Necrosis plays a role in the concentration of mycobacterial antigens in granulomas from Mycobacterium bovis naturally infected cattle.

The dynamics that develop between cells and molecules in the host against infection by Mycobacterium bovis, leads to the formation of granulomas mainly present in the lungs and regional lymph nodes in cattle. Cell death is one of the main features in granuloma organization, however, it has not been characterized in granulomatous lesions caused by M. bovis. In this study we aimed to identify the profiles of cell death in the granuloma stages and its relationship with the accumulation of bacteria. We identified necrosis, activated caspase-3, LC3B/p62 using immunohistochemistry and digital pathology analysis on 484 granulomatous lesions in mediastinal lymph nodes from 23 naturally infected cattle. Conclusions: greater amounts of mycobacterial antigens were identified in granulomas from calves compared with adult cattle. The highest percentage of necrosis and quantity of mycobacterial antigens were identified in granuloma stages (III/IV) from adults. The LC3B/p62 profile was heterogeneous in granulomas between adults and calves. Our data suggest that necrosis is associated with a higher amount of mycobacterial antigens in the late stages of granuloma and the development of autophagy appears to play an heterogeneous effector response against infection in adults and calves. These results represent one of the first approaches in the identification of cell death in the four stages of granulomas in bovine tuberculosis.

In-Silico Selection of Wound-Healing Plant Secondary Molecules and Their Pro-Healing Activities on Experimental Models.

This study explored the potential of plant-derived molecules (PDMs) as a medicinal treatment for skin wounds. To assess their healing properties, 34 potential drug molecules (PDMs) and ten therapeutic targets were subjected to molecular docking and dynamics analysis, with allantoin used as a standard compound. Although aristolochic acid had the most potent inhibitory effect, its toxicity made it unsuitable for testing on cells and mice. Therefore, ß-caryophyllene (BC) and caryophyllene oxide (BCoxide) were chosen for further testing. The results showed that BC-treated HaCat cells had significantly improved scratch area closure, and both BC and BCoxide treatment produced positive effects such as reduced dermal cellularity and mast cells, decreased levels of inflammation markers IL-6 and TNF-α, and an increase in collagen deposition in mice tissues. However, these treatments did not accelerate wound healing. This study suggests that the PDMs selected based on in-silico results have significant potential for pro-healing abilities. It is essential to conduct further research to confirm our findings.

[Regulación de la expresión de IL-33 e IL-17 por la modulación farmacológica de HIF-1 en un modelo murino de inflamación alérgica pulmonar].

Objective: To evaluate the effect of pharmacological modulation of HIF-1 on the expression of IL-33 and IL-17 in a murine model of allergic pulmonary inflam- mation (API) with different degrees of severity. Methods: 5 mice/group received ovalbumin (OVA) 1(mild), 2(moderate) or 3(severe) challenges via i.t. prior to allergen sensitization, in addition to the HIF-1 induction or inhibition groups, received EDHB (OVA+EDHB) i.p. or 2ME (OVA+2ME) i.t. respectively. Control groups received saline solution (SS) in the same way. HE (inflammatory infiltrate), PAS (mucus production) and immunohistochemical staining for HIF-1a, IL-33, IL-17 were performed, quantitatively analyzing by digital pathology. Results: We obtained different degrees of severity with a greater number of challenges, increasing the expression of HIF-1, correlating with the expression of IL-33/IL-17. Increasing or decreasing, respectively by pharmacological modulation. Conclusions: The above suggests that the high expression of HIF-1 favors the production of IL-33 and IL-17 contributing to the damage in lung tissue and the severity of the disease and these can be regulated through the modulation of HIF- 1.

Objetivo: Evaluar el efecto de la modulación farmacológica de HIF-1 en la expresión de IL-33 e IL-17 en un modelo murino de inflamación alérgica pulmonar (IAP) con diferentes grados de severidad. Métodos: 5 ratones/grupo recibieron ovoalbúmina (OVA) 1(leve), 2(moderada) o 3(severa) retos vía i.t. previa sensibilización como alergeno, además los grupos de inducción o inhibición de HIF-1a, recibieron EDHB (OVA+EDHB) i.p. o 2ME (OVA+2ME) i.t. respectivamente. Los grupos controles recibieron solución salina (SS) de igual forma. Se realizaron tinciones de HE (infiltrado inflamatorio), PAS (producción de moco) e inmunohistoquímicas de HIF-1a, IL-33, IL-17, analizando cuantitativamente por patología digital. Resultados: Obtuvimos diferentes grados de severidad a mayor número de retos, incrementando la expresión de HIF-1, correlacionando con la expresión de IL- 33/IL-17. Aumentando o disminuyendo, respectivamente por la modulación farmacológica. Conclusiones: Lo anterior sugiere que la alta expresión de HIF-1 favorece la producción de IL-33 e IL-17 contribuyendo al daño en el tejido pulmonar y la severi- dad de la enfermedad y estas pueden ser reguladas a través de la modulación de HIF-1.

Necrotic Cell Death and Inflammasome NLRP3 Activity in Mycobacterium bovis-Infected Bovine Macrophages.

Mycobacterium bovis is a facultative intracellular bacterium that produces cellular necrosis in granulomatous lesions in bovines. Although M. bovis-induced inflammation actively participates in granuloma development, its role in necrotic cell death and in bovine macrophages has not been fully explored. In this study, we evaluate the effect of M. bovis AN5 and its culture filtrate protein extract (CFPE) on inflammasome activation in bovine macrophages and its consequences on cell death. Our results show that both stimuli induce necrotic cell death starting 4 h after incubation. CFPE treatment and M. bovis infection also induce the maturation of IL-1ß (>3000 pg/mL), oligomerization of ASC (apoptosis-associated speck-like protein containing CARD), and activation of caspase-1, following the canonical activation pathway of the NLRP3 inflammasome. Inhibiting the oligomerization of NLRP3 and caspase-1 decreases necrosis among the infected or CFPE-stimulated macrophages. Furthermore, histological lymph node sections of bovines naturally infected with M. bovis contained cleaved gasdermin D, mainly in macrophages and giant cells within the granulomas. Finally, the induction of cell death (apoptosis and pyroptosis) decreased the intracellular bacteria count in the infected bovine macrophages, suggesting that cell death helps to control the intracellular growth of the mycobacteria. Our results indicate that M. bovis induces pyroptosis-like cell death that is partially related to the NLRP3 inflammasome activation and that the cell death process could control bacterial growth.

BCG∆BCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice.

The efficacy of BCG vaccines against Mycobacterium tuberculosis (Mtb) strains of lineage 2 (Beijing) in preclinical models and humans has been questioned. We have developed BCG∆BCG1419c, by deletion of BCG1419c in BCG Pasteur, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the capacity of BCG and BCG∆BCG1419c to induce autophagy in murine macrophages, modify c-di-GMP content and transcript levels of BCG1416c, encoding the enzyme responsible for c-di-GMP synthesis/degradation, and of BCG1419c, encoding the phosphodiesterase involved in c-di-GMP degradation. Furthermore, we evaluated proteomic differences in vitro and compared protection against TB produced by a low dose of the HN878-Beijing strain at 3- and 6-months post-infection. We found that BCG∆BCG1419c induced more autophagy and produced different levels of c-di-GMP as well as different transcription of BCG1416c with no expression of BCG1419c. BCG∆BCG1419c differentially produced several proteins, including some involved in interaction with host cells. Vaccination with either BCG strain led to control of bacillary burden in lungs and spleen at 3- but not 6-months post-infection, whereas it reduced pneumonic areas compared with unvaccinated controls at 6 months post-infection. Vaccination with BCG∆BCG1419c delayed progression of lung necrosis as this was observed only at 6 months post-infection. Taken together, compared with BCG, BCG∆BCG1419c increased autophagy, presented different levels of c-di-GMP and transcription of BCG1416c in vitro in a growth-phase dependent manner, modified its proteome and delayed progression of lung pathology produced by a highly virulent Beijing strain.

Mycobacterium bovis naturally infected calves present a higher bacterial load and proinflammatory response than adult cattle.

Granulomas are characteristic bovine tuberculosis lesions; studying this structure has improved our understanding of tuberculosis pathogenesis. However, the immune response that develops in granulomas of young cattle naturally infected with Mycobacterium bovis (M. bovis) has not been fully studied. Our previous work described an atypical pattern in granulomatous lesions of cattle younger than 4 months (calves) naturally infected previously M. bovis that did not correspond to the histological classification previously proposed. Histologically, granulomas from calves lack a connective tissue capsule and have fewer multinucleated giant cells (MGCs) and more acid-fast bacilli (AFB) than the classic tuberculosis lesions found in cattle older than 1 year (adults); this suggests a deficient immune response against M. bovis infection in young animals. Therefore, we used IHC and digital pathology analysis to characterize the in situ immune response of granulomas from young and adult cattle. The immunolabeling quantification showed that granulomas from calves had more mycobacteria, CD3+ cells, IFN-γ, TNF-α, and inducible nitric oxide synthase (iNOS) than those of adult cattle. Furthermore, calf granulomas showed lower immunolabeling of MAC387+, CD79+, and WC1+ cells without connective tissue surrounding the lesion and were associated with less vimentin, Alpha Smooth Muscle Actin (α-SMA), and TGF-ß compared with granulomas from adult cattle. Our results suggest that the immune responses in granulomas of cattle naturally infected with M. bovis may be age dependent. This implies that an exacerbated proinflammatory response may be associated with active tuberculosis, producing more necrosis and a lower microbicidal capacity in the granulomas of calves naturally infected with M. bovis.

RRM1 and ERCC1 as biomarkers in patients with locally advanced and metastatic malignant pleural mesothelioma treated with continuous infusion of low-dose gemcitabine plus cisplatin.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is a rare but aggressive neoplasia that usually presents at advanced stages. Even though some advances have been achieved in the management of patients with MPM, this malignancy continuous to impose a deleterious prognosis for affected patients (12-18 months as median survival, and 5-10% 5-year survival rate), accordingly, the recognition of biomarkers that allow us to select the most appropriate therapy are necessary. METHODS: Immunohistochemistry semi-quantitative analysis was performed to evaluate four different biomarkers (ERCC1, RRM1, RRM2, and hENT-1) with the intent to explore if any of them was useful to predict response to treatment with continuous infusion gemcitabine plus cisplatin. Tissue biopsies from patients with locally advanced or metastatic MPM were analyzed to quantitatively asses the aforementioned biomarkers. Every included patient received treatment with low-dose gemcitabine (250 mg/m2) in a 6-h continuous infusion plus cisplatin 35 mg/m2 on days 1 and 8 every 3 weeks as first-line therapy. RESULTS: From the 70 eligible patients, the mean and standard deviation (SD) for ERCC1, RRM1, RRM2 and hENT-1 were 286,178.3 (± 219, 019.8); 104,647.1 (± 65, 773.4); 4536.5 (± 5, 521.3); and 2458.7 (± 4, 983.4), respectively. Patients with high expression of RRM1 had an increased median PFS compared with those with lower expression (9.5 vs 4.8 months, p = < 0.001). Furthermore, high expression of RRM1 and ERCC1 were associated with an increased median OS compared with their lower expression counterparts; [(23.1 vs 7.2 months for RRM1 p = < 0.001) and (17.4 vs 9.8 months for ERCC1 p = 0.018)]. CONCLUSIONS: ERCC1 and RRM1 are useful biomarkers that predict better survival outcomes in patients with advanced MPM treated with continuous infusion of gemcitabine plus cisplatin.

16α-Bromoepiandrosterone as a new candidate for experimental diabetes-tuberculosis co-morbidity treatment.

Tuberculosis (TB) is the leading cause of death from a single bacterial infectious agent and is one of the most relevant issues of public health. Another pandemic disease is type II diabetes mellitus (T2D) that is estimated to affect half a billion people in the world. T2D is directly associated with obesity and a sedentary lifestyle and is frequently associated with immunosuppression. Immune dysfunction induced by hyperglycemia increases infection frequency and severity. Thus, in developing countries the T2D/TB co-morbidity is frequent and represents one of the most significant challenges for the health-care systems. Several immunoendocrine abnormalities are occurring during the chronic phase of both diseases, such as high extra-adrenal production of active glucocorticoids (GCs) by the activity of 11-ß-hydroxysteroid dehydrogenase type 1 (11-ßHSD1). 11-ßHSD1 catalyzes the conversion of inactive cortisone to active cortisol or corticosterone in lungs and liver, while 11-ß-hydroxysteroid dehydrogenase type 2 (11-ßHSD2) has the opposite effect. Active GCs have been related to insulin resistance and suppression of Th1 responses, which are deleterious factors in both T2D and TB. The anabolic adrenal hormone dehydroepiandrosterone (DHEA) exerts antagonistic effects on GC signaling in immune cells and metabolic tissues; however, its anabolic effects prohibit its use to treat immunoendocrine diseases. 16α-bromoepiandrosterone (BEA) is a water miscible synthetic sterol related to DHEA that lacks an anabolic effect while amplifying the immune and metabolic properties with important potential therapeutic uses. In this work, we compared the expression of 11-ßHSD1 and the therapeutic efficacy of BEA in diabetic mice infected with tuberculosis (TB) (T2D/TB) with respect to non-diabetic TB-infected mice (TB). T2D was induced by feeding mice with a high-fat diet and administering a single low-dose of streptozotocin. After 4 weeks of T2D establishment, mice were infected intratracheally with a high-dose of Mycobacterium tuberculosis strain H37Rv. Then, mice were treated with BEA three times a week by subcutaneous and intratracheal routes. Infection with TB increased the expression of 11-ßHSD1 and corticosterone in the lungs and liver of both T2D/TB and TB mice; however, T2D/TB mice developed a more severe lung disease than TB mice. In comparison with untreated animals, BEA decreased GC and 11-ßHSD1 expression while increasing 11-ßHSD2 expression. These molecular effects of BEA were associated with a reduction in hyperglycemia and liver steatosis, lower lung bacillary loads and pneumonia. These results uphold BEA as a promising effective therapy for the T2D/TB co-morbidity.

MicroRNA Profiling in Wilms Tumor: Identification of Potential Biomarkers.

Wilms tumor (WT) is the most frequently diagnosed malignant renal tumor in children. With current treatments, ~90% of children diagnosed with WT survive and generally present with tumors characterized by favorable histology (FHWT), whereas prognosis is poor for the remaining 10% of cases where the tumors are characterized by cellular diffuse anaplasia (DAWT). Relatively few studies have investigated microRNA-related epigenetic regulation and its relationship with altered gene expression in WT. Here, we aim to identify microRNAs differentially expressed in WT and describe their expression in terms of cellular anaplasia, metastasis, and association with the main genetic alterations in WT to identify potential prognostic biomarkers. Expression profiling using TaqMan low-density array was performed in a discovery cohort consisting of four DAWT and eight FHWT samples. Relative quantification resulted in the identification of 109 (48.7%) microRNAs differentially expressed in both WT types. Of these, miR-10a-5p, miR-29a-3p, miR-181a-5p, miR-200b-3p, and miR-218-5p were selected and tested by RT-qPCR on a validation cohort of 53 patient samples. MiR-29a and miR-218 showed significant differences in FHWT with low (P = 0.0018) and high (P = 0.0131) expression, respectively. To discriminate between miRNA expression FHWTs and healthy controls, the receiver operating characteristic (ROC) curves were obtained; miR-29a AUC was 0.7843. Furthermore, low expression levels of miR-29a and miR-200b (P = 0.0027 and P = 0.0248) were observed in metastatic tumors. ROC curves for miR-29a discriminated metastatic patients (AUC = 0.8529) and miR-200b (AUC = 0.7757). To confirm the differences between cases with poor prognosis, we performed in situ hybridization for three microRNAs in five DAWT and 17 FHWT samples, and only significant differences between adjacent tissues and FHWT tumors were found for miR-181a, miR-200b, and miR-218, in both total pixels and nuclear analyses. Analysis of copy number variation in genes showed that the most prevalent alterations were WTX (47%), IGF2 (21%), 1q (36%) gain, 1p36 (16%), and WTX deletion/1q duplicate (26%). The five microRNAs evaluated are involved in the Hippo signaling pathway and participate in Wilms tumor development through their effects on differentiation, proliferation, angiogenesis, and metastasis.

Live Attenuated Salmonella enterica Expressing and Releasing Cell-Permeable Bax BH3 Peptide Through the MisL Autotransporter System Elicits Antitumor Activity in a Murine Xenograft Model of Human B Non-hodgkin's Lymphoma.

The survival of patients with non-Hodgkin's lymphoma (NHL) has substantially improved with current treatments. Nevertheless, the appearance of drug-resistant cancer cells leads to patient relapse. It is therefore necessary to find new antitumor therapies that can completely eradicate transformed cells. Chemotherapy-resistant cancer cells are characterized by the overexpression of members of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein family, such as Bcl-XL, Bcl-2, and Mcl-1. We have recently shown that peptides derived from the BH3 domain of the pro-apoptotic Bax protein may antagonize the anti-apoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. In this study, we investigated the feasibility of releasing this peptide into the tumor microenvironment using live attenuated Salmonella enterica, which has proven to be an ally in cancer therapy due to its high affinity for tumor tissue, its ability to activate the innate and adaptive antitumor immune responses, and its potential use as a delivery system of heterologous molecules. Thus, we expressed and released the cell-permeable Bax BH3 peptide from the surface of Salmonella enterica serovar Typhimurium SL3261 through the MisL autotransporter system. We demonstrated that this recombinant bacterium significantly decreased the viability and increased the apoptosis of Ramos cells, a human B NHL cell line. Indeed, the intravenous administration of this recombinant Salmonella enterica elicited antitumor activity and extended survival in a xenograft NHL murine model. This antitumor activity was mediated by apoptosis and an inflammatory response. Our approach may represent an eventual alternative to treat relapsing or refractory NHL.

Expression of YY1 in Wilms tumors with favorable histology is a risk factor for adverse outcomes.

AIM: To investigate the role of the transcription factor YY1 in Wilms tumor (WT). PATIENTS & METHODS: We measured YY1 expression using tissue microarray from patients with pediatric renal tumors, mainly WT and evaluated correlations with the predicted clinical evolution. YY1 expression was measured using immunohistochemical and protein expression was determined by digital pathology. RESULTS & CONCLUSION: YY1 significantly increased in WT patients. In addition, an increase in YY1 expression had a greater risk of adverse outcomes in WT patients with favorable histology. YY1 expression was higher in the blastemal component of tumors, and high nuclear expression positively correlated with metastasis. YY1 may be considered as a metastasis risk factor in WT.

Dual role of hypoxia-inducible factor 1 α in experimental pulmonary tuberculosis: its implication as a new therapeutic target.

AIM: Investigate the role of hypoxia-inducible factor-1α (HIF-1α) in pulmonary tuberculosis (TB). METHODS & RESULTS: A model of progressive pulmonary TB in BALB/c mice, immunohistochemistry and digital pathology were used. High HIF-1α expression was observed during early TB in activated macrophages. During late TB, even higher HIF-1α expression was observed in foamy macrophages, which are resistant to apoptosis. Blocking HIF-1α during early infection with 2-methoxyestradiol worsened the disease, while during late TB, it induced macrophage apoptosis and decreased bacillary loads. CONCLUSION: HIF-1α has a dual role in experimental TB. This finding could have therapeutic implications because combined treatment with 2-methoxyestradiol and antibiotics appeared to eliminate mycobacteria more efficiently than conventional chemotherapy during advanced disease.

Exercise Induces White Adipose Tissue Browning Across the Weight Spectrum in Humans.

While the effect of exercise on white adipose tissue browning and metabolic improvement in rodents is clear, there are few studies in humans with inconclusive results. Thus, the aim of the study was to assess whether an exercise intervention promotes subcutaneous adipose tissue browning in humans, and whether this response is associated with metabolic improvement in three groups of individuals defined by body mass index (BMI) (kg/m2). Sedentary adult subjects with different BMI were enrolled in a 12-week bicycle-training program (3 times per week, intensity 70-80% HRmax). Brown and beige gene expression in subcutaneous adipose tissue (scWAT) biopsies, and serum glucose, insulin, lipid, adipokine, and myokine levels were compared before and after the exercise intervention. Thirty-three non-diabetic subjects (mean age 30.4 ± 4.6 years; 57.57% female; 13 normal weight, 10 overweight and 10 with obesity) completed the exercise intervention. Without any significant change in body composition, exercise improved several metabolic parameters, most notably insulin resistance and particularly in the overweight group. Circulating adiponectin, apelin, and irisin exercise-induced changes predicted 60% of the insulin sensitivity improvement. After exercise UCP1, TBX1, CPT1B scWAT expression significantly increased, along with P2RX5 significant positive staining. These changes are compatible with scWAT browning, however, they were not associated with glucose metabolism improvement. In conclusion, 12-weeks of exercise training produced brown/beige gene expression changes in abdominal scWAT of non-diabetic individuals with different BMI, which did not contribute to the metabolic improvement. However, this result should not be interpreted as a lack of effect of browning on metabolic parameters. These findings suggest that a bigger effect is needed and should not preclude the development of more effective strategies of browning. Furthermore, exercise-induced changes in adiponectin, apelin, and irisin predicted insulin sensitivity improvement, supporting the important role of adipokines and myokines in metabolism homeostasis.

Association between nuclear expression of retinoic acid receptor alpha and beta and clinicopathological features and prognosis of advanced non-small cell lung cancer.

BACKGROUND: Transcription factors such as retinoic acid receptor alpha (RARα) and beta (RARß) and Yin Yang 1 (YY1) are associated with the progression of non-small cell lung cancer (NSCLC). In particular, a lack of RARß expression is associated with NSCLC development. The aim of this study was to analyze the expression of RARα, RARß and YY1 and their relationship with prognosis in patients with advanced NSCLC. METHODS: The expression of RARα, RARß and YY1 was assessed by immunohistochemistry and quantitative computerized image software. RESULTS: Eighty-five patients treated with platinum-based chemotherapy were included in the analysis. The mean and standard deviation of the nuclear expression of RARα, RARß and YY1 were 184.5 ± 124.4, 18 ± 27 and 16.6 ± 20.5, respectively. The nuclear expression of RARß was associated with the nuclear expression of YY1 (R 2 = 0.28; p value < 0.0001). Patients with high nuclear expression of YY1 were likely to be non-smokers (61.9 vs 40.5 %). Median progression-free survival (PFS) was 5.9 months (3.48-8.28). Low expression of RARα was independently associated with worse PFS following chemotherapy (10.3 vs 5.46 months p = 0.040). Median overall survival (OS) was 15.6 months (4.5-26.7), and lower nuclear expression of RARß was independently associated with shorter OS (27.5 vs 8.7 months; p = 0.037). CONCLUSION: Our study suggests that the loss of RARs is associated with a worse prognosis and these receptors could be a potential molecular target for NSCLC.

A novel role of Yin-Yang-1 in pulmonary tuberculosis through the regulation of the chemokine CCL4.

Mycobacterium tuberculosis (M. tb) is the etiological agent of pulmonary tuberculosis (TB); this disease remains a worldwide health problem. Yin-Yang-1 (YY1) plays a major role in the maintenance and progression of some pulmonary diseases, including pulmonary fibrosis. However, the role of YY1 in TB remains unknown. The aim of this study was to elucidate the role of YY1 in the regulation of CCL4 and its implication in TB. We determined whether YY1 regulates CCL4 using reporter plasmids, ChIP and siRNA assays. Immunohistochemistry and digital pathology were used to measure the expression of YY1 and CCL4 in a mouse model of TB. A retrospective comparison of patients with TB and control subjects was used to measure the expression of YY1 and CCL4 using tissue microarrays. Our results showed that YY1 regulates the transcription of CCL4; moreover, YY1, CCL4 and TGF-ß were overexpressed in the lung tissues of mice with TB during the late stages of the disease and the tissues of TB patients. The expression of CCL4 and TGF-ß correlated with YY1 expression. In conclusion, YY1 regulates CCL4 transcription; moreover, YY1 is overexpressed in experimental and human TB and is positively correlated with CCL4 and TGF-ß expression. Therefore, treatments that decrease YY1 expression may be a new therapeutic strategy against TB.

Diminished expression of CRHR2 in human colon cancer promotes tumor growth and EMT via persistent IL-6/Stat3 signaling.

BACKGROUND & AIMS: Chronic inflammation promotes development and progression of colorectal cancer (CRC). We explored the distribution of Corticotropin-Releasing-Hormone (CRH)-family of receptors and ligands in CRC and their contribution in tumor growth and oncogenic EMT. METHODS: mRNA expression of CRH-family members was analyzed in CRC (N=56) and control (N=46) samples, 7 CRC cell lines and normal NCM460 cells. Immunohistochemical detection of CRHR2 was performed in 20 CRC and 5 normal tissues. Cell proliferation, migration and invasion were compared between Urocortin-2 (Ucn2)-stimulated parental and CRHR2-overexpressing (CRHR2+) cells in absence or presence of IL-6. CRHR2/Ucn2-targeted effects on tumor growth and EMT were validated in SW620-xenograft mouse models. RESULTS: CRC tissues and cell lines showed decreased mRNA and protein CRHR2 expression compared to controls and NCM460, respectively. The opposite trend was shown for Ucn2. CRHR2/Ucn2 signaling inhibited cell proliferation, migration, invasion and colony formation in CRC-CRHR2+ cells. In vivo, SW620-CRHR2+ xenografts showed decreased growth, reduced expression of EMT-inducers and elevated levels of EMT-suppressors. IL-1b, IL-6 and IL-6R mRNAs where diminished in CRC-CRHR2+ cells, while CRHR2/Ucn2 signaling inhibited IL-6-mediated Stat3 activation, invasion, migration and expression of downstream targets acting as cell cycle- and EMT-inducers. Expression of cell cycle- and EMT-suppressors was augmented in IL-6/Ucn2-stimulated CRHR2+ cells. In patients, CRHR2 mRNA expression was inversely correlated with IL-6R and vimentin levels and metastasis occurrence, while positively associated with E-cadherin expression and overall survival. CONCLUSIONS: CRHR2 downregulation in CRC supports tumor expansion and spread through maintaining persistent inflammation and constitutive Stat3 activation. CRHR2low CRC phenotypes are associated with higher risk for distant metastases and poor clinical outcomes.

Inhibition of tumor progression during allergic airway inflammation in a murine model: significant role of TGF-ß.

INTRODUCTION: TGF-ß is an important mediator of pulmonary allergic inflammation, and it has been recently reported to be a potential inhibitor of lung tumor progression. The correlation between cancer and allergic inflammatory diseases remains controversial. Thus, the aim of the present study was to evaluate the effects of pulmonary allergic inflammation and in particular the role of TGF-ß on cancer progression. METHODS: Cancer cells were implanted in a BALB/c mice model of allergic airway inflammation, and tumor growth was measured. Apoptosis was evaluated by TUNEL assay, and TGF-ß was measured by ELISA. Expression of proliferating cell nuclear antigen, TGF-ß, TGF-ß receptors I and II, phospho-Smad2 and phospho-Smad4 was evaluated by immunohistochemistry and quantified using digital pathology. The effect of a TGF-ß activity inhibitor and recombinant TGF-ß on tumor growth was analyzed. The effect of exogenous TGF-ß on cell proliferation and apoptosis was evaluated in vitro. RESULTS: Mice with allergic airway inflammation exhibited decreased tumor volumes due to cell proliferation inhibition and increased apoptosis. TGF-ß was increased in the sera and tumor tissues of allergic mice. TGF-ß activity inhibition increased tumor progression in allergic mice by enhancing proliferation and decreasing apoptosis of tumor cells. The administration of TGF-ß resulted in reduced tumor growth. CONCLUSION: This study is the first to establish an inverse relationship between allergic airway inflammation and tumor progression. This effect appears to be mediated by TGF-ß, which is overexpressed in tumor cells during pulmonary allergic inflammation. This study indicates that TGF-ß is a potential target for antitumor therapy.

Hypoxia inducible transcription factor (HIF-1) regulates CXCL13 expression: Clinical implications in pediatric non-Hodgkin's lymphoma

Background: Lymphomas are B and/or T cell clonal neoplasms in various states of differentiation, characteristically compromising lymph nodes. They are constituted by B and T lymphocytes that reach the node by chemokine-mediated recruitment including CXCL13. Hypoxia-inducible transcription factor (HIF-1α) plays a role in cellular adaptation to oxygen concentration changes. It also regulates expression of chemokines such as CXCL12, CCL20, and CCL5 as well as some of their receptors such as CCR7 and CXCR4. Methods: We performed in silico analysis of the CXCL13 promoter, pharmacologic modulation of HIF-1α activity and, using reporter plasmids, site-directed mutation and DNA-protein interaction analysis we analyzed the relation between HIF-1α activity and CXCL13 expression. Moreover, we did tissue microarray and immunohistochemistry to see the expression of HIF-1α and CXCL13. Results: This study detected three possible HIF-1α binding sites suggesting that this chemokine may be regulated by the CXCL13 transcription factor. We showed that CXCL13 expression is directly dependent, whereby an increase in HIF-1α activity increases CXCL13 expression and decreased HIF-1α activity in turn decreases CXCL13 expression. We proved that HIF-1α transcriptionally regulates the expression of CXCL13 in a direct manner. We established that HIF-1α and CXCL13 are greatly overexpressed in the most aggressive pediatric lymphomas. Conclusions: For the first time, this study showed that HIF-1α directly regulates transcriptional CXCL13 and that both proteins are overexpressed in the most aggressive forms of pediatric lymphoma. This suggests that they may play a significant role in the pathogenesis of pediatric non-Hodgkin's lymphoma.

Virulence and immune response induced by Mycobacterium avium complex strains in a model of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice.

The genus Mycobacterium comprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies with Mycobacterium tuberculosis. The biological differentiation between M. tuberculosis and nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not. Mycobacterium avium complex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after the M. tuberculosis complex. Here, we evaluated the virulence and immune response of M. avium subsp. avium and Mycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue.