The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria.

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

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.

Deletion of GLX3 in Candida albicans affects temperature tolerance, biofilm formation and virulence.

Candida albicans is a predominant cause of fungal infections in mucosal tissues as well as life-threatening bloodstream infections in immunocompromised patients. Within the human body, C. albicans is mostly embedded in biofilms, which provides increased resistance to antifungal drugs. The glyoxalase Glx3 is an abundant proteomic component of the biofilm extracellular matrix. Here, we document phenotypic studies of a glx3Δ null mutant concerning its role in biofilm formation, filamentation, antifungal drug resistance, cell wall integrity and virulence. First, consistent with its function as glyoxalase, the glx3 null mutant showed impaired growth on media containing glycerol as the carbon source and in the presence of low concentrations of hydrogen peroxide. Importantly, the glx3Δ mutant showed decreased fitness at 37°C and formed less biofilm as compared to wild type and a reintegrant strain. At the permissive temperature of 28°C, the glx3Δ mutant showed impaired filamentation as well as increased sensitivity to Calcofluor white, Congo red, sodium dodecyl sulfate and zymolyase, indicating subtle alterations in wall architecture even though gross quantitative compositional changes were not detected. Interestingly, and consistent with its impaired filamentation, biofilm formation and growth at 37°C, the glx3Δ mutant is avirulent. Our results underline the role of Glx3 in fungal pathogenesis and the involvement of the fungal wall in this process.

Micafungin Enhances the Human Macrophage Response to Candida albicans through ß-Glucan Exposure.

Micafungin belongs to the antifungal family of echinocandins, which act as noncompetitive inhibitors of the fungal cell wall ß-1,3-d-glucan synthase. Since Candida albicans is the most prevalent pathogenic fungus in humans, we study the involvement of micafungin in the modulation of the inflammatory response developed by human tissue macrophages against C. albicans The MIC for micafungin was 0.016 µg/ml on the C. albicans SC5314 standard strain. Micafungin induced a drastic reduction in the number of exponential SC5314 viable cells, with the fungicidal effect being dependent on the cellular metabolic activity. Notably, micafungin also caused a structural remodelling of the cell wall, leading to exposure of the ß-glucan and chitin content on the external surface. At the higher doses used (0.05 µg/ml), the antifungal also induced the blowing up of budding yeasts. In addition, preincubation with micafungin before exposure to human tissue macrophages enhanced the secretion of tumor necrosis factor alpha (TNF-α), interleukin-17A (IL-17A), and IL-10 cytokines. Our results strongly suggest that in C. albicans treatment with micafungin, in addition to having the expected toxic antifungal effect, it potentiates the immune response, improving the interaction and activation of human macrophages, probably through the unmasking of ß-glucans on the cell wall surface.

Homozygous deletion of ATC1 and NTC1 genes in Candida parapsilosis abolishes trehalase activity and affects cell growth, sugar metabolism, stress resistance, infectivity and biofilm formation.

A double homozygous atc1Δ/atc1Δ/ntc1Δ/ntc1Δ mutant (atc1Δ/ntc1Δ KO) was constructed in the pathogen opportunistic yeast Candida parapsilosis by disruption of the two chromosomal alleles coding for NTC1 gene (encoding a neutral trehalase) in a Cpatc1Δ/atc1Δ background (atc1Δ KO strain, deficient in acid trehalase). The Cpatc1Δ/ntc1Δ KO mutant failed to counteract the inability of Cpatc1Δ cells to metabolize exogenous trehalose and showed a similar growth pattern on several monosaccharides and disaccharides. However, upon prolonged incubation in either rich medium (YPD) or nutrient-starved medium the viability of Cpatc1Δ cells exhibited a sensitive phenotype, which was augmented by further CpNTC1/NTC1 disruption. Furthermore, Cpatc1Δ/ntc1Δ KO cells had difficulty in resuming active growth in fresh YPD. This homozygous mutant also lacked any in vitro measurable trehalase activity, whether acid or neutral, suggesting that a single gene codes for each enzyme. By contrast, in Cpatc1Δ/ntc1Δ KO strain the resistance to oxidative and heat stress displayed by atc1Δ mutant was suppressed. Cpatc1Δ/ntc1Δ KO cells showed a significant decrease in virulence as well as in the capacity to form biofilms. These results point to a major role for acid trehalase (Atc1p) in the pathobiology of C. parapsilosis, whereas the activity of neutral trehalase can only partially counteract Atc1p deficiency. They also support the use of ATC1 and NTC1 genes as interesting antifungal targets.

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.

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.

Identification of fungal trehalose for the diagnosis of invasive candidiasis by mass spectrometry.

The rapidity of the diagnosis of invasive candidiasis (IC) is crucial to allow the early introduction of antifungal therapy that dramatically increases the survival rate of patients. Early diagnosis is unfortunately often delayed because Candida blood culture, the gold standard diagnostic test, is positive in only 50% of cases of IC and takes several days to obtain this result. Complementary non-culture-based methods relying on the detection of Candida cell wall polysaccharides in the serum, ß-glucans and mannans, by enzymatic and immunological reagents have been successfully developed to allow a more efficient patients care. We have previously demonstrated that detection of circulating glycans by mass spectrometry could provide a reliable and cost-effective early diagnosis method called MS-DS for Mass Spectrometry of Di-Saccharide. Here, by comparing patient's sera and Candida albicans strains deficient in carbohydrates synthesis, we demonstrate that trehalose derived from fungal metabolism can be specifically targeted by MS-DS to allow early diagnosis. In particular, the use of C. albicans strains deficient in the synthesis of trehalose synthesizing enzymes Tps1 and Tps2 show that MS-DS results were correlated to the metabolism of trehalose. Finally, we demonstrate that the performance of the IC diagnosis can be significantly improved by using high resolution mass spectrometry, which opens new perspectives in the management of the disease.

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.

Pga26 mediates filamentation and biofilm formation and is required for virulence in Candida albicans.

The Candida albicans gene PGA26 encodes a small cell wall protein and is upregulated during de novo wall synthesis in protoplasts. Disruption of PGA26 caused hypersensitivity to cell wall-perturbing compounds (Calcofluor white and Congo red) and to zymolyase, which degrades the cell wall ß-1,3-glucan network. However, susceptibility to caspofungin, an inhibitor of ß-1,3-glucan synthesis, was decreased. In addition, pga26Δ mutants show increased susceptibility to antifungals (fluconazol, posaconazol or amphotericin B) that target the plasma membrane and have altered sensitivities to environmental (heat, osmotic and oxidative) stresses. Except for a threefold increase in ß-1,6-glucan and a slightly widened outer mannoprotein layer, the cell wall composition and structure was largely unaltered. Therefore, Pga26 is important for proper cell wall integrity, but does not seem to be directly involved in the synthesis of cell wall components. Deletion of PGA26 further leads to hyperfilamentation, increased biofilm formation and reduced virulence in a mouse model of disseminated candidiasis. We propose that deletion of PGA26 may cause an imbalance in the morphological switching ability of Candida, leading to attenuated dissemination and infection.

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.