Effect of diamond-like carbon doped with chromium on cell differentiation, immune activation and apoptosis.

Diamond-like carbon (DLC) is a biocompatible material that has many potential biomedical applications, including in orthopaedics. DLC layers doped with Cr at atomic percent (at.%) of 0, 0.9, 1.8, 7.3, and 7.7 at.% were evaluated with reference to their osteoinductivity with human bone marrow mesenchymal stromal cells (hMSCs), immune activation potential with RAW 264.7 macrophage-like cells, and their effect on apoptosis in Saos-2 human osteoblast-like cells and neonatal human dermal fibroblasts (NHDFs). At mRNA level, hMSCs on DLC doped with 0.9 and 7.7 at.% of Cr reached higher maximum values of both RUNX2 and alkaline phosphatase. An earlier onset of mRNA production of type I collagen and osteocalcin was also observed on these samples; they also supported the production of both type I collagen and osteocalcin. RAW 264.7 macrophages were screened using a RayBio™ Human Cytokine Array for cytokine production. 10 cytokines were at a concentration more than 2 × as high as the concentration of a positive control, but the values for the DLC samples were only moderately higher than the values on glass. NHDF cells, but not Saos-2 cells, had a higher expression of pro-apoptotic markers Bax and Bim and a lower expression of anti-apoptotic factor BCL-XL in proportion to the Cr content. Increased apoptosis was also proven by annexin V staining. These results show that a Cr-doped DLC layer with a lower Cr content can act as an osteoinductive material with relatively low immunogenicity, but that a higher Cr content can induce cell apoptosis.

Interleukin- 1 gene polymorphisms as assessed in a 10-year study of patients with early-onset periodontitis.

Our 10-year study of early-onset periodontitis (EOP) patients includes repeated clinical observations, microbiological characteristics and analysis of genetic polymorphism of IL-1A and IL-1B genes. Twenty patients (age 15-26 years) were divided according to the clinical status in the 4th year into a group with mean number of teeth with bleeding on probing (BP) 9.8 and mean number of teeth with periodontal pocket (PP) 2.23, and a group with mean number of teeth with BP 5.37 and no PP. Significantly higher values of mean BP and PP were found among the groups during the study but a strong progression of disease was found only in 3 patients. The risk IL-1A allele-2 and IL-1B allele-2 genotype and bacterial presence were analyzed by DNA hybridization methods. No significant differences of bacteria composition (Treponema denticola, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans) were found between the groups. A higher prevalence of composite IL-1 genotype was detected in the group of EOP patients with progressive disease. Early finding of the disease followed by conservative therapy could positively influence the disease progression. EOP treated in early stages may, but need not, develop into the aggressive form in the presence of risk genotype IL-1.

Current trends and perspectives in nutrition and cancer prevention.

There is an increasing evidence that dietary phytochemicals may play important roles as chemopreventive or chemotherapeutic agents in prevention of many diseases, including tumors. The purpose of this study was to examine antimutagenic effects and effect on the immune response of representative series of substances which commonly occur in human diet. Using the Ames bacterial mutagenicity test and in vivo chemiluminescence test, we investigated antigenotoxic and immunomodulatory effects of juices and vegetable homogenates (carrot + cauliflower, cauliflower, red cabbage, broccoli, onion, garlic) on the genotoxicity of AFB1 and pyrolysates of aminoacids. Using the Ames test and in vivo micronucleus, the chemiluminescence test, the blastic transformation test and the comet assay we examined antimutagenic effects of chemically identified chemoprotective substances in the pure form (resveratrol, diallylsulphide, phenethyl isothiocyanate, ellagic acid, epigallocatechin gallate, genistein and curcumin) on mutagenicity induced by three reference mutagens: aflatoxin B1 (AFB1), 2-amino-3-metylimidazo[4,5,-f] chinolin (IQ) and N-nitroso- N-metylurea (MNU) and effect of phytochemicals on the immunosuppression caused by these mutagens. All complete vegetable homogenates and substances of plant origin tested, showed a clear antimutagenic and immunomodulatory activities on mutagenicity and immunosuppression induced by reference mutagens. Only in the Ames test the effect of some phytochemicals against direct mutagen MNU was lower compared to indirect mutagens AFB1 and IQ. Similarly, resveratrol and epigallocatechin gallate had no inhibitory effect on mutagenicity MNU in the Ames test.

Oral administration of probiotic bacteria (E. coli Nissle, E. coli O83, Lactobacillus casei) influences the severity of dextran sodium sulfate-induced colitis in BALB/c mice.

Our study examined whether repeated preventive oral administration of live probiotic bacterial strains Escherichia coli O83:K24:H31 (Ec O83), Escherichia coli Nissle 1917 O6:K5:H1 (Ec Nis) and Lactobacillus casei DN 114001 (Lc) can protect mice against dextran sodium sulfate (DSS)-induced colitis. A significant decrease in average symptom score was observed in Ec O83-, Ec Nis- and Lc-pretreated group (p < 0.05). Significant differences in body mass loss between Lc pretreated mice with DSS-induced colitis were found when compared with nontreated mice (p < 0.05). PBS pretreated mice had a significantly shorter colon than Ec O83-, Ec Nis- and Lc-pretreated mice (p < 0.05). Administration of Lc significantly decreased the severity of DSS induced histological marks of inflammation (p < 0.05). A significant difference (p < 0.05) was also found in specific IgA level against given probiotic in enteral fluid between colitic mice and healthy mice pretreated with Ec 083 and Ec Nis.

Growth hormone treatment increases oestrogen receptor concentration in the guinea-pig uterus.

Growth hormone does not act as a body growth-promoting hormone during the postnatal period in the guinea-pig. To determine whether it affects uterine and mammary growth, guinea-pigs of 8-9 weeks of age were treated with either recombinant bovine GH (bGH; 0.5 mg per animal) or vehicle for 10 days. Uterine and mammary weights were not changed by treating the animals with bGH. The amount of available cytosolic oestradiol receptor per unit of uterine weight or DNA content, or in whole uteri was increased in bGH-treated animals (7.3- to 14.0-fold) when compared with controls. The nuclear uterine oestradiol receptor concentration was 3.3- to 6.7-fold higher than in controls. The dissociation constant values did not differ between control and bGH-treated animals, suggesting that uterine oestradiol receptors are regulated by GH through changes in the number of binding sites rather than alteration of their binding affinity. Mammary growth and oestradiol receptor levels were unaffected by bGH treatment. The results of this investigation demonstrate that injected bGH selectively affects the oestradiol receptor level in guinea-pig uterine tissue.

HLA-association in patients with intolerance to mercury and other metals in dental materials.

A group of selected 25 patients with serious intolerance to heavy metals used for dental restoration were examined for HLA antigens. A significant increase for HLA -- B37, B47 and DR4 was found. The value of the relative risk is not significant after correction for the number of antigens tested and therefore further studies of more patients are needed.

Dose and time dependence of chromosomal aberration yields of bone marrow cells in male Chinese hamsters after a single i.p. injection of aflatoxin B1.

The frequency of chromosomal aberrations in bone marrow cells, after a single i.p. aflatoxin B1 (AFB1) dose, was examined in male Chinese hamsters (Cricetulus griseus). There was a significant increase in aberrant cells within 5 days of administration of a dose of 0.1 micrograms-5 mg AFB1/kg, and on the 36th day. After a single dose of 5 mg AFB1/kg the enhanced frequency of aberrant cells was monitored up to day 104 with no sign of a decrease to control level. The results indicate that the minimum mutagenic effect of an AFB1 dose in this system is 0.1 micrograms/kg. Attention is drawn to the long-term presence of chromosomal aberrations even after a single i.p. exposure to AFB1.

Relationship between experimental results in mammals and man. II. Cytogenetic analysis of bone-marrow cells after treatment of cytembena and cyclophosphamide- cytembena combination.

Cytogenetic analysis and the micronucleus test of bone-marrow cells was used to study the possible extrapolation of results from experimental animals to man. Cytembena was given i.p. in doses of 5, 10, 20, 40 and 80 mg/kg body wt. to Wistar rats and in doses of 20, 40 and 80 mg/kg body wt. to ICR mice and to Chinese hamsters. Five patients with various types of malignancy, so far medically untreated, received 20 mg Cytembena/kg body wt i.v. A combination of Cytembena and cylophosphamide was applied i.p. in single equal doses 1 : 1 of 5,10, 20, and 40 mg/kg body wt to ICR mice, Chinese hamsters and Wistar rats. Patients were given i.v. 20 mg Cytembena and 20 mg cyclophosphamide/kg body wt. Bone-marrow cells were examined 24 h after the administration. The frequency of abnormal metaphases and chromosomal breaks after Cytembena treatment was low; nonetheless, the indicated dose-effect relationship was found in all the rodents used. The frequency of chromosomal breaks was 2--3 times higher in rodents in comparison with man, after treatment with a dose of 20 mg Cytembena/kg body wt. Highest frequencies of induced aberrations were found in mice. The rodents appeared to be 3--4 times more sensitive to the induction of chromosomal breaks and abnormal metaphases than man, after a dose of 20 mg Cytembena and 20 mg cyclophosphamide/kg body wt. The micronucleus test may be regarded as a screening method for assessing mutagenic activity of chemical compounds. Chromosomal analysis and the micronucleus test were about equally convincing in detecting mutagenic effects even with the lowest doses of drugs used. However, the dose-effect relationship was more pronounced in chromosomal aberrations than in the micronucleus test.

The effect of partial hepatectomy on the genotoxicity of aflatoxin B1.

The influence of partial hepatectomy on the genotoxic effect of aflatoxin B1 (AFB1) mycotoxin in male Chinese hamsters (Cricetulus griseus) was studied after application of a single i.p. dose of 1.0 mg AFB1/kg. Changes in the fractions of proliferating bone marrow cells, values of the mitotic index of liver cells and morphologic changes in liver tissue were also monitored. Partial hepatectomy reduced significantly the mutagenic activity of AFB1 measured by the frequency of chromosome aberrations in bone marrow cells during 5 days. In hepatectomized animals AFB1 cytotoxicity was significantly reduced as evaluated by changes in the values of proliferating bone marrow cell fractions. There were no important morphologic changes in the liver. In hepatectomized AFB1 treated animals mitotic activity in liver tissue was substantially lower than in hepatectomized but AFB1 untreated animals.

Autoimmunity: from physiology to pathology. Natural antibodies, mucosal immunity and development of B cell repertoire.

Presence of spontaneously produced immunoglobulins bearing a broad spectrum of "natural" antibody specificities (including autoantibodies) in sera and other body fluids results mainly from inapparent immunization and polyclonal B cell activation by microflora and food antigens occurring mostly on mucosal surfaces. Early postnatal ontogeny in external environment is characterized by rapid growth and functional maturation of secondary lymphatic tissues as a consequence of this "natural" mucosal immunization. Under normal circumstances a state of "oral" tolerance to intestinal antigens is actively established after this period. Studies performed in germ-free, antigen-free and maternal antibody-deprived animals showed that low amounts of natural antibodies (mainly of IgM isotype) are formed without any known cause of stimulation. These "nonstimulated" antibodies, similarly as hybridomas originating from nonimmunized newborns, correspond to the preimmune repertoire of antibodies characterized by poly-specificity, high connectivity and reactivity against self antigens. Together with other innate humoral and cellular factors, they probably represent the first line of anti-infectious resistance. Moreover, due to their connectivity they are supposed to play an important role in B cell repertoire shaping (forming an idiotypic network), through interaction with a broad spectrum of immunological components they act as regulatory molecules, and through their participation in catabolic events they can promote morphogenetic changes during fetal development. Beneficial therapeutic effects of nonspecific gammaglobulin (IVIG) application observed recently in patients with autoimmune diseases suggest that they can influence autoimmune reactivity by a not yet analyzed mechanism. Other functions of natural autoantibodies can be suggested and expected to be found in the near future.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the LFA-1 beta molecule on peripheral blood leukocytes of patients with early-onset periodontitis: effects of dental plaque microbes.

The effect of dental plaque bacteria on LFA-1 beta expression on peripheral blood leukocytes was studied in 20 patients with early-onset periodontitis and in 10 healthy controls. Stimulation of PMN with selected dental plaque bacteria which play a role in the pathogeny of periodontitis significantly increased the expression of LFA-1 beta in the group of patients as compared with the controls.

Stimulation of human blood lymphocyte by different polyclonal B cell activators of bacterial and plant origin: production of IgM, IgG and IgA estimated by the ELISA method.

Lymphocytes isolated from peripheral blood of healthy donors were stimulated in vitro with pokeweed mitogen, concanavalin A, flagellin, Nocardia delipidated cell mitogen (NDCM) and heat-killed bacteria Escherichia coli and Actinomyces viscosus. A simple and sensitive technique, enzyme-linked immunosorbent assay (ELISA) was used for the detection of nanogram levels of IgM, IgA and IgC in media from lymphocyte cultures after polyclonal stimulation, Pokeweed mitogen, NDCM and E. coli were shown to stimulate a high production of IgM; after stimulation with A. viscosus a higher production of IgA was detected. No immunoglobulin production was observed after stimulation with polymerized flagellin.

[Sperm antibodies and immunologic intolerance to metals in infertile couples]. / Protilátky proti spermiím a imunologická intolerance nekterých kovu u neplodných páru.

OBJECTIVE: Verification of the hypothesis of a relationship between the presence of antibodies against sperm cells and immunological reactivity to some metals in infertile couples by the MELISA test. SETTING: Department of Obstetrics and Gynecology, Medical Faculty, Charles University and Faculty Hospital, Plzen. METHOD: From 23 female patients and 21 men (a total of 44 subjects treated for infertility) with confirmed serum antibodies against sperm cells the authors isolated lymphocytyes from the peripheral blood stream, divided them into individual cultures and investigated them by the MELISA test using different metal compounds. RESULTS: The outcome of the MELISA test are values of the stimulation index (SI) by means of which the authors investigated the reactivity of the organism to the given metal. Special attention was devoted to compounds of organic and inorganic mercury. The SI values were subsequently compared with different data obtained from a detailed anamnestic questionnaire which was focused specially on contact with metals and on allergic reactions. In the investigated group of patients the authors detected a positive immune reactivity to inorganic mercury, Ag, Al, Fe. In some subjects they found a very high positive immune reactivity to inorganic mercury, Ni, Al, Cd and Ti. The control groups were formed by healthy fertile subjects without antibodies against sperm cells and with physiological SI values. CONCLUSION: The authors did not prove a direct relationship between the intensity of the laboratory reactivity to metals and the presence of antibodies against sperm cells which cause deterioration of fertility. An exogenous load of metals could in case of genetic predisposition be only one of the factors which participate in the formation of antibodies against sperm cells. The investigation proved that its is not essential, contrary to the view of many stomatologists, to eliminate metal compounds completely from dental practice.

[Effect of heavy metals on immune reactions in patients with infertility]. / Vliv tezkých kovu na imunitní reakci u pacientu s prokázanou neplodností.

BACKGROUND: Heavy metals can negatively influence reproduction because in sensitive persons they are able to alter the immune reactions including autoantibodies production. The altered immune reaction can then cause infertility. METHODS AND RESULTS: The in vitro lymphocyte reaction after stimulation with metals, the production of interferon (IFN-gamma) and antisperm antibodies in supernatants after lymphocyte stimulation in patients with infertility and with the antisperm antibodies present in their serum were investigated. The cause of antisperm antibodies presence was not determined. The diagnosis of metal intolerance was performed by the proliferation method modified for metals (Melisa). In supernatants of tissue cultures of lymphocytes without the antigen stimulation and after stimulation with mercury chloride, the in vitro production of gamma interferon and antisperm antibodies was studied by Elisa. More than 50% of patients did not tolerate mercury, iron, aluminium and silver. When the lymphocyte reaction was compared in patients with and without mercury intolerance we found that lymphocytes of patients with mercury intolerance produced less gamma interferon and more antisperm antibodies in supernatants after mercury stimulation of lymphocytes. CONCLUSIONS: In patients with metal intolerance diagnosed by the Melisa test, metal ions released from the dental materials can represent a factor, that does not cause infertility but is able to influence it negatively.

[Reactions to metals in patients with chronic fatigue and autoimmune endocrinopathy]. / Reakce na kovy u pacientu s chronickou únavou a autoimunitními endokrinopatiemi.

Our study was designed to assess the effect of heavy metals on the severity of fatigue in autoimmune thyroid disease associated with autoantibodies against other endocrine organs. We compared our data with those obtained from other groups of patients. A total of five groups of patients were examined by their medical history, dental examination, and using a modified test of blast transformation of metals (Melisa): a) 10 fatigues female patients with autoimmune thyroidism and polyglandular activation of autoimmunity, b) 12 fatigued patients with autoimmune thyroidism, c) 28 fatigued patients free of endocrinopathy, d) 22 professionals without evidence of autoimmunity, e) 13 controls, a population sample, the individuals did not complain of marked fatigue and their laboratory tests did not show signs of autoimmunity and endocrinopathy. Fatigue regardless of the underlying disease is primarily associated with hypersensitivity to inorganic and organic mercury, nickel, and gold. The groups differed in their hypersensitivity to other metals. In the control group, hypersensitivity--mostly to cadmium and lead--was found in four of the examined individuals only. Statistical analysis of data obtained from professionals and controls revealed a higher incidence of positivity to organic and inorganic mercury and nickel in professionals.

The purple toe syndrome in female with Factor V Leiden mutation successfully treated with enoxaparin.

Purple toe syndrome is a rare complication of warfarin therapy. It occurs usually after 3 to 8 weeks of therapy and it is caused by cholesterol emboli from atheromatous plaque. Sudden onset of pain in affected area, typically in toes and feet, is the main characteristic of the syndrome. We describe a case of a 65-year-old female with purple toe syndrome after 6 weeks of warfarin. Indication of warfarin was a proximal deep venous thrombosis, which developed after prolonged immobilization. Factor V (FV) Leiden and persistent high FVIII activity were found as additional eliciting factors for venous thromboembolism. After warfarin withdrawal and enoxaparin treatment, symptoms disappeared promptly but a slight discoloration of the toe persists.

The interaction of osteoblasts with bone-implant materials: 1. The effect of physicochemical surface properties of implant materials.

This comparative study of various surface treatments of commercially available implant materials is intended as guidance for orientation among particular surface treatment methods in term of the cell reaction of normal human osteoblasts and blood coagulation. The influence of physicochemical surface parameters such as roughness, surface free energy and wettability on the response of human osteoblasts in the immediate vicinity of implants and on the blood coagulation was studied. The osteoblast proliferation was monitored and the expression of tissue mediators (TNF-alpha, IL-8, MMP-1, bone alkaline phosphatase, VCAM-1, TGF-beta) was evaluated after the cell cultivation onto a wide range of commercially available materials (titanium and Ti6Al4V alloy with various surface treatments, CrCoMo alloy, zirconium oxide ceramics, polyethylene and carbon/carbon composite). The formation of a blood clot was investigated on the samples immersed in a freshly drawn whole rabbit blood using scanning electron microscope. The surfaces with an increased osteoblast proliferation exhibited particularly higher surface roughness (here R(a) 3.5 microm) followed by a high polar part of the surface free energy whereas the effect of wettability played a minor role. The surface roughness was also the main factor regulating the blood coagulation. The blood clot formation analysis showed a rapid coagulum formation on the rough titanium-based surfaces. The titanium with an etching treatment was considered as the most suitable candidate for healing into the bone tissue due to high osteoblast proliferation, the highest production of osteogenesis markers and low production of inflammatory cytokines and due to the most intensive blood clot formation.