[Perioperative immunologic changes in colorectal cancer patients]. / Modificari imunologice perioperatorii la pacientii cu cancer colorectal.

The aim of the study was to investigate the perioperative immunological profile in colon cancer patients and possible correlations with disease. To investigate the changes in immune mediators profile induced by tumor resection, we assessed the serum levels of cytokines (IL-6, IL-8, IL-10, IFN-gamma, TNF-alpha), chemokines (MIP-1alpha, MCP-1, ENA-78) and growth factors (VEGF, bFGF) in colon cancer patients before, during and after surgery and compared the results with those measured for a group of healthy controls. We have used XMap profiling technology (Luminex) that allows simultaneous measurement of multiple parameters in small volumes of samples. Circulating levels of proinflammatory cytokines IL-1, IL-6, IL-8 and antiinflamatory cytokine IL-10 were elevated in cancer patients with respect to healthy controls. Before surgery, serum levels of MCP-1 and MIP-1alpha positively correlated with the levels of proinflammatory cytokines. During surgery, an increase in serum concentration of all determined mediators was noticed, with positive correlation between TNF-alpha, IL-8, MCP-1 and MIP-1alpha. Interestingly, these correlations were no more noticed one week after operation. Postoperatively, cytokines levels decreased as compared to those noticed before surgery, but still higher than in control group. These preliminary results suggest that both tumor and surgical act may influence immune mediators' network.

Impact of smoking on the immunological profile of patients with laryngeal carcinoma.

Immunity plays an important role in the prognosis and the natural development of cancer. Previous studies have shown that the presence of tumor in the body could modify the immune response leading to immunosuppression. The aim of this study was to evaluate the immunological changes of patients with larynx squamous cell carcinoma undergoing potentially curative surgery. We assessed the serum levels of cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-gamma, TNF-alpha, GM-CSF), chemokines (MCP-1 and MIP-1alpha) and growth factors (VEGF and bFGF) in laryngeal cancer patients before, during and after surgery. We have used a novel multianalyte XMap profiling technology that allows simultaneous measurement of multiple parameters in small volumes of samples. To investigate the changes in immune mediators' profile induced by tumor resection, we assessed the culture supernatants of the peripheral blood mononuclear cells (PBMC) derived from the patients, before and after surgery. The results suggested a predominance of a Th2 type of immune response associated with the presence of the tumor (especially in the case of heavy smokers who smoke more than 40 pack-years). However, shifts towards a Th1 type of immune response as well as an improvement of monocyte functions were noticed after surgery.

Experimental studies on the bacterial product CANTASTIM derived from Pseudomonas aeruginosa. V. Protective effect in endotoxin shock.

In this paper, we investigated the effect of the treatment with the bacterial immunomodulator CANTASTIM in a model of endotoxin shock in mice. Among the different mouse models described for septic shock, we have chosen the low-dose endotoxin model using D-galactosamine sensitized mice. We noticed a significant increase in the survival rate of the mice treated with CANTASTIM before the endotoxin challenge. This protective effect was correlated with a strong reduction in the level of TNF alpha in the sera of treated mice. Prior exposure to CANTASTIM also attenuated subsequent ex vivo nitric oxide production by peritoneal macrophages of the mice. In this model of endotoxin shock, the major role has been attributed to TNF alpha acting through its receptor TNFRI (p55). A downregulation of this receptor as a consequence of the treatment with CANTASTIM may be hypothesized. However, the intervention of CANTASTIM in other points in the cytokine network involved in endotoxin shock cannot be excluded.

Experimental studies on bacterial product CANTASTIM derived from Pseudomonas aeruginosa. II. Protective effect in Salmonella typhimurium infection.

Stimulation of the host defense system in a nonspecific way may provide effective treatment of recurrent infections. CANTASTIM is a bacterial product that has been successfully used in cancer immunotherapy as well as in chronic infections treatment. The nonspecific protective effect of CANTASTIM was investigated in two models of experimental infection with Salmonella typhimurium in mice. Prophylactic administration of CANTASTIM (three days before challenge) enhanced peritoneal macrophages bactericidal activity and significantly increased survival of treated mice. When CANTASTIM was administered 72 h after bacterial challenge, in a sublethal infection model with Salmonella typhimurium, by activating macrophages, NK and T cells, it increased the survival rate. The cell populations and molecular mechanisms involved in the prophylactic and therapeutic protective effect CANTASTIM seem to be partially different.

Experimental studies on the bacterial product CANTASTIM derived from Pseudomonas aeruginosa. III. Suppression of lipopolysaccharide-induced tumor necrosis factor alpha: are the lipid components involved?

We previously reported that the bacterial immunomodulator CANTASTIM inhibited the LPS-induced TNF-alpha production in murine macrophages both in vivo and in vitro. In this report, we compared the activity of CANTASTIM with that of two phospholipids (cardiolipin and phosphatidylethanolamine) which are among the components of its lipid fraction. We noticed a significant reduction in the production of TNF-alpha upon stimulation with LPS in murine peritoneal macrophages pretreated for at least 3 h with CANTASTIM or cardiolipin. CANTASTIM was active at much lower concentrations than cardiolipin. Preliminary experiments with partially deacylated CANTASTIM indicated some decrease of TNF-alpha secretion. However, further studies are necessary to clarify this matter. Also, while CANTASTIM and its partially deacylated derivative could trigger the TNF-alpha secretion in murine macrophages, individual phospholipids did not. Based on these results, we concluded that CANTASTIM could induce the TNF-alpha suppression by multiple mechanisms, including the induction of regulatory cytokines such as IL-10 and CD14 receptor blockade/downregulation.

Experimental studies on bacterial product CANTASTIM derived from Pseudomonas aeruginosa with immunomodulatory properties. IV. Tyrosine phosphorylation as an effect of stimulation on different cell populations.

The bacterial product derived from Pseudomonas aeruginosa (trade mark-CANTASTIM) proved immunomodulatory effects in different systems, both in vitro and in vivo experimental animal models, as well as in clinical trials. Among the results obtained regarding CANTASTIM, the following immunomodulatory properties could be mentioned: an increase of the activated T cell subpopulations and humoral-mediated immune processes, facilitation of phagocytic processes, stimulation of cytotoxic activity reflected in the improvement of the capacity of defense in several tumoral and infectious diseases. To better elucidate the intimate mechanisms by which CANTASTIM modulates the cellular functions on different cellular populations, we used tyrosine phosphorylation as an estimate of cell activation on peripheral blood lymphocytes (PBL) and a monocyte cell line (THP-1). In PBL, the treatment with CANTASTIM renders them more susceptible to CD3 stimulation than non-treated cells. In monocytes, CANTASTIM and two phospholipid components of CANTASTIM modulated in a different manner the cellular adhesion on fibronectin and tyrosine phosphorylation leading to the conclusion that these phospholipid components do not fully explain CANTASTIM modulatory properties on cell adhesion processes.

In vivo and in vitro effect of Cantastim, an immunomodulatory agent extracted from a highly pathogenic Pseudomonas aeruginosa strain.

Cantastim is a biological product consisting of distinct molecules of phospholipids obtained from a peculiar Pseudomonas aeruginosa strain. It enhanced the IgM and especially the IgG responses of mice pretreated with Cantastim and then intraperitoneally inoculated with T-dependent antigens such as sheep erythrocytes; restored cellular immunocompetence as evidenced by an increase in the in vitro proliferative responses to T-cell mitogens and allogeneic stimuli of murine splenocytes from in vivo pretreated mice activated NK cytotoxicity, and inhibited in vivo growth of the Ehrlich ascites tumor. In vitro it stimulated the mitogenesis of mouse lymphocytes but did not exert such stimulatory effect on guinea pig or human lymphocytes. All these findings allow to define Cantastim as an immunomodulating agent influencing mainly the cell-mediated immune response.

[A nonspecific immunostimulant effect with Cantastim on the cellular and humoral immune responses in mice evaluated by in vivo and in vitro tests]. / Efectul imunostimularii nespecifice cu "Cantastim" asupra raspunsului imun mediat celular si umoral la soarece, apreciat prin teste in vivo si in vitro.

The stimulatory effect was evaluated, of an ethanol extraction obtained from a pathogenic strain of Pseudomonas aeruginosa type XV. It was noted that this extract, commercially known as "Cantastim" has mitogenic effects in vitro for mouse lymphocytes, but not for those of humans or of guinea pigs. It activates the cytostatic functions of macrophages. It is thermoresistant, nonimmunogenic and it is not allergic. A low amount of proteins and carbohydrates are contained in this extract, but it contains over 80% phospholipids. It has a transient stimulating effect of the cytotoxic effects of NK cells, and it also activates the synthesis of antibodies from the IgM and the IgG classes. It probably stimulates the expression of receptors for PHA in the membrane of T lymphocytes, and retards the development of the Ehrlich ascites tumour, protecting at the same time the animals from severe infections with conditionally pathogenic germs. These data suggests that "Cantastim" is a potent immunomodulating agent that could be used successfully in the fight against certain chronic diseases with a bacterial or neoplastic etiology of humans.

Comparative study of the biological characteristics of mucoid and non-mucoid Pseudomonas aeruginosa strains isolated from chronic respiratory infections.

Morphological, culture and enzymatic characteristics, as well as virulence, susceptibility to antimicrobial agents and epidemiological markers, were studied for 100 mucoid and 100 non-mucoid Ps. aeruginosa strains isolated from chronic respiratory infections. For 10 mucoid and 10 non-mucoid strains was performed the active protection test in mice, both with inactivated germs (10(9) germs), and LPS extracted by Westphal method. It was ascertained that mucoid Ps. aeruginosa strains differ from non-mucoid strains by the slow growth on culture media, more reduced proteolytic activity (81% as compared to 99%), slow oxidation of carbohydrates (5-7 days), reduced virulence in mice (8%) or avirulence (92%), higher sensitivity to some antibiotics (amikacin, dibekacin, ticarcillin, tetracycline, cefotaxime, ceftriaxone), lysoresistance (74%) and polyagglutinability (67%). The mucoid strains ensure a reduced active protection in mice, 70% of strains did not protect the mice against the infection with virulent homologous strains, while the non-mucoid strains ensured 80%-100% protection.

Antigenicity and immunogenicity of the polyagglutinable antigen of Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains isolated from cystic fibrosis patients agglutinate in antisera against anti-polyagglutinable antigen (PA). Anti-PA antibodies were formed in rabbits when immunization was carried out with bacteria possessing core-bound PA, independently of whether the strains were of S or R phenotype. For bacterial agglutination with anti-PA antibodies two prerequisites are essential: the bacterial cell must be of R phenotype and must possess the core-linked PA. In contrast, the PA in the isolated LPS's can be demonstrated in passive haemagglutination for both (S or R) phenotypes, provided the PA is core-linked. Two PA forms have been recognized, one found only in P. aeruginosa species, both in free and bound form. The other one is shared by all members of Pseudomonas genus but is present only in a free, unbound form.

Structural relationship between the polyagglutinable antigen and the core polysaccharide of Pseudomonas aeruginosa.

It has been observed that each strain of the Pseudomonas aeruginosa species harbours the so-called polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysaccharide also contains the PA which could possibly play an important role in the patient by protecting P. aeruginosa cells against anti-PA antibodies.

Specific and nonspecific protection of mice by Pseudomonas aeruginosa lipopolysaccharides.

The role of lipopolysaccharide (LPS) in eliciting a predominant cellular or humoral protection of mice is determined by the amount administered and the immunization schedule. Three basically different experimental conditions could be characterized. Five days after immunization with 100 micrograms of LPS, a nonspecific protection was observed due to the immunomodulatory capacity of lipid A. After 14 days, the same amount of LPS results in an O-specific humoral protection. On the other hand, 0.1 micrograms of LPS lead to a specific protection within 5 days after immunization due to a synergistic action of a specific humoral response (induced by the O-side chain) and a nonspecific signal modulated by lipid A. In order to study the role of each LPS region for protection, the free side chain was conjugated to albumin, followed by complexation with lipid A. To induce a nonspecific protection 5 days after immunization, the presence of lipid A was mandatory, in either the conjugated or free form. Removal of ester-linked fatty acids from free lipid A reduced its protective and mitogenic capacities by 50, and 25%, respectively.