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.

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.

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.

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.

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.

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.

Child Head Circumference and Placental MFSD2a Expression Are Associated to the Level of MFSD2a in Maternal Blood During Pregnancy.

Gestational diabetes mellitus (GDM) is a world-wide health challenge, which prevalence is expected to increase in parallel to the epidemic of obesity. Children born from GDM mothers have lower levels of docosahexaenoic acid (DHA) in cord blood, which might influence their neurodevelopment. Recently, the membrane transporter Major Family Super Domain 2a (MFSD2a) was associated with the selective transportation of DHA as lysophospholipids. The expression of the DHA membrane transporter MFSD2a is lower in GDM placentas, which could affect materno-fetal DHA transport. Humans with homozygous inactivating mutations in the MFSD2a gene present severe microcephaly and intellectual impairments. Herein, we intended to identify early blood biomarkers that may be of use during pregnancy to monitor the offspring development and the adequate nutritional interventions, such as nutritional supplementation, that may be selected to improve it. We evaluated MFSD2a expression in maternal blood at the third trimester of pregnancy, and its potential relationship with the expression of placental MFSD2a at delivery and child outcomes. Three groups of pregnant women were recruited: 25 controls, 23 GDM with dietary treatment, and 20 GDM with insulin treatment. Maternal and neonatal anthropometric and biochemical parameters were evaluated. MFSD2a was analyzed in placenta, blood and serum. MFSD2a protein expression in maternal blood was significantly lower in GDM groups and correlated with placental MFSD2a and Z-score neonatal head circumference during the first 6 months of life. The cord/maternal serum ratio of DHA, a solid indicator of materno-fetal DHA transport, was reduced in GDM groups and correlated with MFSD2a in maternal blood at the third trimester and in placenta at delivery. This indicates that altered MFSD2a levels in maternal blood during pregnancy might influence placental nutrient transport and fetal neurodevelopment. Furthermore, MFSD2a levels in maternal blood on the third trimester were inversely correlated to DHA in maternal serum lyso-PL. Thus, the level of MFSD2a in maternal blood could be used as a potential biomarker for the early detection of disturbances of MFSD2a expression during pregnancy and the subsequent consequences for the neurodevelopment of the child, as well as it may help to choose the optimal treatment approach for the affected subjects.

Hypothetical roadmap towards endometriosis: prenatal endocrine-disrupting chemical pollutant exposure, anogenital distance, gut-genital microbiota and subclinical infections.

BACKGROUND: Endometriosis is a gynaecological hormone-dependent disorder that is defined by histological lesions generated by the growth of endometrial-like tissue out of the uterus cavity, most commonly engrafted within the peritoneal cavity, although these lesions can also be located in distant organs. Endometriosis affects ~10% of women of reproductive age, frequently producing severe and, sometimes, incapacitating symptoms, including chronic pelvic pain, dysmenorrhea and dyspareunia, among others. Furthermore, endometriosis causes infertility in ~30% of affected women. Despite intense research on the mechanisms involved in the initial development and later progression of endometriosis, many questions remain unanswered and its aetiology remains unknown. Recent studies have demonstrated the critical role played by the relationship between the microbiome and mucosal immunology in preventing sexually transmitted diseases (HIV), infertility and several gynaecologic diseases. OBJECTIVE AND RATIONALE: In this review, we sought to respond to the main research question related to the aetiology of endometriosis. We provide a model pointing out several risk factors that could explain the development of endometriosis. The hypothesis arises from bringing together current findings from large distinct areas, linking high prenatal exposure to environmental endocrine-disrupting chemicals with a short anogenital distance, female genital tract contamination with the faecal microbiota and the active role of genital subclinical microbial infections in the development and clinical progression of endometriosis. SEARCH METHODS: We performed a search of the scientific literature published until 2019 in the PubMed database. The search strategy included the following keywords in various combinations: endometriosis, anogenital distance, chemical pollutants, endocrine-disrupting chemicals, prenatal exposure to endocrine-disrupting chemicals, the microbiome of the female reproductive tract, microbiota and genital tract, bacterial vaginosis, endometritis, oestrogens and microbiota and microbiota-immune system interactions. OUTCOMES: On searching the corresponding bibliography, we found frequent associations between environmental endocrine-disrupting chemicals and endometriosis risk. Likewise, recent evidence and hypotheses have suggested the active role of genital subclinical microbial infections in the development and clinical progression of endometriosis. Hence, we can envisage a direct relationship between higher prenatal exposure to oestrogens or estrogenic endocrine-disrupting compounds (phthalates, bisphenols, organochlorine pesticides and others) and a shorter anogenital distance, which could favour frequent postnatal episodes of faecal microbiota contamination of the vulva and vagina, producing cervicovaginal microbiota dysbiosis. This relationship would disrupt local antimicrobial defences, subverting the homeostasis state and inducing a subclinical inflammatory response that could evolve into a sustained immune dysregulation, closing the vicious cycle responsible for the development of endometriosis. WIDER IMPLICATIONS: Determining the aetiology of endometriosis is a challenging issue. Posing a new hypothesis on this subject provides the initial tool necessary to design future experimental, clinical and epidemiological research that could allow for a better understanding of the origin of this disease. Furthermore, advances in the understanding of its aetiology would allow the identification of new therapeutics and preventive actions.

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.

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.

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.

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.

Anti-leukemia activity of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines.

Pteridines are bicyclic heterocyclic compounds with a pyrazino[2,3-d]pyrimidine nucleus that have shown a wide range of therapeutic utilities. Concretely, 4-aminopteridine derivatives have demonstrated both anti-inflammatory and anti-cancer properties, and some of them, such as methotrexate, are profusely used in medical practice. We have recently synthesized and tested the biological activity of a novel series of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines, finding that they present anti-inflammatory properties, as they were able to inhibit in vitro the production of pro-inflammatory cytokines TNF-α and IL-6. Now, we have evaluated the anti-tumor potential of these compounds on HL-60 and K562 leukemia cell lines. Cells growing at exponential rate were exposed to decreasing doses of each compound, from 50 to 0.39 µM, for 24, 48, and 72 h. Cell viability was tested by MTT assay and cell death fashion determined by annexin V/propidium iodide assay. The cytotoxicity of the compounds was determined in differentiated macrophage-like HL-60 cells and in human peripheral blood mononuclear cells to evaluate the potential side effects on quiescent tumor cells and normal cells, respectively. Among the series, compounds 1a, 1b, 1g, 1j, and 1k showed anti-proliferative activity. Compounds 1j and 1k were active against both HL-60 and K562 cells, with a lower IC50 against HL-60 cells. Compounds 1a, 1b, and 1g had a great cytotoxic activity against HL-60, but they were far less potent against K562 cells. None had side effects in differentiated tumor cells or in human peripheral blood mononuclear cells. In conclusion, our results demonstrate that some compounds of this series of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines have anti-cancer properties in vitro.

Intracellular signaling modifications involved in the anti-inflammatory effect of 4-alkoxy-6,9-dichloro[1,2,4]triazolo[4,3-a]quinoxalines on macrophages.

Inflammation is part of a complex biological response directed by the immune system to fight pathogens and maintain homeostasis. Dysregulation of the inflammatory process leads to development of chronic inflammatory or autoimmune diseases. Several cell types, such as macrophages, and cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) are involved in the regulation of inflammation. The important role played by these cytokines as mediators of the inflammatory process and the side effects of current therapies have promoted the search of new therapeutic alternatives. Quinoxalines are important compounds allowing a wide range of chemical modifications in order to provide an extensive repertoire of biological activities. We have previously shown that a series of 4-alkoxy-6,9-dichloro[1,2,4]triazolo[4,3-a]quinoxalines exhibit potent anti-inflammatory activity, inhibiting the production of TNF-α and IL-6. Our aim here was to study the mechanism thereby this series of compounds act upon different intracellular signaling pathways to uncover their potential molecular targets. By using immunoblotting assays, we found that these compounds inhibit ERK 1/2 and JNK/c-Jun cascades, and reduce c-Fos expression, while activate the anti-inflammatory PI3K/Akt route. These results provide further information on their effect upon the intracellular signal transduction mechanisms leading to inhibition of TNF-α and IL-6 secretion. Our results may be of great interest for the pharmaceutical industry, and could be used as a starting point for the development of new and more potent anti-inflammatory drugs derived from the quinoxaline core.

Placental MFSD2a transporter is related to decreased DHA in cord blood of women with treated gestational diabetes.

BACKGROUND & AIMS: Maternal-fetal transfer of docosahexaenoic acid (DHA) is impaired by gestational diabetes mellitus (GDM), but the underlying mechanisms are still unknown. MFSD2a was recently recognized as a lyso-phospholipid (lyso-PL) transporter that facilitates DHA accretion in brain. The role of this transporter in placenta is uncertain. We evaluated effects of GDM and its treatment (diet or insulin) on phospholipid species, fatty acid profile in women, cord blood and placental fatty acid carriers. METHODS: Prospective observational study of pregnant women recruited in the third trimester (25 controls, 23 GDM-diet, 20 GDM-insulin). Fetal ultrasound was performed at gestational week 38. At delivery, maternal and neonatal anthropometry was performed, and fatty acids in total lipids and phospholipid species were analyzed in placenta, maternal and venous cord blood. Western-blot analyses were performed for placental fatty acid carriers. RESULTS: Fetal abdominal circumference z-score at 38 weeks tended to higher values in GDM (P = 0.071), pointing toward higher fetal fat accretion in these babies. DHA percentage in cord serum total lipids (P = 0.029) and lyso-PL (P = 0.169) were reduced in GDM. Placental MFSD2a was reduced in both GDM groups and was positively correlated to DHA values in cord serum total lipids (r = 0.388, P = 0.003). Among established placental lipid carriers, only FATP4 was correlated to DHA concentration in placental lyso-PL. In all compartments, DHA percentage was inversely correlated to fetal abdominal circumference. CONCLUSIONS: In offspring of women with GDM treated either with diet or insulin, higher fetal fat accretion and lower placental MFSD2a contribute to reduce DHA availability. Lyso-PL appear to contribute to materno-fetal DHA transport.

Attenuated JNK signaling in multidrug-resistant leukemic cells. Dual role of MAPK in cell survival.

Having found previously that leukemic cells with multidrug resistant (MDR) phenotype, but not their sensitive counterparts, exhibit collateral sensitivity to cold stress in a P-gp-dependent manner, our aim was to study the signaling pathways involved in this phenomenon in sensitive (L1210) and resistant cells (L1210R and CBMC-6). It was observed that the acquisition of MDR phenotype by leukemic cells or their transfection with the extrussion pump, P-gp, modifies the activation profile and regulation of Mitogen-Activated Protein Kinases (MAPK) in cells exposed to low temperatures. More specifically, cold stress provoked the activation of c-Jun N-terminal kinase (JNK) in sensitive cells, while attenuated JNK signaling was observed in MDR cells. This effect was also observed, although with less intensity, in P-gp-transfected cells. Using pharmacological inhibitors to determine the role of MAPK in leukemic cell survival in physiological conditions or under cold stress, a dual temperature-dependent role was observed for JNK in MDR cell survival. At 37°C JNK is necessary for the survival of parental, resistant and P-gp-transfected cells; however, the use of inhibitors of either extracellular signal-regulated protein kinase (ERK) or JNK significantly counteracts cold-induced death of resistant and P-gp-transfected cells, supporting a role for ERK and JNK in cold-stress induced cell death. Finally, a connectivity model concerning MAPK is proposed, summarizing how cold stress and MDR-1 might trigger apoptosis in resistant cell lines. These findings on MDR cells may assist in the design of specific therapeutic strategies to complement current chemotherapy.