Membrane Vesicles for Nanoencapsulated Sulforaphane Increased Their Anti-Inflammatory Role on an In Vitro Human Macrophage Model.

At present, there is a growing interest in finding new non-toxic anti-inflammatory drugs to treat inflammation, which is a key pathology in the development of several diseases with considerable mortality. Sulforaphane (SFN), a bioactive compound derived from Brassica plants, was shown to be promising due to its anti-inflammatory properties and great potential, though its actual clinical use is limited due to its poor stability and bioavailability. In this sense, the use of nanocarriers could solve stability-related problems. In the current study, sulforaphane loaded into membrane vesicles derived from broccoli plants was studied to determine the anti-inflammatory potential in a human-macrophage-like in vitro cell model under both normal and inflammatory conditions. On the one hand, the release of SFN from membrane vesicles was modeled in vitro, and two release phases were stabilized, one faster and the other slower due to the interaction between SFN and membrane proteins, such as aquaporins. Furthermore, the anti-inflammatory action of sulforaphane-loaded membrane vesicles was demonstrated, as a decrease in interleukins crucial for the development of inflammation, such as TNF-α, IL-1ß and IL-6, was observed. Furthermore, these results also showed that membrane vesicles by themselves had anti-inflammatory properties, opening the possibility of new lines of research to study these vesicles, not only as carriers but also as active compounds.

Anti-Leukemic Activity of Brassica-Derived Bioactive Compounds in HL-60 Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a cancer of the myeloid blood cells mainly treated with chemotherapy for cancer remission, but this non-selective treatment also induces numerous side effects. Investigations with bioactive compounds from plant-derived foods against cancer have increased in the last years because there is an urgent need to search for new anti-leukemic agents possessing higher efficacy and selectivity for AML cells and fewer negative side effects. In this study, we analyzed the anti-leukemic activity of several phytochemicals that are representative of the major classes of compounds present in cruciferous foods (glucosinolates, isothiocyanates, hydroxycinnamic acids, flavonols, and anthocyanins) in the human acute myeloid leukemia cell line HL-60. Our results revealed that among the different Brassica-derived compounds assayed, sulforaphane (SFN) (an aliphatic isothiocyanate) showed the most potent anti-leukemic activity with an IC50 value of 6 µM in dose-response MTT assays after 48 h of treatment. On the other hand, chlorogenic acid (a hydroxycinnamic acid) and cyanidin-3-glucoside (an anthocyanin) also displayed anti-leukemic potential, with IC50 values of 7 µM and 17 µM after 48 h of incubation, respectively. Importantly, these compounds did not show significant cell toxicity in macrophages-like differentiated cells at 10 and 25 µM, indicating that their cytotoxic effects were specific to AML cancer cells. Finally, we found that these three compounds were able to induce the NRF2/KEAP1 signaling pathway in a dose-dependent manner, highlighting SFN as the most potent NRF2 activator. Overall, the present evidence shed light on the potential for using foods and ingredients rich in anticancer bioactive phytochemicals from Brassica spp.

Potential of Sulforaphane and Broccoli Membrane Vesicles as Regulators of M1/M2 Human Macrophage Activity.

Macrophages have emerged as important therapeutic targets in many human diseases. The aim of this study was to analyze the effect of broccoli membrane vesicles and sulphoraphane (SFN), either free or encapsulated, on the activity of human monocyte-derived M1 and M2 macrophage primary culture. Our results show that exposure for 24 h to SFN 25 µM, free and encapsulated, induced a potent reduction on the activity of human M1 and M2 macrophages, downregulating proinflammatory and anti-inflammatory cytokines and phagocytic capability on C. albicans. The broccoli membrane vesicles do not represent inert nanocarriers, as they have low amounts of bioactive compounds, being able to modulate the cytokine production, depending on the inflammatory state of the cells. They could induce opposite effects to that of higher doses of SFN, reflecting its hormetic effect. These data reinforce the potential use of broccoli compounds as therapeutic agents not only for inflammatory diseases, but they also open new clinical possibilities for applications in other diseases related to immunodeficiency, autoimmunity, or in cancer therapy. Considering the variability of their biological effects in different scenarios, a proper therapeutic strategy with Brassica bioactive compounds should be designed for each pathology.

Crohn's Disease, Host-Microbiota Interactions, and Immunonutrition: Dietary Strategies Targeting Gut Microbiome as Novel Therapeutic Approaches.

Crohn's disease (CD) is a complex, disabling, idiopathic, progressive, and destructive disorder with an unknown etiology. The pathogenesis of CD is multifactorial and involves the interplay between host genetics, and environmental factors, resulting in an aberrant immune response leading to intestinal inflammation. Due to the high morbidity and long-term management of CD, the development of non-pharmacological approaches to mitigate the severity of CD has recently attracted great attention. The gut microbiota has been recognized as an important player in the development of CD, and general alterations in the gut microbiome have been established in these patients. Thus, the gut microbiome has emerged as a pre-eminent target for potential new treatments in CD. Epidemiological and interventional studies have demonstrated that diet could impact the gut microbiome in terms of composition and functionality. However, how specific dietary strategies could modulate the gut microbiota composition and how this would impact host-microbe interactions in CD are still unclear. In this review, we discuss the most recent knowledge on host-microbe interactions and their involvement in CD pathogenesis and severity, and we highlight the most up-to-date information on gut microbiota modulation through nutritional strategies, focusing on the role of the microbiota in gut inflammation and immunity.

The Role of Peritoneal Macrophages in Endometriosis.

Endometriosis is an estrogen-dependent gynecological disorder, defined as the growth of endometrial stromal cells and glands at extrauterine sites. Endometriotic lesions are more frequently located into the abdominal cavity, although they can also be implanted in distant places. Among its etiological factors, the presence of immune dysregulation occupies a prominent place, pointing out the beneficial and harmful outcomes of macrophages in the pathogenesis of this disease. Macrophages are tissue-resident cells that connect innate and adaptive immunity, playing a key role in maintaining local homeostasis in healthy conditions and being critical in the development and sustainment of many inflammatory diseases. Macrophages accumulate in the peritoneal cavity of women with endometriosis, but their ability to clear migrated endometrial fragments seems to be inefficient. Hence, the characteristics of the peritoneal immune system in endometriosis must be further studied to facilitate the search for new diagnostic and therapeutic tools. In this review, we summarize recent relevant advances obtained in both mouse, as the main animal model used to study endometriosis, and human, focusing on peritoneal macrophages obtained from endometriotic patients and healthy donors, under the perspective of its future clinical translation to the role that these cells play on this pathology.

Isolation of functional mature peritoneal macrophages from healthy humans.

Macrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.

Brassica Bioactives Could Ameliorate the Chronic Inflammatory Condition of Endometriosis.

Endometriosis is a chronic, inflammatory, hormone-dependent disease characterized by histological lesions produced by the presence of endometrial tissue outside the uterine cavity. Despite the fact that an estimated 176 million women are affected worldwide by this gynecological disorder, risk factors that cause endometriosis have not been properly defined and current treatments are not efficient. Although the interaction between diet and human health has been the focus of many studies, little information about the correlation of foods and their bioactive derivates with endometriosis is available. In this framework, Brassica crops have emerged as potential candidates for ameliorating the chronic inflammatory condition of endometriosis, due to their abundant content of health-promoting compounds such as glucosinolates and their hydrolysis products, isothiocyanates. Several inflammation-related signaling pathways have been included among the known targets of isothiocyanates, but those involving aquaporin water channels have an important role in endometriosis. Therefore, the aim of this review is to highlight the promising effects of the phytochemicals present in Brassica spp. as major candidates for inclusion in a dietary approach aiming to improve the inflammatory condition of women affected with endometriosis. This review points out the potential roles of glucosinolates and isothiocyanates from Brassicas as anti-inflammatory compounds, which might contribute to a reduction in endometriosis symptoms. In view of these promising results, further investigation of the effect of glucosinolates on chronic inflammatory diseases, either as diet coadjuvants or as therapeutic molecules, should be performed. In addition, we highlight the involvement of aquaporins in the maintenance of immune homeostasis. In brief, glucosinolates and the modulation of cellular water by aquaporins could shed light on new approaches to improve the quality of life for women with endometriosis.

Therapeutic potential of pteridine derivatives: A comprehensive review.

Pteridines are aromatic compounds formed by fused pyrazine and pyrimidine rings. Many living organisms synthesize pteridines, where they act as pigments, enzymatic cofactors, or immune system activation molecules. This variety of biological functions has motivated the synthesis of a huge number of pteridine derivatives with the aim of studying their therapeutic potential. This review gathers the state-of-the-art of pteridine derivatives, describing their biological activities and molecular targets. The antitumor activity of pteridine-based compounds is one of the most studied and advanced therapeutic potentials, for which several molecular targets have been identified. Nevertheless, pteridines are also considered as very promising therapeutics for the treatment of chronic inflammation-related diseases. On the other hand, many pteridine derivatives have been tested for antimicrobial activities but, although some of them resulted to be active in preliminary assays, a deeper research is needed in this area. Moreover, pteridines may be of use in the treatment of many other diseases, such as diabetes, osteoporosis, ischemia, or neurodegeneration, among others. Thus, the diversity of the biological activities shown by these compounds highlights the promising therapeutic use of pteridine derivatives. Indeed, methotrexate, pralatrexate, and triamterene are Food and Drug Administration approved pteridines, while many others are currently under study in clinical trials.

Increased Alkaline Phosphatase in Cord Blood of Obese Diabetic Mothers Is Associated to Polyunstaurated Fatty Acid Levels.

INTRODUCTION: Recent studies indicate that alkaline phosphatase (ALP) may affect expression and activity of fatty acid (FA) transport proteins in placenta and other tissues. OBJECTIVE: To evaluate if disturbed FA profile in offspring of gestational diabetes mellitus (GDM) with different maternal pregestational weight could be related to maternal or neonatal ALP. METHODS: Prospective observational study of pregnant women recruited in the third trimester (25 controls, 23 lean-GDM, 20 obese-GDM). Fetal ultrasound was performed. At delivery, FAs were analyzed in placenta, maternal, and venous cord blood. Western blotting analysis of lipid carriers was performed in placenta. RESULTS: Newborns from obese-GDM tended to higher birthweight (p = 0.059) than those from both lean-GDM and controls. ALP in maternal blood tended to be lower in GDM (p = 0.170) while increased significantly in cord blood of obese-GDM with respect to controls (p = 0.039). Saturated FA percentages in cord blood were significantly higher (p < 0.000), while polyunsaturated FA (PUFA) percentages were lower (p = 0.003) in both GDM, which could be due to a lower expression of major family domain 2a receptor (MFSD2a) in the placenta. Plasma ALP in the offspring of obese-GDM was inversely associated to cord essential PUFAs (ß = -6.18, p = 0.005) and to placental MFSD2a (ß = -38.46, p = 0.014). CONCLUSIONS: Cord PUFA and placental MFSD2a are decreased in both lean and obese-GDM pregnancies. Higher ALP in cord blood of obese-GDM could play a role in the FA levels in these pregnancies.

Decreased Blood Level of MFSD2a as a Potential Biomarker of Alzheimer's Disease.

The protein Major Facilitator Superfamily Domain containing 2A (MFSD2a) was recently described as the primary carrier for docosahexaenoic acid (DHA) into the brain. Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by lower DHA levels in blood lipids. The aim of this study was to investigate the expression of MFSD2a in the whole blood and brain as a potential biomarker of AD. Three groups were established: 38 healthy controls, 48 subjects with moderate AD (GDS4), and 47 with severe AD (GDS6). We analyzed postmortem brain samples from the hippocampus of 11 healthy controls and 11 severe AD patients. Fatty acid (FA) was determined in serum and brain by gas chromatography. Blood and brain MFSD2a protein expression was analyzed by Western blotting. We found a significant and progressive decline of MFSD2a levels in blood of AD patients (Control 0.83 ± 0.13, GDS4 0.72 ± 0.09, GDS6 0.48 ± 0.05*, p ˂ 0.01). We also corroborated a significant reduction of DHA and other n-3 long-chain polyunsaturated FA in serum of AD. No differences were found in MFSD2a expression or FA levels in brain of controls and AD subjects. MFSD2A carrier was analyzed in AD patients for the first time and the level of MFSD2a in the whole blood could be a potential biomarker of this disease.

Role of MAP kinases and PI3K-Akt on the cytokine inflammatory profile of peritoneal macrophages from the ascites of cirrhotic patients.

AIMS: Several new approaches targeting inflammation associated with different diseases are in clinical development. OBJECTIVE: To explore the role played by MAPK and PI3K-Akt pathways on the release of cytokines in monocyte-derived macrophages (M-DM) obtained from the ascites of cirrhotic patients to identify novel targets for pharmaceutical intervention to prevent hepatic damage. METHODS: M-DM were isolated from the ascites of cirrhotic patients and stimulated in vitro with LPS and heat-killed Candida albicans in the presence or absence of the inhibitors for MEK1, p38 MAPK, JNK and PI3K. The MAPK phosphorylation levels were determined by Western Blot. Cell culture supernatants were assayed by ELISA for TNF-α, IL-6 and IL-10. RESULTS: The release of the pro-inflammatory cytokines IL-6 and TNF-α at baseline was more effectively reduced by the MAPK inhibitors, while the basal IL-10 anti-inflammatory cytokine secretion was only and strongly (90.3%) affected by the PI3K inhibitor. The incubation of peritoneal M-DM in the presence of LPS and C. albicans increased the release of IL-6, TNF-α and IL-10. LPS-induced pro-inflammatory cytokines secretion was more sensitive to MAPK inhibitors, whereas that induced by C. albicans was more susceptible to inhibition of PI3K. Finally, inhibition of PI3K almost completely suppressed the secretion of IL-10 in stimulated M-DM. CONCLUSIONS: These results demonstrate that pro-inflammatory cytokines release in M-DM from this clinical setting strongly depends on the MAPK signalling pathways, differs depending on the microbial stimulus added and confirms the prominent role of the PI3K-Akt pathway in the modulation of IL-10-mediated anti-inflammatory function.

Anti-inflammatory potential of digested Brassica sprout extracts in human macrophage-like HL-60 cells.

Cruciferous vegetables have been reported to be a great source of anti-inflammatory compounds. Specifically, sprouts from the Brassicaceae family stand out for their high content of glucosinolates (and their bioactive derivatives, isothiocyanates), phenolic acids, and anthocyanins. Despite the evident anti-inflammatory activity of certain Brassica phytochemicals such as sulforaphane or phenolic acids, the effect of digested Brassica vegetables on inflammation remains understudied. In this work, we aimed to evaluate the anti-inflammatory potential of the bioaccessible forms of cruciferous bioactives (from red cabbage sprouts (RCS) and red radish sprouts (RRS)) obtained upon in vitro gastrointestinal digestion in the HL-60 macrophage-like differentiated human cell line. The study was performed under basal conditions or stimulated with a low dose of LPS for 24 hours as a validated in vitro model of chronic inflammation. The cell viability was determined by MTT assay. The gene expression and production of pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß were determined by RT-qPCR and ELISA respectively. Our results revealed no cytotoxicity with any of the treatments in LPS-stimulated macrophage-like HL60 cells. Regarding cytokine production, digestates significantly decreased the production of the three pro-inflammatory cytokines at concentrations of 50 and 100 µg mL-1 except for IL-1ß treated with RCS digestates. Furthermore, the RT-qPCR analysis showed a decrease in the relative expression of pro-inflammatory cytokines in LPS-stimulated cells treated with RRS digestates at 100 µg mL-1 but not with red cabbage digestates. In conclusion, RRS bioaccessible compounds in the extracts could be used as dietary coadjuvants given their potential anti-inflammatory effect on this in vitro model of chronic inflammation.

NAD+ Precursors and Intestinal Inflammation: Therapeutic Insights Involving Gut Microbiota.

The oxidized form of nicotinamide adenine dinucleotide (NAD+) is a critical metabolite for living cells. NAD+ may act either as a cofactor for many cellular reactions as well as a coenzyme for different NAD+-consuming enzymes involved in the physiological homeostasis of different organs and systems. In mammals, NAD+ is synthesized from either tryptophan or other vitamin B3 intermediates that act as NAD+ precursors. Recent research suggests that NAD+ precursors play a crucial role in maintaining the integrity of the gut barrier. Indeed, its deficiency has been associated with enhanced gut inflammation and leakage, and dysbiosis. Conversely, NAD+-increasing therapies may confer protection against intestinal inflammation in experimental conditions and human patients, with accumulating evidence indicating that such favorable effects could be, at least in part, mediated by concomitant changes in the composition of intestinal microbiota. However, the mechanisms by which NAD+-based treatments affect the microbiota are still poorly understood. In this context, we have focused specifically on the impact of NAD+ deficiency on intestinal inflammation and dysbiosis in animal and human models. We have further explored the relationship between NAD+ and improved host intestinal metabolism and immunity and the composition of microbiota in vivo. Overall, this comprehensive review aims to provide a new perspective on the effect of NAD+-increasing strategies on host intestinal physiology.

Inherited Epigenetic Hallmarks of Childhood Obesity Derived from Prenatal Exposure to Obesogens.

Childhood obesity has reached epidemic levels in developed countries and is becoming a major cause for concern in the developing world. The causes of childhood obesity are complex and multifactorial, involving the interaction between individual genetics and environmental and developmental factors. Among the environmental factors, there is a growing interest in understanding the possible relationship between the so-called environmental obesogens and the development of obesity in children. Exposure to these obesogens such as phthalates, bisphenol A, or parabens, has been identified as a promoter of obesity through different mechanisms such as the alteration of adipocyte development from mesenchymal progenitors, the interference with hormone receptors, and induced inflammation. However, less attention has been paid to the inheritance of epigenetic modifications due to maternal exposure to these compounds during pregnancy. Thus, the aim of this review is to summarize the current knowledge of epigenetic modifications due to maternal exposure to those obesogens during pregnancy as well as their potential implication on long-term obesity development in the offspring and transgenerational inheritance of epiphenotypes.

Plasma short-chain fatty acid changes after bariatric surgery in patients with severe obesity.

BACKGROUND: Obesity has reached epidemic dimensions in recent decades. Bariatric surgery (BS) is one of the most effective interventions for weight loss and metabolic improvement in patients with obesity. Short-chain fatty acids (SCFA) are gut microbiota-derived metabolites with a key role in body weight control and insulin sensitivity. Although BS is known to induce significant changes in the gut microbiota composition, its impact on the circulating levels of certain metabolites produced by the gut microbiota such as SCFA remains poorly understood. OBJECTIVE: To determine the impact of BS on the circulating SCFA levels in patients with severe obesity. SETTING: University hospital. METHODS: An observational, prospective study was performed on 51 patients undergoing Roux-en-Y gastric bypass. Plasma samples were collected at baseline (1 day before surgery) and at 6 and 12 months after BS. Plasma SCFA levels were determined by liquid chromatography-mass spectrometry. RESULTS: The results revealed significant changes in the circulating levels of SCFA after BS. A marked increase in propionate, butyrate, isobutyrate, and isovalerate levels and a decrease in acetate, valerate, hexanoate, and heptanoate levels were observed 12 months after BS. Furthermore, the changes in the levels of propionate, butyrate, and isobutyrate negatively correlated with changes in body mass index, while those of isobutyrate correlated negatively with changes in the homeostatic model assessment for insulin resistance index. CONCLUSION: These results suggest that propionate, butyrate, and isobutyrate levels could be related to weight loss and improved insulin sensitivity in patients with severe obesity after BS.

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.

The peritoneal macrophage inflammatory profile in cirrhosis depends on the alcoholic or hepatitis C viral etiology and is related to ERK phosphorylation.

BACKGROUND: The development of ascites in cirrhotic patients generally heralds a deterioration in their clinical status. A differential gene expression profile between alcohol- and hepatitis C virus (HCV)-related cirrhosis has been described from liver biopsies, especially those associated with innate immune responses. The aim of this work was to identify functional differences in the inflammatory profile of monocyte-derived macrophages from ascites in cirrhotic patients of different etiologies in an attempt to extrapolate studies from liver biopsies to immune cells in ascites. To this end 45 patients with cirrhosis and non-infected ascites, distributed according to disease etiology, HCV (n=15) or alcohol (n=30) were studied. Cytokines and the cell content in ascites were assessed by ELISA and flow cytometry, respectively. Cytokines and ERK phosphorylation in peritoneal monocyte-derived macrophages isolated and stimulated in vitro were also determined. RESULTS: A different pattern of leukocyte migration to the peritoneal cavity and differences in the primed status of macrophages in cirrhosis were observed depending on the viral or alcoholic etiology. Whereas no differences in peripheral blood cell subpopulations could be observed, T lymphocyte, monocyte and polymorphonuclear cell populations in ascites were more abundant in the HCV than the alcohol etiology. HCV-related cirrhosis etiology was associated with a decreased inflammatory profile in ascites compared with the alcoholic etiology. Higher levels of IL-10 and lower levels of IL-6 and IL-12 were observed in ascitic fluid from the HCV group. Isolated peritoneal monocyte-derived macrophages maintained their primed status in vitro throughout the 24 h culture period. The level of ERK1/2 phosphorylation was higher in ALC peritoneal macrophages at baseline than in HCV patients, although the addition of LPS induced a greater increase in ERK1/2 phosphorylation in HCV than in ALC patients. CONCLUSIONS: The macrophage inflammatory status is higher in ascites of alcohol-related cirrhotic patients than in HCV-related patients, which could be related with differences in bacterial translocation episodes or regulatory T cell populations. These findings should contribute to identifying potential prognostic and/or therapeutic targets for chronic liver diseases of different etiology.

Peritoneal macrophage priming in cirrhosis is related to ERK phosphorylation and IL-6 secretion.

BACKGROUND: Bacterial infections are common complications arising in patients with cirrhosis and ascites. Translocation of bacterial DNA is a dynamic process that is associated with an increased inflammatory response and a poor prognosis in this setting. The aim of this study was to study whether peritoneal macrophages remain in a chronic primed status to allow a rapid response to subsequent events of bacterial translocation. PATIENTS AND METHODS: Peritoneal monocyte-derived macrophages were isolated from 25 patients with cirrhosis and non-infected ascites and compared with donor's blood monocytes. Activation cell-surface markers were screened using flow-cytometry, and the phosphorylation state of ERK 1/2, p38 MAP Kinase, PKB/Akt and transcription factors c-Jun and p65 NFκB were evaluated using Western blot. Synthesis of tumour necrosis factor alpha, interleukin 6 (IL-6) and interleukin-10 (IL-10) at baseline and in response to bacterial stimuli was evaluated using ELISA. RESULTS: A high expression of CD54, CD86 and HLA-DR at baseline was displayed by peritoneal macrophages. Increased phosphorylated levels of ERK1/2, protein kinase B (PKB) and c-Jun, together with IL-6 production, were observed in peritoneal macrophages at baseline compared with donors' blood monocytes. A positive correlation was established between basal IL-6 levels and extracellular signal-regulated kinase (ERK) phosphorylation in peritoneal macrophages from patients with cirrhosis (r=0·9; P=0·005). Addition of lipopolysaccharide induced higher phosphorylation levels of all studied signalling intermediates than synthetic-oligodeoxydinucleotides, but similar end-stage p65 NFκB. CONCLUSIONS: A sustained immune response is present in ascitic fluid of cirrhotic patients, even in the temporal absence of bacterial antigens. This would facilitate a fast response, probably controlled by IL-6, against repeated bacterial-DNA translocation or in liver chronic inflammation.

Norfloxacin modulates the inflammatory response and directly affects neutrophils in patients with decompensated cirrhosis.

BACKGROUND & AIMS: Patients with cirrhosis undergoing selective intestinal decontamination with norfloxacin show a reduction in serum cytokine levels, probably because of a combined effect of norfloxacin on bowel flora and neutrophils. METHODS: Thirty-one patients with cirrhosis receiving norfloxacin (400 mg/day) were included. Blood samples were collected at 0.5-4 hours (peak samples group, n = 47) and at 22-24 hours (trough samples group, n = 84) after dose. Fifty-nine ascitic fluid samples were obtained. Single doses of norfloxacin and trimethoprim/sulfamethoxazole were administered to 13 and 5 patients, respectively, (temporal profile group) and samples were collected at 0, 0.5, 1, 1.5, 2, 4, and 24 hours. Norfloxacin, trimethoprim/sulfamethoxazole, cytokines, nitric oxide, expression levels of nuclear factor (NF)-kappaB and inhibitor of NF-kappaB (IkB-alpha), neutrophil oxidative burst, and rate of apoptotic events were determined. RESULTS: All samples were bacterial DNA negative and had no significant levels of lipopolysaccharide. Serum and ascitic levels of tumor necrosis factor-alpha, interferon-gamma, interleukin-12, and nitric oxide were significantly lower in peak than in trough samples. A correlation was present between serum norfloxacins concentrations and tumor necrosis factor-alpha (r = -0.68; P < .001), interferon-gamma (r = -0.66; P < .001), interleukin-12 (r = -0.66; P < .001), and nitric oxide (r = -0.68; P < .001). Serum norfloxacin's highest concentrations (1 +/- 0.5 microg/mL) were achieved at 1-2 hours and concurred in time with the lower levels of cytokines and nitric oxide. Intracellular norfloxacin's highest levels (2 +/- 1 microg/mL/10(7) cells) were observed at 2 hours and concurred with a lower NF-kappaB expression, a reduced anion superoxide generation, and apoptotic rate in response to phorbol myristate acetate. Trimethoprim/sulfamethoxazole did not significantly modulate cytokine expression. CONCLUSIONS: Norfloxacin but not trimethoprim/sulfamethoxazole modulates inflammatory response and directly affects neutrophils in patients with cirrhosis.

Insulin resistance in polycystic ovary syndrome is associated with defective regulation of ERK1/2 by insulin in skeletal muscle in vivo.

Insulin resistance is a recognized feature of PCOS (polycystic ovary syndrome). However, the molecular reason(s) underlying this reduced cellular insulin sensitivity is not clear. The present study compares the major insulin signalling pathways in skeletal muscle isolated from PCOS and controls. We measured whole-body insulin sensitivity and insulin signalling in skeletal muscle biopsies taken before and after acute exposure to hyperinsulinaemia in nine women diagnosed with PCOS and seven controls. We examined the expression, basal activity and response to in vivo insulin stimulation of three signalling molecules within these human muscle samples, namely IRS-1 (insulin receptor substrate-1), PKB (protein kinase B) and ERK (extracellular-signal-regulated kinase) 1/2. There was no significant difference in the expression, basal activity or activation of IRS-1 or PKB between PCOS and control subjects. However, there was a severe attenuation of insulin stimulation of the ERK pathway in muscle from all but two of the women with PCOS (the two most obese), and an accompanying trend towards higher basal phosphorylation of ERK1/2 in PCOS. These results are striking in that the metabolic actions of insulin are widely believed to require the IRS-1/PKB pathway rather than ERK, and the former has been reported as defective in some previous PCOS studies. Most importantly, the molecular defect identified was independent of adiposity. The altered response of ERK to insulin in PCOS was the most obvious signalling defect associated with insulin resistance in muscle from these patients.