Optimal combined use of rifampicin and immunomodulator of microbial origin in experimental Q-Rickettsia infection

Multifactorial analysis of the combined use of rifampicin and an immunomodulator of the microbial origin, such as peptidoglycan, was performed on a model of experimental Q fever in albino mice. On the basis of the experimental results, statistic polynomial models describing the weight of the murine spleens and the titers of the complement-binding antibodies were designed. It was shown that the action of the immunomodulator and antibiotic was highly synergistic with respect to the chemotherapeutic activity and antibody titers. The preventive use of the immunomodulator yielded a 30-fold decrease in a rifampicin therapeutic dose. The use of the immunomodulator also provided a pronounced immunomodulating effect with respect to humoral immunity. Nomographs for optimizing the dose-time parameters of the antibacterial and immunomodulating therapy were plotted.     

Immunomodulating polysaccharide fractions of Menyanthes trifoliata L

Looking for new plant sources of immunomodulating agents polysaccharide-rich fractions (PS) from Menyanthes trifoliata L. (Menyanthaceae) have been isolated. The herb of Menyanthes trifoliata L. was sequentially extracted with water, 0.1 M NaOH, 8% CH3COOH, and 1 M NaOH. After dialysis and resolution on Biogel P-10 four homogenic (B-4, B-5, C-4, D-5) and two nonhomogenic (A-3 and D-4) PS were isolated. About 0.5% of PS over 3500 Da were found in the dry plant material. They were characterized through chemical analysis, NMR and vibrational spectroscopy. Speciation analysis of chosen metal/metaloid elements was performed and an exceptionally high concentration of Se was found in PS of a pure water extract (A-3). The biological tests on the immunomodulating influence with human blood-derived lymphocytes and granulocytes revealed that two fractions, B-4 and B-5, were strong stimulators of immune cells, whereas fractions D-5 and A-3 were found as potent suppressive and anti-inflammatory agents. The applied isolation procedures led to the separation of active compounds into stimulatory and inhibitory fractions.     

Clustered ergot alkaloids modulate cell-mediated cytotoxicity.

Dimers of agroclavine (1) and terguride (2), as well as a series of terguride oligomers, for example trimers (5, 6), tetramer (7), hexamer (8) and functionalized tergurides for further complex clustering were synthesized. Terguride oligomers were screened for their direct cellular toxicity on lymphoma cell lines in vitro and for their immunomodulating activities, represented by the natural killer (NK) cell-mediated cytotoxicity, as the most sensitive screening marker during immune responses. Dimers linked via aromatic spacer showed a high toxicity (1 microM) to lymphoma cells, which was not detected in other derivatives. In vitro and ex vivo experiments performed on mouse spleen lymphocytes in the presence of terguride oligomers demonstrated an immunosuppressive effect of dimers with aromatic spacer (4c-d) and NK cell stimulatory effect of terguride hexamer (8) and trimer with aliphatic spacer (5c). There is a considerable evidence that indolic part of molecule contributes to immunosuppressive action of terguride, which is potentiated in dimers carrying aromatic linker. This effect can be reversed by higher oligomerization of the respective alkaloids.     

Regulatory effects of human mononuclear cells treated with diucifon may be dependent on membrane-associated form of interleukin-2]

The immunomodulating activity of diucifon--activated and glutaraldehyde fixed human mononuclear cells (MNC-DGA) was studied in the test--system of autologous and allogenic mononuclear cells proliferative response. It was shown that MNC-DGA had the same stimulating activity as the diucifon treated unfixed cells. The immunomodulating capacity of MNC-DGA was fully abolished in the case MNC-DGA. So, the interaction between interleukin 2 receptor (Tac--antigen) and the membrane form of the mediator, expressed on MNC after diucifon treatment may play an important role in MNC-DGA immunomodulating activity. The regulating activity of MNC-DGA was revealed both in autologous and allogenic systems. The speculation is that construction of new immunomodulating drugs as complexes of cell-like carrier and immunomodulators fixed on their surface may be perspective.     

Enzymatic preparation of immunomodulating hydrolysates from soy proteins

Soy protein hydrolysates with lower molecular weight were enzymatically prepared by several commercially available proteases (Alcalase 2.4L, Flavourzyme, Trypsin, Papain, Protease A and Peptidase R) with protein recovery varied from 42.59% to 79.87%. Relative content of positively charged peptides was determined on SP Sephadex C-25 using gradient sodium chloride solution as eluents. Immunomodulating properties were evaluated by measuring their effect on in vitro proliferation of murine spleen lymphocytes and phagocytic activity of peritoneal macrophages. The results showed that soy protein hydrolysates (SPHs) prepared with Alcalase and insoluble soy protein (InSP), preferable to other enzymes and soy proteins, have the highest immunomodulating activity and the optimum conditions were determined as follows: E/S=2% (Alcalase), 60 degrees C, pH 8.0, InSP concentration 6% and 225min. Positive correlations were obtained between the immunomodulating activity and content of positively charged peptides. The results suggested that lower molecular weight and positively charged peptides released from soy protein were effective in stimulating immunomodulating activity, thus provided insights into the preparation of potent immunomodulating products.     

RNA-based drug screening using automated RNA purification and real-time RT-PCR1

A new system has been developed for RNA-based drug screening, and the feasibility of this approach has been demonstrated by the identification of new immunomodulating compounds. Peripheral blood mononuclear cells were chosen as the cellular assay system. Cells were either stimulated by TPA/ionomycin to produce T cell cytokines as asthma targets or stimulated by lipopolysaccharide to produce proinflammatory cytokines as targets for chronic obstructive pulmonary disease (COPD). The authors developed a new fully automated system for RNA purification from cells grown in 96-well plates. Gene expression was determined in 384-well plates using real-time quantitative one-tube RT-PCR. Small interdonor variation could be demonstrated. The assay system was validated with known immunosuppressants cyclosporine and dexamethasone. Screening of 800 compounds resulted in 9.5% compounds inhibiting the induction of at least 1 T cell derived cytokine and 6.8% compounds inhibiting at least 1 cytokine relevant for COPD. All these compounds were retested by analyzing remaining RNA from the 1st round of screening. The reproducibility of hits was between 56% and 74% for different cytokines. One compound selectively inhibited TNF, which was confirmed by IC(50) determination. Analyzing its effect on cells from different donors revealed little interdonor variation. In conclusion, the authors established fully automated RNA isolation and precise gene expression profiling using real-time RT-PCR for drug screening.     

Effects of L-156,602, a C5a receptor antagonist, on mouse experimental models of inflammation.

L-156,602, a C5a receptor antagonist, was found as an immunosuppressant with preferential effects on delayed-type hypersensitivity (DTH) in our screening program and it was shown that L-156,602 suppressed the efferent phase of DTH. Here, we tested its effects on experimental models of inflammation induced in mice. L-156,602 did not suppress serotonin- and carrageenan- induced inflammation while it completely suppressed concanavalin A-induced inflammation 4 h after elicitation. The inflammation appeared 24 h after the elicitation with concanavalin A and it was significantly suppressed by L-156,602. Muramyl dipeptide (MDP)-induced acute joint inflammation was also significantly suppressed by L-156,602. These results demonstrated the unique immunomodulating properties of L-156,602 in mouse experimental models of inflammation.     

低氘水对小鼠免疫调节作用的研究

探讨低氘水(deuterium-depleted water,DDW)对小鼠免疫调节的作用。实验组小鼠以低氘水作为饮用水,对照组饮自来水,饲养6周,分别测定免疫蛋白IgG,,IgM的含量,脾脏指数和胸腺指数(mg/g),脾淋巴细胞增殖能力,迟发型变态反应,NK细胞活性以及T细胞亚群CD4+和CD4+/CD8+的变化,观察低氘水对小鼠非特异性免疫和特异性免疫功能的影响。结果低氘水能显著提高IgM,脾脏指数的水平和CD4+,CD4+/CD8+的含量。低氘水对小鼠非特异性免疫和特异性免疫功能具有一定的增强作用。     

Immunomodulating effect of protein fractions isolated from bifidobacteria

The screening of the immunomodulating activity (IMA) of different protein fractions isolated from bifidobacteria was carried out and the capacity of these fractions for changing the proliferative activity of immunocompetent cells was evaluated. Soluble proteins were extracted from lyophilized and sonicated bacterial mass of B. bifidum strain 1 in Na2HPO4 (pH 8) in a water bath at 65 degrees C for 30 minutes. After the formation and removal of nucleic acid sediment the resulting supernatant fluid was dialyzed, its adsorption spectra were analyzed and the fluid was fractionated in a specially proposed device for preparative electrophoresis. Protein fractions were tested for IMA on spleen cells of CBA mice in the reaction of lymphocyte blast-transformation by the level of the inclusion 3H thymidine. The analysis of IMA of protein fractions revealed that their high-molecular components produced a pronounced dose-dependent effect on the proliferative activity of spleen cells. The fractions containing low-molecular components were either inactive (fraction 4) or active only in the maximum dose (fraction 5).     

Immunostimulating and anticoagulating activity of fucoidan from brown algae Fucus evanescens of Okhotskoe sea]

Fucoidan--nontoxic sulfated polysaccharide was isolated from brown algae Fucus evanescens in Okhotskoe Sea. Chemical analysis of the compound was performed, it was shown that fucoidan is freely soluble in water and acid solutions. Immunotropic and anticoagulating properties of the compound were evaluated in comparison with heparin. It was demonstrated that fucoidan in wide range of doses stimulated phagocytic and bactericidic activity at leucocytes of mice peritoneal exudate. Heparin on the contrary demonstrated depressive effect on these functions at high dose. It was shown that fucoidan has dose-dependent anticoagulating activity in vitro and in vivo comparable with heparin activity. The results of investigation demonstrated possibility of fucoidan application as immunomodulating and anticoagulating agent of plant origin.     

Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist.

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.     

Correlation of immunomodulating effects induced by lysozyme and naphthoquinones in acute blood loss]

The role of erythrocytes in realization of interrelation of the immunomodulating effects of lysozyme and naphthoquinones in normal and under conditions of acute hemorrhage was studied. Interaction of lysozyme and menadione at the level of intact erythrocytes and their stroma resulted in formation of highly efficient immunomodulating factors. The effect of such factors under conditions of acute hemorrhage was mediated by cytokines of the spleen macrophagael cells.     

Immunochemotherapy of breast cancer with Propionibacterium granulosum

Summary Eighty-two patients with metastatic breast cancer were treated with adriamycin, cyclophosphamide, and 5-fluorouracil (FAC). These patients were divided by randomization into two groups. One was receiving cytostatic drugs only, while another received the identical cytostatic medication plus additional therapy consisting of IV infusions of gamma-radiation-sterilized cells of Propionibacterium granulosum strain KP-45. Metastases were localized in six different sites and/or organs. Multiple parameters for monitoring changes in metastatic lesions and laboratory tests were performed serially in all patients. The addition of P. granulosum to standard (FAC) chemotherapy of metastatic breast cancer influenced metastates in the liver. It also resulted in increased survival time within the observation period. Possible mechanisms of immunomodulating activity of P. granulosum in this disease are discussed.     

The action of pertussis vaccines and GMDP on the change in the enzymatic activity of macrophages from peritoneal exudate

The influence of whole-cell and acellular pertussis vaccines, introduced both alone and in combination with N-acetylglucosaminylmuramyl-2-alanine-D-isoglutamine (GMDP) on the activity of two enzymes of peritoneal exudate macrophages (5'-nucleotidase and Na+K(+)-adenosine triphosphatase) was studied. The study revealed that both pertussis vaccines exhibited immunomodulating properties, these properties being most pronounced in whole-cell pertussis vaccine. The use of GMDP in combination with pertussis vaccines led to changes in the enzymatic activity of peritoneal exudate macrophages, which was indicative of a decrease in the immunomodulating action of pertussis preparations.     

Immunomodulating effect of cyclophosphamide on cytotoxic activity of rats and mice splenocytes

This study examines the immunomodulating effect of cytostatic drug--cyclophosphamide (Cy)--on natural cytotoxic activity of rats and mice splenocytes. The cytotoxicity of the effector cells against the confluent monolayer cell lines of rat hepatoma Zajdela and HTC and mice hepatoma MH-22a was estimated by means the morphometric analysis. It was shown, that 48 h after single intraperitoneal injection of Cy produced a immunomodulating effect on the activity of splenocytes--suppressor action on cells of Zajdela hepatoma and immunopotentiating action on the target cells of HTC and MH-22a hepatomas. The possible mechanisms of immunopotentiating effect of Cy are discussed.     

Immunomodulating activity of polyunsaturated phospholipids and regulators of energy metabolism in toxic anemia]

Possible potentiation of the immunomodulating effects of carbohydrate and lipid metabolism regulators by their use in combination with polyunsaturated phospholipids was studied. The polyunsaturated phospholipids in toxic anemia icreased the immunomodulating effects of thiamine and inosine which activated glucose catabolism in erythrocytes. The combined use of the polyunsaturated phospholipids and thiamine normalized the oxidation--energy status and lowered manifestation of the immunosuppressing properties of light erythrocytes in laboratory rats exposed to hemolytic poison. The use of the combination of the polyunsaturated phospholipids and inosine normalized the oxidation--energy status and induced manifestation of the immunomodulating properties in heavy erythrocytes of the poisoned rats. The globulin fraction of the rat serum containing antibodies to erythrocytes of the poisoned rats exposed to the polyunsaturated phospholipids and inosine increased the immunity status of the poisoned rats treated with the above mentioned agents. Carnitine and biotin in combination with the polyunsatured phospholipids showed no effect on the phagocytic and metabolic activity of leukocytes and the immunity status of the rats exposed to hemolytic poison.     

Preferential Suppression of Delayed-type Hypersensitivity by L-156,602, a C5a Receptor Antagonist

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.     

Immunomodulating action of interferon in congenital and experimental thymus-dependent immunodeficiency

A study of immunomodulating action of mouse alpha/beta interferon (IFN) was performed in conditions of congenital and experimental thymus-dependent immune deficiency. The action of IFN was assessed in vivo, with IFN injected intraperitoneally. It was shown that IFN did not change killer effect of lymphocytes in the reaction of stem cell inactivation in "nude" mice, but enhanced antibody and rosette formation in the spleen of these animals. In addition, IFN did not change these parameters in the spleen of HRS mice with abnormal differentiation of T lymphocytes, but enhanced antibody and rosette formation in the spleen of mice, thymectomized in old age. It is concluded that the normal function of T-system is essential for the action of IFN.     

Immunomodulating effect of cyclophosphane under different modes of administration to mice

A study was made of the effect of injection of cyclophosphamide (CP) immunomodulating to BALB/c mice, according to 3 schemes of injection with the whole dose being 200 mg/kg. In all, the state of peripheral blood and spleen, the cellular content of lymph nodes and the number of DNA-synthesizing cells in them were studied, in addition to the ability of lymph node lymphocytes to proliferative reaction on T and B cells mitogens in vivo. It was determined that a 2-fold injection of CP, in contrast to 10- and 5-fold injections, resulted in splenomegaly and leukocytosis in the experimental animals. By means of quantitative tests in vivo, in was demonstrated that the dynamics of restoration of proliferative responses to mitogens depends on the CP injection scheme. These tests proved to be more informative for registration of immunomodulating effect of CP, than the study of cellular content of lymph nodes and the number of DNA-synthesizing cells in them.     

Preparation and purification of an immunoregulatory peptide from Stolephorus chinensis of the East Sea of China

Immunomodulatory peptides derived from marine organisms are potential sources of new immunomodulating drugs. This study aimed to investigate the optimal enzymatic hydrolysis conditions of immunoregulatory peptides from Stolephorus chinensis. The relative proliferation rate (RPR) of RAW264.7 macrophages was set as the screening index. The immunoregulatory peptides from S. chinensis was prepared via process optimization, ultrafiltration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (RP-HLPC). The amino acid sequence of the immunoregulatory peptide from S. chinensis (IPSC) was identified to be Tyr-Val-Met-Arg-Phe (YVMRF; MW 715.4 Da) using Edman degradation and mass spectrometry. In addition, the macrophages became larger with more pseudopods after IPSC treatment, indictating that IPSC stimulated RAW 264.7 differentiation. IPSC also increased productions of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Our results provide a theoretical basis for preparing immunomodulatory functional food from S. chinensis in future.