Marcela A. Hermoso

Chronic Inflammation and Cytokines in the Tumor Microenvironment

Acute inflammation is a response to an alteration induced by a pathogen or a physical or chemical insult, which functions to eliminate the source of the damage and restore homeostasis to the affected tissue. However, chronic inflammation triggers cellular events that can promote malignant transformation of cells and carcinogenesis. Several inflammatory mediators, such as TNF-α, IL-6, TGF-β, and IL-10, have been shown to participate in both the initiation and progression of cancer. In this review, we explore the role of these cytokines in important events of carcinogenesis, such as their capacity to generate reactive oxygen and nitrogen species, their potential mutagenic effect, and their involvement in mechanisms for epithelial mesenchymal transition, angiogenesis, and metastasis. Finally, we will provide an in-depth analysis of the participation of these cytokines in two types of cancer attributable to chronic inflammatory disease: colitis-associated colorectal cancer and cholangiocarcinoma.

ClC-3 is a fundamental molecular component of volume-sensitive outwardly rectifying Cl- channels and volume regulation in HeLa cells and Xenopus laevis oocytes.

Volume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Native VSOACs are believed to be a major pathway for regulatory volume decrease (RVD) through efflux of chloride and organic osmolytes. ClC-3 has been proposed to encode native VSOACs in Xenopus laevis oocytes and in some mammalian cells, including cardiac and vascular smooth muscle cells. The relationship between the ClC-3 chloride channel, the native volume-sensitive osmolyte and anion channel (VSOAC) currents, and cell volume regulation in HeLa cells andX. laevis oocytes was investigated using ClC-3 antisense. In situ hybridization in HeLa cells, semiquantitative and real-time PCR, and immunoblot studies in HeLa cells and X. laevis oocytes demonstrated the presence of ClC-3 mRNA and protein, respectively. Exposing both cell types to hypotonic solutions induced cell swelling and activated native VSOACs. Transient transfection of HeLa cells with ClC-3 antisense oligonucleotide or X. laevis oocytes injected with antisense cRNA abolished the native ClC-3 mRNA transcript and protein and significantly reduced the density of native VSOACs activated by hypotonically induced cell swelling. In addition, antisense against native ClC-3 significantly impaired the ability of HeLa cells and X. laevis oocytes to regulate their volume. These results suggest that ClC-3 is an important molecular component underlying VSOACs and the RVD process in HeLa cells and X. laevis oocytes.

Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

1 Intracellular dialysis of NIH/3T3 cells with a commercially available anti‐ClC‐3 polyclonal antibody (Ab) for ≈30 min completely inhibited expressed guinea‐pig ClC‐3 currents (IgpClC‐3), while intracellular dialysis with antigen‐preabsorbed anti‐ClC‐3 Ab failed to affect IgpClC‐3. 2 Anti‐ClC‐3 Ab was used as a selective probe to examine the relationship between endogenous ClC‐3 expression and native volume‐sensitive outwardly rectifying anion channels (VSOACs) in guinea‐pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti‐ClC‐3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen‐preabsorbed anti‐ClC‐3 Ab. 3 It is suggested that endogenous ClC‐3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti‐ClC‐3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC‐3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

Soluble ST2: A new and promising activity marker in ulcerative colitis

AIM To correlate circulating soluble ST2 (sST2) levels with the severity of ulcerative colitis (UC) and serum levels of pro-inflammatory cytokines, and to demonstrate the predictive power of sST2 levels for differentiation between active and inactive UC. METHODS We recruited 153 patients: 82 with UC, 26 with Crohns disease (CD) and 43 disease controls [non-inflammatory bowel disease (IBD)]. Subjects were excluded if they had diagnosis of asthma, autoimmune diseases or hypertension. The serum levels of sST2 and pro-inflammatory cytokines [pg/mL; median (25th-75th)] as well as clinical features, endoscopic and histological features, were subjected to analyses. The sST2 performance for discrimination between active and inactive UC, non-IBD and healthy controls (HC) was determined with regard to sensitivity and specificity, and Spearmans rank correlation coefficient (r). To validate the method, the area under the curve (AUC) of receiver-operator characteristic (ROC) was determined (AUC, 95% CI) and the total ST2 content of the colonic mucosa in UC patients was correlated with circulating levels of sST2. RESULTS The serum sST2 value was significantly higher in patients with active [235.80 (90.65-367.90) pg/mL] rather than inactive UC [33.19 (20.04-65.32) pg/mL], based on clinical, endoscopic and histopathological characteristics, as well as compared with non-IBD and HC (P < 0.001). The median level of sST2 in CD patients was 54.17 (35.02-122.0) pg/mL, significantly higher than that of the HC group only (P < 0.01). The cutoff was set at 74.87 pg/mL to compare active with inactive UC in a multicenter cohort of patients. Values of sensitivity, specificity, and ability to correctly classify UC, according to activity, were 83.33%, 83.33% and 83.33%, respectively. The AUC of the ROC curve to assess the ability of this molecule to discriminate between active vs inactive UC was 0.92 (0.86-0.97, P < 0.0001). The serum levels of sST2 in patients with UC significantly correlated with endoscopic and histopathological scores (r = 0.76 and r = 0.67, P < 0.0001, respectively), and with the pro-inflammatory cytokine, tumor necrosis factor-α (r = 0.69 and r = 0.61, respectively, P < 0.0001). Interestingly, we found a direct correlation between total intestinal ST2 content and serum levels of sST2, adjusted to endoscopic activity score in patients with mild (r = 0.44, P = 0.004), moderate (r = 0.59, P = 0.002) and severe disease (r = 0.82, P = 0.002). Only patients with inactive UC showed no significant correlation (r = 0.45, P = 0.267). CONCLUSION sST2 levels correlated with disease severity and inflammatory cytokines, are able to differentiate active from inactive UC and might have a role as a biomarker.

Severe alterations in expression and localisation of α6β4 integrin in salivary gland acini from patients with Sjögren syndrome

Objectives: In salivary glands from patients with Sjögren syndrome, overexpression of laminins 1 and 5 and disorganisation of the acinar basal lamina have been reported. Laminin 5 mediates association of the basal lamina with epithelial cells by forming adhesion complexes upon interaction with α6β4 integrin. In the present work, mRNA and protein levels of α6β4 integrin were determined and its localisation in salivary glands evaluated in patients with Sjögren syndrome. Methods: Salivary glands of 12 patients with Sjögren syndrome and 8 controls were studied. The mRNA and protein levels of α6β4 were determined by semiquantitative reverse transcriptase (RT)-PCR and western blot analysis, respectively. The subcellular localisation of α6β4 and laminin were evaluated by confocal microscopy. Results: In patients, no significant differences in α6 and β4 mRNA levels were detected. However, β4 integrin protein levels were significantly lower, whereas, changes in α6, were highly variable. In controls, α6β4 was detected in the basolateral and basal surface of serous and mucous acini, respectively. In patients, alterations in α6β4 distribution were particularly dramatic for acini with strong basal lamina disorganisation. α6β4 was also detected in the cytoplasm and lateral plasma membrane in serous and mucous acini. Conclusion: Mild alterations in the basal lamina correlated with lateral redistribution of α6β4 integrin and the formation of new cell–cell adhesions that help maintain acinar organisation and promote cell survival. Conversely, in cases with severe basal lamina alterations, lateral α6β4 redistribution was no longer sufficient to maintain acinar cell survival. Thus, maintenance of equilibrium between cell–cell and cell–basal lamina attachment is required to sustain gland cell survival.

Innate Immunity Modulation by the IL-33/ST2 System in Intestinal Mucosa

Innate immunity prevents pathogens from entering and spreading within the body. This function is especially important in the gastrointestinal tract and skin, as these organs have a large surface contact area with the outside environment. In the intestine, luminal commensal bacteria are necessary for adequate food digestion and play a crucial role in tolerance to benign antigens. Immune system damage can create an intestinal inflammatory response, leading to chronic disease including inflammatory bowel diseases (IBD). Ulcerative colitis (UC) is an IBD of unknown etiology with increasing worldwide prevalence. In the intestinal mucosa of UC patients, there is an imbalance in the IL-33/ST2 axis, an important modulator of the innate immune response. This paper reviews the role of the IL-33/ST2 system in innate immunity of the intestinal mucosa and its importance in inflammatory bowel diseases, especially ulcerative colitis.

Glucocorticosteroid therapy in inflammatory bowel diseases: From clinical practice to molecular biology

Inflammatory bowel diseases (IBDs), such as ulcerative colitis and Crohn’s disease, are chronic pathologies associated with a deregulated immune response in the intestinal mucosa, and they are triggered by environmental factors in genetically susceptible individuals. Exogenous glucocorticoids (GCs) are widely used as anti-inflammatory therapy in IBDs. In the past, patients with moderate or severe states of inflammation received GCs as a first line therapy with an important effectiveness in terms of reduction of the disease activity and the induction of remission. However, this treatment often results in detrimental side effects. This downside drove the development of second generation GCs and more precise (non-systemic) drug-delivery methods. Recent clinical trials show that most of these new treatments have similar effectiveness to first generation GCs with fewer adverse effects. The remaining challenge in successful treatment of IBDs concerns the refractoriness and dependency that some patients encounter during GCs treatment. A deeper understanding of the molecular mechanisms underlying GC response is key to personalizing drug choice for IBDs patients to optimize their response to treatment. In this review, we examine the clinical characteristics of treatment with GCs, followed by an in depth analysis of the proposed molecular mechanisms involved in its resistance and dependence associated with IBDs. This thorough analysis of current clinical and biomedical literature may help guide physicians in determining a course of treatment for IBDs patients and identifies important areas needing further study.

Endoplasmic reticulum stress in autoimmune diseases: Can altered protein quality control and/or unfolded protein response contribute to autoimmunity? A critical review on Sjögren's syndrome

For many years, researchers in the field of autoimmunity have focused on the role of the immune components in the etiopathogenesis of autoimmune diseases. However, some studies have demonstrated the importance of target tissues in their pathogenesis and the breach of immune tolerance. The immune system as well as target tissue cells (plasmatic, β-pancreatic, fibroblast-like synoviocytes, thyroid follicular and epithelial cells of the lachrymal glands, salivary glands, intestine, bronchioles and renal tubules) share the characteristic of secretory cells with an extended endoplasmic reticulum (ER). The function of these cells depends considerably on a normal ER function and calcium homeostasis, so they can produce and secrete their main components, which include glycoproteins involved in antigenic presentation such as major histocompatibility complex (MHC) class I and II. All these proteins are synthesized and modified in the ER, and for this reason disturbances in the normal functions of this organelle such as protein folding, protein quality control, calcium homeostasis and redox balance, promote accumulation of unfolded or misfolded proteins, a condition known as ER stress. Autoimmune diseases are characterized by inflammation, which has been associated with an ER stress condition. Interestingly, patients with these diseases contain circulating auto-antibodies against chaperone proteins (such as Calnexin and GRP94), thus affecting the folding and assembly of MHC class I and II glycoproteins and their loading with peptide. The main purpose of this article is to review the involvement of the protein quality control and unfolded protein response (UPR) in the ER protein homeostasis (proteostasis) and their alterations in autoimmune diseases. In addition, we describe the interaction between ER stress and inflammation and evidences are shown of how autoimmune diseases are associated with an ER stress condition, with a special emphasis on the second most prevalent autoimmune rheumatic disease, Sjögrens syndrome.

Identification of Intracellular Escherichia Coli (IEC), Associated With Crohn’s Disease, in Other Intestinal Pathologies: P-260

markers and greater volatility in gut microbiota that eventuated in colitic T5KO mice having a markedly different microbiota than their non-colitic siblings, whose microbiota stabilized to a similar composition as WT mice. One striking feature of gut microbiota of colitic T5KO mice was the transient presence of high levels of Proteobacteria, and especially Enterobacteria species including E. coli, which could be observed in close proximity to the gut epithelium. Administration of a Crohn’s disease-associated E. coli strain induced chronic colitis in T5KO, but not WT, mice that persisted well past the time by which both strains of mice had largely eliminated the bacteria. Of interest, gut microbiota composition analysis revealed modification in T5KO mice compare to WT after LF82 clearance. This reveal that LF82 strain could act as an instigator able to modify microbiota to colitogenic, as suggested by analysis of LPS and flagellin loads. CONCLUSION(S): Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring Proteobacteria can drive and/or instigate chronic colitis.