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.

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.

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.

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.

Correlation between inhibited alternative complement activity and the protective effect induced by Nocardia lysozyme digest (NLD) during Klebsiella pneumoniae infection in mice.

Nocardia lysozyme digest (NLD) was administered intraperitoneally (i.p.) at a dose of 500 micrograms/kg to normal and immunosuppressed mice for 3 consecutive days prior to inoculation with Klebsiella pneumoniae.

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.

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).

Modulating the expression of some biological membrane glycoconjugates by a Nocardia opaca fraction.

By some hemagglutination (HA) studies it was proved that NLD (the lysozyme digest fraction of Nocardia opaca) interacts with the Sendai virus envelope glycoproteic receptors, but not with those of the Beijing 353/89 H3N2) Influenza virus. Very likely the inhibition is due to the presence of some GAL lectins in NLD: The erythrocytes agglutinability (by the Sendai virus and by the Beijing 353/89 H3N2) Influenza virus is enhanced by their incubation with galactose.

Flow cytometric study of the epirubicine accumulation modulation in the human erythrocytes by a Nocardia opaca fraction.

The effect of the "Nocardia lysozyme digest" (NLD) obtained from Nocardia opaca on the Epirubicine accumulation in erythrocytes was studied by flow cytometry. It was shown that the number of fluorescent cells increased under the NLD action due to the increased membrane permeability for epirubicine. An opposite effect was also recorded consisting in the decrease of the relative fluorescence intensity per cell.

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.

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.

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.

Stimulation of enterocyte enzymatic activities, MHC class II expression and other immunological factors after oral treatment with Nocardia delipidated cell mitogen in germ-free rats.

Nocardia delipidated cell mitogen (NDCM) was given intragastrically (200 micrograms/animal) to 2-month-old germ-free (GF) and conventionally (CV) reared AVN rats. On day 4, enzymatic activities of enterocyte brush border membrane vesicles (BBMV) isolated from jejunal scraping were measured. The results indicated that activities of sucrase, lactase and glucoamylase in BBMV were stimulated following NDCM treatment.

Treatment of radiation disease by Nocardia fraction: possible effect of inflammatory cytokines.

Nocardia delipidated cell mitogen (NDCM) derived from Nocardia opaca, given 2 h after whole-body gamma irradiation (2.5 Gy) to germ-free piglets in amounts of 1 mg/kg via a stomach tube, prevented the loss of B-cells in the spleen, intestinal mucosa, and mesenteric lymph nodes when recorded on day 8 after irradiation. NDCM and/or formolized Enterococcus faecalis strain (Ef) applied intraperitoneally also 2 h after irradiation increased the survival rate in mice irradiated with 8.

Antitumoral activity of an immunomodulatory fraction of Nocardia opaca: mechanism of action.

Immunomodulatory substances have been used as antineoplastic agents in experimental and human systems. Many of these agents were derived from microorganisms. Several biologically active fractions have been isolated from Nocardia.

Immunomodulation by bacterial fractions.

The cells of Nocardia opaca are a source of potent immunostimulating substances, differing in solubility and in the presence or absence of peptidoglycan. Three classes of N. opaca fractions have been investigated: (1) NDCM (Nocardia delipidated cell mitogen), the starting fraction; (2) PG (peptidoglycan)-containing fractions: CW (cell walls), PG, and fractions containing soluble PG derivatives such as NWSM (Nocardia water soluble mitogen) and NSPD (Nocardia soluble peptidoglycan derivative); and (3) soluble fractions devoid of PG, such as NWSMP (NWSP-pellet) and CyI (cytoplasmic membrane).

Protein tyrosine kinases and TNF alpha secretion in human monocytes.

The role of protein tyrosine kinases (PTK) in TNF alpha secretion by human monocytes was investigated in this report. We showed that an immunomodulator such as Nocardia lysozyme digest (NLD) and a particulate agonist, zymosan, stimulated an increase in tyrosine phosphorylation of several endogenous substrates including 53-56 kDa protein which was the predominant phosphoprotein. In addition, NLD and zymosan induced TNF alpha secretion which was impaired by a PTK inhibitor, tyrphostin.

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.

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.

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.

Macrophage involvement in the antitumoral effect of Nocardia-delipidated cell mitogen (NDCM).

Several immunomodulatory fractions derived from Nocardia have been found to inhibit the growth of several experimental tumors, including Lewis lung carcinoma (3LL). An involvement of both macrophages and lymphocytes in the antitumoral effect of Nocardia fractions has been suggested. The mechanism of the Nocardia delipidated Cell Mitogen (NDCM)-induced tumor-inhibiting effect was investigated further in the present study.

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.