The Vβ8 Gene Family is Preferentially Used by''Naturally''Activated T Cells

Receptors encoded by the V beta 8 gene family detected by the monoclonal antibody F23.1 are expressed among 'naturally' activated T cells in normal spleen at frequencies significantly higher than in the total CD4+ and CD8+ cell populations. The positive selection of these clones into 'natural' T-cell activity could be the reason for the high frequencies of cells expressing V beta 8 genes. This phenotype is strain-dependent.     

Antibody Responses to λ1J558 and λ2315 Light Chains

Specificity of BALB/c antibody responses to lambda chains of isologous myeloma proteins 315 and J558 was explored by enzyme-linked immunosorbent assay. lambda-chain binding antibodies were not detected when immunizing with assembled (H + L) myeloma proteins. However, relatively high titred IgG antibodies were elicited by free lambda 2(315) immunization. Antibodies were directed to 'hidden' determinants since binding was abrogated upon H + L assembly of chains. At least a portion of antibodies bound antigenic determinants in the variable region and cross-reacted with lambda 1 land lambda 3 chains. Free lambda 1J558 immunization induced low-titred, predominantly IgM antibodies that also only reacted with 'hidden' determinants. These determinants were most probably located in the constant (C) region and no cross-reaction to lambda 2 or lambda 3 was observed. An artefact of technical importance was noted: myeloma proteins exposed 'hidden' determinants on their lambda chains when coated directly to polystyrene walls. This artefactual exposition was lost when anti C-region antibody spacer molecules were inserted between the wall and the myeloma proteins. Antibody and T helper cell (Th) responses to free lambda 2(315) covaried significantly in various strains while antibody and Th responses to free lambda 1J558 did not. In some strains, weak antibody responses were detected without detectable Th.     

Increased expression of collagen receptors: α1β1 and α2β1 integrins on blood eosinophils in bronchial asthma

BACKGROUND: Eosinophils are one of the major effector cells in bronchial asthma. Their infiltration of airways correlates with the asthma severity. Recruitment and activation of eosinophils are partially mediated by integrins alpha4beta1 and alpha4beta7. Collagens type I and IV constitute important components of extracellular matrix and vascular basement membrane, respectively. Therefore, collagen-binding integrins (alpha1beta1 and alpha2beta1) may also play a role in eosinophil lung infiltration. OBJECTIVE: To evaluate the possible presence of alpha1beta1 and alpha2beta1 integrins on peripheral blood eosinophils from asthmatic subjects. METHODS: Collagen receptors were studied on eosinophils separated by immunomagnetic CD16-negative method from healthy donors (n=13) and patients with moderate persistent atopic bronchial asthma (n=15). Surface receptor identification was performed by flow cytometry and cell adhesion assay. RESULTS: Eosinophils isolated from the patients showed increased expression of both alpha1beta1 and alpha2beta1 integrins as compared with healthy controls. Moreover, adhesive function of eosinophils to collagen type IV was inhibited by snake venom disintegrins: viperistatin and obtustatin. These disintegrins contain KTS active motif and are specific inhibitors of alpha1beta1 integrin. CONCLUSION: We demonstrated for the first time that collagen receptors: alpha1beta1 and alpha2beta1 integrins are overexpressed on the surface of peripheral blood eosinophils of asthmatic subjects. Further studies may reveal potential application of KTS-disintegrins or their structural analogs for therapy of bronchial asthma.     

Urinary leukotriene E4 and 9α, 11β-prostaglandin F2 concentrations in mild, moderate and severe asthma, and in healthy subjects

BACKGROUND: Airway inflammation in asthma is associated with cysteinyl leukotriene and prostaglandin D(2) production. Measurement of urinary metabolites of these eicosanoids may be useful for monitoring asthma patients. However, the influence of asthma phenotype and severity on basal urinary excretion of these metabolites is unknown. OBJECTIVE: To compare urinary leukotriene (LT)E(4) and 9 alpha, 11 beta-prostaglandin (PG)F(2) concentrations in large groups of mild, moderate and severe asthmatic patients and healthy control subjects. METHODS: Asthma severity, treatment and aspirin sensitivity were assessed by questionnaire in 168 asthmatic patients. Basal LTE(4) and 9 alpha, 11 beta-PGF(2) concentrations were measured in urine samples from these patients and from 175 control subjects using enzyme immunoassays. RESULTS: Urinary LTE(4) was correlated with 9 alpha, 11 beta-PGF(2) in both control subjects and asthmatic patients (P<0.002). Median LTE(4) and 9 alpha, 11 beta-PGF(2) concentrations in patients with severe asthma were significantly reduced compared with mild asthmatic patients (P<0.05 and <0.001, respectively). Urinary 9 alpha, 11 beta-PGF(2), but not LTE(4) was lower in asthmatic patients using inhaled corticosteroids (P<0.02). Multiple regression analysis indicated that urinary 9 alpha, 11 beta-PGF(2) concentration was negatively correlated with asthma severity (P=0.003) and also with % predicted FEV(1) (forced expiratory volume in 1 s) (P=0.005). CONCLUSIONS: Baseline urinary LTE(4) and 9 alpha, 11 beta-PGF(2) concentrations are of limited value in discriminating between patients with different severities of asthma. Reduced urinary LTE(4) and 9 alpha, 11 beta-PGF(2) in patients with severe asthma suggest that direct or indirect effects of high-dose corticosteroid therapy combined with other factors associated with severe asthma may influence eicosanoid production. However, the negative association of urinary 9 alpha, 11 beta-PGF(2) with lung function suggests an adverse effect of chronic PGD(2) production on lung function in asthma, irrespective of severity.     

Fast IPSCs in rat thalamic reticular nucleus require the GABAA receptor β1 subunit

Synchrony within the thalamocortical system is regulated in part by intranuclear synaptic inhibition within the reticular nucleus (RTN). Inhibitory postsynaptic currents (IPSCs) in RTN neurons are largely characterized by slow decay kinetics that result in powerful and prolonged suppression of spikes. Here we show that some individual RTN neurons are characterized by highly variable mixtures of fast, slow and mixed IPSCs. Heterogeneity arose largely through differences in the contribution of an initial decay component (τ_D∼10 ms) which was insensitive to loreclezole, suggesting involvement of the GABA_A receptor β₁ subunit. Single-cell RT-PCR revealed the presence of β₁ subunit mRNA only in those neurons whose IPSCs were dominated by a rapid and prominent initial decay phase. These data show that brief, β₁-dependent, loreclezole-insensitive IPSCs are present in a subpopulation of RTN neurons, and suggest that striking differences in IPSC heterogeneity within single neurons can result from of the presence or absence of a single GABA_A receptor subunit.     

Gastric motility in soluble guanylate cyclase α1 knock-out mice

The principal target of the relaxant neurotransmitter nitric oxide (NO) is soluble guanylate cyclase (sGC). As the α₁β₁-isoform of sGC is the predominant one in the gastrointestinal tract, the aim of this study was to investigate the role of sGC in nitrergic regulation of gastric motility in male and female sGCα₁ knock-out (KO) mice. In circular gastric fundus muscle strips, functional responses and cGMP levels were determined in response to nitrergic and non-nitrergic stimuli. sGC subunit mRNA expression in fundus was measured by real-time RT-PCR; in vivo gastric emptying of a phenol red meal was determined. No changes were observed in sGC subunit mRNA levels between wild-type (WT) and KO tissues. Nitrergic relaxations induced by short trains of electrical field stimulation (EFS) were abolished, while those by long trains of EFS were reduced in KO strips; the latter responses were abolished by 1 H [1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The relaxations evoked by exogenous NO and the NO-independent sGC activator BAY 41-2272 were reduced in KO strips but still sensitive to ODQ. Relaxations induced by vasoactive intestinal peptide (VIP) and 8-bromo-cGMP were not influenced. Basal cGMP levels were decreased in KO strips but NO, long train EFS and BAY 41-2272 still induced a moderate ODQ-sensitive increase in cGMP levels. Gastric emptying, measured at 15 and 60 min, was increased at 15 min in male KO mice. sGCα₁β₁ plays an important role in gastric nitrergic relaxation in vitro , but some degree of nitrergic relaxation can occur via sGCα₂β₁ activation in sGCα₁ KO mice, which contributes to the moderate in vivo consequence on gastric emptying.     

Fibronectin Co-Stimulates via the α5β1 Receptor IL-2, IL-4 Production by Splenic, Granuloma Lymphocytes of Schistosoma mansoni Infected Mice

In murine Schistosomiasis mansoni, soluble worm egg antigens (SEA) induce L3T4⁺ T helper cell-mediated chronic granulomatous inflammations around parasite eggs. Within the fully developed granuloma lymphocytes, macrophages, and eosinophils, fibroblasts are embedded in extracellular matrix (ECM) composed of fibronectin, laminin, glycosaminoglycans and collagens. The present study examined in vitro the putative co-stimulatory role of fibronectin (FN) in acute and chronic infection splenic and granuloma lymphocyte responses. Plate-bound FN enhanced the anti-CD3 MoAb stimulated normal and acute or chronic infection splenic lymphoproliferation by 20–32%. The co-stimulatory effect was evident in SEA stimulated acute but not chronic infection spleen cells. Proliferation of stimulated granuloma lymphocytes could not be up-regulated by immobilized FN. Plate-bound FN significantly enhanced IL-2 and IL-4 production by SEA-stimulated acute, but not chronic, infection granuloma lymphocytes. However, FN had no influence on the high level of IL-2, IL-4 production of anti-CD3 MoAb stimulated acute or chronic infection splenic or granuloma lymphocytes. Because in the antigen-stimulated acute infection spleen or granuloma cultures the co-stimulatory effect by FN was abrogated by the tripeptide (RGD) arg-gly asp, and anti α₅β₁ antibody, enhancement is attributed to signalling via the α₅β₁ integrin receptor of lymphocytes.     

Phenotypic and Functional Analysis of Activated B Cells of Autoimmune NZB × NZW F1 Mice

Polyclonal B-cell activation is the central theme in the production of autoantibodies and possible activation of autoreactive T cells in both human and murine lupus. The abnormal expansion of CD5⁺ B cells in murine lupus has been suggested, in particular, to be one of the most characteristic findings in these mice. Activated B cells can be separated from the B cells of resting stage by the difference in cell density. The aim of this study was to investigate the characteristics of different densities of the spleen cells separated by gradient density. Furthermore, the ability of anti-DNA antibody secretion in each percoll gradient fraction of B cells was also analysed. The results showed: a higher percentage of CD5⁺ B cells, which corresponded to the activated B-cell population, in percoll gradient 1 and 2 fractions; that splenic B cells of NZB/W F₁ mice had proliferative response to interleukin (IL)-4 or IL-5 but not to IL-10 or interferon-γ (IFN-γ); and that B cells isolated by percoll gradient produced anti-DNA antibody after stimulation with lipopolysaccharide (LPS) plus IL-5 and IFN-γ, but not IL-4 and IL-10. These data suggest that B cells at different stages of activation express differential characteristics and functions.     

Biochemical and Biosynthetic Studies of a Crystallizable Human γ1 Heavy-Chain Disease Protein

We have studied the structure of a crystallizable gamma 1 heavy-chain disease protein that lacks the entire VH and C gamma 1 domains. The protein starts within the hinge region at aspartic acid 221 (Eu numbering). The native protein is a disulphide-linked dimer with an apparent molecular weight of 52,000, consistent with the biochemical data obtained on the whole protein and its cyanogen bromide fragments. The carbohydrate content of this protein was 6.8%. As shown by biosynthesis experiments intracytoplasmic gamma chains synthesized by neoplastic cells had an apparent molecular weight similar to that of the serum heavy-chain disease protein. These data are compared with those obtained for other gamma 1 heavy-chain disease proteins beginning in the hinge region, and the mechanisms leading to those abnormal Ig products are discussed.     

Target Cell Lysis by Natural Killer Cells is Influenced by β2-Microglobulin Expression

Natural killer (NK) cells form part of the vertebrate defence against viruses and tumours, but show only limited specificity. The molecule(s) recognized by NK cells on target cells are at present unknown. Major histocompatibility complex (MHC) class I antigen concentration on target cells is inversely correlated with NK cell lysis. Here we show that MHC class I-unassociated beta 2-microglobulin (beta 2-m) expression is involved in NK cell-target cell interaction. Two human MHC class I negative cell lines, Daudi and K562, are differentially susceptible to NK cell lysis. Daudi cells are beta 2-m-negative and resistant to NK lysis, K562 are beta 2-m-positive and highly susceptible to lysis by NK cells. Interferon (IFN) treatment augments beta 2-m expression and NK lysis of K562, but not in Daudi cells. NK cell lysis of K562, but not YAC-1 cells, can be inhibited by monoclonal anti-human beta 2-m antibody. Furthermore, susceptibility of mouse embryo fibroblasts (MEF) to NK lysis can be increased by infection with recombinant vaccinia virus expressing the human beta 2-m gene.     

Further studies on serum α1 lipoprotein in famiIiaI lecithin:cholesteroI acyltransferase deficiency

Serum high density lipoprotein (HDL) from two patients with LCAT deficiency has been compared with HDL from normal subjects and HDL from a presumed heterozygous carrier. By two different immunological methods the concentration of (α₁-lipoprotein in seruin of the patients was found to be about 25–30 % of the normal concentration.
Patient HDL is composed of two fractions, as shown previously. The ht fraction contains particles of high molecular weight with an electrophoretic mobility slightly slower than that of normal HDL. The lipid content is 70 %, and the concentrations of unesterified cholesterol and phospholipids are about 10–15 and 3–4 times that of normal HDL, respectively. The second fraction consists of particles of relatively low molecular weight with electrophoretic mobility faster than albumin. This fraction exhibits close to normal concentrations of unesterified cholesterol and phospholipids. Esterified cholesterol or lysolecithin could be demonstratcd in none of the fractions.
HDL from the presumed heterozygous carrier was normal as judged from immunoelectro-phoresis, gel filtration, electron microscopy, and lipid analyses.