Seroprevalence of rickettsial infection in northern Cyprus: A study among hunters.

This study was conducted to investigate rickettsial seropositivity among hunters, a high-risk population for tick-borne diseases in northern Cyprus. Serum samples were collected from 300 hunters from different locations during the 2017-2018 hunting season (November 2017 - February 2018). The samples were analyzed by indirect immunofluorescence assay (IFA) using slides coated with Rickettsia slovaca, a species belonging to the spotted fever group (SFG). During the sample collection, a questionnaire was also applied to evaluate possible risk factors for rickettsial seropositivity. Of the 300 serum samples, six (2.0%) were found to be IgG-positive with a titer of 1:64. While all seropositive individuals were male, the statistical analysis revealed no significant association of gender with rickettsial seropositivity (p=1.000). Other factors including age (p=0.414), residential places of the participants (p=0.347), hunting years (p=0.694) or hunting abroad (p=1.000) did not significantly affect the IgG positivity. Also, no statistical correlation was found between a history of an arthropod (tick, louse, or flea) bite and rickettsial seropositivity (p=1.000). To our knowledge, this is the first study that demonstrates rickettsial seropositivity among human population in northern Cyprus. Our study suggests that awareness should be raised among the people especially involved in outdoor activities such as hunting, and control programs should be implemented to prevent possible rickettsiosis cases. Further serological studies using other Rickettsia spp. antigens, as well as molecular studies that search for Rickettsia spp. in humans, animals and arthropods are needed to obtain more comprehensive data on rickettsiosis in northern Cyprus.

Natural killer cell activity, serum immunoglobulins, complement proteins, and zinc levels in patients with psoriasis vulgaris.

The role of the immune system in patients with psoriasis vulgaris (PV) was investigated. The genetic and immunological basis for psoriasis is still unknown. Because of the reports of immunological defects in this disease, we investigated serum levels of immunoglobulins IgG, IgM, IgA, complement proteins C3, C4, serum zinc (Zn) levels and natural killer (NK) cell activities. Skin lesions of the psoriatic patients involved in the study comprised less than 10 % of the total body and the disease was in a stationary period. Zn levels were measured by atomic absorption spectrophotometry. NK cell activity was measured by 51Cr (Na2 51CrO4). IgG, IgA, IgM, C3 and C4 assays were done by liquid-phase immunoprecipitation assay with nephelometric endpoint detection. IgG, IgA, C3 and C4 levels were significanty higher in patients with PV than in healty controls (p < 0.05). However, NK cell activity, serum Zn and IgM levels did not show significant differences between these two groups. There are changed immunological responses, which may play an important role in the pathogenesis of the disease. Many controversial results have been related to immunological parameters in psoriatic patients. Therefore, more detailed studies in this field need to be done to determine the relationship between psoriasis and the immune system.

Development of a rapid, single-step procedure using protein G affinity chromatography to deplete fetal calf serum of its IgG and to isolate murine IgG1 monoclonal antibodies from supernatants of hybridoma cells.

Fetal calf serum (FCS) was depleted of its immunoglobulin G (IgG) in a rapid procedure using protein G affinity chromatography. 20 ml of FCS was depleted of its IgG in less than 80 min by applying 5 ml of FCS to a 1 ml HiTrap protein G Sepharose column followed by appropriate elution. Various concentrations of IgG-depleted FCS (G-FCS) were used in RPMI-1640 medium to grow the mouse hybridoma cell lines CAy-G (anti-HBs IgG1 mAb producing hybridoma cell) and CAy-M (anti-HBs IgM mAb producing hybridoma cell), which secreted hepatitis B virus surface antigen (HBsAg)-reactive IgG1 and IgM monoclonal antibodies (mAbs), respectively. Antibody production and cell growth were used as indices to compare the efficacy of RPMI/G-FCS with that of RPMI/FCS and serum/protein-free Hybri Max (Sigma, MO, USA) hybridoma medium. MAb production and cell growth of CAy-G and CAy-M hybridoma cell lines in RPMI/G-FCS were similar to culture in RPMI/FCS and significantly better than culture in Hybri Max. We found that G-FCS was superior to whole FCS as a culture supplement for the purification of IgG1 mAbs. IgG1 mAbs were isolated in a single-step procedure using protein G affinity chromatography, from the supernatant of CAy-G hybridoma cells cultured in RPMI/10% G-FCS (RPMI-1640 medium supplemented with 10% G-FCS). SDS-PAGE analysis revealed that the purity of IgG isolated from the supernatant of CAy-G cells cultured in RPMI/10% G-FCS was more than 99%.

Effects of occupational chronic co-exposure to n-hexane, toluen, and methyl ethyl ketone on NK cell activity and some immunoregulatory cytokine levels in shoe workers.

1. To evaluate the effects of occupational long-term co-exposure to n-hexane, toluen, and methyl ethyl ketone (MEK) on NK cell activity and serum IL-2, gamma-IFN levels, we studied a group of workers employed in a shoe factory where the jobs include use of glues and adhesives containing mainly n-hexane, and at low concentrations, toluen and MEK. 2. No differences were found in these parameters even in those workers with 3.3-fold higher mean levels of urine, 2,5-Hxdn and approximately twofold higher mean levels of urine hippuric acid as compared to controls. 3. We conclude that chronic co-exposure to n-hexane, toluen, and MEK at these levels is not associated with an impairment on either NK cell activity or serum IL-2 and gamma-IFN levels.

Proliferative response of peripheral blood lymphocytes and lymphocyte subpopulations in n-Hexane, toluen, and methyl ethyl ketone co-exposed workers.

To estimate the quantitative relation between chronic co-exposure to airborne n-hexane, toluen, methyl ethyl ketone (MEK) and various markers of immune function such as proliferative response of peripheral blood lymphocytes and lymphocyte subpopulations, a group of workers employed in a shoe factory were examined and compared with the unexposed controls. A significant increase was observed in the proliferative response of the peripheral lymphocytes to 2.5 and 5 µg PHA in the exposed group compared with that of the control group. There was no significant change in the percentage of circulating CD3(+), CD4(+), CD8(+), CD19(+), CD16(+) lymphocytes even in those workers with 3.3-fold higher mean levels of urine 2,5-hexanedione (2,5-Hxdn) and approximately twofold higher mean levels of urine hippuric acid (HA) as compared to controls. No difference was also observed between the mean granulocyte, monocyte, lymphocyte percentages of the groups, but a significant increase was observed in mean serum C3 level of the workers. Our results suggest that while lymphocyte subpopulations and leucocyte percentages are not affected, the proliferative response of the peripheral lymphocytes is stimulated after chronic co-exposure to n-hexane, toluen and MEK at the defined levels.

Comparison of the effects of Salmonella minnesota Re595 lipopolysaccharide, lipid A and monophosphoryl lipid A on nitric oxide, TNF-alpha, and IL-6 induction from RAW 264.7 macrophages.

Lipopolysaccharide (LPS) exhibits a wide variety of bioactivities. Although it was generally proposed that the lipid A component represented the active center responsible for most of the bioactivities of LPS, a variety of lipid A partial structures and analogues were reported to have different properties. Lipopolysaccharide of the Re595 mutant of Salmonella minnesota is lack of O and part of the core polysaccharide (2 keto-3-deoxyoctanate (KDO) left on lipid A). Re595 lipid A (LA) and Re595 monophosphoryl lipid A (MPLA) differ in structure from Re595 LPS by lacking KDO and KDO plus phosphoryl group respectively. Whether these lipid A-common Re595 LPS preparations differed in activities, we investigated their effects on nitric oxide (NO), TNF-alpha, IL-6, and IL-12 induction from murine macrophage cell line RAW 264.7. RAW 264.7 cells (2 x 10(5) cells ml(-1)) were stimulated with these LPS preparations at 1 microg ml(-1) for 48 h. Re595 LPS, Re595 LA and Re595 MPLA significantly induced NO, TNF-alpha and IL-6 production; NO, TNF-alpha and IL-6 inducing capacities were in the order of LPS = LA > MPLA, LPS = LA = MPLA, and LPS = LA > MPLA respectively. However, these preparations did not induce IL-12 production from RAW cells even when stimulated in combination with IFN-gamma (20 U ml(-1)). IFN-gamma itself also induced NO, TNF-alpha and IL-6 production from RAW 264.7 cells. When RAW 264.7 cells were stimulated with IFN-gamma plus any of these preparations, effects were additive and synergistic for NO and IL-6 responses respectively. But TNF-alpha responses of RAW cells against these preparations were almost equal when cultured alone or in combination with IFN-gamma. Pre-treatment of RAW cells either with LPS, LA or MPLA at low concentration (0.1 microg ml(-1)) for 60 min before pulsing with IFN-gamma (20 IU ml(-1)) plus LPS (1 microg ml(-1)) for an additional 48 h, significantly (P < 0.01) decreased NO response. Although to a lesser extent, TNF-alpha and IL-6 responses were also decreased. Complete inhibition of NO inducing effect of these LPS preparations was achieved with polymyxin B at 40 microg ml(-1). But the concentration of polymyxin B to get a significant (P < 0.05) inhibitory effect on LPS was four times higher than that for LA or MPLA. Unexpectedly, polymyxin B also inhibited INF-gamma-induced NO production from RAW cells in a dose-dependent fashion. These findings suggested that effect of LPS was dependent, at least in part, on both the LPS polysaccharide chain length and the hydrophilic portion of LPS. In addition, not only LPS but also LA and MPLA exert either enhancing or suppressive effects, depending on their concentrations and the timing of their addition with respect to co-stimulators.

Involvement of the Fas/Fas ligand pathway in activation-induced cell death of mycobacteria-reactive human gamma delta T cells: a mechanism for the loss of gamma delta T cells in patients with pulmonary tuberculosis.

Although the identity of T cells involved in the protection against Mycobacterium tuberculosis (Mtb) in humans remain unknown, patients with pulmonary tuberculosis (TB) have reduced numbers of Mtb-reactive, V gamma 9+/V delta 2+ T cells in their blood and lungs. Here we have determined whether this gamma deltaT loss is a consequence of Mtb Ag-mediated activation-induced cell death (AICD). Using a DNA polymerase-mediated dUTP nick translation labeling assay, 5% or less of freshly isolated CD4+ alpha beta or gamma delta T cells from normal healthy individuals and TB patients were apoptotic. However, during culture Mtb Ags induced apoptosis in a large proportion of V gamma 9+V delta 2+ peripheral blood T cells from healthy subjects (30-45%) and TB patients (55-68%); this was increased further in the presence of IL-2. By contrast, anti-CD3 did not induce any significant level of apoptosis in gamma delta T cells from healthy subjects or TB patients. Mtb Ag stimulation rapidly induced Fas and Fas ligand (FasL) expression by gamma delta T cells, and in the presence of metalloproteinase-inhibitors >70% of gamma delta T cells were FasL+. Blockade of Fas-FasL interactions reduced the level of Mtb-mediated gamma delta T cell apoptosis by 75 to 80%. Collectively, these findings demonstrate that Mtb-reactive gamma delta T cells are more susceptible to AICD and that the Fas-FasL pathways of apoptosis is involved. AICD of gamma delta T cells, therefore, provides an explanation for the loss of Mtb-reactive T cells during mycobacterial infection.

Alpha interferon treatment in myasthenia gravis: effects on natural killer cell activity.

The efficacy of recombinant interferon-alpha (rIFN alpha), on natural killer (NK) cell cytotoxic activity, CD3+, CD4+, CD8+, CD56+, HLA-DR+ lymphocyte counts, anti-acetylcholine receptor antibody (AChR Ab) levels, single fibre electromyography findings (SFEMG) and clinical course were evaluated in patients with myasthenia gravis (MG). During the IFN alpha treatment (3 mu, subcutaneous, 3 times a week), NK cell cytotoxicity and CD4+/8+ ratio increased, NK cell count remarkably decreased, and no significant clinical or SFEMG changes were observed. This preliminary open study in MG patients has demonstrated enhanced NK activity per unit NK cell after IFN alpha therapy. Although lymphocyte phenotypes and NK function approached normal levels during therapy, a higher dose of IFN alpha may be required for a significant clinical response. It has been also concluded that 6 months of IFN alpha therapy seems to be safe in MG, though in patients with malignancy, IFN alpha may cause increased autoimmunity, AChR positivity and MG.

Effects of some antigenic fractions of Leishmania major on nitric oxide production and IL-12 secretion by murine peritoneal macrophages.

Nitric oxide (NO) and IL-12 are important mediators of the immune response to Leishmania major. In this study, the effects of L. major promastigotes, crude antigenic fraction (CAF) and its subfractions on NO production and IL-12 secretion by BALB/c mice peritoneal macrophages is investigated. The subfractions of CAF, namely, fractions 1, 2 and 3, that were in the molecular weight range of 97.4-66, 66-45 and below 45 kDa, respectively, were separated by SDS-PAGE. NO production was determined by using Griess reagent and IL-12 was measured by ELISA. It was found that NO production was stimulated by promastigotes but not by CAF or its subfractions. IL-12 secretion was stimulated by promastigotes, CAF and fraction 1 while fractions 2 and 3 did not have any effect.

Effects of occupational lead and cadmium exposure on some immunoregulatory cytokine levels in man.

The levels of serum interleukin-1beta (IL-1beta), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-alpha) and gamma-interferon (gamma-IFN) were assessed in the workers who were occupationally exposed to lead and cadmium. The values were compared with the age-matched control group. Blood lead and cadmium levels were significantly raised. Our findings suggest that chronic lead and cadmium exposure in humans resulted in significant suppression of the serum IL-1beta level, but did not alter IL-2 and TNF-alpha levels. The gamma-IFN level was also reduced in lead workers. In contrast, a significant enhancement was observed in the cadmium-exposed group. We conclude from these results that lead and cadmium exposure at chronically high level may affect some cytokine levels in humans.

Simultaneous effects of lead and cadmium on NK cell activity and some phenotypic parameters.

Lead and cadmium are common environmental contaminants that alter the immune response. Natural killer cell (NK) cytotoxicity and phenotypic parameters are crucial immune responses, but little is known about the effects of these metals on these responses. In the present paper, we investigated the simultaneous effects of lead and cadmium on NK cell activity and CD4+, CD20+ cell percentages in the workers and compared the data with control and lead-exposed groups. We conclude that simultaneous exposure to lead and cadmium is not associated with an impairment of either NK cell function of CD4+ cell percentage. On the other hand, CD20+ cell percentage was found higher in lead+cadmium exposed group than controls.

Effects of high-level exposure to lead on NK cell activity and T-lymphocyte functions in workers.

1. NK and T cell functions were measured in peripheral blood lymphocytes of workers occupationally exposed to lead. 2. No differences were found in these functions even in those workers with higher levels of blood and urine lead and urinary delta-ALA than the currently accepted biological limit values as compared to controls. 3. We conclude that high chronic exposure to lead is not associated with an impairment or either T- or NK cell functions in man.

Defibrotide augments the anticandidial activity of NK cells.

1. The mechanism of action of Defibrotide, a fibrinolytic agent on Natural Killer (NK) cell cytotoxicity, was investigated through verapamil, TMB-8 cells and pertussis toxin. 2. Defibrotide increased the activity against Candida albicans cells (anticandidial activity), and it is determined that the calcium channels have a role in this effect. 3. Blockage of calcium channels reduced the anticandidial effect by 39.2%. Pertussis toxin led to a 10.7% inhibition, whereas the application of TMB-8 resulted in the stimulation of anticandidial activity. 4. It is concluded that defibrotide is a potent activator of NK cells.

Effects of crude antigenic fractions of Leishmania major on natural killer cell cytotoxicity, interferon-gamma and interleukin-4 secretion from peripheric blood lymphocytes of unexposed individuals.

The crude antigenic fraction (CAF) isolated from Leishmania major was fractionated into three subfractions by sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). The effects of CAF and its subfractions on NK cell cytotoxicity is investigated by chromium release assay. These subfractions designated as fractions 1, 2 and 3 correspond to 97.4-66 kD, 66-45 kD and 29 kD and below respectively. Although both CAF and its subfractions have inhibited the cytotoxicity of natural killer (NK) cells, the effects of fractions 2 and 3 were more pronounced. The effect of the fractions on the Interferon-gamma (IFN-gamma) and Interleukin-4 (IL-4) secretion by peripheric blood lymphocytes was also analyzed. It was found that CAF and fraction 1 induce IFN-gamma secretion while on the other hand IL-4 secretion was mostly suppressed by fraction 2. Therefore, further research is being executed which focuses on the effects of CAF, fractions 1 and 2 on macrophage effector functions.

Aqueous transforming growth factor-beta-I levels in rabbit eyes after excimer laser photoablation.

Transforming growth factor beta (TGF-beta) plays an important role in anterior segment wound healing, by controlling the cell proliferation and differentiation, angiogenesis, extracellular matrix composition and mediating the immunosuppressive properties of the aqueous humor. The present study was undertaken to clarify the possible changes of aqueous humor TGF-betaI levels after excimer laser photoablation. Twenty-eight New Zealand rabbits were divided into four groups of 7 rabbits each. Group 1 served as control, the central 7 mm of corneal epithelium was removed in groups 2, 3 and 4. We performed 50-microm corneal photoablation in group 3, and 100-microm ablation in group 4. After 48 h we measured the TGF-betaI levels of the aqueous humor by ELISA method. The mean TGF-betaI value of the aqueous humor was found to be 162.94+/-13.73 pg/ml in the control group. Mechanical deepithelialization did not change the TGF-betaI levels of the aqueous humor (p > 0.05). There was no significant difference between the 50-microm photoablated group and the controls (p > 0.05), but the TGF-betaI levels of the 100-microm photoablated group were found to be significantly higher than those of both the control group and 50-microm photoablated group (p < 0.05). Many factors and cytokines may induce corneal haze and myopic regression after excimer laser photoablation; our study demonstrated that TGF-betaI is one of these factors and there is a positive correlation between the depth of corneal photoablation and aqueous TGF-betaI concentrations.

Effect of a Staphylococcus epidermidis-extracted slime factor on human natural killer cell activity.

A slime factor produced by Staphylococcus epidermidis was a complex glycoconjugate extracted by the phenol extraction method. The potential stimulatory or inhibitory capacity of the phenol-extracted slime (PES) was tested on human natural killer cell cytotoxic activity. Various concentrations of the PES preparation were incubated with the effector cells 30 min before and during the assay period. The PES factor inhibited natural killer cell cytotoxic activity at a concentration of 250 micrograms/ml and at higher concentrations (p < 0.05). The inhibition of natural killer cell cytotoxic activity may probably be related to the complex composition of the slime substance.

Disease-specific changes in gammadelta T cell repertoire and function in patients with pulmonary tuberculosis.

Although gammadelta T cells are known to contain the highest frequency of mycobacteria-reactive cells in humans and numerous studies have suggested that they play an important role in the initial immune response to Mycobacterium tuberculosis (Mtb), very few studies have attempted to analyze these cells in patients with active pulmonary tuberculosis. The aim of the present study was therefore to evaluate the consequences of infection on the number and activity of mycobacteria-reactive gammadelta T cells. Three-color flow cytometric analysis of blood and bronchoalveolar lavage gammadelta T cells of patients diagnosed with active pulmonary tuberculosis showed that compared with normal healthy subjects and patients with the unrelated pulmonary granulomatous diseases sarcoidosis and berylliosis the size of the mycobacteria-reactive Vgamma9+/Vdelta2+ gammadelta T cell subset in both the blood and lung was dramatically reduced. In addition, the Vgamma9+/Vdelta2+ cells left intact in patients with tuberculosis were refractory to in vitro stimulation by Mtb Ags, which are potent stimuli for these cells in normal subjects. Our results demonstrate for the first time a strong correlation between the absence or loss of the major Vgamma9+/Vdelta2+ Mtb-reactive subset of gammadelta T cells and manifestations of disease, consistent with the hypothesis that these gammadelta T cells play a role in the protective immune response to Mtb infection.

Effect of allergen specific immunotherapy (IT) on natural killer cell activity (NK), IgE, IFN-gamma levels and clinical response in patients with allergic rhinitis and asthma.

Allergen specific immunotherapy (IT) has been widely used for many years as a specific treatment of allergic diseases. A variety of changes in immunological parameters have been described but it still remains uncertain as to which of them is responsible for the improvement of symptoms. The aim of our study was to evaluate the effect of IT on natural killer (NK) cell activity, IL-4, IFN-gamma, IgE levels and skin test reactivity in addition to clinical efficacy. Thirty-one patients with allergic rhinitis and asthma were selected according to positive history, skin prick tests to Dermatophagoides pteronyssinus (Der p) or grass pollens, presence of specific IgE antibodies in sera and clinical findings, and were submitted to one year of placebo-controlled IT. Total IgE, specific IgE, IL-4 and IFN-gamma levels were measured by using ELISA method. Standard chromium 51 release assay was used to measure NK cell cytotoxic activity against the human leukemic cell line, K562 target cells. Mean symptom and medication scores, skin test reactivity and histamine sensitivity were significantly decreased in the patients given IT at the end of the first year when compared with the placebo group. However, there was neither a significant reduction in total and specific IgE levels nor a significant increase in IFN-gamma levels at the first year of IT. IL-4 levels were only measured at the beginning of the study because of the very low levels. A decrease in NK cell activity was found in patients treated with grass pollen extracts after 12 months when compared with Der p and placebo group. No signs of major local or systemic side effects due to IT were seen in patients during the study. Although significant clinical efficacy of specific IT with standardized extracts has been demonstrated in allergic rhinitis and asthmatic patients at the end of the first year of IT, no significant changes in immunological parameters were observed. However we conclude that a decrease in NK cell cytotoxic activity during IT has to be taken into account in the follow-up of patients.

Anti-candidial activity of natural killer (NK) and lymphokine activated killer (LAK) lymphocytes in vitro.

The natural cytotoxic effects of peripheral blood lymphocytes (PBL) on Candida stellatoidea and several other Candida species were examined by a colony forming inhibition (CFI) assay. Peripheral blood mononuclear cells (PBMC), were incubated with C. stellatoidea yeast cells. After the incubation period the colony-forming ability of the yeast was significantly reduced. In similar experiments, six different Candida species (C. albicans, C. krusei, C. stellatoidea, C. tropicalis, C. pseudotropicalis, C. guillermondii) were used as target cells. There was no statistically significant difference in the anticandidial activities of PBL against the Candida species used. It was demonstrated that a fraction of lymphocytes, natural killer cells (NK), had the major natural anti-candidial activity by using anti-Leu M1 (CD 15) and anti-Leu 11b (CD 16) monoclonal antibodies (mAbs) plus complement (C'). It was observed that inhibition of colony-forming ability of C. stellatoidea was significantly (78-96%) reduced when anti-Leu 11b plus C' were used. In addition, the colony formation inhibition capacity of NK cells was increased by recombinant human interleukin-2 (rhIL-2) while anti-interferon-gamma (IFN-gamma) had no effect. Besides the fact that NK cells are among those responsible for natural immunity against Candida species, this colony-forming inhibition assay performed with C. stellatoidea yeast cells as target and monocyte-depleted PBMC as effector cells, is a simple method to assess NK cell activity.