Hanna Antushevich

Competition of Lactobacillus paracasei with Salmonella enterica for Adhesion to Caco-2 Cells

Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. In this study, we examined the ability of Lactobacillus paracasei to inhibit the adhesion of pathogenic Salmonella enterica to human colon adenocarcinoma Caco-2 cells. Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively. In adhesion experiments, bacteria were added to the cells for 2 or 4 hours. The number of attached bacteria was expressed as colony-forming units (CFUs), Caco-2 cells were counted in hematocytometer. Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to L. paracasei and almost 27-fold higher after 4 hours of contact. Two types of experiments were done: coincubation (both bacteria were added to Caco-2 cells simultaneously), and preincubation (L. paracasei was incubated with Caco-2 cells first, and then S. enterica was added). In coincubation experiment, the presence of L. paracasei decreased S. enterica adhesion by 4-fold and in preincubation experiment even 7-fold. Generally, Lactobacillus spent culture supernatants (SCSs) acted weaker as inhibitors of Salmonella adhesion in comparison to the whole L. paracasei culture in coincubation experiment. In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation.

Peripheral Injection of SB203580 Inhibits the Inflammatory-Dependent Synthesis of Proinflammatory Cytokines in the Hypothalamus

The study was designed to determine the effects of peripheral injection of SB203580 on the synthesis of interleukin- (IL-) 1β, IL-6, and tumor necrosis factor (TNF) α in the hypothalamus of ewes during prolonged inflammation. Inflammation was induced by the administration of lipopolysaccharide (LPS) (400 ng/kg) over 7 days. SB203580 is a selective ATP-competitive inhibitor of the p38 mitogen-activated protein kinase (MAPK), which is involved in the regulation of proinflammatory cytokines IL-1β, IL-6 and TNFα synthesis. Intravenous injection of SB203580 successfully inhibited (P < 0.01) synthesis of IL-1β and reduced (P < 0.01) the production of IL-6 in the hypothalamus. The p38 MAPK inhibitor decreased (P < 0.01) gene expression of TNFα but its effect was not observed at the level of TNFα protein synthesis. SB203580 also reduced (P < 0.01) LPS-stimulated IL-1 receptor type 1 gene expression. The conclusion that inhibition of p38 MAPK blocks LPS-induced proinflammatory cytokine synthesis seems to initiate new perspectives in the treatment of chronic inflammatory diseases also within the central nervous system. However, potential proinflammatory effects of SB203580 treatment suggest that all therapies using p38 MAPK inhibitors should be introduced very carefully with analysis of all expected and unexpected consequences of treatment.

The effect of rivastigmine on the LPS-induced suppression of GnRH/LH secretion during the follicular phase of the estrous cycle in ewes

This study was designed to determine the effect of a potent subcutaneously injected acetylcholinesterase inhibitor, rivastigmine (6mg/animal), on the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release during inflammation induced by an intravenous lipopolysaccharide (LPS) (400ng/kg) injection in ewes during the follicular phase of the estrous cycle. The results are expressed as the mean values from -2 to -0.5h before and +1 to +3h after treatment. Rivastigmine decreased the acetylcholinesterase concentration in the blood plasma from 176.9±9.5 to 99.3±15.1μmol/min/ml. Endotoxin suppressed LH (5.4±0.6ng/ml) and GnRH (4.6±0.4pg/ml) release; however, the rivastigmine injection restored the LH concentration (7.8±0.8ng/ml) to the control value (7.8±0.7ng/ml) and stimulated GnRH release (7.6±0.8pg/ml) compared to the control (5.9±0.4pg/ml). Immune stress decreased expression of the GnRH gene and its receptor (GnRH-R) in the median eminence as well as LHβ and GnRH-R in the pituitary. In the case of the GnRH and LHβ genes, the suppressive effect of inflammation was negated by rivastigmine. LPS stimulated cortisol and prolactin release (71.1±14.7 and 217.1±8.0ng/ml) compared to the control group (9.0±5.4 and 21.3±3.5ng/ml). Rivastigmine also showed a moderating effect on cortisol and prolactin secretion (43.1±13.1 and 169.7±29.5ng/ml). The present study shows that LPS-induced decreases in GnRH and LH can be reduced by the AChE inhibitor. This action of the AChE inhibitor could result from the suppression of pro-inflammatory cytokine release and the attenuation of the stress response. However, a direct stimulatory effect of ACh on GnRH/LH secretion should also be considered.

Central Interleukin-1β Suppresses the Nocturnal Secretion of Melatonin

In vertebrates, numerous processes occur in a rhythmic manner. The hormonal signal reliably reflecting the environmental light conditions is melatonin. Nocturnal melatonin secretion patterns could be disturbed in pathophysiological states, including inflammation, Alzheimers disease, and depression. All of these states share common elements in their aetiology, including the overexpression of interleukin- (IL-) 1β in the central nervous system. Therefore, the present study was designed to determine the effect of the central injection of exogenous IL-1β on melatonin release and on the expression of the enzymes of the melatonin biosynthetic pathway in the pineal gland of ewe. It was found that intracerebroventricular injections of IL-1β (50 µg/animal) suppressed (P < 0.05) nocturnal melatonin secretion in sheep regardless of the photoperiod. This may have resulted from decreased (P < 0.05) synthesis of the melatonin intermediate serotonin, which may have resulted, at least partially, from a reduced expression of tryptophan hydroxylase. IL-1β also inhibited (P < 0.05) the expression of the melatonin rhythm enzyme arylalkylamine-N-acetyltransferase and hydroxyindole-O-methyltransferase. However, the ability of IL-1β to affect the expression of these enzymes was dependent upon the photoperiod. Our study may shed new light on the role of central IL-1β in the aetiology of disruptions in melatonin secretion.

Involvement of prolactin in the meloxicam-dependent inflammatory response of the gonadotropic axis to prolonged lipopolysaccharide treatment in anoestrous ewes

An immune challenge can affect the reproductive process in females. Peripheral administration of bacterial endotoxin (lipopolysaccharide; LPS) decreases LH secretion and disrupts ovarian cyclicity. The aim of the present study was to determine the effects of a cyclo-oxygenase (COX)-2 inhibitor (meloxicam) on gonadotropin-releasing hormone (GnRH) and LH secretion in anoestrous ewes during systemic inflammation induced by LPS. LPS (400ngkg-1 per day) suppressed LH release. In three individuals, meloxicam (500μgkg-1, i.v.) abolished LPS-induced LH suppression. In another three ewes LH was ineffective. Similar changes were observed in hypothalamic GnRH expression. The effect of meloxicam depended on the circulating level of prolactin: meloxicam abolished inflammatory-dependent suppression of GnRH and LH secretion when plasma prolactin levels were similar to those in untreated animals, but was ineffective in those with elevated levels of prolactin. We conclude that COX-2 inhibitors minimise the negative effect of inflammation on the reproductive system but that this effect may be antagonised by prolactin.

Interleukin-1 β Modulates Melatonin Secretion in Ovine Pineal Gland: Ex Vivo Study

The study was designed to determine the effect of proinflammatory cytokine, interleukin- (IL-) 1β, on melatonin release and expression enzymes essential for this hormone synthesis: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) in ovine pineal gland, taking into account the immune status of animals before sacrificing. Ewes were injected by lipopolysaccharide (LPS; 400 ng/kg) or saline, two hours after sunset during short day period (December). Animals were euthanized three hours after the injection. Next, the pineal glands were collected and divided into four explants. The explants were incubated with (1) medium 199 (control explants), (2) norepinephrine (NE; 10 µM), (3) IL-1β (75 pg/mL), or (4) NE + IL-1β. It was found that IL-1β abolished (P < 0.05) NE-induced increase in melatonin release. Treatment with IL-1β also reduced (P < 0.05) expression of AA-NAT enzyme compared to NE-treated explants. There was no effect of NE or IL-1β treatment on gene expression of HIOMT; however, the pineal fragments isolated from LPS-treated animals were characterized by elevated (P < 0.05) expression of HIOMT mRNA and protein compared to the explants from saline-treated ewes. Our study proves that IL-1β suppresses melatonin secretion and its action seems to be targeted on the reduction of pineal AA-NAT protein expression.

Effect of apelin on mitosis, apoptosis and DNA repair enzyme OGG 1/2 expression in intestinal cell lines IEC-6 and Caco-2.

Apelin is a regulatory peptide, identified as an endogenous ligand of the Apelin receptor (APJ). Both the apelin and the APJ were detected in brain, lung, heart, mammary gland, kidney, placenta, adipose tissues and the gastrointestinal tract. Apelin is considered an important regulatory gut peptide with a potential physiological role in gastrointestinal cytoprotection, regulation of food intake and drinking behaviour. The aim of the present study was to assess the effect of the apelin on mitosis, apoptosis and the expression of DNA repair enzyme (OGG 1/2), and APJ receptor in intestinal cell lines: rat crypt (IEC-6) and human enterocyte model (Caco-2). The cell cultures were incubated with the apelin-12 (10-8 M) for 4, 6, 12, 24 and 48 h and the apoptosis (caspase 3), mitosis (Ki-67) and DNA repair enzyme (OGG1/2) markers were studied by Real-Time qRT-PCR and immunofluorescent methods. The results of Real-Time qRT-PCR and immunocytochemical analysis showed that the levels of mRNAs were inversely related to the expression level of corresponding proteins. Immunofluorescent studies revealed inhibitory effect of apelin-12 on apoptosis, mitosis and the expression of OGG1/2 in the intestinal crypt cell line IEC-6. However, in the enterocyte model Caco-2 cells apelin stimulated apoptosis and mitosis, and reduced OGG1/2 expression. These findings suggest that apelin may be involved in the control of epithelial cell turnover in the gastrointestinal tract.

Modifications of Western-type diet regarding protein, fat and sucrose levels as modulators of steroid metabolism and activity in liver

The aim of the study was to evaluate whether the modification of the Western-type diet (high-fat, high-sucrose diet rich in saturated fatty acids) considering macronutrients content would influence hepatic metabolism and activity of steroids. For 3 weeks Wistar rat were fed the Western-type diet (21% fat, 35% sucrose, 19% protein, lard) and its modifications regarding dietary protein (10 and 19%), fat (5 and 21%) and sucrose (0 and 35%) levels. The steroid 5α-reductase type 1 (Srd5a1) and androgen receptor (Ar) gene expression as well as testosterone (T) conversion towards 5α-reduced derivatives in liver were positively correlated with body weight gain. The Western-type diets with decreased protein content regardless of the sucrose level exerted the most negative effect on the antioxidant system decreasing catalase (Cat), sodium dismutase (Sod1) and glutathione peroxidase (Gpx1) gene expression as well as Cat and Gpx activity and total antioxidant status, simultaneously intensifying lipid peroxidation. The impaired antioxidant system was accompanied by decreased level of hepatic T metabolism towards estrogens: 17β-estradiol (E2) and estriol, and increased estrogen receptor type 1 (Esr1) gene expression. Liver Esr1 mRNA level was differently correlated with T (positively) and E2 (negatively) plasma levels. Whereas the fat reduction in Western-type diet restored the plasma proportion between T and E2. In conclusion it could be stated that Western-type diet modification relating to protein, sucrose and fat content can influence hepatic steroid metabolism and activity; however the estrogens and androgens metabolism in liver would be connected with impairment of liver function or catabolic activity, respectively.

Peripheral Inhibitor of AChE, Neostigmine, Prevents the Inflammatory Dependent Suppression of GnRH/LH Secretion during the Follicular Phase of the Estrous Cycle

The study was designed to test the hypothesis that the inhibition of acetylcholinesterase (AChE) activity at the periphery by Neostigmine (0.5 mg/animal) will be sufficient to prevent inflammatory dependent suppression of the gonadotropin-releasing hormone (GnRH)/luteinising hormone (LH) secretion in ewes in the follicular phase of the estrous cycle, and this effect will be comparable with the systemic AChE inhibitor, Donepezil (2.5 mg/animal). An immune/inflammatory challenge was induced by peripheral administration of lipopolysaccharide (LPS; 400 ng/kg). Peripheral treatment with Donepezil and Neostigmine prevented the LPS-induced decrease (P < 0.05) in LHβ gene expression in the anterior pituitary gland (AP) and in LH release. Moreover, Donepezil completely abolished (P < 0.05) the suppressory effect of inflammation on GnRH synthesis in the preoptic area, when pretreatment with Neostigmine reduced (P < 0.05) the decrease in GnRH content in this hypothalamic structure. Moreover, administration of both AChE inhibitors diminished (P < 0.05) the inhibitory effect of LPS treatment on the expression of GnRH receptor in the AP. Our study shows that inflammatory dependent changes in the GnRH/LH secretion may be eliminated or reduced by AChE inhibitors suppressing inflammatory reaction only at the periphery such as Neostigmine, without the need for interfering in the central nervous system.

Endotoxin-induced inflammation disturbs melatonin secretion in ewe

Objective The study examined the effect of intravenous administration of bacterial endotoxin—lipopolysaccharide (LPS) —on the nocturnal secretion of melatonin and on the expression of enzymes of the melatonin biosynthetic pathway in the pineal gland of ewes, taking into account two different photoperiodic conditions: short-night (SN; n = 12) and long-night (LN; n = 12). Methods In both experiments, animals (n = 12) were randomly divided into two groups: control (n = 6) and LPS-treated (n = 6) one. Two hours after sunset, animals received an injection of LPS or saline. Blood samples were collected starting one hour after sunset and continuing for 3 hours after the treatment. The ewes were euthanized 3 hours after LPS/saline treatment. The concentration of hormones in plasma was assayed by radioimmunoassay. In the pineal gland, the content of serotonin and its metabolite was determined by HPLC; whereas the expression of examined genes and protein was assayed using real-time polymerase chain reaction and Western Blot, respectively. Results Endotoxin administration lowered (p<0.05) levels of circulating melatonin in animals from LN photoperiod only during the first hour after treatment, while in ewes from SN photoperiod only in the third hour after the injection. Inflammation more substantially suppressed biosynthesis of melatonin in ewes from SN photoperiod, which were also characterised by lower (p<0.05) cortisol concentrations after LPS treatment compared with animals from LN photoperiod. In the pineal gland of ewes subjected to SN photoperiod, LPS reduced (p<0.05) serotonin content and the expression of melatonin biosynthetic pathway enzymes, such as tryptophan hydroxylase and arylalkylamine-N-acetyltransferase. Pineal activity may be disturbed by circulating LPS and proinflammatory cytokines because the expression of mRNAs encoding their corresponding receptors was determined in this gland. Conclusion The present study showed that peripheral inflammation reduces the secretion of melatonin, but this effect may be influenced by the photoperiod.