Mimosine is a non-toxic plant aminoacid which is an effective inhibitor of DNA replication by acting at the S-phase. In this study we infected mice with T. spiralis, a nematode parasite, and studied the inflammatory response through the determination of MIP-2, a C-X-C chemokine and MCP-1, a C-C chemokine in the inflamed area around the parasitic cyst. The animals were infected and their diaphragms were tested for inflammatory response. MCP-1 and MIP-2 was tested after 1, 10, 20, 30, and 40 days post inoculation, before and after mimosine treatment. The inflammatory index was calculated by counting the white blood cells around the nematode cysts, while expression of MIP-2 and MCP-1 was calculated by ELISA method and transcription by Northern blot and RT-PCR. Here we found that mimosine strongly inhibited the inflammatory index in the diaphragmatic tissue at 10, 20, 30 and 40 days post-treatment. In these experiments, mimosine had no effect on the number of cysts produced. In addition, we found that MCP-1 transcription and translation was completely inhibited by mimosine, while MIP-2 transcription and translation was partially inhibited at 30 and 40 days; yet it was totally inhibited after 10 and 20 days in encysted diaphragm tissue infected by T. spiralis. Our studies suggest that mimosine has an inhibitory effect through the inhibition of cytoplasmatic serine hydroxymethyltransferase altering the cell cycle of white blood cells. This study suggests for the first time the premise that mimosine acts as an anti-inflammatory compound.

The aim of this study was to investigate the effect of pyridoxine (Vitamine B6) deficiency on the immunological response of BALB/c mice infected with the parasite T. spiralis. Specific anti-parasite IgM and IgG immunoglobulins were detected by ELISA method in the serum of treated animals at different periods for 60 days post infection.

Animals fed diets deficient in vitamin B6 develop microcytic anemia, alterations of growth, and other pathologies. 4-deoxypirydoxine is a potent antagonist of vitamin B6 coenzyme which depresses IL-1, TNF and IL-6 and has anti-inflammatory properties. The aim of this study was to show the anti-infl ammatory effects of 4-DPD on chronic inflammation caused by the nematode parasite T. spiralis, specifically on the recruitment and the activation of inflammatory cells. Two groups of mice, 6 weeks of age, were used: one was maintained on a vitamin B6-deficient synthetic pellet diet for 15 days before injection of the nematode, and administered an intraperitoneal injection (i.p.) of 4-DPD (250 μg/mouse) for 15 days (the first, 5 days before infection), and the second group was maintained on a normal diet for the total duration of the experiment. These two groups were then injected with 150 larvae (L1-T. spiralis) per os.

MCP-1 is a small (8-10 KDa) protein and a prototype member of the CC chemokine β subfamily, which plays a critical role in acute and chronic inflammation. Recent evidence suggests an important role for MCP-1, MCP-2 and MCP-3 in a number of pathological states, including delayed type hypersensitivity conditions, parasitic infections and rheumatoid arthritis. Forty BALB-c mice were treated with the parasite Trichinella spiralis. After the infection the animals were sacrificed at different periods from the initial infection and MCP-1 and TNFα were quantified in the mouse serum. The level of MCP-1 in the serum of mice infected with 100 larvae increase 27.5 ± 7.0 pg/ml at day 23, to a maximum level of 31.5 ± 5.0 pg/ml at day 33, from then decreased to 14.6 ± 2.0 pg/ml at day 47. When the mice were infected with 200 larvae of T. spiralis the maximum increase was 34.4 ± 2.5 pg/ml found on day 23. From day 33 to day 47 MCP-1 levels were decreased. In addition, in infected mice levels of TNFα were detectable in the serum as early as day 1. The level of TNFα was maximum at day 35 (3812 ± 224 pg/ml). Serum from non-infected mice contained no detectable levels of either MCP-1 or TNFα. However, even if MCP-1 seems to be implicated in Trichinellosis, its exact role and function in inflammatory parasitic diseases remains to be determined.

MCP-1 is a prototype member of the CC chemokine subfamily, having chemoattractant properties for monocytes, T lymphocytes, basophils, but not for neutrophils. Twenty BALB-c mice were treated with the parasite Trichinella spiralis. After the infection the animals were sacrificed at different periods from the initial infection and MCP-1 and TNF alpha were quantified ire the mouse serum. The level of MCP-1 in the serum of mice infected with 100 larvae increases 27.5 +/- 7.0 pg/ml at day 23, reaching a maximum level of 31.5 +/- 5.0 pg/ml at day 33, and decreases to 14.5 +/- 2.0 pg/ml at day 47. When the mice were infected with 200 larvae of T. spiralis the maximum increase was 34.4 +/- 2.5 pg/ml found on day 23. From day 33 to day 47 MCP-1 levels were decreased. In addition, in infected mice levels of TNF alpha were detectable in the serum as early as day 1. The level of TNF alpha was maximum at day 35 (3812 +/- 224 pg/ml). Serum from non-infected mice contained no detectable levels of either MCP-1 or TNF alpha. Our data provides new evidence for MCP-1, suggesting an involvement of this chemokine in mediating parasitic diseases.

Regulated upon activation normal T expressed and secreted (RANTES) is a new inducible protein member of the human CC branch of chemokines. RANTES is a potent monocyte and lymphocyte chemoattractant and is a mediator of inflammatory responses. In these studies we found that RANTES 10 ng50 μl chemoattracts basophilic cells in a dose-dependent manner 4 h after an intradermal injection in rat skin sites, as revealed by optic microscopy. Moreover, in biopsy specimens from rat skin injection sites histamine release was significantly higher (P < 0.05) than in controls (PBS 50 μl) after 4 h from RANTES treatment. The presence of basophilic cells in rat skin injection sites after RANTES-treatment was also confirmed by electron microscopy studies. In addition, histidine decarboxylase (HDC) mRNA was increased in rat skin sites injected with RANTES compared to sites injected with PBS (controls). Our report describes additional biological activities for RANTES, suggesting that this human chemoattractant protein may play a fundamental role in histamine and HDC generation, along with basophilic cell recruitment.

Pyridoxine deficiency leads to impairment of immune responses. It appears that the basic derangement is the decreased rate of production of one-carbon units necessary for the synthesis of nucleic acids. The key factor is a pyridoxine enzyme, serine hydroxymethyltransferase. This enzyme is very low in resting lymphocytes but increases significantly under the influence of antigenic or mitogenic stimuli, thus supplying the increased demand for nucleic acid synthesis during an immune response. Serine hydroxymethyltransferase activity is depressed by deoxypyridoxine, a potent antagonist of pyridoxal phosphate, and also by known immunosuppressive or antiproliferative agents. The combination of these agents is additive. Our results lead us to suggest the following medical applications: (a) combination of deoxypyridoxine with immunosuppressive or chemotherapeutic drugs may be effective in cases of immunosuppressive therapy or organ transplantation, (b) the development of special agents directed against the serine hydroxymethyltransferase apoprotein may prove to be a valuable medical tool, since this enzyme presents an excellent target for chemotherapy, (c) lymphocytes of individual patients could be used to design tailor-made specific immunosuppressive or chemotherapeutic treatment, and (d) the serine hydroxymethyltransferase activity of lymphocyte culture presents an excellent indicator for the evaluation of potency of immunosuppressive, chemotherapeutic or genotoxic compounds in a simple and rapid test.

The effect of infra-red laser irradiation has been experimented on various biological systems and particularly in human tissues, in vitro as well as in vivo. In order to examine the influence of laser irradiation on cells of the monocytic lineage we have irradiated human peripheral blood mononuclear cells with an infra-red laser at a wavelength of 904 nm, at 2000 Hz frequency and 15 mW for 2 min. Here, we report that laser irradiation for 2 min. at different preincubation times (T = 0 and T = 30 min) enhances LPS (10 μg/ml or PHA (10 μg/ml, suboptimal concentration) -stimulated monocytes by modifying cell proliferation, as judged by [3H] thymidine incorporation. IL-1 receptor antagonist (IL-1ra) along with an increased release of [3H] Arachidonic acid production, is also influenced by laser irradiated monocytes when treated for 2 min after 1 h incubation. IL-1RA production increased 4-5-fold after laser irradiation, while 3H-arachidonic acid incorporated from PMA-stimulated cellsased and the effect was significant at T = 0 and T = 30 min; while at T = 1 h the effect was negligible. These results may provide new information regarding the effect of laser irradiation on the immune system.

A 21 year old woman from Thrace with clinical and laboratory findings of mild hemolysis, was found to be homozygous for haemoglobin O Arab. Blood samples from 15 members of her family and from 42 inhabitants of her village were examined for the presence of Hb O Arab. A high incidence of this variant allele was also detected among the patient's family members as well as in other inhabitants of the village (p = 0.274 +/- 0.049). The possibility that Pomaks, a culturally unique tribe with controversial ethnic origin, may represent one of the original pools for the geographic distribution of the gene in the broader area of the Mediterranean basin is discussed.

Chemokines are a family consisting of at least ten distinct novel 8-10 kD cytokines. Monocyte chemoattractant protein-1 (MCP-1) is the prototype of the CC chemokine subfamily, purified from different sources with chemoattractant and activator properties. In this study we found that supernatants derived from TNF-alpha (10 ng/ml) or LPS (10 mu g/ml)-activated rat peritoneal mast cell cultures (5 x 10(5)/ml), incubated for 18 h, produced levels in the order of 560 and 420 pg/ml of MCP-1, respectively. This data describes an additional mode of generation of MCP-1.

Prostaglandins and thromboxanes (T×s) are produced by polymorphonuclears (PMNs) and macrophages (MØs) in response to various stimuli. PMNs were separated from other human blood cells and MOs were separated from rat peritoneal lavage. In this paper we show that human recombinant interleukin-1 (hrIL-1) can stimulate the release of thromboxane B2(T×B2) by PMNs and MØs. In addition, we have shown that aggregation of PMNs may occur when calcium ions (7 mM) and hrIL-1 (100 ng/ml) are added to the cell preparation, but not when Ca2+ alone, hrIL-1 alone, or first hrIL-1 then calcium are added to the cell preparation. The treatment of human platelets with hrIL-1 shows that after 15 min incubation T×B2 is released. In addition, we compared the aggregation of platelets caused by ADP with that caused by hrIL-1. Human recombinant IL-1 at a concentration of 100 ng/ml also causes little aggregation of platelets, in this case the aggregation is reversible. In conclusion, hrIL-1β stimulates T×B2 release in PMNs, MØs and platelets and this effect increases with addition of Ca2+ ions. The mixture of hrIL-1 and Ca2+ causes little aggregation of PMNs. In monocyte suspensions, pretreated with human recombinant IL-1 receptor antagonist (IL-Ira) 500 ng/ml for 10 min and then treated with LPS or hrIL-1β 10 μg/ml, the release of T×B2 was partially inhibited. IL-Ira may play a significant role in the control of IL-1 and LPS induction in the release of T×B2.

Redox cycling compounds such as daunorubicin have been assumed to be toxic because they stimulate reactive oxygen-mediated lipid peroxidation. Furthermore, both DT-diaphorase and glutathione (GSH) have been regarded as protective cellular compounds against daunorubicin cardiotoxicity, but their role in daunorubicin nephrotoxicity remains unclear. To investigate this issue, 10 adult Wistar rats were twice injected with a single dose of 20 mg/kg body weight daunorubicin into the tail vein; the interval between injections was 48 h. A control group of 10 rats were injected with normal saline. One day after the second injection, all the animals were sacrificed and their kidneys were analyzed for malondialdehyde (MDA) as an index of lipid peroxidation, DT-diaphorase activity, and GSH and glutathione disulphide (GSSG) content. A significant increase of MDA concentration (2.41 vs. 1.64 p < 0.001) and DT-diaphorase activity (0.2 vs. 0.12, p < 0.001) was found in the renal tissue of daunorubicin injected rats. In contrast, GSH and GSSG levels were decreased in those animals (566 vs. 1282, p < 0.001 and 115 vs 187, p < 0.01, respectively). The results of this study give evidence that a high dosage of daunorubicin induces lipid peroxidation in renal tissue of rats stimulating the activation of DT-diaphorase and the detoxificative depletion of GSH.

Infections caused by the nematode Trichinella spiralis is characterized in the host by an inflammatory response with cytokine production. In these studies we have detected TNF alpha, IL-6, IFN gamma, IL-4 and IL-10 in the serum of 10 mice infected with T. spiralis. Moreover, we detected, for the first time, these cytokines in the serum of mice treated with 4-DPD, a potent antagonist of vitamin B6 coenzyme which has anti-inflammatory properties. 4-DPD was used at 100, 400, 800 micrograms/bolus for 20 days, starting one day before the infection. After 15 days of T. spiralis infection, TNF alpha reached a maximum level, while IL-6 was maximal after 7 days, IFN gamma at 20 days and IL-4 at 14 days. IL-10 was not affected by the T. spiralis infection. When the animals were treated with 4-DPD at the reported dosages and infected with T. spiralis the inhibition of TNF alpha and IL-6, were dose-dependent in the first 7 days while IL-4 was reduced only at 400-800 micrograms/bolus. 4-DPD-treated mice did not statistically (P > 0.05) affect the generation of IFN gamma. In healthy animals the production of cytokines were not measurable, just as it was in non-infected animals treated with 4-DPD. The increase of cytokines such as, TNF alpha and IL-6 may be related to the severity of the disease, boosting the host's resistance to the pathogen and inhibiting parasite survival. In addition, the augmentation of IL-4 production enhances T and B cells and macrophage responses and may stimulate T-cell antibody-mediated response to the pathogen. 4-DPD, an inhibitor of IL-1 and inflammatory reactions, proved to be most effective on TNF alpha and IL-6, which are mainly produced by macrophages.

A heparin binding fibroblast growth factor with a molecular weight of approximately 45 kDa has been detected in human placenta. The protein was extracted from placenta and purified by a combination of ammonium sulfate precipitation, DE-52 anion exchange chromatography, G100 gel exclusion and heparin sepharose affinity chromatography. The molecule was shown to be monomeric after treatment with beta-mercaptoethanol, and had an acidic isoelectric point. The properties of the polypeptide described in this paper (45 kDa, Pi 4-5, monomeric, heparin binding fibroblast growth factor), suggest that it may represent an as yet undescribed growth factor.

Measurements of Serine Hydroxymethyltransferase (SHMT) in resting lymphocte cultures showed that the activity level of this enzyme is very low. Under the influence of mitogenic stimuli SHMT activity is induced 5-20 fold. Addition in the cultures of antiproliferative (AP) or immunosuppressive (IMS) agents, namely D-actinomycin, cytarabine, L-asparaginase and cyclosporin led to the following observations:1. All AP and IMS agents, added to the lymphocyte culture 48 hrs after stimulation, cause a decrease in the mitogen induced activity of SHMT. The higher the concentration of the AP or IMS compound, the greater the decrease of the enzyme activity.2. The observed activity changes are not, as one would expect, the same in the case of all four drugs. Higher inhibition of SHMT activity is produced by actinomycin (about 50%) and lower by cyclosporin (about 30%). Probably, each compound causes a different degree of inhibition, reflecting its specific mechanism of action.Dose titration experiments established the limit between active and inactive concentrations of the tested agents.The development of a simple test, based on SHMT activity estimation, for rapidly assessing a compound's antiproliferative or immunosuppressive potency is proposed. Also, this test could be valuable for-the titration of substances tested and provide preliminary data concerning the doses that would be administered during clinical trials.

Tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6), along with interleukin 1 (IL-1) are cytokines involved in the acute phase response and play a central role in parasitic inflammatory states, 4-Deoxypyridoxine (4-DPD) an antagonist of vitamin B6 inhibits IL-1 and inflammation, In this study are found that 4-DPD (100, 400, and 800 mu g/animal) strongly reduces (p < 0.05) in a dose-dependent manner, TNF alpha and IL-6, but not interferon gamma (IFN gamma) in the serum of T, spiralis-infected mice.The inhibitory effect of 4-DPD on TNF alpha was studied from the day before T, spiralis infection until the 35th day, The present data may prove and suggest that 4-DPD, a novel anti-inflammatory agent, may be beneficial in clinical applications in T, spiralis infection.

1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography. 2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin-dependent protein kinase and casein kinases. 3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis. 4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca2+/calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

The serological identification of HLA class II alleles is often doubtful. Since accurate HLA typing is essential for the matching of donor-recipient pairs in allogeneic transplantation, an effort was made to establish DNA restriction fragment length polymorphism (RFLP) typing and to assess the correlation between the serological and RFLP techniques in the population of Northern Greece. One hundred and two healthy individuals (204 HLA-DR alleles) from Northern Greece were HLA-DR, DQ typed with both the microcytotoxicity and the Taq I RFLP method, using three exon-specific probes. DNA-RFLP typing revealed (1) concordant results with serology in 69.9% (142/204) of the alleles and (2) at least one HLA-DR allele discrepant to serology in 30.4% (62/204) of the alleles. Incorrect serological DR types (weak reactions or inability to distinguish between two alleles with a common epitope) were identified in 54 alleles (26.5%), while 3.9% (8/204) of serological "blank" alleles turned out to be definable alleles by RFPL. Of the individuals tested, 10.8% (11/102) were DR-homozygous by RFLP. This comparison of results obtained by serology and RFLP demonstrated the necessity of the clinical application of DNA typing, especially for organ transplantation where accurate HLA typing has an important influence on graft survival.

Measurements of serine hydroxymethyltransferase (SHMT) in resting lymphocyte cultures showed that the level of activity of this enzyme is very low. Under the influence of mitogenic stimuli serine hydroxymethyltransferase activity is induced 5-20-fold. Addition in the cultures of 4-deoxypyridoxine (dB6), a potent antagonist of vitamin B6 coenzymes, concurrently with the mitogen, inhibits the induction of SHMT. Separate addition in the cultures of four anti-proliferative (AP) and immunosuppressive (IMS) agents, namely actinomycin, cytarabine, asparaginase and cyclosporine, led to the following observations. (1) The AP and IMS agents produce a decrease in the mitogen-induced activity of SHMT. The higher the concentration of the AP/IMS compound, the greater the decrease of enzymatic activity. (2) When a AP/IMS agent is combined with dB6 its effect on SHMT is considerably greater. (3) Ineffective concentrations of AP/IMS agents become effective when combined with dB6. (4) The observed changes in SHMT activity are not, as one would expect, the same in the case of all four drugs. (5) The combination makes it possible to use much smaller doses of these agents with much better results, at least as far as the decrease of enzymic activity is concerned. This is very promising for clinical use of AP agents in cancer chemotherapy and IMS agents in transplantation especially of the heart and lungs, because combining these compounds with dB6 will make possible to use smaller doses over a longer period of time with greater effectiveness.(ABSTRACT TRUNCATED AT 250 WORDS)