Biomedical subjects
Publications and source records attributed to Michał Zimecki.
Structural and biological investigations on a new 7-amino-3,5-dimethylisoxazole [5,4e][1,3,4]-triazepin-4-one were performed. The compound was shown to stimulate concanavalin A-induced mouse splenocyte proliferation and cytokine production by macrophage P388D1 cell line.
A human lactoferrin-specific cell line was generated in CBA mice, sensitized with 200 microg HLF in Freund's complete adjuvant. HLFK1 cells derived from the lymph nodes of these mice were maintained using HLF as the antigen. HLF was added at the beginning of each 14-day restimulation cycle, at a concentration of 100 microg/ml. The presentation of the antigen to HLFK1 was demonstrated using glass-adherent lymphocytes from spleens (GAL) as the antigen-presenting cells (APC). The presentation of HLF by GAL was highly efficient; a very low concentration of the antigen (1 microg/ml) was enough to stimulate proliferation of the HLFK1 cell line. HLFK1 did not proliferate in the presence of ovalbumin or bovine lactoferrin (BLF), which is structurally related to HLF. However, we found that BLF caused a reduction in the proliferation of the HLFK1 cell line when BLF was added to the cultures together with the antigen - (HLF). On the other hand, proliferation of the HLFK1 cell line was not inhibited by pretreatment of the antigen-presenting cells or T cells with BLF. Therefore, we suggest that bovine lactoferrin may interfere with the binding or uptake of the antigen (HLF). Alternatively, BLF may nonspecifically inhibit the activation of the HLFK1 cell line.
The aim of this investigation was to reveal the regulatory properties of bacteriophage preparations in a model of mitogen-induced splenocyte proliferation in mice. We showed that sepharose 4B-purified preparations of the Staphylococcus aureus phage A20/R exhibited costimulatory activity in splenocyte proliferation induced by suboptimal (0.25 microg/ml) concentrations of ConA. On the other hand, the purified phage fraction was regulatory with regard to splenocyte proliferation induced by the optimal (2.5 microg/ml) ConA concentration. We also showed that the phage preparation can elicit IL-6 production in splenocyte cultures and enhance ConA-induced production of that cytokine. Furthermore, the phages preferentially induced IL-6 production in adherent splenocytes and increased levels of that cytokine in cultures of peritoneal cells from mice and rats. This phenomenon may explain the costimulatory activity of phages in the model described.
The aim of this investigation was to establish the impact of phage therapy on the turnover and function of circulating neutrophils in 37 patients with suppurative bacterial infections. We determined the levels of circulating neutrophils and their precursors before therapy, after 3 weeks of therapy, and at a distant time interval (3 months) following the beginning of therapy. In addition, we measured the ability of neutrophils to phagocytize Staphylococcus aureus in vitro. Eight healthy blood donors served as a control group. The results showed that, among the studied parameters, the significant changes involved neutrophil precursor count and the ability of neutrophils to phagocytize bacteria. The percentage of neutrophils in patients before therapy was lower than in healthy donors (mean 58.0, versus 61.4). This value dropped further in patients after 3 months of following the therapy (mean 55.6). The content of neutrophil precursors, on the other hand, was lower in healthy donors than in patients before therapy (mean 2.5, versus 3.8). After 3 weeks of the therapy and after 3 months, the levels of neutrophil precursors were significantly higher (mean 4.8 and 4.9, respectively) than in control donors. The phagocytic index was lower in patients before therapy than in control donors (mean 66.3, versus 70.1) and decreased further after 3 weeks of therapy (mean 59.0) and after 3 months (mean 59.6). The results of this investigation indicate that successful phage therapy accelerates the turnover of neutrophils, accompanied by a decrease in their ability to phagocytize bacteria.
The mannose receptor (MR) mediates clearance of pathogenic microorganisms and potentially harmful glycoproteins by recognition of the carbohydrate structures. This manuscript describes the significance of the MR in mediation of the lactoferrin (LF) effects in delayed type hypersensitivity (DTH) in mice. Here we demonstrate that the inhibitory effect of LF on the effector phase of DTH to sheep red blood cells (SRBC) was abolished by intraperitoneal (i.p.) pretreatment of mice with 50 mg of methyl-alpha-D-mannopyranoside (MMan). Also, we report that the inhibition of the effector DTH response to ovalbumin (OVA) was reversed by MMan but not by D-galactose (Gal). MMan was found to reduce the adjuvant activity of LF given together with OVA, however, did not interfere with the upstimulatory effect of the complete Freund's adjuvant. Comparative studies between bovine and human lactoferrins (BLF and HLF), revealed that the adjuvant effect of BLF was stronger than that of HLF, nevertheless, both effects were inhibited by MMan. Thus, we postulate, that MR is a primary receptor for lactoferrin in mediation of its some immunotropic activities.
Lactoferrin (LF) is an iron-binding glycoprotein present in the cytoplasmic granules of neutrophils and in external secretions of mammals. Although the biological role of human and bovine lactoferrin has been extensively studied, there is still uncertainty as to the nature and function of lactoferrin receptors. We recently determined that methyl-alpha-D-mannopyranoside given intraperitoneally (i.p.) could suppress the adjuvant activity of LF in the generation of delayed-type hypersensitivity (DTH) to ovalbumin (OVA). We concluded that the lactoferrin effects in DTH are mediated by carbohydrate-recognizing receptors like the mannose receptor (MR). This study indicates that subcutaneous (s.c.) administration of very small doses of the Man-bovine serum albumin (Man-BSA) complex, together with a sensitizing dose of the antigen, gives the same effects as i.p. administration of methyl-alpha-D-mannopyranoside. The latter is also a blocker of MR, although of a much lower affinity to the receptor than Man-BSA. The blocking of the adjuvant effect of LF by the Man-BSA complex (when given together with the sensitising dose of antigen) suggests that the function of antigen-presenting cells in the skin (presumably immature dendritic cells expressing MR) is inhibited. The results of our study indicate that a receptor with an affinity for mannose is essential for the mediation of adjuvant lactoferrin function in the generation of DTH.
Homeostasis is the maintenance of equilibrium in a biological system by means of positive and negative feedback control mechanisms that counteract influences tending toward physiological dissonance. At the molecular level, homeostasis is controlled by the network of the neuro-endocrine-immune system, in which lactoferrin (LF) plays a central role. The purpose of this review is to provide a comprehensive summary of a collaborative study established between the Hirszfeld Institute of Immunology and Experimental Therapy (Wrocław, Poland) and the University of Texas Health Science Center (Houston, USA) regarding LF and its role in homeostasis. In our studies we focused on the immunoregulatory functions of LF, both in vitro and in vivo. We investigated the immune status of individuals subjected to different insults, including experimental endotoxemia in mice and surgery in humans. We also studied a LF-dependent delayed type hypersensitivity (DTH) response to evaluate some of the mechanisms by which LF can effectively substitute an adjuvant in vaccine.
The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the beta chain (164-172) and having the sequence VPRSGEVYT, suppresses the immune response. On the basis of the three-dimensional structure of the HLA-DR superdimer, we designed new dimeric analogs in which the VPRSGEVYT peptides are linked through their N-termini by poly(ethylene glycol) linkers of different lengths and are able to mimic the dimeric nature of the immunosuppressive fragments of HLA class II molecules. The analogs were synthesized using standard solid-phase peptide synthesis protocols. The dimerization was achieved by cross-linking the N-terminal positions of the peptides, attached to an MBHA resin, with alpha,omega-bis(acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the amino-terminal dimerization of the peptide results in enhanced immunosuppressive activity and that the potency of the conjugates depends on the length of the poly(ethylene glycol) linker. MS/MS analysis of the obtained dimeric peptides is also presented.
A new series of 5-substituted 3-methylisoxazole[5,4-d] 1,2,3-triazin-4-one derivatives was prepared and investigated. The immunological activities of the studied compounds were investigated in two murine models of the immune response to sheep erythrocytes (SRBC)--the humoral immune response and delayed type hypersensitivity (DTH). Quantum-chemical calculations were carried out using AM1, a semiempirical method for geometry optimization, estimation of descriptors values and localization of the HOMO and LUMO orbitals.
Looking for active immunosuppressant, a series of substituted phenylamides of 5-aminomethinimino-3-methylisoxazole-4-carboxylic acid was obtained, which showed immunosuppressory activities in the in vitro and in vivo tests, comparable with that of cyclosporine A. Rentgenostructural studies of three most representative derivatives were performed and the molecular modelling of compounds, demonstrating most characteristic biological activities, was performed. In the next stage, quantum-chemical investigations were conducted in order to determine structure-activity relationships.
The aim of this investigation was to evaluate immunotropic properties of a new isoxazolotriazepine (compound RM33) in mice. We found that RM33 significantly inhibited induction of the humoral immune response to sheep red blood cells (SRBC), given 2 h prior to immunization. The development of the cellular immune response (delayed type hypersensitivity--DTH) to SRBC and to ovalbumin (OVA) was also suppressed, as well as the effector phase of the DTH to OVA. The compound was also effective when administered per os. The suppressive effects of RM33 on the immune response were comparable to those of cyclosporine A(CsA). We also showed that RM33 inhibited DTH to OVA if admixed with the sensitizing dose of an antigen and complete Freund's adjuvant (cFa) suggesting that the compound may affect initial events of antigen presentation. Such a hypothesis was supported by finding that RM33 significantly inhibited foot pad edema elicited by administration of cFa. The effects of RM33 on the activities of cytokines relevant to development and control of the immune response and inflammation such as: tumor necrosis factor alpha (TNF-alpha), interleukin 6 and 10 (IL-6 and IL-10) were also studied. The compound markedly (by 63%) inhibited lipopolysaccharide (LPS)-induced TNF-alpha serum level whereas IL-6 activity was lowered to a lesser extent (by 17%). The inducible IL-10 level in the splenocyte cultures was not affected at all. In summary, the presented results revealed immunosuppressive properties of RM33, which could be associated with its selective interference with co-stimulatory signals provided by adjuvant at initiation of the immune response.
Effects of orally administered lactoferrin (LF) on the cellular and humoral immune responses in mice subjected to immobilization stress (IS) were investigated. Here, we demonstrate that long-term IS induced significant suppression of cellular and humoral immune responses in CBAmice. The suppression was attenuated by LF given to mice in drinking water as determined by the number of antibody-forming cells (AFC) in the spleen and the magnitude of delayed type of hypersensitivity (DTH). On the other hand, LF lowered the elevated DTH response in mice exposed to short-term IS (5 h only) on the day of elicitation of the DTH reaction. We also showed that LF up-regulated spontaneous transforming growth factor beta (TGF-beta) production in the cultures of mesenteric lymph node cells derived from short-term stressed mice. This is the first report on the regulatory effect of LF on the immune response modified by the psychic stress and is consistent with other reports on antinociceptive and analgesic actions of LF in experimental animals.
The aim of this investigation was to evaluate effectiveness of RM-33, a new isoxazolotriazepine, in the model of carrageenan-induced inflammation in rats. Wistar rats were pretreated with intraperitoneal (ip) or oral (po) doses of RM-33, at daily doses ranging from 250 to 1000 microg, administered 1-3 days before elicitation of the carrageenan reaction. We showed that both routes of RM-33 administration were effective in significantly diminishing the footpad edema. The effects were dose-dependent and better pronounced at the ip administration of the compound. We found a lower production of tumor necrosis factor alpha (TNF-alpha) by mitogen-stimulated splenocytes isolated from rats pretreated with RM-33 and injected with carrageenan, as well as lower serum TNF-alpha levels in these rats, as compared to the respective control. Histological analysis of the skin reaction site revealed that in the rats pretreated with RM-33, the carrageenan-induced inflammation was reduced, as reflected by a lesser damage of mast cells, smaller infiltration by macrophages and a diminished edema of the connective tissue. Together with our previous data, indicating the antagonistic action of RM-33 in the adjuvant-induced footpad inflammation in mice, the present results confirm the anti-inflammatory activity of RM-33 compound.