Methylprednisolone concentrations in the vitreous and the serum after pulse therapy.

PURPOSE: Intravenous (i.v.) pulse of corticosteroids has been used to treat severe eye inflammation from different origins. Whether such large doses result in vitreous levels that differ either in magnitude or duration from more conventional corticotherapy remain unsolved issues. The authors therefore determined levels of methylprednisolone hemisuccinate and methylprednisolone in the vitreous and serum of patients at different times after a single i.v. perfusion of methylprednisolone hemisuccinate. METHODS: Fifty patients scheduled for a first vitrectomy received an i.v. injection of 500 mg hemisuccinate methylprednisolone at different times before surgery (from 15-24 hours). Patients were divided into two groups: those with (n = 21) and without (n = 29) retinal detachment (RD). Pure vitreous samples were analyzed by high-pressure liquid chromatography. RESULTS: Both the ester and the nonester methylprednisolone forms were sampled in the vitreous, showing a slower rate of hydrolysis compared to the serum. On average, the highest concentration of total methylprednisolone in the vitreous was found at 2.5 hours and rapidly decreased for the group of patients with RD. In the group of patients without RD, the highest concentration was reached at 6 hours and then slowly decreased. The antiinflammatory potency in the nondetached retina eyes was approximately 500 times more than in the physiologic vitreous, but despite the route of administration (i.v. or oral), only 1/10 of the corticosteroid serum concentration was measured in the vitreous. CONCLUSION: High concentration of methylprednisolone is achieved by i.v. pulse therapy without changing the kinetic of entry in the vitreous of nondetached retina eyes when compared to conventional oral corticotherapy. Hydrolysis occurs in the vitreous resulting in high rate of active form. Pulse therapy could be considered in cases of severe ocular inflammation involving the posterior segment of the eye.

Establishment of an In Vitro Photoallergy Test Using NCTC2544 Cells and IL-18 Production

Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of eratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously demonstrated the possibility to use the human keratinocyte cell line NCTC2455 and the production of interleukin-18 (IL-18) to screen low molecular weight sensitizers. The purpose of this work was to explore the possibility to use the NCTC2544 assay to identify photoallergens and discriminate from phototoxic chemicals. First, we identified suitable condition of UV-irradiation (3.5 J/cm2) by investigating the effect of UVAirradiation on intracellular IL-18 on untreated or chloropromazine (a representative phototoxic compound)- treated NCTC2544 cells. Then, the effect of UVA-irradiation over NCTC2544 cells treated with increasing concentrations of 15 compounds including photoallergens (benzophenone, 4-ter-butyl-4-methoxydibenzoylmethane, 2-ethylexyl-p-methoxycinnamate, ketoprofen, 6-methylcumarin); photoirritant and photoallergen (4-aminobenzoic acid, chlorpromazine, promethazine); photoirritants (acridine, ibuprofen, 8-methoxypsoralen, retinoic acid); and negative compounds (lactic acid, SDS and p-phenilendiamine) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay or LDH leakage, while ELISA was used to measure the production of IL-18. At the maximal concentration assayed with non-cytotoxic effects (CV80 under irradiated condition), all tested photoallergens induced a significant and a dose-dependent increase of intracellular IL-18 following UVA irratiation, whereas photoirritants failed. We suggest that this system may be useful for the in vitro evaluation of the photoallergic potential of chemicals.

Postmortem serum protein growth arrest-specific 6 levels in sepsis-related deaths.

Growth arrest-specific 6 (Gas6) is widely expressed in leukocytes, platelets, endothelial cells, and monocytes. It regulates various processes including granulocyte adhesion to the endothelium, cell migration, thrombus stabilization, and cytokine release. In humans, increased plasma Gas6 levels have been described in patients with sepsis and septic shock. In this study, Gas6 concentrations were measured in postmortem serum from femoral blood in a series of sepsis-related fatalities and control cases. The aims were twofold: first, to determine whether Gas6 can be reliably determined in postmortem serum; and second, to assess its diagnostic potential in identifying sepsis-related deaths. Two study groups were prospectively formed, a sepsis-related fatalities group (24 cases) and a control group (24 cases) including cases of deep vein thrombosis and fatal pulmonary embolism, cases of systemic inflammatory response syndrome in severe trauma, cases of end-stage renal failure, and cases of hanging (non-septic, non-SIRS, non-end stage renal failure cases). The preliminary results of this study seem to indicate that Gas6 can be effectively measured in postmortem serum. However, Gas6 levels in sepsis-related fatalities do not appear to be clearly distinguishable from concentrations in pulmonary embolism, severe trauma, and end-stage renal failure cases. These findings tend to support previous reports that indicated that Gas6 behaves as an acute phase reactant and can be considered a general marker of inflammation rather than a specific biomarker of sepsis.

Serum Vitamin D Concentrations Are Not Associated with Insulin Resistance in Swiss Adults.

BACKGROUND: Low vitamin D status has been associated with an increased risk of developing type 2 diabetes and insulin resistance (IR), although this has been recently questioned. OBJECTIVE: We examined the association between serum vitamin D metabolites and incident IR. METHODS: This was a prospective, population-based study derived from the CoLaus (Cohorte Lausannoise) study including 3856 participants (aged 51.2 ± 10.4 y; 2217 women) free from diabetes or IR at baseline. IR was defined as a homeostasis model assessment (HOMA) index >2.6. Fasting plasma insulin and glucose were measured at baseline and at follow-up to calculate the HOMA index. The association of vitamin D metabolites with incident IR was analyzed by logistic regression, and the results were expressed for each independent variable as ORs and 95% CIs. RESULTS: During the 5.5-y follow-up, 649 (16.9%) incident cases of IR were identified. Participants who developed IR had lower baseline serum concentrations of 25-hydroxyvitamin D3 [25(OH)D3 (25-hydroxycholecalciferol); 45.9 ± 22.8 vs. 49.9 ± 22.6 nmol/L; P < 0.001], total 25(OH)D3 (25(OH)D3 + epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3]; 49.1 ± 24.3 vs. 53.3 ± 24.1 nmol/L; P < 0.001), and 3-epi-25(OH)D3 (4.2 ± 2.9 vs. 4.3 ± 2.5 nmol/L; P = 0.01) but a higher 3-epi- to total 25(OH)D3 ratio (0.09 ± 0.05 vs. 0.08 ± 0.04; P = 0.007). Multivariable analysis adjusting for month of sampling, age, and sex showed an inverse association between 25(OH)D3 and the likelihood of developing IR [ORs (95% CIs): 0.86 (0.68, 1.09), 0.60 (0.46, 0.78), and 0.57 (0.43, 0.75) for the second, third, and fourth quartiles compared with the first 25(OH)D3 quartile; P-trend < 0.001]. Similar associations were found between total 25(OH)D3 and incident IR. There was no significant association between 3-epi-25(OH)D3 and IR, yet a positive association was observed between the 3-epi- to total 25(OH)D3 ratio and incident IR. Further adjustment for body mass index, sedentary status, and smoking attenuated the association between 25(OH)D3, total 25(OH)D3, and the 3-epi- to total 25(OH)D3 ratio and the likelihood of developing IR. CONCLUSION: In the CoLaus study in healthy adults, the risk of incident IR is not associated with serum concentrations of 25(OH)D3 and total 25(OH)D3.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

The mediterranean diet pattern and its main components are associated with lower plasma concentrations of tumor necrosis factor receptor 60 in patients at high risk for cardiovascular disease

Adherence to aMediterranean diet (MD) is associated with a reduced risk of coronary heart disease. However, themolecular mechanisms involved are not fully understood. The aim of this studywas to compare the effects of 2MD with those of a lowfat- diet (LFD) on circulating inflammatory biomarkers related to atherogenesis. A total of 516 participants included in the PreventionwithMediterraneanDiet Studywere randomized into 3 intervention groups [MD supplementedwith virgin olive oil (MD-VOO); MD supplemented with mixed nuts (MD-Nuts); and LFD]. At baseline and after 1 y, participants completed FFQ and adherence to MD questionnaires, and plasma concentrations of inflammatory markers including intercellular adhesion molecule-1(ICAM-1), IL-6, and 2 TNF receptors (TNFR60 and TNFR80) were measured by ELISA. At 1 y, the MD groups had lower plasma concentrations of IL-6, TNFR60, and TNFR80 (P , 0.05), whereas ICAM-1, TNFR60, and TNFR80 concentrations increased in the LFD group (P , 0.002). Due to between-group differences, participants in the 2 MD groups had lower plasma concentrations of ICAM-1, IL-6, TNFR60, and TNFR80 compared to those in the LFD group (P # 0.028). When participants were categorized in tertiles of 1-y changes in the consumption of selected foods, those in the highest tertile of virgin olive oil (VOO) and vegetable consumption had a lower plasma TNFR60 concentration compared with those in tertile 1 (P,0.02).Moreover, the only changes in consumption thatwere associated with 1-y changes in the geometricmean TNFR60 concentrations were those of VOO and vegetables (P = 0.01). This study suggests that a MD reduces TNFR concentrations in patients at high cardiovascular risk.

Oxidative stress in gastric mucosa of asymptomatic humans infected with Helicobacter pylori: effect of bacterial eradication.

BACKGROUND: Inducible nitric oxide synthase (iNOS) and interleukin 8 (IL-8) are positive in approximately 50% of Helicobacter pylori-related diseases but it is not clear whether oxidative stress is also present in H. pylori asymptomatic humans. Our aim was to study the expression of iNOS, superoxide dismutase, catalase and IL-8 production in H. pylori-infected asymptomatic humans, and to investigate the effect of eradication of H. pylori. MATERIALS AND METHODS: Biopsies of corpus and antrum of asymptomatic H. pylori positive and negative humans served for determination of the gastritis score and H. pylori status; iNOS was measured by reverse transcriptase polymerase chain reaction and immunohistochemistry and superoxide dismutase and catalase by immunohistochemistry. IL-8 in biopsies was assessed by enzyme-linked immunosorbent assay. RESULTS: Immunostaining of iNOS, catalase and superoxide dismutase was significantly associated with H. pylori infection and was localized to inflammatory cells. IL-8 concentrations were greater in the H. pylori positive than H. pylori negative group and decreased after bacterial eradication. A decrease in staining for iNOS and catalase was observed after H. pylori eradication. CONCLUSIONS: INOS and antioxidant enzymes are present in gastric biopsies of asymptomatic H. pylori positive humans. Eradication caused a significant decrease in staining for iNOS and catalase. These results indicate that oxidative stress occurs in asymptomatic patients and can be modulated by H. pylori eradication.

Bone Morphogenetic Protein-6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro

This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.

Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822)

Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L) for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphorylase, alkaline phosphatase (ALP), acid phosphatase (AcP) were assessed in different tissues (liver, muscle and gills). Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05) time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.

Behavior of Bradyrhizobium japonicum strains under different herbicide concentrations

The Bradyrhizobium japonicun strains SEMIA 5073, SEMIA 5074, SEMIA 5079 and SEMIA 5080 were grown in vitro using Vincent medium combined with different rates of the herbicides imazaquin (0, 0.04, 0.12, 0.24, 0.36 mg a.i. g-1), clomazone (0, 0.4, 0.8, 1.6 and 3.2 mg a.i. g-1) and sulfentrazone (0, 0.2, 0.4, 0.8 and 1.6 mg a.i. g-1) to evaluate the strains tolerance to herbicides. The three herbicides drastically inhibited all the rhizobium strains tested, showing a significant decrease of the CFU number as a function of herbicide rates. The rhizobium strains presented a differentiated tolerance to the herbicides. The herbicide rates that reduced 50% (I50) of the growth or survival of the rhizobium strains were below the recommended sprayed rates for weed control in the soybean crop, for all the three herbicides studied; however, sulfentrazone I50 was smaller than imazaquin and clomazone I50.

Bradykinin enhances membrane electrical activity of pancreatic beta cells in the presence of low glucose concentrations

In most of cells bradykinin (BK) induces intracellular calcium mobilization. In pancreatic beta cells intracellular calcium is a major signal for insulin secretion. In these cells, glucose metabolism yields intracellular ATP which blocks membrane potassium channels. The membrane depolarizes, voltage-dependent Ca2+ channels are activated and the intracellular calcium load allows insulin secretion. Repolarization occurs due to activation of the Ca2+-dependent K+ channel. The insulin secretion depends on the integrity of this oscillatory process (bursts). Therefore, we decided to determine whether BK (100 nM) induces bursts in the presence of a non-stimulatory glucose concentration (5.6 mM). During continuous membrane voltage recording, our results showed that bursts were obtained with 11 mM glucose, blocked with 5.6 mM glucose and recovered with 5.6 mM glucose plus 100 nM BK. Thus, the stimulatory process obtained in the presence of BK and of a non-stimulatory concentration of glucose in the present study suggests that BK may facilitate the action of glucose on beta cell secretion.

Induction of interleukin-10 by HIV antigens in peripheral mononuclear cells of health care workers after occupational exposure to HIV-1-positive blood

Evaluation of HIV-induced IL-2 production by peripheral blood mononuclear cells (PBMC) and HIV-specific T helper and cytotoxic T lymphocyte (CTL) responses in health care workers (HCW) occupationally exposed to HIV reveals a high rate of response to HIV among non-seroconverters. IL-10 is also known to interfere with HIV infection in vitro. To evaluate the induction of IL-10 by HIV antigens in HCW occupationally exposed to HIV, 18 HCW with percutaneous injury were enrolled in this study, 9 of them exposed to HIV-contaminated blood, and 9 exposed to HIV-negative blood. PBMC were incubated on plates coated with HIV-1 antigens, and IL-10 was measured in supernatants by ELISA. Five of nine HCW exposed to HIV-contaminated blood presented HIV-induced IL-10. Two of nine HCW exposed to HIV-negative source patients also had detectable levels of HIV-induced IL-10, one of them in the sample obtained on the day of accidental exposure. There was a relationship between the type of device involved in injury and IL-10 production. Individuals exposed to hollow needles or scalpels presented HIV-induced IL-10, whereas those exposed to solid needles and to digital puncture did not, suggesting a relationship between infectious load and IL-10. Although occupational exposure to HIV leads to a low rate of seroconversion, these individuals can develop an antigen-specific immune response characterized in our study by induction of IL-10 in PBMC in vitro.

High concentrations of KCl release noradrenaline from noradrenergic neurons in the rat anococcygeus muscle

The aim of the present study was to investigate the effects of high concentrations of KCl in releasing noradrenaline from sympathetic nerves and its actions on postsynaptic alpha-adrenoceptors. We measured the isotonic contractions induced by KCl in the isolated rat anococcygeus muscle under different experimental conditions. The contractile responses induced by KCl were inhibited by alpha-adrenoceptor antagonists in 2.5 mM Ca2+ solution. Prazosin reduced the maximum effect from 100 to 53.9 ± 10.2% (P<0.05) while the pD2 values were not changed. The contractile responses induced by KCl were abolished by prazosin in Ca2+-free solution (P<0.05). Treatment of the rats with reserpine reduced the maximum effect induced by KCl as compared to the contractile responses induced by acetylcholine from 339.5 ± 157.8 to 167.3 ± 65.5% (P<0.05), and increased the pD2 from 1.57 ± 0.01 to 1.65 ± 0.006 (P<0.05), but abolished the inhibitory effect of prazosin (P<0.05). In contrast, L-NAME increased the contractile responses induced by 120 mM KCl by 6.2 ± 2.3% (P<0.05), indicating that KCl could stimulate the neurons that release nitric oxide, an inhibitory component of the contractile response induced by KCl. Our results indicate that high concentrations of KCl induce the release of noradrenaline from noradrenergic neurons, which interacts with alpha1-adrenoceptors in smooth muscle cells, producing a contractile response in 2.5 mM Ca2+ (100%) and in Ca2+-free solution, part of which is due to a direct effect of KCl on the rat anococcygeus muscle.