Haluk Ataoglu

Evaluation of in vitro Probiotic Potential of Pediococcus pentosaceus OZF Isolated from Human Breast Milk

This study was conducted to evaluate the probiotic properties of Pediococcus pentosaceus OZF isolated from human breast milk. The results obtained so far suggest that the strain is resistant to low pH, bile salt, pepsin and pancreatin, so it could survive while passing through the upper part of the gastrointestinal tract and reveal its potential probiotic action on host organism. The strain was non-pathogenic (γ-hemolytic), produced anti-Listerial bacteriocin, exhibited a strong autoaggregating phenotype (85.71%) and demonstrated 6.26 and 12.99% coaggregation with Salmonella enterica serotype Typhimurium SL 1344 and Escherichia coli LMG 3083 (ETEC), respectively. The degree of adhesion of Ped. pentosaceus OZF to the human Caco-2 cell line was investigated and when compared to the adhesion of pathogenic strains tested, it was shown to inhibit the growth of human enterotoxigenic E. coli LMG 3083 (ETEC) and of Salm. Typhimurium SL 1344. Ped. pentosaceus OZF seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and lipid factors on the bacteria and eukaryotic cell surface. The percentage of adhesion to n-hexadecane was 34% showing that the surface was rather hydrophilic. Higher affinity displayed by Ped. pentosaceus OZF for chloroform demonstrates the basic property of a cell, which may be due to the presence of carboxylic groups on the cell surface.

Effects of selective cyclooxygenase enzyme inhibitors on lipopolysaccharide-induced dual thermoregulatory changes in rats

The effects of selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors (valeryl salicylate and SC-58236, respectively) on Escherichia coli O111:B4 lipopolysaccharide (LPS)-induced dual thermoregulatory changes and serum tumor necrosis factor-alpha elevation were investigated in rats. LPS (50 microg/kg, intraperitoneal) produced an initial hypothermia that was then followed by fever. Serum tumor necrosis factor-alpha levels elevated at the initial phase of hypothermia. Valeryl salicylate injections (20, 40, and 80 mg/kg, subcutaneous [s.c.]) completely inhibited hypothermia without any effect on the elevated serum tumor necrosis factor-alpha levels and on the subsequent fever. On the other hand, SC-58236 injections (10, 20, and 40 mg/kg, s.c.) only partially abolished the hypothermia. SC-58236 had no effect on the initiation of fever, however completely inhibited the maintenance of fever. The serum tumor necrosis factor-alpha elevation was not reduced by SC-58236 treatment. The combination of valeryl salicylate and SC-58236 also failed to inhibit the initiation of fever. These findings suggest that cycloxygenase-1 may have a predominant role for the development of LPS-induced hypothermia, but cyclooxygenase-1 does not seem to be involved in the mediation of LPS-induced fever. Meanwhile, cyclooxgenase-2 may be critical for the late phase rather than the initiation of the fever response in rats.

Erythropoietin reduces lipopolysaccharide-induced cell Damage and midkine secretion in U937 human histiocytic lymphoma cells

IntroductionErythropoietin (EPO) is a haematopoietic stimulatory protein that is used to treat anaemia in patients on dialysis. In addition, EPO has been shown to have anti-inflammatory properties, which may be important as dialysis patients tend to exist within a chronic, low-grade inflammatory state, and tend to be more susceptible to infections. It has been suggested that EPO has direct immunomodulatory potency on monocytes/macrophages.MethodsIn this study, we aimed to clarify the effects of EPO during the inflammatory processes in human mononuclear phagocytic cells by monitoring the secretion of the following cytokines; midkine, tumour necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). For this purpose, U937 human histiocytic lymphoma cell lines were used. Time-dependent effects of varying doses of EPO (0.1 to 50 IU/ml) treatment during lipopolysaccharide (LPS)-mediated cytotoxicity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide test. LPS-stimulated midkine secretion was measured immunohistochemically and quantification of TNF-α, IL-6 and midkine secretion was achieved by ELISA.ResultsEPO treatment prevented the direct toxic effects of LPS on the U937 cells. TNF-α, IL-6 and midkine secretions were found to increase in the U937 cells in response to LPS treatment. Interleukin-6 response was varied in a doseand time-dependent manner.ConclusionTreatment with EPO significantly inhibits the LPS-induced secretion of midkine and TNF-α regardless of the dosage. The data presented here provide the first evidence to indicate that EPO treatment directly reverses some of the cytotoxic and secretory effects of LPS in mononuclear cells. Inhibition of midkine secretion might be responsible for the anti-inflammatory role of EPO.

Nimesulide and diclofenac inhibit lipopolysaccharide-induced hypothermia and tumour necrosis factor-alpha elevation in rats.

The effects of nimesulide and diclofenac on lipopolysaccharide (LPS)‐induced rectal temperature changes and serum tumour necrosis factor (TNF)‐α elevation were investigated in rats. LPS (Escherichia coli O111:B4; 50 µg/kg, intraperitoneally) produces a dual body temperature response, in which initial hypothermia precedes fever. Serum TNF‐α levels rise during the initial phase of the induced hypothermia. Nimesulide, a preferential inhibitor of cyclooxygenase‐2 (0.05, 0.5 or 1 mg/kg, subcutaneously) completely abolished the hypothermia, resulting in an acceleration of the fever phase. However, the peak and plateau phases of fever were not changed by nimesulide treatment. Nimesulide (0.5 mg/kg) partially prevented serum TNF‐α elevation. The non‐selective cyclooxygenase inhibitor diclofenac inhibited hypothermia at all doses tested (0.03, 0.3 or 3 mg/kg, subcutaneously) although fever was completely abolished at the 3 mg/kg dose only. Diclofenac also partially abolished the elevation in serum TNF‐α levels, but at the highest dose only (3 mg/kg). These data suggest that nimesulide and diclofenac can preferentially inhibit LPS‐induced hypothermia at doses that do not abolish fever in rats. Both these drugs also reduced elevated TNF‐α levels, a fact which may, at least partly, explain the antihypothermic effect of nimesulide.

Experimental otitis media induced by coagulase negative staphylococcus and its L-forms☆

In a previous study, we found 15% L-forms of bacteria (predominately coagulase negative staphylococci (CNS)) in ears which gave negative cultures by conventional methods. In this study, we used an animal model to test whether CNS and its L-forms can be pathogenic and whether L-forms have a crucial role in the tendency to secretory otitis media (SOM). We inoculated the tympanic bullas of guinea pigs, in 2 groups, with CNS and its L-forms (revertant forms). We observed that both CNS and its L-forms had the capability of causing infection. However, it was milder for the L-forms than CNS. We clearly noticed that on day 30 60% of the ears inoculated with L-forms had effusion and/or retraction of the tympanic membrane. These ears were histopathologically characterized by hypertrophied pseudostratified epithelium or stratified squamous epithelial metaplasia. The ears inoculated with the original form of CNS had only 16.66% effusion. On day 60 we observed similar findings. Thus, it might be proposed that L-forms could be responsible for chronic irritation to middle ear mucosa leading to SOM.

Effect of monophosphoryl lipid A on antibody response to diphtheria toxin and its subunits

Monophosphoryl lipid A (MPL) was evaluated for its ability to enhance the antibody response to diphtheria toxin and its fragment A and fragment B subunits. BALB/c mice were immunized subcutaneously with 1 Lf of diphtheria toxoid in the presence of 25 μg of MPL on days 0 and 14. Two weeks after the second immunization, sera were obtained from the mice and analysed for antibody response to diphtheria toxin and its subunits. A new ELISA method, developed in our laboratory, was used to measure antibody levels against the toxin, fragment A, and fragment B. It was observed that MPL significantly enhanced antibody responses to diphtheria toxin and its subunits. However, there was no statistical difference between anti‐A and anti‐B responses. The results indicated that MPL seems to be a potential candidate as an adjuvant for future diphtheria vaccine formulation.

Preliminary report on L-forms: possible role in the infectious origin of secretory otitis media.

Infection and inflammation of the middle ear cleft are important factors in the pathogenesis of secretory otitis media. Although high percentages of negative cultures are confronted in many studies, strong evidence pointing to the infectious nature of this disease could not be overlooked. Many authors agree about the failure of conventional culture methods in identifying the responsible pathogen or pathogens. Besides, some agents, such as some kinds of antibiotics, lysozyme, and perhaps some undetected materials, are capable of changing bacterial behavior and consequently the clinical course. Effusions taken from 40 ears with secretory otitis media were cultured by means of conventional brain-heart infusion broth and special hypertonic thioglycollate broth. Strikingly, bacterial L-forms were detected in 6 specimens in thioglycollate broth, with no growth in the conventional broth. We concluded that these atypical forms of bacteria, the L-forms, may play an important role in the bacteriologic aspect of secretory otitis media.

The effect of K-ATP channel blockage during erythropoietin treatment in renal ischemia-reperfusion injury.

ATP dependent K channels (K-ATP) take part in the Erythropoietin (EPO) induced cardioprotection but these channel activations have role in cytoprotective role of EPO in the renal ischemia reperfusion (IR) damage is still unknown. For this purpose rats were pretreated with EPO (500 IU/kg) and/or K-ATP channel blocker glibenclamide (40mM/kg) i.p. before bilateral renal IR damage. Renal tissues were used for histological examination and measurement of caspase-3 and TNF-α levels. Renal functions were evaluated by glomerular filtration rate (GFR) fractional excretion of sodium (FENa) and potassium (FEK). Renal TNF-α and caspase-3 levels were decreased in both glibenclamide and EPO-treated IR rats compared to untreated rats. The protection afforded by the pretreatment with EPO alone was greater than that of administering glibenclamide alone. Application of glibenclamide at the same time partly abolished the cytoprotective effect of EPO treatment. K-ATP mediated cytoprotection is not the main mechanism of protective effect of EPO.

Polysaccharide mannan components of Candida albicans and Saccharomyces cerevisiae cell wall produce fever by intracerebroventricular injection in rats.

The cell wall mannan components of Candida albicans and Saccharomyces cerevisiae produced hyperthermic responses when injected intracerebroventricularly at doses of 10 microg in rats. Indomethacin treatment (5 mg/kg subcutaneously) completely abolished these responses. Serum interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta levels showed an upward trend during the initial phase of the hyperthermic response induced by S. cerevisiae mannan. Meanwhile, serum levels of these proinflammatory cytokines did not increase at all at the initial phase of C. albicans mannan-induced hyperthermia. Histopathological examination of the brain tissue samples revealed no specific change throughout the parenchyma of rats given either mannan. These results indicate that the polysaccharide mannan components of yeasts, regardless of the pathogenicity, produce a pyrogenic response by a direct injection into the brain in rats. This response is not accompanied by proinflammatory cytokine induction in the periphery.

Inhibition of endothelium-dependent relaxation by Candida albicans.

This study examines the effects of Candida albicans on acethylcholine-induced, endothelium-dependent relaxation of thoracic aorta of rabbits, precontracted by phenylephrine (10(-7) M). Isolated vessel rings were incubated with C. albicans, Saccharomyces cerevisiae, or their mannans, and endothelium-dependent relaxation was measured by the induction of acethylcholine. Endothelium-dependent relaxation remained unaffected after 3 hours by either C. albicans or S. cerevisiae, or their mannans. After 24 hours, however, incubation with C. albicans had completely abolished relaxation, whereas relaxation was decreased by mannan of C. albicans and continued unaffected by S. cerevisiae. In contrast, no change was registered with a 24 hours incubation of C. Albicans in a sodium nitroprusside-induced, endothelium-independent, vascular smooth muscle relaxation. Microscopical investigation of the morphological structure of vessel walls revealed penetration of C. albicans on the intimal surface after 3 hours incubation and infiltration of the yeast through the vessel wall after 24 hours. No changes in vessel morphology occurred after 3 or 24 hours with S. cerevisiae or the mannan of C. albicans. These results show the ability of C. albicans to inhibit endothelium-dependent, but not endothelium-independent, relaxation of vascular smooth muscle and may have important implications for functional damage to endothelial cells and the regulation of vessel tone and blood flow.