Marjorie De la Fuente

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.

Escherichia coli isolates from inflammatory bowel diseases patients survive in macrophages and activate NLRP3 inflammasome.

Crohns disease (CD) is a multifactorial pathology associated with the presence of adherent-invasive Escherichia coli (AIEC) and NLRP3 polymorphic variants. The presence of intracellular E. coli in other intestinal pathologies (OIP) and the role of NLRP3-inflammasome in the immune response activated by these bacteria have not been investigated. In this study, we sought to characterize intracellular strains isolated from patients with CD, ulcerative colitis (UC) and OIP, and analyze NLRP3-inflammasome role in the immune response and bactericidal activity induced in macrophages exposed to invasive bacteria. For this, intracellular E. coli isolation from ileal biopsies, using gentamicin-protection assay, revealed a prevalence and CFU/biopsy of E. coli higher in biopsies from CD, UC and OIP patients than in controls. To characterize bacterial isolates, pulsed-field gel electrophoresis (PFGE) patterns, virulence genes, serogroup and phylogenetic group were analyzed. We found out that bacteria isolated from a given patient were closely related and shared virulence factors; however, strains from different patients were genetically heterogeneous. AIEC characteristics in isolated strains, such as invasive and replicative properties, were assessed in epithelial cells and macrophages, respectively. Some strains from CD and UC demonstrated AIEC properties, but not strains from OIP. Furthermore, the role of NLRP3 in pro-inflammatory cytokines production and bacterial elimination was determined in macrophages. E. coli strains induced IL-1β through NLRP3-dependent mechanism; however, their elimination by macrophages was independent of NLRP3. Invasiveness of intracellular E. coli strains into the intestinal mucosa and IL-1β production may contribute to CD and UC pathogenesis.

The IL-33/ST2 axis: Role in health and disease

IL-33, an IL-1 family member, is expressed by many cell types and can regulate gene transcription. IL-33 is released upon cell necrosis and the precursor form is enzymatically processed, and then drives inflammation as a damage-associated molecular pattern. The IL-33 receptor ST2, encoded by IL1RL1, is expressed as both a membrane-anchored receptor (ST2L) activated by IL-33, and as a soluble variant (sST2) that exhibits anti-inflammatory properties. The IL-33/ST2 axis is involved in the pathogenesis of atopic and autoimmune diseases, cancer, and central nervous system disorders. Here, we review recent findings on the role of the IL-33/ST2 axis in health and disease.

Spectrum of MLH1 and MSH2 mutations in Chilean families with suspected Lynch syndrome.

PURPOSE: Lynch syndrome is the most common inherited syndrome of colorectal cancer, caused principally by germline mutations in MLH1 and MSH2. We report our experience with genetic screening in the diagnosis of Lynch syndrome in Chile, a country previously underserved in the capacity to diagnose hereditary colorectal cancer. METHODS: Families from our Familial Colorectal Cancer Registry were selected for this study if they fulfilled either Amsterdam I/II or Bethesda criteria for classification of Lynch syndrome. Analysis of colorectal tumors from probands included a microsatellite instability study and immunohistochemical evaluation for MLH1 and MSH2. Screening of germline mutations was performed by single-strand conformation polymorphism analysis and DNA sequencing. RESULTS: A total of 21 families were evaluated, 14 meeting Amsterdam criteria and 7 meeting Bethesda criteria. Tumors in 20 families (95%) showed microsatellite instability (19 high and 1 low) and 9 of these 20 families (45%) harbored a germline mutation (7 of 13 Amsterdam and 2 of 7 Bethesda families). Of the 9 mutations identified, 6 were in MLH1 and 3 in MSH2. Two of the mutations were novel, 3 were previously found in 1 to 2 European populations, and 4 were previously found in various ethnic populations worldwide. Only 2 mutations were previously found in another Latin American population (Colombia). In our probands, colorectal cancer was located mainly (57%) in the right or transverse colon. Pedigree information from 104 family affected members of 21 studied families showed endometrial cancer to be the most frequent primary extracolonic tumor, accounting for 15.1% of total cases, followed by stomach (13.2%) and breast cancer (11.3%). Analysis of mitochondrial DNA haplotypes showed a strong Amerindian genetic component in 15 (71.4%) of the 21 families analyzed. CONCLUSION: The study of Lynch syndrome in families of different ethnic origins contributes to the definition of genetic and clinical differences among populations. Wide distribution in other ethnic populations strongly suggests varying origins of 4 the mutations found. Although cancer phenotype was consistent with those from other Latin American populations, only 2 of 9 mutations were shared with other South American populations and 2 novel mutations were found. The Chilean population is considered to be an admixture of Amerindian and European—mainly Spanish—populations, producing an ethnic group with significant genetic differences from populations previously studied.

Increased production of soluble TLR2 by lamina propria mononuclear cells from ulcerative colitis patients.

Toll-like receptor 2 (TLR2) is a type I pattern recognition receptor that has been shown to participate in intestinal homeostasis. Its increased expression in the lamina propria has been associated with the pathogenesis in inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohns disease (CD). Recently, soluble TLR2 (sTLR2) variants have been shown to counteract inflammatory responses driven by the cognate receptor. Despite the evident roles of TLR2 in intestinal immunity, no study has elucidated the production and cellular source of sTLR2 in IBD. Furthermore, an increase in the population of activated macrophages expressing TLR2 that infiltrates the intestine in IBD has been reported. We aimed first to assess the production of the sTLR2 by UC and CD organ culture biopsies and lamina propria mononuclear cells (LPMCs) as well as the levels of sTLR2 in serum, and then characterize the cell population from lamina propria producing the soluble protein. Mucosa explants, LPMCs and serum were obtained from UC, CD patients and control subjects. The level of sTLR2 was higher in conditioned media from organ culture biopsies and LPMCs from UC patients in comparison to CD and controls. Moreover, an inverse correlation between the content of intestinal and serum sTLR2 levels was observed in UC patients. Additionally, when characterizing the cellular source of the increased sTLR2 by LPMCs from UC patients, an increase in TLR2(+)/CD33(+) cell population was found. Also, these cells expressed CX3CR1, which was related to the increased levels of intestinal FKN in UC patients, suggesting that a higher proportion of TLR2(+) mononuclear cells infiltrate the lamina propria. The increased production of sTLR2 suggests that a differential regulating factor of the innate immune system is present in the intestinal mucosa of UC patients.

Metalloproteinase-Dependent TLR2 Ectodomain Shedding is Involved in Soluble Toll-Like Receptor 2 (sTLR2) Production

Toll-like receptor (TLR) 2, a type I membrane receptor that plays a key role in innate immunity, recognizes conserved molecules in pathogens, and triggering an inflammatory response. It has been associated with inflammatory and autoimmune diseases. Soluble TLR2 (sTLR2) variants have been identified in human body fluids, and the TLR2 ectodomain can negatively regulate TLR2 activation by behaving as a decoy receptor. sTLR2 generation does not involve alternative splicing mechanisms, indicating that this process might involve a post-translational modification of the full-length receptor; however, the specific mechanism has not been studied. Using CD14+ peripheral human monocytes and the THP-1 monocytic leukemia-derived cell line, we confirm that sTLR2 generation increases upon treatment with pro-inflammatory agents and requires a post-translational mechanism. We also find that the constitutive and ligand-induced release of sTLR2 is sensitive to pharmacological metalloproteinase activator and inhibitors leading us to conclude that metalloproteinase TLR2 shedding contributes to soluble receptor production. By expressing human TLR2 in ADAM10- or ADAM17-deficient MEF cells, we find both enzymes to be implicated in TLR2 ectodomain shedding. Moreover, using a deletion mutant of the TLR2 juxtamembrane region, we demonstrate that this domain is required for sTLR2 generation. Functional analysis suggests that sTLR2 generated by metalloproteinase activation inhibitsTLR2-induced cytokine production by this monocytic leukemia-derived cell line. The identification of the mechanisms involved in regulating the availability of soluble TLR2 ectodomain and cell surface receptors may contribute further research on TLR2-mediated processes in innate immunity and inflammatory disorders.

Genetic diversity and virulence determinants of Escherichia coli strains isolated from patients with Crohn's disease in Spain and Chile

Adherent-invasive Escherichia coli (AIEC) strains are genetically variable and virulence factors for AIEC are non-specific. FimH is the most studied pathogenicity-related protein, and there have been few studies on other proteins, such as Serine Protease Autotransporters of Enterobacteriacea (SPATEs). The goal of this study is to characterize E. coli strains isolated from patients with Crohns disease (CD) in Chile and Spain, and identify genetic differences between strains associated with virulence markers and clonality. We characterized virulence factors and genetic variability by pulse field electrophoresis (PFGE) in 50 E. coli strains isolated from Chilean and Spanish patients with CD, and also determined which of these strains presented an AIEC phenotype. Twenty-six E. coli strains from control patients were also included. PFGE patterns were heterogeneous and we also observed a highly diverse profile of virulence genes among all E. coli strains obtained from patients with CD, including those strains defined as AIEC. Two iron transporter genes chuA, and irp2, were detected in various combinations in 68–84% of CD strains. We found that the most significant individual E. coli genetic marker associated with CD E. coli strains was chuA. In addition, patho-adaptative fimH mutations were absent in some of the highly adherent and invasive strains. The fimH adhesin, the iron transporter irp2, and Class-2 SPATEs did not show a significant association with CD strains. The V27A fimH mutation was detected in the most CD strains. This study highlights the genetic variability of E. coli CD strains from two distinct geographic origins, most of them affiliated with the B2 or D E. coli phylogroups and also reveals that nearly 40% of Chilean and Spanish CD patients are colonized with E.coli with a characteristic AIEC phenotype.

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.

EGFR pathway subgroups in Chilean colorectal cancer patients, detected by mutational and expression profiles, associated to different clinicopathological features

Colorectal cancer is a multistep process affecting several signaling pathways including EGFR (epidermal growth factor receptor), a therapeutic target for metastatic disease. Our aim was to characterize the mutational and expression profiles of the EGFR pathway in colorectal tumors and to integrate these results according to five previously defined groups. We screened seven genes for mutations (KRAS-BRAF-PIK3CA-PIK3R1-AKT1-MAP2K1-PTEN) and six proteins (EGFR-p110α-p85α-PTEN-phosphoAKT-phosphoMEK1) by immunohistochemistry, PTEN deletion, and MSI. At least one mutated gene was observed in 68% of tumors (KRAS 45%, PIK3CA 21%, BRAF 14%, and PTEN 7%). PTEN deletion was observed in 10.7% of tumors and 19.6% were MSI-High. In all, 54% of tumors showed a high EGFR expression, 48% p110α, 4.4% phosphoAKT, and 22% phosphoMEK1; and 43% showed low PTEN expression and 22% p85α. In total, five groups of tumors were defined based on MSI, BRAF, and KRAS mutations. Three groups gather mainly early-stage tumors, whereas a fourth group is mostly conformed by advanced tumors. We described here that 71.4% of tumors from one group have a mutated PI3K/PTEN pathway, in comparison to other groups having 32%, 27%, and 25%. In addition, the five groups are differentiated by molecular features such as EGFR, p85α, p110α, and PTEN, showing variable expression among tumor groups. In conclusion, alterations on the EGFR pathway were found in a high percentage of colorectal cancer patients. Using the integration of diverse molecular markers, we ratified previous classification in an ethnic group having relevant genetic differences and living in a different environmental background, adding complementary molecular targets related to therapy.

Mutación homocigota en la línea germinal del gen MUTYH en una paciente chilena con poliposis adenomatosa familiar

Recently, MUTYH mutations have been reported to predispose to the development of polyposis. However, polyposis caused by mutations in MUTYH has been characterized as an autosomal recessive hereditary disease, different from the autosomal dominant pattern observed in polyposis caused by APC mutations. We report a 41-year-old female consulting for anemia. Colonoscopy detected multiple sessile polyps and a cecal carcinoma. The patient was operated and in the surgical piece, the tumor invaded serosa and there was lymph node involvement. Approximately 100 polyps were found. The patient received 5-fluorouracil, as adjuvant therapy. The patient had a sister (of a total of 12 brothers) with a colorectal carcinoma. The genetic study identified a homozygous mutation of the MUTYH gene, called c.340T > C, that produces an amino acid change of tyrosine for histidine called p.Y114H. The sister with colorectal cancer was a heterozygous carrier of this mutation.