New Potential Pharmacological Options for Endometriosis Associated Pain.

Endometriosis is a chronic inflammatory disorder characterized by the abnormal growth of endometrial-like tissue outside the uterine cavity, affecting 10-15% of women of reproductive age. Pain is the most common symptom. Treatment options include surgery, which has limited effectiveness and high recurrence rates, and pharmacotherapy. Hormonal therapies, commonly used for symptom management, can have side effects and contraceptive outcomes, contributing to the infertility associated with endometriosis, with pain and lesions often reappearing after treatment cessation. Among its etiological factors, immunological and inflammatory dysregulation plays a significant role, representing an interesting target for developing new therapeutic strategies. This review critically analyzes recent studies to provide an updated synthesis of ongoing research into potential new pharmacotherapies focusing on lesion progression, pain relief, and improving quality of life. Immunotherapy, natural anti-inflammatory and antioxidant compounds and drug repurposing show promise in addressing the limitations of current treatments by targeting immunological factors, potentially offering non-invasive solutions for managing pain and infertility in endometriosis. Promising results have been obtained from in vitro and animal model studies, but clinical trials are still limited. More effort is needed to translate these findings into clinical practice to effectively reduce disease progression, alleviate pain symptoms and preserve the reproductive capacity, improving patients' overall wellbeing.

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.

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.

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.

Collateral sensitivity to cold stress and differential BCL-2 family expression in new daunomycin-resistant lymphoblastoid cell lines.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells is one of the main causes of chemotherapy failure in cancer, and, usually, is due to the increased expression of P-glycoprotein (MDR-1, P-gp, ABCB1), a pump that expels chemotherapeutics from the cell and/or regulates apoptosis. Thus, it is fundamental to find drugs or stress stimuli with a capacity to induce apoptosis in such cells and to identify the mechanisms involved. We address this matter in human cells and establish new daunomycin (DNM)-resistant cell lines (IM-9R) by exposing the parental lymphoblastic cells (IM-9) to increasing doses of the anti-neoplastic drug, daunomycin. The resistance level of IM-9R cell lines, MDR-1 expression and functionality, collateral sensitivity and Bcl-2 and caspases protein expression are analyzed. As a result, we show for the first time that, unlike the parental cells, human lymphoblastic resistant cells exhibit collateral sensitivity to cold stress, confirming that this phenomenon is not exclusive to murine leukemic cells, but a broader one associated with the acquisition of drug resistance. Furthermore, the new resistant cell lines undergo a significant increase in active caspase-3 and -9 levels and drastic changes in Bcl-2 family protein expression during the process of MDR phenotype acquisition.

Mathematical modelling and computational study of two-dimensional and three-dimensional dynamics of receptor-ligand interactions in signalling response mechanisms.

Cell signalling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signalling pathways. But the binding process still presents many enigmas and, by analogy with surface catalytic reactions, two different mechanisms can be conceived: the first mechanism is related to the Eley-Rideal (ER) mechanism, i.e. the bulk-dissolved ligand interacts directly by pure three-dimensional (3D) diffusion with the specific surface receptor; the second mechanism is similar to the Langmuir-Hinshelwood (LH) process, i.e. 3D diffusion of the ligand to the cell surface followed by reversible ligand adsorption and subsequent two-dimensional (2D) surface diffusion to the receptor. A situation where both mechanisms simultaneously contribute to the signalling process could also occur. The aim of this paper is to perform a computational study of the behavior of the signalling response when these different mechanisms for ligand-receptor interactions are integrated into a model for signal transduction and ligand transport. To this end, partial differential equations have been used to develop spatio-temporal models that show trafficking dynamics of ligands, cell surface components, and intracellular signalling molecules through the different domains of the system. The mathematical modeling developed for these mechanisms has been applied to the study of two situations frequently found in cell systems: (a) dependence of the signal response on cell density; and (b) enhancement of the signalling response in a synaptic environment.

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.

Optimization of peritoneal fluid and leukocyte collection in patients with endometriosis.

OBJECTIVE: To propose a standardized protocol for peritoneal free fluid and leukocyte sample collection in women with endometriosis suitable for biomedical research on the basis of the surgical procedure, the clinical and technical conditions, and the quality of the samples obtained. DESIGN: Video showing the step-by-step collection procedure and the suitability of samples obtained for biomedical research. SUBJECTS: This study included 103 women with confirmed endometriosis by pathology analysis, who signed informed consent and were recruited from the Hospital "Virgen de la Arrixaca", Murcia, Spain. The study was approved by the Ethics Committee of University of Murcia (CEI 3156/2020). MAIN OUTCOME MEASURES: We analyzed the presence of free fluid in the peritoneal cavity and its relationship with hormonal treatment intake. In addition, the presence of blood contamination, the number of viable leukocytes and macrophages in free peritoneal fluid and lavages as well as their relationship with the lavage volume used, the body mass index, and the age of patients were analyzed. RESULTS: The presence of free peritoneal fluid, in which cells and molecules could be quantified, was scarce in the patients (21%), and it was not significantly related to hormonal treatment intake. The cell viability was higher than 98% in all collected samples; although 54% showed good quality and enough cellularity to be used in biomedical research, 40% were contaminated with blood and 6% had low cellularity. The number of leukocytes and macrophages recovered from the peritoneal lavages correlated positively with the lavage volume used and negatively with the body mass index and was independent of the age of the patients. CONCLUSION: We describe a standardized step-by-step procedure for peritoneal fluid and leukocyte collection in women with endometriosis, suitable for biomedical research, taking into account that not all women present free fluid in the peritoneal cavity. We propose to increase the lavage volume recommended by the World Endometriosis Research Foundation from 10 mL to at least 40 mL of sterile saline solution and its mobilization for at least 30 seconds within the peritoneal cavity, especially in patients with higher body mass index, to improve the efficiency of the procedure.

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.

Analysis of the anti-inflammatory potential of Brassica bioactive compounds in a human macrophage-like cell model derived from HL-60 cells.

BACKGROUND: Chronic inflammatory diseases are major causes of global morbidity and mortality. Acute inflammation is meant to protect the body against foreign agents, but it also plays a major role in tissue repairment. Several mediators are involved in this process, including pro-inflammatory cytokines produced by macrophages. Occasionally, if the inflammatory response is not resolved, the acute inflammatory process can evolve into a chronic inflammation. Natural compounds from vegetables are considered as an important source of active agents with potential to treat or prevent inflammatory related pathologies and could be used as an alternative of the therapeutic agents currently in use, such as non-steroidal anti-inflammatory drugs (NSAIDs), which present several side effects. METHODS: In this research work we evaluated in vitro the anti-inflammatory activity of a series of ten phytochemicals present in Brassica, measured as the potential of those compounds to reduce the production of key pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) by a human macrophage-like cell model of HL-60 cells RESULTS: Most of the tested phytochemicals (including the most representative bioactive molecules of the major classes of compounds present in cruciferous foods such as glucosinolates, isothiocyanates, hydroxycinnamic acids, flavonols and anthocyanins) demonstrated significant anti-inflammatory activity at micromolar level in the absence of cytotoxic effects in this human macrophage-like cell model. CONCLUSION: These data confirm that phytochemicals commonly obtained from Brassica may be potential therapeutic leads to treat or prevent human chronic inflammation and related diseases.

Glycoconjugate expression on the cell wall of tps1/tps1 trehalose-deficient Candida albicans strain and implications for its interaction with macrophages.

The yeast Candida albicans has developed a variety of strategies to resist macrophage killing. In yeasts, accumulation of trehalose is one of the principal defense mechanisms under stress conditions. The gene-encoding trehalose-6-phosphate synthase (TPS1), which is responsible for trehalose synthesis, is induced in response to oxidative stress, as in phagolysosomes. Mutants unable to synthesize trehalose are sensitive to oxidative stress in vitro. In mice, the TPS1-deficient strain, tps1/tps1, displays a lower infection rate than its parental strain (CAI4). We have previously demonstrated the reduced binding capacity of tps1/tps1 and its lower resistance to macrophages. At the same time, its outer cell wall layer was seen to be altered. In this study, we show that depending on the culture conditions, the tps1/tps1 strain regulates the carbohydrate metabolism in a different way to CAI4, as reflected by the enhanced ß-mannosylation of cell wall components, especially at the level of the 120 kDa glycoprotein species, accessible at the cell surface of tps1/tps1 when cultured in liquid medium, but not on solid medium. This leads to changes in its surface properties, as revealed by decreased hydrophobicity, and the lower levels of ERK1/2 phosphorylation and tumor necrosis factor-α (TNF-α) production in macrophages, thus increasing the resistance to these cells. In contrast, in solid medium, in which over-glycosylation was less evident, tps1/tps1 showed similar macrophage interaction properties to CAI4, but was less resistant to killing, confirming the protective role of trehalose. Thus, the lack of trehalose is compensated by an over-glycosylation of the cell wall components in the tps1/tps1 mutant, which reduces susceptibility to killing.

Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells.

We have tested the usefulness of several commercial anti-CD33 monoclonal antibodies (mAb) to determine the expression and localization of the two CD33 isoforms on several hematopoietic cell lines. The expression of the isoform CD33m, a CD33 transmembrane splice variant lacking the ligand-binding V immunoglobulin (Ig)-like domain, was detected by RT-polymerase chain reaction, western blot, confocal microscopy and flow cytometry on the membrane of several human cell types. CD33m was only detected by the anti-CD33 mAb HIM3-4 on the cell surface, whereas WM53, P67.6, 4D3, HIM3-4, WM54, D3HL60.251 or MY9 detected the CD33M isoform, indicating that HIM3-4 is the only mAb recognizing CD33 C(2) Ig domain. Accordingly, HIM3-4 binding to CD33 did not interfere with the binding of other antibodies against the CD33 V-domain. P67.6 mAb interfered with recognition by the rest of antibodies specific for the V domain. HIM3-4 staining could be increased after the sialidase treatment of all CD33(+) cells. However, this increase was stronger in activated T cells, suggesting a CD33 masking state in this cell population. Confocal microscopy analysis of CD33m HEK 293T-transfected cells revealed that this protein is expressed on the cell membrane and also detected in the Golgi compartment. CD33 is constitutively located outside the lipid raft domains, whereas cross-linked CD33 is highly recruited to this signaling platform. The unique ability of HIM3-4 mAb to detect the masking state of CD33 on different cell lineages makes it a good tool to improve the knowledge of the biological role of this sialic acid-binding Ig-like lectin.

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.

Recent insights into the characteristics and role of peritoneal macrophages from ascites of cirrhotic patients.

Macrophages are a diverse myeloid cell population involved in innate and adaptive immune responses, embryonic development, wound repair, and regulation of tissue homeostasis. These cells link the innate and adaptive immunities and are crucial in the development and sustainment of various inflammatory diseases. Macrophages are tissue-resident cells in steady-state conditions; however, they are also recruited from blood monocytes after local pathogen invasion or tissue injury. Peritoneal macrophages vary based on their cell complexity, phenotype, and functional capabilities. These cells regulate inflammation and control bacterial infections in the ascites of decompensated cirrhotic patients. Our recent work reported several phenotypic and functional characteristics of these cells under both healthy and pathological conditions. A direct association between cell size, CD14/CD16 expression, intracellular level of GATA-6, and expression of CD206 and HLA-DR activation/maturation markers, indicate that the large peritoneal macrophage CD14highCD16high subset constitutes the mature phenotype of human resident peritoneal macrophages during homeostasis. Moreover, elevated expression of CD14/CD16 is related to the phagocytic capacity. The novel large CD14highCD16high peritoneal subpopulation is increased in the ascites of cirrhotic patients and is highly sensitive to lipopolysaccharide (LPS)-induced activation, thereby exhibiting features of inflammatory priming. Thus, phosphorylation of ERK1/2, PKB/Akt, and c-Jun is remarkably increased in response to LPS in vitro, whereas that of p38 MAPK is reduced compared with the monocyte-derived macrophages from the blood of healthy controls. Furthermore, in vitro activated monocyte-derived macrophages from ascites of cirrhotic patients secreted significantly higher levels of IL-6, IL-10, and TNF-α and lower amounts of IL-1ß and IL-12 than the corresponding cells from healthy donor's blood. Based on these results, other authors have recently reported that the surface expression level of CD206 can be used to identify mature, resident, inflammatory peritoneal macrophages in patients with cirrhosis. Soluble CD206 is released from activated large peritoneal macrophages, and increased concentrations in patients with cirrhosis and spontaneous bacterial peritonitis (SBP) indicate reduced odds of survival for 90 d. Hence, the level of soluble CD206 in ascites might be used to identify patients with SBP at risk of death. In conclusion, peritoneal macrophages present in ascites of cirrhotic patients display multiple phenotypic modifications characterized by reduced ratio of cells expressing several membrane markers, together with an increase in the ratios of complex and intermediate subpopulations and a decrease in the classic-like subset. These modifications may lead to the identification of novel pharmaceutical targets for prevention and treatment of hepatic damage.

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.

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.