a non-zoocin A producing-zoocin A sensitive cell to a zoocin A producing-zoocin A resistant cell. zif has sequence homology to femA (factor essential for methicillin resistance) and lif (lysostaphin immunity factor). No differences were observed in amino acid or amino sugar compositions of peptidoglycan purified from zoocin A sensitive vs. zoocin A immune cells.

Amino Acids↗
PubMed1989

Characterization of lambda class antibodies from the BALB/c memory response to a [glucosyl-alpha(1----3) glucosyl]-protein conjugate.

Both dextran B-1355 and nigerosyl-KLH (N-KLH) contain alpha(1----3) diglucosyl moieties and both induce lambda class serum antibodies which recognize the alpha(1----3) linkage. However, as shown here, some of the antibodies induced by the thymus-dependent form, N-KLH, have a distinct fine specificity. It is known that B-1355 induces antibodies which resemble the anti-alpha(1----3) myeloma proteins MOPC 104E and J558. The new fine specificity which does not resemble such antibodies is found in the serum of N-KLH primed mice challenged with N-KLH. The two fine specificity types are distinguished by their sensitivity to inhibition by nigerose in ELISA using B-1355 or N-BSA as bound antigen. Twelve hybridomas were produced from N-KLH primed mice boosted with either N-KLH or B-1355. Six of the 12 had the new fine specificity; only two out of the 12 expressed the IdX determinant commonly associated with the B-1355 response and neither of these possessed the new reactivity pattern. Comparative inhibition with the disaccharide nigerose and the tetrasaccharide nigerantetraose indicated that 11 out of the 12 hybridoma proteins have combining sites larger than a disaccharide. Southern and Northern blot analysis of eight hybridomas revealed that all expressed VH genes from the J558 family, but that at least two distinct VH genes from this family were used. The data support the hypothesis that immunization with an alpha(1----3) diglucosyl-protein conjugate alters the composition of the B cell pool capable of producing lambda class antibodies from that ordinarily observed following immunization with B-1355.

Animals↗
PubMed2000

Targeted inactivation of the tetraspanin CD37 impairs T-cell-dependent B-cell response under suboptimal costimulatory conditions.

CD37 is a membrane protein of the tetraspanin superfamily, which includes CD9, CD53, CD63, CD81, and CD82. Many of these molecules are expressed on leukocytes and have been implicated in signal transduction, cell-cell interactions, and cellular activation and development. We generated and analyzed mice deficient for CD37. Despite the high expression of CD37 on cells of the immune system, no changes in development and cellular composition of lymphoid organs were observed in mice lacking CD37. Analyses of humoral immune responses revealed a reduced level of immunoglobulin G1 (IgG1) in the sera of nonimmunized mice and an alteration of responses to T-cell-dependent antigens. Antibody responses to model antigen administered in the absence of adjuvant and to viral infections were generally poor in CD37-deficient mice. These poor antibody responses could be overcome by the immunization of antigen together with adjuvant. These results suggest a role for CD37 in T-cell-B-cell interactions which manifests itself under suboptimal costimulatory conditions.

Animals↗
PubMed2004

[Depression, myocardial infarction and the immune system--the chicken before the egg problem].

Major depression is a risk factor, associated with a twofold increase in the incidence of ischemic heart disease (IHD). One of every 6 patients suffers from major depression following acute myocardial infarction (AMI). This connection is of major concern, considering that major depression is an independent risk factor for cardiac morbidity and mortality after AMI, increasing overall mortality fourfold. Activation of the immune system has a significant role in the pathogenesis of IHD and depression. Vast physiological responses, mediated mostly by activation of the immune system, accompany post MI depression and may account for increased prevalence of arrhythmias and high mortality. This includes activation of the hypothalamic-pituitary-adrenocortical axis, endothelial dysfunction, platelets activation and alterations of phospholipid composition in cell membranes. On the other hand, activation of the immune system after AMI includes elevated levels of interleukin-1 and interleukin-6. which induce "sickness behavior", characterized by symptoms similar to those observed in major depression. The key question raised by this data, whether inflammation is the common ground for both AMI and depression, or if it is accompanying one and sets the ground for the other, remains unanswered at this time. The significance of major depression as an independent risk factor for post MI mortality and morbidity raises the practical question, whether treatment of depression can reduce mortality after AMI. Several recent studies that evaluated this presumption, failed to prove it. In this review we present an overview of the cross interaction between depression, AMI and inflammation and its diagnostic and therapeutic implications.

Acute Disease↗
PubMed2026

Single-cell transcriptional profiling identifies the swimming crab Portunus trituberculatus in response to bacterial infection.

Crustaceans rely entirely on innate immunity, yet the cellular composition, functional specialization, and pathogen-induced remodeling of their immune system remain poorly resolved. Here, we generated a high-resolution single-cell transcriptomic atlas of hemocytes from the swimming crab Portunus trituberculatus following Vibrio parahaemolyticus infection using 10× Genomics scRNA-seq. Seven putatively distinct hemocyte clusters were identified, including granulocytes, semigranular hemocytes, prohemocytes, unresolved hemocytes, hyalinocyte-like hemocytes, biosynthetically active secretory hemocytes, and regulatory hemocytes. Although the overall cellular composition remained relatively stable after infection, hemocytes exhibited pronounced cluster-specific transcriptional reprogramming involving Toll/NF-κB signaling, antimicrobial peptide synthesis and metabolic rewiring. By integrating single-cell and bulk transcriptomes, we identified multiple anti-lipopolysaccharide factors (ALFs) as key secretory effectors and experimentally validated their antibacterial activities. FITC-based bacterial engulfment assays and RNA-seq of sorted phagocytes demonstrated that phagocytic capability was shared across multiple hemocyte clusters. Notably, the immunoglobulin superfamily receptor DSCAM displayed extensive alternative splicing and strong infection-induced activation in unresolved hemocytes. Immune-training experiments showed that prior bacterial exposure was associated with altered DSCAM expression and reduced early cumulative mortality upon secondary challenge, suggesting a memory-like immune phenotype. These findings provide a foundational framework for understanding crustacean immunity and advancing disease-resistant breeding in aquaculture.

Antimicrobial peptides↗
PubMed1987

Dietary fat, plasma lipoproteins, and immune function in middle-aged American men.

Dietary fat has been incriminated as a positive risk factor for the development of neoplasia in human populations. We used adipose tissue fatty acid analysis as an index of dietary fat intake to study the association between dietary fat and immune function in a group of 94 free-living American males (avg age 47 years). Immunocompetence was tested by a battery of T- and B-lymphocyte stimulation tests and also by natural killer (NK) cell activity. Correlations were sought between fatty acid composition, plasma lipids, and immune responsivity. The degree of unsaturation of the diet over a polysaturated-to-saturated fat ratio range of 0.54-1.01 had no predictable effect on the immune function. Stepwise regression analysis showed that the concentrations of plasma triglycerides and cholesterol and its subfractions did not explain any of the variance in the immune tests. Palmitic acid (16:0) was associated with 7% of the variance of the response to C. albicans and E. coli, perhaps through influencing B-cell activity. Stearic acid (18:0) was correlated negatively to concanavalin A responsivity (18% of the variance) and positively to NK activity (20% of the variance). If impaired in vitro immune function is a marker of increased risk for carcinogenesis, then our data do not support a role for dietary fat influencing in any systematic manner lymphocyte function in vitro, as reflected by proliferative response or NK activity. Further, plasma lipoproteins, in particular cholesterol levels, did not appear to affect any immune function test. It remains to be studied whether dietary fat, lipoproteins, or fat-soluble substances may influence membrane structure and function and prostaglandin formation as alternative pathways in the promotion of neoplasia.

Adipose Tissue↗
PubMed2005

Transcriptional responses in mouse lungs induced by vaccination with Mycobacterium bovis BCG and infection with Mycobacterium tuberculosis.

Transcriptome analyses enable the assessment of signature alterations in whole tissues and organs undergoing pathological processes. We analyzed gene expression profiles of lungs from mice infected with Mycobacterium tuberculosis or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). We compared high-dose systemic and low-dose aerosol M. tuberculosis infections as well as systemic BCG vaccination. Expression profiles in lungs were analyzed at day (d) 1 and d 30 post infection / vaccination using a custom tailored 'in situ' synthesized 60-mer oligonucleotide microarray with focus on immunologically relevant genes. At d 1, a small number of genes were differentially regulated, whereas at d 30, a discrete expression pattern was identified in the lung. Differential gene expression profiles between M. tuberculosis infection and BCG vaccination indicate differences in naturally and vaccine induced pulmonary immune responses. The shared signature of systemic and aerosol M. tuberculosis infection revealed dominance of genes related to or controlled by interferon gamma (IFN-gamma). We assume that differential gene expression profiles after M. tuberculosis infection are strongly influenced by differences in cellular composition of the lung due to migration of immune cells, primarily neutrophils, basophils, eosinophils and monocytes to the site of infection.

Animals↗
PubMed2001

Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human gamma delta T cells.

Small phosphorylated metabolites from mycobacteria stimulate human gammadelta T lymphocytes. Although such phosphoantigens could prove useful in the composition of vaccines involving gammadelta T cell-mediated immunity, their very low abundance in natural sources limits such applications. Here, we describe the chemical production, purification, and bioactivity of a phosphorylated bromohydrin (BrHPP) analogue that mimics the biological properties of natural phosphoantigens. This compound can be obtained in gram amounts, is easy to detect, and is of high stability in aqueous solutions. Whereas unspecific binding of BrHPP to a wide panel of cell surface receptors is not detected even at micromolar concentrations, nanomolar concentrations specifically trigger effector responses of human gammadelta T lymphocytes. Thus, BrHPP is a novel molecule enabling potent immunostimulation of human gammadelta T lymphocytes.

Alcohols↗
PubMed1989

Prospects for the future of bone marrow transplantation from the experimental studies on cells and animals.

Despite accumulated extensive clinical experience bone marrow transplantation (BMT) remains a largely experimental procedure with high risk and numerous limitations. Part of the progress has to come from improvements in clinical care, but still as in former years there is a need of implementation of results of experiments on cells and animals. In the area of better engineering of posttransplant haemopoietic and immune cell production, this include variable pretransplant modifications of composition of inoculated cells as well as utilization of new drugs and growth factors. In the area of BM donor selection this includes identification of minor histocompatibility antigens. In the area of applications except for being a replacement therapy of haematologic disorders BMT may be considered as an adjunct therapy for transplantation of various organs. Finally, combination of cellular engineering of BM inoculum with genetic modifications of cells may offer therapy for some patients.

Animals↗
PubMed2000

Antigenic analysis of gamonts of Hepatozoon canis purified from leukocytes.

Hepatozoon canis is a tick-borne apicomplexan parasite of dogs that infects neutrophils and parenchymal tissues. To study the antigenic characteristics of this parasite, a technique was devised for the purification of gamonts from peripheral blood neutrophils. White blood cells were separated on Ficoll-Hypaque density gradients and the gamonts were released from the host neutrophils by nitrogen cavitation. The blood used for purification originated from dogs with natural or experimental infections of H. canis with a parasitemia of 1.4-33%. The number of parasites collected ranged from 1.5 X 10(6) to 4.2 X 10(7). Portions of purified gamonts were separated and examined under phase and scanning electron microscopy, and the remaining purified parasites were then used as a source of antigens to characterize the humoral immune response by western blot analysis. Serum antibodies from infected dogs recognized more than 15 gamont antigens, and the antigenic patterns observed with sera from naturally and experimentally infected dogs were nearly similar. Four immunodominant protein bands of relative molecular weights of 107, 88, 63, and 28 kDa were recognized by all of the sera examined. The technique applied here for the isolation of host cell-free gamonts will facilitate studies on antigenic composition and immune responses against H. canis and on antigenic relationships between Hepatozoon from different host species and geographic regions.

Animals↗