Immunostimulatory effects of dimerized lysozyme (KLP-602) on the nonspecific defense mechanisms and protection against furunculosis in salmonids.

Utilization of natural immunostimulants in fish culture offers a wide range of attractive methods for inducing and building protection against diseases. Lysozyme is an enzyme with bacteriolytic properties and is ubiquitous in its distribution among living organisms. This enzyme has antiviral, antibacterial and anti-inflammatory properties. In nature, lysozyme is found as a monomer. Lysozyme dimer is significantly less toxic than its monomer, and its high biological activity has been ascertained in cases of both viral and bacterial infections. In our study, we examined the influence of dimerized lysozyme (KLP-602) on the nonspecific cellular and humoral defense mechanisms and protection against furunculosis in rainbow trout (Oncorhynchus mykiss). We have analyzed the immunomodulatory effects of KLP-602 after experimental infection by Aeromonas salmonicida. Application of dimerized lysozyme (KLP-602) by injection stimulated the cellular and humoral defense mechanisms and provided protection against furunculosis. By contrast, mortality rate was reduced to 45% (one injection) and 25% (three injections) using 10 or 100 microg/kg KLP-602. Mortality in the untreated control group was 85%.

Effects of lysozyme dimer on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunosuppressed mice.

The effects of lysozyme dimer on humoral response to sheep erythrocytes (SRBC) and restoration of the response impaired by a single cyclophosphamide dose (200 mg/kg) were tested on mice. The effect of lysozyme dimer on the humoral response to SRBC in non-treated with cyclophosphamide mice was determined in relation to doses (0.2, 2, 20 or 200 micrograms/kg) and the time of the drug administration with respect to the antigen before or after SRBC immunization. Moreover, the effect of lysozyme dimer on the humoral response in cyclophosphamide-treated mice was studied depending on the dose applied and time of exposure to the drug in relation to SRBC. It has been found that lysozyme dimer potentiates the humoral response to SRBC in mice, resulting in an increased number of splenocytes producing haemolytic antibodies (PFC) and the total and 2-mercaptoethanol resistant level of anti-SRBC antibodies. A single exposure to lysozyme dimer gave the strongest stimulating action on SRBC when the doses of 2 or 20 micrograms/kg were administered 2 h prior to the antigen. The potentiating effect of the drug was reduced when it was administered 24 h before the antigen and also when single doses were as high as 200 micrograms/kg and as low as 2 micrograms/kg. Exposure to four doses of lysozyme dimer at 24 h intervals was more activating than a single injection. A strong potentiating effect on the specific response to SRBC was noted after four injections of lysozyme dimer at doses from 0.2 to 20 micrograms/kg. The effect of the drug did not depend on the time of exposure to the antigen. It has also been found that lysozyme dimer significantly reduces the suppressive effect of a high cyclophosphamide dose (200 mg/kg) on the humoral response of SRBC-immunized mice. The protective action of lysozyme dimer was dose- and time-dependent. The strongest protection was observed after three doses of 20 micrograms/kg administered prior to pharmacological immunosuppression. Reduction in the dose to 2 micrograms/kg and shorter treatment resulted in reduced protective effects. We have also found that the protective action of three doses of lysozyme dimer (2 or 20 micrograms/kg each) administered between cyclophosphamide injection and the antigen, or after antigen administration is weaker than such a treatment prior to cyclophosphamide immunosuppression.

Reciprocal effects of prostaglandin E2 (PGE2) and of thymic factor (TFX-thymomodulin) on bactericidal activity of human neutrophils.

Studies were performed on phagocytosis and on bactericidal capacity of human neutrophils (HN), isolated from healthy volunteers. Effects of indomethacin, PGE2, and TFX-Thymomodulin on function of the cells were examined. Only in a proportion of cases indomethacin was significantly reducing bactericidal activity of HN indicating that the activity might require the presence of endogenous prostaglandin. On the other hand, exogenous PGE2, inhibited bacterial killing in all cases and the suppression was abolished by TFX-Thymomodulin. The results might indicate antagonistic action on target cells of PGE2 on the one hand and of TFX-Thymomodulin on the other, which could point to the presence of receptors for thymic factors on HN.

Reciprocal effects of prostaglandin E2 (PGE2) and of thymic factor (TFX-thymomodulin) on bactericidal activity of human neutrophils.

Studies were performed on phagocytosis and on bactericidal capacity of human neutrophils (HN), isolated from healthy persons. Effects of indomethacin, PGE2 and TFX-Thymomodulin on function of the cells were examined. Only in a proportion of cases indomethacin was significantly reducing bactericidal activity of HN indicating that the activity might require the presence of endogenous prostaglandin. On the other hand, exogenous PGE2 inhibited bacterial killing in all cases and the suppression was abolished by TFX-Thymomodulin. The results might indicate antagonistic action on target cells of PGE2 on one hand and of TFX-Thymomodulin on the other, which could point to the presence of receptors for thymic factors on HN.

Suppression of monocyte mediated anti U562 cytotoxicity by soluble factors in the serum of cancer patients and in the supernatant of K562 cell culture.

Anti U562 cytotoxicity of monocytes isolated from blood of patients with alimentary tract cancer was shown to be depressed compared to monocytes of healthy individuals. A serum factor was demonstrated in the patients which depressed the cytotoxicity of healthy donor monocytes. Such suppressive factor was also produced by the K562 cell line and its production was blocked by indomethacin, a prostaglandin synthesis inhibitor, as well as by thymus preparations TFX and Thymex L.

Studies on natural cytotoxicity of human monocytes in viral hepatitis.

Studies on natural cytotoxicity of peripheral blood monocytes were conducted in patients with acute viral hepatitis B, patients with chronic aggressive hepatitis etiopathogenically linked to viral hepatitis B, and in asymptomatic carriers of HBs antigen. In the majority of cases of viral hepatitis in the acute stage of the disease and in patients with chronic aggressive hepatitis a significant reduction in the examined function of monocytes was noted which became normalized during convalescence. Results obtained for HBs antigen carriers did not differ from those obtained for normal blood donors. The observations may indicate that restricted natural cytotoxicity of monocytes in the course of viral hepatitis B is related to liver injury. The disturbed monocyte natural cytotoxicity was becoming normal after in vitro incubation with thymic preparations (TFX, Thymex L).

The influence of thymus extract (TFX) on the phagocytosis and the bactericidal capacity of polymorphonuclear neutrophils from patients with insulin-dependent diabetes mellitus.

The influence of thymus extract (TFX) on phagocytosis and bactericidal capacity of polymorphonuclear neutrophils (PMN) from patient with insulin-dependent diabetes was studied. In previous observations PMN from these patients revealed the serum-related defect of bacterial killing. The preincubation of cells with standard dose of TFX significantly increased ingestion rate and recovered the bactericidal capacity to normal values. The augmentation of bactericidal capacity due to preincubation with TFX appeared to be dose-dependent and not related to other blood cells participation. The increase of bacterial killing was also evident, when TFX was applied after phagosome formation. The presented studies indicate that, among numerous peptides, thymus extracts contain those exerting their hormone-like activity on polymorphonuclear neutrophils. The observations may be of clinical importance.

Studies on pokeweed mitogen-induced lymphocyte proliferation in viral hepatitis.

Lymphocyte reactivity to pokeweed mitogen (PWM) was examined in patients with acute viral hepatitis type B and in patients with chronic aggressive hepatitis etiopathogenically linked with hepatitis type B virus (HBV). The proportion of cells containing immunoglobulins in their cytoplasm was normal in PWM-stimulated lymphocyte cultures, originating from patients at an acute stage of viral hepatitis but was significantly lowered in the cases of chronic hepatitis. The findings were interpreted as a result of long term in vivo induction of lymphocyte proliferation which would restrict the in vitro reactivity of the lymphocytes to PWM.

Studies on leukocyte migration inhibitory factor production by T lymphocytes in viral hepatitis.

The response of peripheral blood T lymphocytes to concanavalin A (Con A) assessed by the production of leukocyte migration inhibitory factor (LMIF), was studied in patients with acute viral hepatitis type B and in patients with chronic aggressive hepatitis carrying hepatitis B surface antigen (HBsAg). Lymphocytes from patients in the acute period of the disease and in severely active chronic aggressive hepatitis showed the ability to respond by LMIF production while lymphocytes from patients with moderately active chronic aggressive hepatitis were unable to do so. In parallel, it was shown that lymphocytes from the latter group of patients were capable of suppressing Con-A induced LMIF production. Thus, mitogen-induced mediator production may be a useful parameter in the further characterization of chronic viral hepatitis.

Cell-mediated immunity in response to HBs antigen.

The response of peripheral blood leukocytes to purified HBsAg was studied in vitro by means of the leukocyte migration test in patients with acute viral hepatitis B, patients with chronic aggressive hepatitis carrying GBsAg, and in asymptomatic carriers of HBs antigen. In the acute period of the disease no leukocyte migration inhibition could be observed. Patients with chronic aggressive hepatitis also frequently failed to develop cell-mediated immunity to HBsAg. Results obtained for HBs antigen carriers did not differ from those of normal blood donors. The observations may indicate participation of cell-mediated immunity in the development of liver damage.