Bożena Obmińska-Mrukowicz

Isoxazole ring as a useful scaffold in a search for new therapeutic agents

Due to its relatively easy synthesis, isoxazole ring has been as an object of interest for chemists and pharmacologists from research groups all over the world. Its chemical modifications include both connection of isoxazole with other aromatic, heteroaromatic or non aromatic rings and substitution with different alkyl groups. Thanks to their usually low cytotoxicity, isoxazole derivatives are still popular scaffolds for the development of new agents with variable biological activities, such as antimicrobial, antiviral, anticancer, anti-inflammatory, immunomodulatory, anticonvulsant or anti-diabetic properties. This review discusses the chemical structure of recently developed isoxazole derivatives with regards to their activity and potential therapeutic use.

Influence of polysaccharide fractions isolated from Caltha palustris L. on the cellular immune response in collagen-induced arthritis (CIA) in mice. A comparison with methotrexate

ETHNOPHARMACOLOGICAL RELEVANCE The extracts from Caltha palustris have been used in traditional Canadian and Asian medicine to treat arthritis and rheumatism. AIMS The aim of the study was to investigate the anti-arthritis and immunomodulatory activity of the polysaccharide fractions B and C of Caltha palustris L. herbal extracts in collagen-induced arthritis (CIA) mice, an animal model of rheumathoid arthritis. The results were compared with those of methotrexate (MTX) treatment. MATERIALS AND METHODS CIA was induced in male and female DBA/1J mice by intradermal injection of chicken type II collagen in Freunds complete adjuvant (cFA). Booster injection of collagen (in incomplete Freunds adjuvant) was given on day 21 of the experiment. Mice were treated daily for 21 consecutive days with investigated fractions B or C at a dose of 10mg/kg (the first dose was given 24h after the booster) or phosphate buffered saline (PBS) (negative and positive control group). MTX was administered in parallel, intraperitoneally at three weekly cycles-every 48 h for 3 weeks at a dose of 6.6 mg/kg, the first dose was given on day 22 of the experiment. The severity of arthritis was evaluated by arthritic scores. Flow cytometry was used to investigate subsets of T lymphocytes in the thymus, and T and B lymphocytes in the spleen, and in mesenteric lymph nodes. T regulatory lymphocytes in the spleen were also quantified by means of flow cytometry. The levels of IL-2, IL-6, IL-10, IFN-γ and TNF-α in serum were also measured. RESULTS The results revealed that fraction B significantly reduced the severity of joint swelling and erythema to a similar degree as MTX. It was also found that B fraction and MTX inhibited leucocytosis in peripheral blood caused by CIA, however the inhibitory effect of MTX persisted longer than that of fraction B. The analysis of lymphocyte T subsets demonstrated that both investigated fractions and MTX caused a partial or complete normalization in the percentage and the absolute number of CD4(-)CD8(-) thymocytes (immature, double-negative cells), and increased the percentage of CD8(+) T cells in peripheral lymphoid organs of mice with CIA. Moreover, an increase in the percentage of CD4(+) thymic cells was observed after treatment with fraction B or MTX. Fraction C showed the weakest effect in normalization of the percentage and the absolute number of CD4(-)CD8(-) thymus lymphocytes in mice with CIA. The potency of fraction B was comparable to MTX. A significant decrease in the percentage and the absolute count of splenic T-regulatory cells (CD4(+)CD25(+)FOXP3(+)) was observed after treatment with both Caltha palustris fractions. The inhibiting influence of investigated fractions on TNF-α serum concentration was significant and lasted longer in the case of fraction C. Production of other cytokines was modulated slightly (increase in IFN-γ) or markedly (decrease in IL-2). CONCLUSION The results of the experiment suggested that the administration of polysaccharide B fraction from Caltha palustris extract significantly suppressed the progression of CIA. These results are similar to those obtained in the case of MTX treatment. This indicates that fraction B may be a potent candidate for botanical anti-arthritic agent.

Modulating effects of nonselective and selective phosphodiesterase inhibitors on lymphocyte subsets and humoral immune response in mice

Phosphodiesterase (PDE) inhibitors can regulate the activity of immune cells by increasing intracellular levels of cyclic nucleotides. The aim of this study was to determine the effects of milrinone, a selective PDE3 inhibitor, sildenafil, a selective PDE5 inhibitor, and aminophylline, a nonselective PDE inhibitor, on lymphocyte subsets and humoral immune response in mice when administered in vivo. Aminophylline (20 mg/kg, i.m.), milrinone (1 mg/kg, i.m.) or sildenafil (1 mg/kg, p.o.) were administered to mice either once or five times at 24 h intervals. Some mice were immunized with a sheep red blood cell (SRBC) suspension administered i.p. either 2 h after the single dose or 2 h after the second of the five doses. In non-immunized mice treated five times with PDE inhibitors, the subsets of T lymphocytes in the thymus and T and B lymphocytes in the spleen and mesenteric lymph nodes were determined 12, 24 or 72 h after the last dose. The humoral immune response was determined on days 4, 7 and 14 after SRBC injection in SRBC-immunized mice treated with PDE inhibitors. A modulating effect of the drugs on lymphocyte subpopulations was observed. The greatest impact was observed in splenocyte subpopulations, and resulted in decreased percentages of B cells (CD19(+)) and increased percentages of T cells (CD3(+), CD4(+), CD8(+)). No effect or slight influence of the drugs on anti-SRBC hemagglutinins was observed, but the number of plaque-forming splenocytes was increased. The drugs under investigation did not show a significant immunosuppressive effect.

Immunomodulatory effects of betulinic acid from the bark of white birch on mice.

The objective of this study was to explore the immunomodulatory effects of betulinic acid (BA) extracted from the bark of white birch on mice. Female mice were orally administered BA for 14 days in doses of 0, 0.25, 0.5, and 1 mg/kg body weight. We found that BA significantly enhanced the thymus and spleen indices, and stimulated lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide as shown by MTT assay. Flow cytometry revealed that BA increased the percentage of CD4+ cells in thymus as well as the percentage of CD19+ and the ratios of CD4+/CD8+ in spleen. BA increased the number of plaque-forming cell and macrophage phagocytic activity as indicated by a neutral red dye uptake assay, and the peritoneal macrophages levels of TNF-α were also increased. In contrast, serum levels of IgG and IgM and serum concentrations of IL-2 and IL-6 were significantly decreased in BA-treated mice compared to the control as assayed by haemagglutination tests and ELISA, respectively. Taken together, these results suggest that BA enhances mouse cellular immunity, humoral immunity, and activity of macrophages. Thus, BA is a potential immune stimulator and may strengthen the immune response of its host.

In Vivo Studies on the Influence of Bacteriophage Preparations on the Autoimmune Inflammatory Process

Phage preparations used for phage therapy may have not only direct antibacterial action but also immunomodulating effects mediated by phages themselves as well as by bacterial antigens. Therefore phage application in patients with immune disorders, and especially with autoimmune diseases, requires special attention. The aim of this study was to investigate the effect of phage lysates (staphylococcal phages A3/R, phi200, and MS-1 cocktail, enterococcal phage 15/P, Pseudomonas phage 119x, and E. coli T4 phage) as well as purified T4 phage on the course of murine collagen-induced arthritis (CIA), commonly used as an animal model of rheumatoid arthritis. Intraperitoneal application of phage lysates or purified T4 phage did not aggravate the course of autoimmune joint disease. Moreover, although endotoxins are known to potentiate CIA, the systemic administration of phage lysate of Pseudomonas aeruginosa, which contains debris of this Gram-negative bacillus, did not significantly influence CIA although the sonicate of the corresponding bacterial strain did. Interestingly, a purified T4 phage revealed some anti-inflammatory activity when applied under the therapeutic scheme. Our preliminary results do not suggest that phages may aggravate the symptoms of rheumatoid arthritis. In contrast T4 phage may even exert an immunosuppressive effect.

Modulation of Th1/Th2 cytokine production by selective and nonselective phosphodiesterase inhibitors administered to mice.

Phosphodiesterase (PDE) inhibitors can modulate the functions of immune cells, including T lymphocytes, due to increased intracellular levels of cyclic nucleotides. The drugs (aminophylline, milrinone and sildenafil) were administered once or five times at 24 h intervals at the following doses: 20 mg/kg, i.m., 1 mg/kg, i.m. and 1 mg/kg, p.o., respectively. Th1 and Th2 cytokine levels (IL-2, IFN-γ, IL-4, IL-5, TNF) were determined 12, 24 or 72 h after the last administration of the drugs. A commercial BD™ Cytometric Bead Array Mouse Th1/Th2 Cytokine Kit (CBA) was used to determine the levels of Th1/Th2 cytokines in the serum. Neither of the PDE inhibitors under investigation administered once changed IFN-γ, TNF and IL-4 production. A single dose of aminophylline decreased the production of IL-2 (after 12 h). A single dose of milrinone did not affect Th1/Th2 cytokine secretion. Sildenafil administered once decreased the production of IL-2 (after 72 h). A temporary enhancement in the level of IL-5 was observed 12 h after a single dose of sildenafil. No changes in Th1 and Th2 cytokine production were observed after five doses of PDE inhibitors under investigation. These results indicate that nonstimulated lymphocytes Th1 and Th2 exhibited a slight sensitivity to aminophylline and sildenafil. The drugs under investigation were ineffective inhibitors of Th1/Th2 cytokine production.

Restoration of immune system function is accelerated in immunocompromised mice by the B-cell-tropic isoxazole R-11

BACKGROUND Restoration of impaired immune response in immunocompromised patients is a crucial problem. In this study we evaluated the efficacy of isoxazole R-11 in reconstitution of the immune response in immunosuppressed mice. METHODS Mice were given a sublethal dose (250 mg/kg b.w.) of cyclophosphamide (CP). The cellular immune response to ovalbumin (OVA) and the humoral immune response to sheep erythrocytes (SRBC) were generated. R-11 was administered at repetitive, intraperitoneal doses (20 μg/mouse) until determination of the immune responses: 7 and 15 doses on alternate days for cellular and humoral immune response, respectively. For phenotypic studies R-11 was given per os, at a single dose of 20 μg/mouse. The ability of R-11 to affect interleukin- 6 (IL-6) production was determined in the whole human blood cell culture. RESULTS R-11 increased the content of CD19+ cells in the spleens and lymph nodes with a concomitant decrease of CD3+ and CD4+ cells. The compound significantly accelerated restoration of both cellular and humoral immune responses, elevated the numbers of circulating leukocytes and splenocytes and normalized the blood cell picture. Supplementary experiments showed that R-11 was not toxic with regard to human peripheral blood mononuclear cells (PBMC) and that it upregulated IL-6 production in blood cell culture stimulated with lipopolysaccharide (LPS). CONCLUSIONS We demonstrated that R-11 is likely a B-cell tropic agent which can restore both cellular and humoral immune responses in immunocompromised mice and may have a potential to be applied in therapy of immunocompromised patients.

In vitro immunomodulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the cellular immune response

Abstract The aim of this study was to determine the immunomodulatory activity of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide in vitro. This compound was used for the synthesis of a series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides with documented immunotropic activity. The performed measurements assessed the cytotoxic effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the murine macrophages (cell line J774E.1) and lymphoblasts (cell line D10.G4.1), the influence of this compound on the proliferation of murine lymphocytes isolated from peripheral lymphatic organs and murine peritoneal macrophages stimulated with mitogens (concanavalin A(ConA), lipopolysaccharide (LPS), phytohemagglutinin A (PHA)). Moreover, the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β by the murine peritoneal macrophages stimulated with LPS from Escherichia coli was assessed. It was found that 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide displayed no cytotoxic effects in the murine J774E.1 and D10.G4.1 cell lines in a wide range of concentrations (0.5–200 μg/ml). Furthermore, the compound stimulated proliferation of lymphocytes isolated from the spleen and mesenteric lymph nodes when used alone and in combination with mitogens (ConA and PHA). This effect was stronger in the nonstimulated cells, and it followed a dose–response relationship. The same phenomenon was observed for the proliferation of the murine peritoneal macrophages. The investigated hydrazide, at the highest used concentration of 150 μg/ml, increased the LPS-induced production of IL-1β and did not affect the level of TNF-α. These results confirmed the immunomodulatory properties of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide and indicated that this compound could be useful in further research aimed at finding novel functional drugs.

Immunophenotypic characterization of canine malignant lymphoma: a retrospective study of cases diagnosed in Poland Lower Silesia, over the period 2011–2013

Lymphoma is the most frequently diagnosed cancer of the canine haematopoietic system. In this study, the flow cytometry and polymerase chain reaction (PCR) analysis were used to characterize a series of canine lymphomas in detail. The aim of this study was to determine the incidence of B- and T-cell high-grade lymphomas and their immunophenotypic characterization in Lower Silesia, Poland. The results show that the frequency of each type of lymphoma is 71% for B-cell and 17% for T-cell lymphomas. In two cases the PCR techniques confirmed the presence of simultaneous double gene rearrangements of the BCR and TCR receptors.

The effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in SRBC-immunized mice

Abstract 5-Amino-3-methyl-4-isoxazolecarboxylic acid hydrazide is a non-cytotoxic synthetic isoxazole derivative with considerable immunomodulatory properties demonstrated in in vitro experiments. The aim of this study was to investigate the influence of this compound, depending on the dosage and schedule of treatment, on lymphocyte subsets in non-immunized mice and humoral immune response in SRBC (sheep red blood cells)-immunized mice. An analysis of lymphocyte subsets was carried out by flow cytometry, using specific monoclonal antibodies stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). In the SRBC-immunized mice, the influence of the compound on the humoral response was determined, depending on the time of administration relative to the antigen. The number of plaque forming cells (PFC) was determined by a local hemolysis technique in an agar gel. Total and 2-mercaptoethanol resistant serum agglutination titers were defined by active hemagglutination test carried out on microplates. The investigated hydrazide was able to modulate the percentage and absolute number of T lymphocyte subsets in the thymus, and T and B lymphocytes in the peripheral lymphatic organs. It also enhanced humoral immune response in SRBC-immunized mice by increasing the number of cells producing hemolytic anti-SRBC antibodies (PFC) and by augmenting the level of total and 2-mercaptoethanol resistant hemagglutinin. The present study showed modulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in mice. This compound could be potentially useful for the treatment of autoimmune diseases, infections or as an adjuvant for boosting the efficacy of vaccines.