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Biomedical subjects

R Barot-Ciorbaru

Publications and source records attributed to R Barot-Ciorbaru.

At least 19 recordsLinked to original sources
PubMed1998

Effect of controlled antigenic stimulation on lymphocyte subsets in pigs and pig fetuses.

The effect of controlled antigenic stimulation in immunologically virgin organisms, i.e. pig fetuses treated with NDCM (Nocardia delipidated cell mitogen) and germ-free (GF) piglets associated with a non-pathogenic E. coli 086, on peripheral blood lymphocyte subsets defined by the expression of CD5 and CD8 was studied by double color flow cytometry. Stimulation of both fetuses and GF piglets increased the frequency of CD8low+ lymphocytes. A prominent subset of CD5-CD8low+NK cells was present in GF and E. coli associated piglets and their frequency was slightly higher in E. coli associated animals. The most pronounced difference between stimulated and non-stimulated animals was in a relative proportion of an ill-defined lymphocyte subset with an unusual CD5low+CD8low+ expression. Both NDCM injection into fetal blood circulation and association of GF piglets with E. coli resulted in a marked increase of frequency of CD5low+CD8low+ lymphocytes in peripheral blood.

Adjuvants, Immunologic↗
PubMed1998

Stimulation of macrophages by Bacillus firmus: production of nitric oxide and cytokines.

Immunostimulatory properties of gram-positive Bacillus firmus were investigated under in vitro conditions using murine peritoneal macrophages. B. firmus stimulated in a concentration and time dependent manner the secretion of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10), but it had no influence upon interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) production. It also substantially augmented production of nitric oxide (NO) induced by exogenous IFN-gamma. Inhibitory experiments using neutralizing antibodies against TNF-alpha and/or IL-10 have demonstrated that these cytokines are responsible for triggering the underlying mechanism(s) leading to enhanced NO production. The cytokine-stimulatory and NO-costimulatory properties could participate in the antiinfectious and anticancer effects of B. firmus, detected previously in the in vivo experiments.

Animals↗
PubMed1998

Immunomodulatory properties of Nocardia lysozyme digest (NLD) in complement normal and C5-deficient mice.

The constantly increasing number of substances with adjuvant activity outpaces the elucidation of their mode of action. This problem is of great importance as the immunomodulatory action of an adjuvant is time- and route-dependent, which implies that administration at a different moment or site may result in a reduced immune response. In the present work the possibility to achieve dual effect (stimulatory or inhibitory) is regarded in the light of the complement system. The object of the study is a preparation obtained by lysozyme digestion of Nocardia opaca cell walls (NLD). According to the results, the administration of NLD to mice (i.p. at a daily dose of 0.5 mg kg-1) during 3 days prior to the antigen resulted in an inhibition of serum antibody level against sheep red blood cells (SRBC) and lipopolysaccharide (LPS). At the same time, the preparation stimulated the antibody response to SRBC if it was applied after the antigen. The ability of NLD to ensure protection against experimental infection with Klebsiella pneumoniae was comparatively studied in complement-normal mice (strain ICR) and in C5-deficient mice (strain DBA/2). Firstly, it was established that complement-deficient mice were more resistant to infection than complement-normal. Secondly, the preparation expressed a protective effect in C5-deficient animals; nevertheless the inoculation was done s.c. or i.v. The departure of the infection depended on the rate of opsonization of K. pneumoniae. Under certain conditions NLD can provoke excessive C3 activation, which might aggravate the course of the infection. The preparation augmented the host response to second challenge with K. pneumoniae of complement-normal and C5-deficient mice.

Adjuvants, Immunologic↗
PubMed1996

Occurrence and specificity of human natural and in vitro induced antibodies to Nocardia opaca antigens.

Nocardia opaca, a Gram-positive bacterium, is a potent source of immunostimulatory substances. Screening of sera of adult human donors revealed that all sera tested contained antibodies reactive with isolated Nocardia fractions (Nocardia delipidated cell mitogen, NDCM; Nocardia lysozyme digest, NLD; Nocardia water-soluble mitogen, NWSM; and fraction B). The respective values of reciprocal titres for IgM and IgG were in the range of 100 to 12,800, and 10 to 320 for IgA antibody isotypes, when NLD or fraction B were used as antigens in enzyme-linked immunosorbent assay (ELISA) tests. The level of antibodies directed to NDCM, a potent polyclonal B cell activator, was found to be the lowest. In vitro spontaneous as well as NDCM-induced production of antibodies to NDCM by human peripheral blood lymphocytes involved mainly the IgM class. Western-blot analysis demonstrated that antibodies in normal human sera react with nocardial antigens of molecular mass approximately 60, 40, 20 and 15-10 kDa. The same antigens were also recognized by rabbit and mouse hyperimmune sera, also confirming the immundominancy of these nocardial antigens in other species. The presence of anti-nocardia antibodies in human sera and their production by both stimulated and non-stimulated lymphocytes points to the natural sensitization of humans either by ubiquitous no-cardial components or by cross-reactive bacterial or food antigens.

Adult↗
PubMed1996

Macrophage nitric oxide synthase (NOS) activation by Nocardia opaca fractions and 15- and 56-kD isolated antigens.

The Gram-positive bacterium, Nocardia opaca, is a source of substances with adjuvant effect, ability to stimulate macrophages and natural killer cells for enhanced cytotoxity and cytokine production and B lymphocytes for polyclonal immunoglobulin secretion. We determined the immunogenicity of isolated N. opaca fractions and prepared MoAbs against immunogenic water-soluble mitogen (NWSM). Two main proteins of molecular mass 15 and 56 kD were detected in western blot analysis and isolated by affinity chromatography using anti-NWSM MoAb B7/7. Both these isolated nocardial antigens were found to stimulate mouse peritoneal macrophage NOS. The effect of 5 micrograms NWSM was comparable to that of 5 micrograms lipopolysaccharide (LPS) or 20 U of interferon-gamma (IFN-gamma) added to cell cultures. The MoAb B7/7 decreased No2- production induced by NWSM or by isolated nocardial antigens, but did not significantly influence the production elicited by LPS or IFN-gamma. On the other hand, NOS activation by NWSM was not affected by anti-IFN-gamma MoAb. The possible independent pathway for IFN-gamma and NWSM macrophage activation is discussed.

Animals↗
PubMed1996

Isotype and antibody specificity of spontaneously formed immunoglobulins in pig fetuses and germ-free piglets: production by CD5- B cells.

Pig fetuses, colostrum-deprived newborns and germ-free (GF) piglets, animals in which B-cell development is not influenced by maternal regulatory factors, were employed to study the occurrence and specificity of natural antibodies (NAb). Serum immunoglobulins of all isotypes were found in 44-day-old fetuses (the gestation period in pigs lasts 114 days) and their level, with predominating IgM, was increased during fetal ontogeny. In sera of fetuses at the end of embryonic life as well as of newborns and older GF piglets, antibody activity against autoantigens (thyroglobulin, hormones, ssDNA), phylogenetically conserved proteins (myosin), haptens (trinitrophenyl; TNP) and bacterial components (Escherichia coli O86, tetanic anatoxin) was detected by enzyme-linked immunosorbent assay. The antigen-biding activity of IgM NAb increased after isolation of the serum immunoglobulins on a Staphylococcus Protein A (SPA)-Sepharose column. IgM reactivity similar to that detected in serum was found in supernatants from polyclonally stimulated cultures of spleen of 8- and 12-day-old GF piglets. Pig fetal liver IgM+ B cells, which were able to produce IgM after polyclonal stimulation, did not express the CD5 molecule. Our results indicate that pig preimmune repertoire is comparable to that described in humans and mice, although in contrast to these species pig B-1 cells do not express CD5.

Animals↗
PubMed1995

Expression of TNF-alpha in pig fetal cells stimulated in vitro.

Macrophages and lymphocytes of pig fetuses stimulated in vitro with bacterial mitogens such as lipopolysaccharide and Nocardia opaca delipidated cell mitogen showed a high TNF-alpha cytoplasmic expression. TNF-alpha was detected by immunofluorescence in peripheral blood lymphocytes and lymphocytes from the thymic region as early as at 34 d of gestation. Macrophages were the main producers of TNF-alpha at later developmental stages.

Animals↗
PubMed1995

Polyclonal immunoglobulin response of thymic, hepatic and splenic lymphocytes from fetal, germ-free and conventionally reared pigs to different B-cell activators.

Immunoglobulin (Ig) response to different polyclonal B-cell activators was measured by ELISA in cell culture media of thymocytes, splenocytes and liver cells isolated from pig fetuses, 8-d-old germ-free piglets and conventionally reared pigs. Both in fetal and in postnatal life polyclonally stimulated lymphocytes were found to produce predominantly the IgM isotype; the first IgM formation was detected in 50-d-old fetal liver (gestation in pigs lasts 114 d). Surprisingly, 73-d-old fetal thymic cells were shown to be induced to Ig synthesis and secretion. In contrast to splenocytes of the same age, which secreted exclusively IgM, fetal thymocytes produced IgM, IgG and IgA. Polyclonally stimulated splenic cells as compared with thymic cells started to produce IgA later in fetal ontogeny, whereas the IgG response was not detectable in splenic cell culture media during the whole embryonal development and appeared only after birth. The earliest and the highest Ig stimulation was found after cultivation of lymphocytes with Nocardia delipidated cell mitogen. Interestingly, the moderate stimulatory effect of 65-kDa heat shock protein (Hsp-65) in polyclonal IgM response of fetal splenocytes was observed. We showed that thymic B lymphocytes represent probably the first maturing B cell population detectable in fetal life, which is able to differentiate after polyclonal stimulation into IgM as well as IgA and IgG producing cells.

Animals↗
PubMed1994

Protective effects of Nocardia delipidated cell mitogen on the mucosa of the small intestine after irradiation of germ-free piglets.

The radioprotective effect of the bacterial immunomodulator Nocardia delipidated cell mitogen (NDCM) on intestinal mucosa and disaccharidase activities was studied in irradiated germ-free piglets. Three-week-old germ-free (GF) piglets were intragastrically pretreated with 1 mg NDCM per 1 kg body weight. The piglets were whole-body irradiated with 2.5 Gray five days after the NDCM pretreatment and sacrificed eight days after irradiation. In the non-irradiated group of GF piglets, NDCM application stimulated lactase activity and markedly increased sucrase activity. This stimulatory effect of NDCM disappeared after irradiation and the piglets exhibited a normal activity of lactase in the jejunal brush-border membrane vesicles, while the sucrase activity decreased to the level found in irradiated controls. NDCM-pretreated intestinal mucosa contained some infrequent lymphocytes which disappeared from the control irradiated tissue. It also exhibited less injury of the epithelium and stroma cells.

Animals↗
PubMed1994

Depression of cytochrome P-450 in mouse liver induced by fractions from Nocardia opaca.

We measured the liver cytochrome P-450 content of mice 24 h after they had been injected with the following immunoadjuvants: Nocardia opaca derivatives and peptidoglycans from several bacterial strains. The cell wall fraction was not active, the others diminished liver cytochrome P-450 levels. The dose-response activity varied with the bacterial origin of the peptidoglycans. These findings indicate that the toxicity and efficiency of immunochemotherapeutic protocols can be modified by altering drug metabolism.

Adjuvants, Immunologic↗
PubMed1993

Nocardia fractions, NLD and NWSM, induce tumor necrosis factor-alpha secretion in human monocytes: role of protein kinase C.

Nocardia lysozyme digest (NLD) and Nocardia water-soluble mitogen (NWSM) are two fractions derived from Nocardia opaca. In this report, we demonstrated that both fractions elicited significant secretion of tumor necrosis factor-alpha (TNF-alpha) in human monocytes. Supernatants from monocytes stimulated with NWSM and low concentrations of NLD displayed a cytotoxic activity against TNF-alpha-sensitive L929 cells, but supernatants from monocytes stimulated with high concentrations of NLD failed to lyse L929 cells. This latter phenomenon might be related to the secretion of an inactive form of TNF-alpha or the release of an inhibitor of TNF-alpha cytotoxic activity. Since it is well established that protein kinase C (PKC) plays a major role in the signaling of several monocyte activators, we investigated the putative role of PKC in cytokine synthesis induced by NLD and NWSM fractions. TNF-alpha secretion in response to both Nocardia fractions was inhibited by sphingosine, staurosporine and calphostin C, known PKC inhibitors, as well as by a PKC depletion procedure. In addition, NLD and NWSM induced a transient increase in [3H]phorbol dibutyrate binding, which assessed the activation of PKC. The data suggest the involvement of PKC in the signaling of NLD and NWSM fractions leading to the synthesis and the secretion of TNF-alpha by human monocytes.

Adjuvants, Immunologic↗
PubMed1993

Induction of inflammatory cytokines by Nocardia fractions.

NDCM and NLD fractions of Nocardia opaca cell walls were used for in vitro stimulation of human and porcine peripheral blood mononuclear cells. Inflammatory cytokines IL-1, IL-6, tumour necrosis factor (TNF) alpha and interferon (IFN) gamma were detected at the single-cell level using paraformaldehyde-fixed and saponin-permeabilized mononuclears stained with cytokine-specific antibodies and indirect immunofluorescence method. IL-1, IL-6 and TNF were produced by human monocytes stimulated for 2 h, IFN gamma-positive lymphocytes were detected later. IFN gamma was produced also by activated porcine lymphocytes. Cells expressing apoptotic features were found among blood mononuclears treated with Nocardia fractions.

Animals↗
PubMed1990

Intrinsic B lymphocyte defect in untreated patients with Hodgkin's disease.

In vivo and in vitro humoral and cell-mediated immunological defects have been described in untreated patients with Hodgkin's disease (HD). The cellular basis of the recently described in vitro reduction of mitogen-induced immunoglobulin synthesis has not been elucidated so far. In this study, we attempted to dissect T and B lymphocyte function in untreated HD patients. Mitogen-induced in vitro immunoglobulin synthesis was assessed in the presence of pokeweed mitogen, the mitogenic anti-CD3 monoclonal antibody OKT3 and the relatively T-cell-independent B cell mitogen Nocardia opaca delipidated mitogen (NDCM). Mitogen-induced Ig synthesis by HD peripheral blood mononuclear cells was significantly reduced compared to that in control peripheral blood mononuclear cells. In coculture assays, T cells of HD patients exerted an adequate helper function to control B cells. However, normal donor T cells did not restore Ig synthesis by B cells of HD patients. Finally, B cells of HD patients were unresponsive to NDCM, which is able to induce Ig synthesis in control B cells even in the absence of T cells. These data provide evidence for an intrinsic functional B lymphocyte defect in HD patients, and suggest that increased activation of suppressor HD T lymphocytes may not play a significant role in the suppression of in vitro Ig synthesis.

Antibody Formation↗
PubMed1990

Cystic fibrosis patients' B-lymphocyte response is resistant to the in vitro enhancing effect of corticosteroids.

Cystic fibrosis is associated with an cAMP-regulated channel defect, which has been evidenced in many cell types including B lymphocytes. To document a B-cell dysfunction potentially related to this defect, we studied the in vitro IgG production by lymphocytes from 11 cystic fibrosis patients. B lymphocytes were co-cultured with autologous monocytes and stimulated with Staphylococcus aureus Cowan or with Nocardia-delipidated cell mitogen in the presence of low concentrations of IL2. Cystic fibrosis patients' cells produced amounts of IgG comparable with that of normal and control patients' cells. However, dexamethasone (10(-7) mol l-1) had no effect on the response of cystic fibrosis patients' cells, whereas it enhanced that of the latter two groups. This resistance of cystic fibrosis cells was true with concentrations of dexamethasone up to 10(-6) mol l-1, whereas this agent induced a dose-related enhancement from 10(-8) to 10(-6) mol l-1 in cultures of normal cells. Co-culture experiments showed that cystic fibrosis B lymphocytes themselves are resistant to the effect of dexamethasone. In contrast dexamethasone normally suppressed the anti-CD3 antibody-induced response of cystic fibrosis T cells in the presence of IL2 and the IL1 alpha- or beta-induced collagenase production of cystic fibrosis fibroblast cell lines. Thus cystic fibrosis B lymphocytes exhibit a selective defect which may interfere with the normal interactions between the hormonal and immune systems and may participate in the sensitivity of cystic fibrosis patients to bacterial bronchopulmonary infections.

Adolescent↗
PubMed1989

Enhancement of natural killer cell activity by Nocardia opaca fractions.

Three molecules derived from Nocardia opaca bacteria, NDCM, NWSMP, and PG, have been shown to express immunomodulating properties. The present study was aimed at assessing the effects of these derivatives on natural killer (NK) activity. Two experimental protocols were adopted, consisting of incubating whole or Percoll fractionated NK cells in vitro with those substances, and the other in which the derivatives were administered in vivo to mice and the activity assessed later. Incubation of spleen cells in vitro with NWSMP or its precursor NDCM promoted NK activity. This effect could be observed after only 2 h of incubation and continued until day 2. Percoll fractions 1-3, which contain most of the NK activity, were enhanced to a similar extent. Band 4, which is usually devoid of such activity, remained unresponsive even after contact with the N. opaca derivatives. PG was practically ineffective upon all the subsets. The results of experiments in vivo correlated with those obtained in vitro in that NWSMP and NDCM, but not PG, promoted NK activity. Bands 1-3 were similarly enhanced, the effect was observed after short treatment times, and could be partially cancelled by the concomitant administration of anti-interferon antibodies (anti-IFN Ab). All these findings suggest that the promoting effects of N. opaca derivatives are mediated through alpha/beta IFN. In contrast to the results observed on spleen NK cells, NK cells from the peritoneum displayed susceptibility mainly to PG, and much less to NWSMP or NDCM. The administration of PG to mice in vivo had a particularly marked promoting effect upon the cytotoxic activity of peritoneal cells. One logical explanation for the difference observed between PG and NWSMP or NDCM may be related to the specific IFN inducing properties of these compounds as well as to the different responsiveness of the NK cells present in the spleen and peritoneal cavity.

Animals↗