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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 086, tetanic anatoxin) was detected by enzyme-linked immunosorbent assay. The antigen-binding 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-α cytoplasmic expression. TNF-α 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-α at later developmental stages. [ABSTRACT FROM AUTHOR]

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.

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. A protective effect was observed when the pathogen was injected subcutaneously and intravenously, as opposed to an aggravating effect obtained in the case of intraperitoneal inoculation The i.p. administration of NLD partially restored the immunosuppression caused by cyclophosphamide but did not change cobra venom-induced deterioration of the infection. The results obtained could be regarded as a consequence of the lowered alternative pathway serum complement activity and the crucial role of the diminished level of complement in the peritoneal cavity.

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.

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. This effect was attributed to the fluorescence quenching by the oxygen metabolites produced by NDL. In this paper we present some results of the study of the NLD effect on the epirubicine (Epir) accumulation in the erythrocytes (E). It is shown by flow cytometry that the Epir accumulation in the red cells is modified by the presence of the NLD fraction.

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 8.3 Gy and 8.5 Gy doses. The number of nucleated bone marrow cells and the content of RNA in the spleen were significantly higher in rats given immunostimulators NDCM and Ef after irradiation with 5.0 Gy. The effect is presumed to be a consequence of stimulated production of inflammatory cytokines.

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). This report summarizes our results on the relationship between the structure of N. opaca fractions and their stimulatory activities on mouse, rabbit, germ-free piglet and human lymphocytes and monocytes/macrophages. The studies of biological properties of these compounds are the result of an extensive collaborative effort of several teams.

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. We suggest that a cascade of kinases including PTK is involved in NLD and zymosan signalling.

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. These derivatives were shown to induce interferon production, to activate natural killer cells and macrophages and to exert an antitumoral effect. We attempted to examine the mechanism of the antitumoral activity of the Nocardia water-soluble mitogen (NWSM). The tumor tested was the Lewis lung carcinoma (3LL). Regular histological examination and identification of the cellular immune reaction by monoclonal antibodies against macrophages (Mac 1 antigen), B- (IgG expressing) and T-lymphocytes (anti-Lyt-1), analysed by flow cytometry, were performed on samples of the tumor site and of the spleen. Intratumoral administration of the immunomodulators resulted in a massive accumulation of inflammatory cells around the tumor in mice treated with NWSM. The thick rim of infiltrating cells consisted of macrophages and lymphocytes, while the nontreated tumor was found to provoke only a scanty lymphocyte infiltration. Macrophages were, therefore, present at the tumor site and were directly implicated in the antitumoral effect of the Nocardia immunomodulator. T-lymphocytes were also observed at the site of the tumor. The spleen reaction consisted of marked extramedullary hematopoiesis and enlarged follicles containing prominent germinal centres (assessed also by a FACS-demonstrated increase in B-lymphocytes). In view of the inefficiency of chemotherapy in the treatment of advanced cancer, it is of major importance to explore alternative cancer treatment modalities. Immunotherapy is a particularly interesting alternative since it can potentially affect metastatic disease.

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. The most pronounced increase of enzymatic activities was found in GF rats. In contrast to CV rats, GF rats do not express MHC class II on epithelial cells during their life. NDCM treatment induced MHC class II expression in enterocytes from GF rats. The levels of IgA and IgG in sera from NDCM-treated CV rats did not change significantly. The level of IgG in sera of GF rats was found to be enhanced after NDCM treatment. Markedly increased incorporation of 3H-thymidine by spleen lymphocytes stimulated with Con A in vitro was observed only in NDCM-treated GF rats. 3H-uridine incorporation into Con A-stimulated lymphocytes of GF rats was decreased after NDCM treatment.

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.

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.

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.

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. Macrophages activated by NDCM exerted a cytotoxic effect on 3LL cells in vitro, indicating a direct influence of macrophages on the tumor cells. These results correlate with previous findings which showed a local accumulation of macrophages (but also lymphocytes) at the tumor site in NDCM-treated mice. In tumor-bearing mice--both treated and non-treated with NDCM--a splenomegaly due to a pronounced extramedullary hematopoiesis was seen. Concomitant with the gradual evolution of the extramedullary hematopoiesis in the red pulp, a depletion in white pulp component was observed, more pronounced in the control 3LL-bearing mice than in the 3LL-inoculated NDCM-treated animals. The disappearance of the lymphatic follicles in 3LL-bearing mice may be responsible for the failure to cope with the tumor. It is therefore possible that by delaying white pulp depletion, NDCM favors a better host defense against the tumor. Examination of lungs in 3LL-bearing mice treated by NDCM showed a rich infiltration of macrophages in the vicinity of isolated tumor cells, probably indicating a defensive role of NDCM-activated macrophages against metastatic spread of the tumor. Although the macrophage appears to be of major importance in the NDCM-induced host response against the tumor, other components of the immune system are probably system are probably also activated by the Nocardia fraction in defense against the neoplasm.

Various Nocardial fractions have been shown to exert inhibitory effects against several experimental tumors, via a host response mechanism. With the aim of obtaining further information on the mechanism of action of these immunomodulators, we examined in the present study the local cellular host response induced by intratumoral administration of one of these fractions, the Nocardia-delipidated cell mitogen (NDCM), at the site of the tumor (Lewis lung carcinoma-3LL). The tumor itself was found to provoke a scanty cellular infiltration surrounding the tumor mass, mainly composed of lymphocytes. The site of the tumor in NDCM-treated mice was surrounded by a very thick rim of inflammatory cells, consisting of macrophages and lymphocytes. Although recognized decades ago, the existence of tumor infiltrating leukocytes still constitutes a puzzle. The composition and significance of these host cells, in both non-treated and treated tumors has not yet been elucidated. Since lymphocytes were also found around non-treated tumors and they apparently did not prevent tumor development, it is possible that macrophages play a more important role in the NDCM-elicited host defense against the Lewis lung carcinoma.

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.

Lipid-free cells of Nocardia opaca and a cell extract (NWSM) are interferon inducers in syrian hamsters: peak interferon appears 2 h after injection as compared to 8 h in the case of interferon induced by inactivated NDV virus. The interferon induced by Nocardia product is inactivated by a treatment for 30 min at 56 degrees C or 24 h at pH 2: NDV-induced interferon is more stable under these conditions. Hamsters inoculated with NWSM before and after infection with rabies virus are partially protected: their survival time is slightly increased.

Scintigraphic imaging due to its sensitivity is in many cases one of the most powerful techniques for demonstrating metastases. Severe limitations still exist in cancer when it is necessary to detect the presence of a few tumour cells in the residual minimal disease. In preliminary experiments it had been observed that an immunomodulator isolated from Nocardia bacteria (Nocardia Soluble Peptidoglycan Derivative: NSPD) electively bound to a model of activated macrophages. An hypothesis has been put forward that the enhanced detection of macrophages that are usually present in the vicinity or inside tumours should represent a polyspecific test for scintigraphy of a variety of metastases. NSPD radiolabelled with 99mTechnetium is not usable when injected intravenously due to its physiochemical properties. It has therefore been encapsulated into liposomes then administered via the respiratory tract as an aerosol. Amphiphilic properties, as well as its low molecular weight allow a rapid diffusion of NSPD in blood. Scintigraphy of metastases was possible from 1.5 to 6 hours after inhalation. The first stage of the study was carried out on 5 patients bearing known metastases (skin, lymph nodes, bone) from malignant melanoma that all were imaged with 99mTc-NSPD. The test was then applied to patients with a high risk of recurrent cancers (melanoma: 6, breast tumour: 7) based on the detection in their plasmas of high Lipid Associated Sialic Acid (LASA) concentrations. The association of these two sensitive techniques has resulted in the detection of very small metastases that were not seen using conventional scintigraphy; they were then confirmed histologically.

Cell walls from Nocardia opaca induce the production of mitogenic factors by mouse peritoneal macrophages in vitro. These factors stimulate thymocytes from C3H/HeJ mice. Supernatants of peritoneal cell culture exhibiting this activity were fractionated by chromatographic procedures such as gel filtration and metal chelate affinity chromatography and the biological activities assayed. These fractionation studies indicate that several biologically active products occur in the supernatant. Four factors monokines (M) with different apparent molecular masses M1 (100,000), M2 (50,000), M3 (16,000) and M4 (7000) were obtained, one of which (M3) was identical to interleukin 1 (IL1). Several of the biochemical parameters of one of these factors, M2, were analyzed. It was found that this monokine had many properties in common with IL1: stimulation of proliferation of thymocytes from C3H/HeJ mice, similar amino acid composition and mobility during isoelectric focusing.

Of 14 asymptomatic hemophilia A patients receiving factor VIII concentrates, 11 severe hemophilia patients had an inverted T-helper/suppressor ratio but 3 moderate patients had a normal ratio. Most hemophilia patients showed poor lymphocyte proliferative responses to mitogens and diminished in vitro immunoglobulin-producing ability of lymphocytes. One important finding was that most patients were found to have increased numbers of Ia-like antigen-positive suppressor T cells, suggesting that circulation activated suppressor T cells. In addition, OKT8+ suppressor T cells from severe hemophilia patients showed strong suppressor activity on B-cell differentiation in a Nocardia delipidated cell mitogen-driven system, whereas those from normal age-matched controls showed no suppressor function. These results suggested that suppressor T cells in hemophilia patients treated with factors might be already activated in vivo by undetermined mechanisms, implying the presence of a peculiar immunoregulatory status in this disease.

Nocardia delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, injected i.p. in an oil/water emulsion to F6 rhabdomyosarcoma-bearing rats, inhibited the development of pulmonary metastases; 6 out of 10 rats were protected. Repeated i.p. administration of emulsified NDCM and of two other compounds, a Nocardia water soluble mitogen (NWSM a hydrosoluble fraction) and purified cell walls (CW, an insoluble macromolecular fraction) in Lewis lung carcinoma (LLC)-bearing mice resulted in a significant reduction of lung metastases. The efficiency of these fractions was enhanced by association with monokines. A combination regimen of NDCM, NWSM, and CW (100 micrograms/0.1 ml) and monokines (0.1 ml), injected i.p. in LLC-bearing mice, yielded a greater antimetastatic effect than either therapy alone. Peritoneal macrophages from mice which had been injected i.p. with NWSM or CW, when triggered either by TPA (tetradecanoyl phorbol acetate) or by zymosan, released large quantities of hydrogen peroxide and had a high rate of glucose consumption. These macrophages were activated as judged by their cytostatic activity against syngeneic P815 mastocytoma growth; they expressed biochemical markers which have been reported to characterize the activated state. Incubation of thioglycollate-elicited peritoneal macrophages with NWSM, and monokines for 72 h resulted in a cytotoxic activity against labeled LLC cells; addition of macrophage activating factor significantly increased the cytotoxic capacity of these macrophages. In view of this we postulate that the antimetastatic effect of soluble and insoluble N. opaca fractions and monokines might be mediated by activated peritoneal macrophages.

The axial organ (AO-cells) of the starfish Asterias rubens is a primitive immune organ. The total population was fractionated into two populations: adherent (B-like) and non-adherent (T-like) to nylon wool. Nocardia-delipidated cell mitogen (NDCM), Nocardia water soluble mitogen (NWSM), NWSM-pellet (NWSMP) and purified cell walls (CW) from Nocardia opaca induce the proliferation of B-like cells from the axial organ. The Nocardia immunomodulators studied are inactive on the total and T-like population. The mitogenic activity of B-like cells requires the presence of phagocytic cells.