Genetic Variant of DNAM-1 rs763361 C>T Is Associated with Ankylosing Spondylitis in a Mexican Population.

DNAM-1 (CD226) is an activating receptor expressed in CD8+ T cells, NK cells, and monocytes. It has been reported that two SNPs in the DNAM-1 gene, rs763361 C>T and rs727088 G>A, have been associated with different autoimmune diseases; however, the role of DNAM-1 in ankylosing spondylitis has been less studied. For this reason, we focused on the study of these two SNPs in association with ankylosing spondylitis. For this, 34 patients and 70 controls were analyzed using endpoint PCR with allele-specific primers. Our results suggest that rs763361 C>T is involved as a possible protective factor under the CT co-dominant model (OR = 0.34, 95% CI = 0.13-0.88, p = 0.022) and the CT + TT dominant model (OR = 0.39, 95% CI = 0.17-0.90, p = 0.025), while rs727088 G>A did not show an association with the disease in any of the inheritance models. When analyzing the relationships of the haplotypes, we found that the T + A haplotype (OR = 0.31, 95% CI = 0.13-0.73, p = 0.0083) is a protective factor for developing the disease. In conclusion, the CT and CT + TT variants of rs763361 C>T and the T + A haplotype were considered as protective factors for developing ankylosing spondylitis.

Uncovering the Expression Pattern of the Costimulatory Receptors ICOS, 4-1BB, and OX-40 in Exhausted Peripheral and Tumor-Infiltrating Natural Killer Cells from Patients with Cervical Cancer.

Cervical cancer (CC) poses a significant health burden, particularly in low- and middle-income countries. NK cells play a crucial role against CC; however, they can become exhausted and lose their cytotoxic capacity. This work explores the expression of costimulatory receptors (ICOS, 4-1BB, OX-40) in exhausted NK cells from CC patients. Peripheral blood and tumor biopsies were collected, and flow cytometry was used to evaluate the expression of costimulatory receptors in exhausted NK cells. There is an increase of peripheral exhausted NK cells (PD-1+TIGIT+) in CC patients; this subpopulation has a selectively increased expression of the costimulatory receptors ICOS and 4-1BB. An exhausted population is also highly increased in tumor-infiltrating NK cells, and it shows a dramatically increased expression of the costimulatory receptors ICOS (>15×) and 4-1BB (>10×) compared to peripheral NK cells. The exhausted cells, both in the periphery and in the tumor infiltrating lymphocytes (TILs), are also more likely than non-exhausted NK cell populations (PD-1-TIGIT-) to express these costimulatory receptors; increases ranging from 2.0× ICOS, 2.4× 4-1BB, and 2.6× OX-40 in CD56dim PBMCs to 1.5× ICOS, 5× 4-1BB, and 10× OX-40 in TILs were found. Our study demonstrates for the first time the increased expression of the costimulatory receptors ICOS, 4-1BB, and OX-40 in peripheral CD56dim, CD56bright, and tumor-infiltrating NK cells in CC. Targeting these receptors for stimulation could reverse exhaustion and be a promising immunotherapy strategy.

Non-fitness status of peripheral NK cells defined by decreased NKp30 and perforin, and increased soluble B7H6, in cervical cancer patients.

The NKp30 receptor is one of the three natural cytotoxic receptors reported in NK cells. This receptor is codified by the NCR3 gene, which encodes three isoforms, a consequence of the alternative splicing of exon 4. A greater expression of the three isoforms (A, B, and C), along with low levels of the NKp30 ligand B7H6, has been reported as a positive prognostic factor in different cancer types. Here, in patients with cervical cancer and precursor lesions, we report an altered immune-phenotype, characterized by non-fitness markers, that correlated with increased disease stage, from CIN 1 to FIGO IV. While overall NK cell numbers increased, loss of NKp30+ NK cells, especially in the CD56dim subpopulation, was found. Perforin levels were decreased in these cells. Decreased expression of the NKp30 C isoform and overexpression of soluble B7H6 was found in cervical cancer patients when compared against healthy subjects. PBMCs from healthy subjects downregulated NKp30 isoforms after co-culture with B7H6-expressing tumour cells. Taken together, these findings describe a unique down-modulation or non-fitness status of the immune response in cervical cancer, the understanding of which will be important for the design of novel immunotherapies against this disease.

Low-diversity microbiota and an increased metabolism of arginine and aromatic amino acids: a hallmark of hepatic encephalopathy in western Mexican patients with alcohol-associated cirrhosis.

AIMS: To evaluate the composition and functions of the gut microbiota in patients with decompensated alcohol-associated cirrhosis, with and without hepatic encephalopathy (HE). METHODS AND RESULTS: Faecal samples from 31 inpatients (20 with HE, 11 without HE), and from 18 age-balanced healthy controls (HC), were included. Microbial composition was determined by 16S rRNA amplicon sequencing and analysed using QIIME2. Metabolic pathways were inferred by PICRUSt2, and short-chain fatty acids (SCFAs) quantification was performed by gas chromatography. The gut microbiota of patients with HE was characterized by a diminished α-diversity, compared to no-HE (P < 0.01) and HC (P < 0.001) groups; ß-diversity also differed between HE vs no-HE patients (P < 0.05), and between HE vs HC (P < 0.001). In patients with HE, Escherichia/Shigella, Burkholderiales and Lactobacillales taxa predominated. In contrast, patients without HE were characterized by Veillonella and Bacteroides. Reduced levels of faecal SCFAs in both groups correlated with a depletion of beneficial taxa, such as Ruminococcus or Faecalibacterium. PICRUSt2 analysis showed both an enhanced catabolism of arginine through ammonia-producing pathways and chorismate biosynthesis in HE patients, a key precursor of aromatic amino acids. CONCLUSIONS: The gut microbiota of HE patients exhibits a proinflammatory dysbiotic profile, plus metabolic pathways that produce potentially neurotoxic byproducts.

Gut microbiota from Mexican patients with metabolic syndrome and HIV infection: An inflammatory profile.

AIM: A remarkable increase in metabolic syndrome (MetS) has occurred in HIV-infected subjects. Gut dysbiosis is involved in the pathogenesis of metabolic disorders. Therefore, the aim is to explore the profile of the gut microbiota in Mexican population with HIV infection and MetS. METHODS AND RESULTS: In all, 30 HIV-infected patients with MetS were compared to a group of 30 patients without MetS, treated with integrase inhibitors and undetectable viral load were included in the study. Stool samples were analysed by 16S rRNA next-generation sequencing. High-sensitivity C-reactive protein >3 mg L-1 and higher scores in cardiometabolic indices were associated with MetS. The group with MetS was characterized by a decrease in α-diversity, higher abundance of Enterobacteriaceae and Prevotella, as well as a dramatic decrease in bacteria producing short-chain fatty acids. Prevotella negatively correlated with Akkermansia, Lactobacillus and Anaerostipes. Interestingly, the group without MetS presented higher abundance of Faecalibacterium, Ruminococcus, Anaerofilum, Oscillospira and Anaerostipes. Functional pathways related to energy metabolism and inflammation were increased in the group with MetS. CONCLUSIONS: HIV-infected patients with MetS present a strong inflammatory microbiota profile; therefore, future strategies to balance intestinal dysbiosis should be implemented.

Positive staining of the immunoligand B7-H6 in abnormal/transformed keratinocytes consistently accompanies the progression of cervical cancer.

BACKGROUND: B7-H6 has been revealed as an endogenous immunoligand expressed in a variety of tumors, but not expressed in healthy tissues. Heretofore, no studies have been reported describing B7-H6 in women with cervical cancer. To investigate this question, our present study was conducted. RESULTS: This retrospective study comprised a total of 62 paraffinized cervical biopsies, which were distributed in five groups: low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), squamous cervical carcinoma (SCC), uterine cervical adenocarcinoma (UCAC), and a group of cervicitis (as a control for non-abnormal/non-transformed cells). Cervical sections were stained by immunohistochemistry to explore the expression of B7-H6, which was reported according to the immunoreactive score (IRS) system. We observed a complete lack of B7-H6 in LSIL abnormal epithelial cells. Interestingly, B7-H6 began to be seen in HSIL abnormal epithelial cells; more than half of this group had B7-H6 positive cells, with staining characterized by a cytoplasmic and membranous pattern. B7-H6 in the SCC group was also seen in the majority of the sections, showing the same cytoplasmic and membranous pattern. Strong evidence of B7-H6 was notably found in UCAC tumor columnar cells (in 100% of the specimens, also with cytoplasmic and membranous pattern). Moreover, consistent B7-H6 staining was observed in infiltrating plasma cells in all groups. CONCLUSIONS: B7-H6 IRS positively correlated with disease stage in the development of cervical cancer; additionally, B7-H6 scores were found to be even higher in the more aggressive uterine cervical adenocarcinoma, suggesting a possible future therapeutic target for this cancer type.

B7-H6, an immunoligand for the natural killer cell activating receptor NKp30, reveals inhibitory effects on cell proliferation and migration, but not apoptosis, in cervical cancer derived-cell lines.

BACKGROUND: Although great progress has been made in treatment regimens, cervical cancer remains as one of the most common cancer in women worldwide. Studies focusing on molecules that regulate carcinogenesis may provide potential therapeutic strategies for cervical cancer. B7-H6, an activating immunoligand expressed by several tumor cells, is known to activate NK cell-mediated cytotoxicity once engaged with its natural receptor NKp30. However, the opposite, that is, the effects in the tumor cell triggered by B7-H6 after interacting with NKp30 has not yet been well explored. METHODS: In this study, we evaluated the surface expression of B7-H6 by flow cytometry. Later, we stimulated B7-H6 positive cervical cancer derived-cell lines (HeLa and SiHa) with recombinant soluble NKp30 (sNKp30) protein and evaluated biological effects using the impedance RTCA system for cell proliferation, the scratch method for cell migration, and flow cytometry for apoptosis. Cellular localization of B7-H6 was determined using confocal microscopy. RESULTS: Notably, we observed that the addition of sNKp30 to the cervical cancer cell lines decreased tumor cell proliferation and migration rate, but had no effect on apoptosis. We also found that B7-H6 is selectively maintained in tumor cell lines, and that efforts to sort and purify B7-H6 negative or positive cells were futile, as negative cells, when cultured, regained the expression of B7-H6 and B7-H6 positive cells, when sorted and cultivated, lost a percentage of B7-H6 expression. CONCLUSIONS: Our results suggest that B7-H6 has an important, as of yet undescribed, role in the biology of the cervical tumor cells themselves, suggesting that this protein might be a promising target for anti-tumor therapy in the future.

IMMUNOLOGY OF CERVICAL CANCER.

Optimal function of the immune system allows the recognition and elimination of infected and tumor cells. However, these cells can develop mechanisms to evade the cellular immune response. In human papillomavirus (HPV) infection, dysregulation of major histocompatibility complex Class I molecules and other components of the innate immune system promote the survival of infected cells by allowing the infection to persist which, in turn, favors the development of cancer. Further, tumor cells possess inherent mechanisms designed to block the recognition and activation of cytotoxic lymphocytes: particularly, HPV proteins such as E1 and E2 and oncoproteins E5, E6, and E7 that inhibit immune mechanisms and/or stimulate the expression of immunosuppressive cytokines. These mechanisms include a decrease in receptor activation and costimulating molecules on the surface of immune cells, as well as the constitutive expression of molecules that inhibit their function, which allow HPV persistence and tumor progression. Immunotherapy-based therapeutic options are positioned as excellent candidates for the treatment of cervical cancer.

Bacterial Translocation Is Linked to Increased Intestinal IFN-γ, IL-4, IL-17, and mucin-2 in Cholestatic Rats.

Background and rationale for the study. Bacterial translocation is an important triggering factor of infection and mortality in cirrhosis. In a rat model using bile duct ligation (BDL), bacterial translocation appears within 24 h after ligation. The dynamic between TH1/TH2/TH17 cytokines and the integrity of the colonic mucosa in the context of cirrhosis is little known. This study aims to determine the link between bacterial translocation and intestinal inflammation in a cholestasis model. Additionally, alterations of the colonic mucus layer and the bacterial load were also addressed. RESULTS: Bacterial translocation detected by microbiological cultures and MALDI-TOF showed that Escherichia coli predominates in mesenteric lymph nodes of BDL rats. Intestinal bacterial load analyzed by qPCR indicates a dramatic Escherichia/Shigella overgrowth at 8 and 30 days post-BDL. IFN-γ, IL-4, and IL-17 evaluated by Western blotting were increased at 8 and 30 days in the small intestine. In the colon, in contrast, only IFN-γ was significantly increased. The colonic mucus layer and mucin-2 expression determined by Alcian blue staining and immunohistochemistry surprisingly showed an increase in the mucus layer thickness related to increased mucin-2 expression during the entire process of liver damage. Hepatic enzymes, as well as collagen I, collagen III, TNF-α, and IL-6 liver gene expression were increased. In conclusion, bacterial overgrowth associated with bacterial translocation is linked to the over-expression of IFN-γ, IL-4, IL-17 and mucin-2. These molecules might facilitate the intestinal permeability through exacerbating the inflammatory process and disturbing tight junctions, leading to the perpetuation of the liver damage.

Liver fibrosis in bile duct-ligated rats correlates with increased hepatic IL-17 and TGF-ß2 expression.

UNLABELLED: BACKGROUND AND RATIONALE FOR THE STUDY: IL-17, TGF-ß1/2 are cytokines involved in the development of kidney, pulmonary and liver fibrosis. However, their expression kinetics in the pathogenesis of cholestatic liver fibrosis have not yet been fully explored. The aim of the study was to analyze the expression of IL-17, RORγt, NKp46, TGF-ß1, and TGF-ß2 in the liver of rats with bile duct ligation (BDL). RESULTS: Hepatic IL-17A gene expression analyzed by qRT-PCR showed a dramatic increase of 350 and 10 fold, at 8 and 30 days post BDL, respectively. TGFß1 and TGFß2 gene expression significantly increased throughout the whole fibrotic process. At the protein level in liver homogenates, IL-17, TGF-ß1, and RORγt significantly increased at 8 and 30 days after BDL. Interestingly, a significant increase in the protein levels of TGF-ß2 and decrease of NKp46 was observed only 30 days after BDL. Unexpectedly, TGF-ß2 exhibited stronger signals than TGF-ß1 at the gene expression and protein levels. Histological analysis showed bile duct proliferation and collagen deposition. CONCLUSIONS: Our results suggest that pro-fibrogenic cytokines IL-17, TGF-ß1 and, strikingly, TGF-ß2 might be important players of liver damage in the pathogenesis of early and advanced experimental cholestatic fibrosis. Th17 cells might represent an important source of IL-17, while NK cell depletion may account for the perpetuation of liver damage in the BDL model.

Prolactin modulates cytokine production induced by culture filtrate proteins of M. bovis through different signaling mechanisms in THP1 cells.

The immunomodulatory functions of prolactin (PRL) are well recognized. Augmented PRL plasma levels were observed in patients with advanced tuberculosis (TB). Recently, we have reported that LPS and Mycobacterium bovis (M. bovis) induced differential expression of PRL receptor (PRLR) isoforms in THP-1 cells and bovine macrophages, respectively. The aim of this work was to determine whether PRL should be considered as a potential modulator of the signaling pathways and cytokine synthesis, induced by culture filtrate protein (CFP) from M. bovis in THP-1 monocytes. The THP-1 cells were stimulated with PRL (20ng/mL), M. bovis CFP (50µg/mL). PRLR as well as phosphorylated STAT3, STAT5, Akt1/2/3, ERK1/2 and p38 expression were evaluated by Western blot. IL1-ß, TNF-α, IL-6, IL-12, IL-8, and IL-10 concentrations were measured by ELISA. Our results demonstrated that the expression pattern of PRLR short isoforms is induced by M. bovis CFP. M bovis CFP induced phosphorylation of Akt2, ERK1/2, p38, STAT3, and STAT5 pathways. In turn, PRL only activated the JAK2/STAT3-5 signaling pathway. However, when combined both stimuli, PRL significantly increased STAT3-5 phosphorylation and downregulated Akt2, ERK1/2, and p38 phosphorylation. As expected, M. bovis CFP induced substantial amounts of IL1-ß, IL-6, TNF-α, IL-8, IL-12, and IL-10. However, the PRL costimulation considerably decreased IL1-ß, TNF-α, and IL-12 secretion, and increased IL-10 production. This results suggest that up-regulation of IL-10 by PRL might be modulating the pro-inflammatory response against mycobacterial antigens through the MAPK pathway.

An approach to the immunophenotypic features of circulating CD4⁺NKG2D⁺ T cells in invasive cervical carcinoma.

BACKGROUND: NKG2D, an activating immunoreceptor, is primarily restricted to NK cells and CD8(+) T cells. The existence of an atypical cytotoxic CD4(+)NKG2D(+) T cell population has also been found in patients with autoimmune dysfunctions. Nonetheless, contradictory evidence has categorized this population with a regulatory rather than cytotoxic role in other situations. These confounding data have led to the proposal that two distinct CD4(+)NKG2D(+) T cell subsets might exist. The immune response elicited in cervical cancer has been characterized by apparent contradictions concerning the role that T cells, in particular T-helper cells, might be playing in the control of the tumor growth. Interestingly, we recently reported a substantial increase in the frequency of CD4(+)NKG2D(+) T cells in patients with cervical intraepithelial neoplasia grade-1. However, whether this particular population is also found in patients with more advanced cervical lesions or whether they express a distinctive phenotype remains still to be clarified. In this urgent study, we focused our attention on the immunophenotypic characterization of CD4(+)NKG2D(+) T cells in patients with well-established cervical carcinoma and revealed the existence of at least two separate CD4(+)NKG2D(+) T cell subsets defined by the co-expression or absence of CD28. RESULTS: Patients with diagnosis of invasive cervical carcinoma were enrolled in the study. A group of healthy individuals was also included. Multicolor flow cytometry was used for exploration of TCR alpha/beta, CD28, CD158b, CD45RO, HLA-DR, CD161, and CD107a. A Luminex-based cytokine kit was used to quantify the levels of pro- and anti-inflammatory cytokines. We found an increased percentage of CD4(+)NKG2D(+) T cells in patients with cervical cancer when compared with controls. Accordingly with an increase of CD4(+)NKG2D(+) T cells, we found decreased CD28 expression. The activating or degranulation markers HLA-DR, CD161, and CD107a were heterogeneously expressed. The levels of IL-1beta, IL-2, TNF-alpha, and IL-10 were negatively correlated with the percentages of CD4(+)NKG2D(+) T cells in patients with cervical carcinoma. CONCLUSIONS: Taken together, our results reveal the existence of two separate CD4(+)NKG2D(+) T cell subsets defined by the co-expression or absence of CD28, the latter more likely to be present in patients with cervical cancer.

Cataloging circulating CD3+CD56+ NKT-like cells through a series of stimulating (NKG2D and DNAM-1) and inhibitory (PD-1, TIGIT, and Tim-3) immune checkpoint receptors in women diagnosed with precancerous cervical lesions or invasive cervical carcinoma.

Persistent human papillomavirus infection is associated with the development of premalignant lesions that can eventually lead to cervical cancer. In this study, we evaluated the expression of activating (NKG2D, DNAM-1) and inhibitory immune checkpoints receptors (PD-1, TIGIT, and Tim-3) in peripheral blood NKT-like (CD3+CD56+) lymphocytes from patients with cervical carcinoma (CC, n = 19), high-grade lesions (HG, n = 8), low-grade lesions (LG, n = 19) and healthy donors (HD, n = 17) using multiparametric flow cytometry. Dimensional data analysis showed four clusters within the CD3+CD56+ cells with different patterns of receptor expression. We observed upregulation of CD16 in CC and HG patients in one of the clusters. In another, TIGIT was upregulated, while DNAM-1 was downregulated. Throughout manual gating, we observed that NKT-like cells expressing activating receptors also co-express inhibitory receptors (PD-1 and TIGIT), which can affect the activation of these cells. A deeper characterization of the functional state of the cells may help to clarify their role in cervical cancer, as will the characterization of the NKT-like cells as cytotoxic CD8+ T cells or members of type I or type II NKT cells.

Intestinal Dysbiosis in Subjects with Obesity from Western Mexico and Its Association with a Proinflammatory Profile and Disturbances of Folate (B9) and Carbohydrate Metabolism.

Obesity is a public health problem with a growing prevalence worldwide. In Mexico, it is estimated that one out of three adults suffer from obesity. In these patients, the intestinal microbiota (IM) undergoes pathological changes that are associated with a dysbiotic state; however, the microbiota profile of adult subjects with obesity from western Mexico has not been described. To assess this, fecal samples were obtained from 65 participants (Obese = 38; Control = 27). The microbial composition was characterized by 16S rRNA amplicon sequencing. The IM of the group with obesity revealed a clear decrease in richness and diversity (p < 0.001), as well as a significant increase in proinflammatory bacterial groups, mainly genera belonging to the Negativicutes class, Escherichia/Shigella, and Prevotella. Likewise, an increase in short-chain fatty acid-producing bacteria was found, especially the genus Lachnoclostridium. Additionally, PICRUSt2 analysis showed a depletion of vitamin B9 metabolism and an increase in saccharolytic pathways. The IM of patients with obesity possesses a dysbiotic, proinflammatory environment, possibly contributing to lipogenesis and adiposity. Thus, assessing the IM will allow for a better understanding of the pathophysiology of metabolic diseases of high prevalence, such as obesity. These findings are described for the first time in the adult population of western Mexico.

Unraveling the non-fitness status of NK cells: Examining the NKp30 receptor and its isoforms distribution in HIV/HCV coinfected patients.

BACKGROUND: HIV/HCV coinfection is associated with a rapid progression to liver damage. Specifically, NK cell population dysregulation is of particular interest, as these cells have been shown to block HCV replication effectively and have an anti-fibrogenic activity. The NKp30 receptor is linked to tumor cell lysis and has a crucial role during viral infections. In the present study, we determined the subpopulations of NK cells based on CD56 and CD16 expression, NKp30 receptor expression, its isoforms A, B, and C, along with the cytotoxicity molecules in patients with HIV/HCV. RESULTS: evidenced by the APRI and FIB-4 indices, the HCV-infected patients presented greater liver damage than the HIV and HIV/HCV groups. The HCV group presented a decreased expression of NKp30 isoform A, and NK cell frequency was not different between groups; however, CD56brigth subpopulation, NKp30 receptor, and CD247 adaptor chain were decreased in HIV/HCV patients; further, we described increased levels of soluble IL-8, IL-10, IL-12, and IL-23 in the serum of HIV/HCV patients. CONCLUSIONS: HCV and HIV/HCV patients have multiple parameters of non-fitness status in NK cells; awareness of these dysfunctional immunological parameters in HIV/HCV and HCV patients can elucidate possible novel therapeutics directed towards the improvement of NK cell fitness status, in order to improve their function against liver damage.

New Advances in the Study of CMTM6, a Focus on Its Novel Non-Canonical Cellular Locations, and Functions beyond Its Role as a PD-L1 Stabilizer.

CMTM6 is a membrane protein that acts as a regulator of PD-L1, maintaining its expression on the cell surface, and can prevent its lysosome-mediated degradation. It is unknown if CMTM6 is present in the plasma of patients with cervical cancer, and if it has non-canonical subcellular localizations in cell lines derived from cervical cancer. Our objective was to determine whether CMTM6 is found in plasma derived from cervical cancer patients and its subcellular localization in cell lines. Patient plasma was separated into exosome-enriched, exosome-free, and total plasma fractions. The levels of CMTM6 in each fraction were determined using ELISA and Western blot. Finally, for the cellular model, HeLa, SiHa, CaSki, and HaCaT were used; the subcellular locations of CMTM6 were determined using immunofluorescence and flow cytometry. Soluble CMTM6 was found to be elevated in plasma from patients with cervical cancer, with a nearly three-fold increase in patients (966.27 pg/mL in patients vs. 363.54 pg/mL in controls). CMTM6 was preferentially, but not exclusively, found in the exosome-enriched plasma fraction, and was positively correlated with exosomal PD-L1; CMTM6 was identified in the membrane, intracellular compartments, and culture supernatant of the cell lines. These results highlight that CMTM6, in its various presentations, may play an important role in the biology of tumor cells and in immune system evasion.

High expression of prolactin receptor is associated with cell survival in cervical cancer cells.

BACKGROUND: The altered expression of prolactin (PRL) and its receptor (PRLR) has been implicated in breast and other types of cancer. There are few studies that have focused on the analysis of PRL/PRLR in cervical cancer where the development of neoplastic lesions is influenced by the variation of the hormonal status. The aim of this study was to evaluate the expression of PRL/PRLR and the effect of PRL treatment on cell proliferation and apoptosis in cervical cancer cell lines. RESULTS: High expression of multiple PRLR forms and PRLvariants of 60-80 kDa were observed in cervical cancer cell lines compared with non-tumorigenic keratinocytes evaluated by Western blot, immunofluorecence and real time PCR. Treatment with PRL (200 ng/ml) increased cell proliferation in HeLa cells determined by the MTT assay at day 3 and after 1 day a protective effect against etoposide induced apoptosis in HeLa, SiHa and C-33A cervical cancer cell lines analyzed by the TUNEL assay. CONCLUSIONS: Our data suggests that PRL/PRLR signaling could act as an important survival factor for cervical cancer. The use of an effective PRL antagonist may provide a better therapeutic intervention in cervical cancer.

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1.

BACKGROUND: The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8+ cytotoxic T cells and is classically considered absent in CD4+ T cells. However, recent studies have identified a distinctive population of CD4+ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4+ T cell-immunocompromised individuals suggests that CD4+ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4+ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4+NKG2D+ T cell expansion were also measured. RESULTS: Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4+ T cells and CD8+ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4+ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4+NKG2D+ T cells was seen in patients with CIN 1, despite the overall levels of CD4+ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4+NKG2D+ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group. CONCLUSIONS: Taken together, our results suggest that the significant increase within the CD4+NKG2D+ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion.

Recombinant p40 Protein Promotes Expression of Occludin in HaCaT Keratinocytes: A Brief Communication.

The ability of epithelial barriers to perform as the first defense line against external damage derives from tight junctions, protein complexes that block microorganisms through the paracellular space. Indeed, disturbances of barrier permeability caused by bacterial metabolites and other inflammatory stimuli are the consequence of changes in protein expression in these complexes. Postbiotics, molecules derived from bacteria with beneficial effects on the host, improve barrier function through the activation of survival pathways in epithelial cells. Lacticaseibacillus rhamnosus GG secretes the muramidase p40, which protects intestinal barriers through an EGFR-dependent pathway. In this work, we cloned, expressed, and purified the recombinant p40 protein from L. rhamnosus GR-1 to evaluate its effect on cell viability, cell cytotoxicity, TEER, and protein levels of tight junctions, as well as EGFR activation via Western blot on HaCaT keratinocytes subjected to LPS. We found a novel mutation at residue 368 that does not change the structure of p40. Our protein also reduces the LPS-induced increase in cell cytotoxicity when it is added prior to this stimulus. Furthermore, although LPS did not cause changes in barrier function, p40 increased TEER and occludin expression in HaCaT, but unlike previous work with p40 from LGG, we found that recombinant p40 did not activate EGFR. This suggests that recombinant p40 enhances epithelial barrier function through distinct signaling pathways.

Inhibition of Tumor Growth and Metastasis by Newcastle Disease Virus Strain P05 in a Breast Cancer Mouse Model.

PURPOSE: Conventional therapies and surgery remain the standard treatment for breast cancer. However, combating the eventual development of metastasis is still a challenge. Newcastle disease virus (NDV) is one of the various species of viruses under clinical evaluation as a vector for oncolytic, gene-, and immune-stimulating therapies. The purpose of this study was to evaluate the antitumor activity of a recombinant NDV (rNDV-P05) in a breast cancer murine model. METHODS: Tumors were induced by injecting the cellular suspension (4T1 cell line) subcutaneously. The virus strain P05 was applied three times at intervals of seven days, starting seven days after tumor induction, and was completed 21 days later. Determination of tumor weight, spleen index, and lung metastasis were done after sacrificing the mice. Serum levels of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, and TNF-related apoptosis-inducing ligand (TRAIL) were quantified by enzyme-linked immunosorbent assay. CD8+ infiltrated cells were analyzed by immunofluorescence. RESULTS: rNDV-P05 showed a route-of-administration-dependent effect, demonstrating that the systemic administration of the virus significantly reduces the tumor mass and volume, spleen index, and abundance of metastatic clonogenic colonies in lung tissue, and increases the inhibition rate of the tumor. The intratumoral administration of rNDV-P05 was ineffective for all the parameters evaluated. Antitumor and antimetastatic capability of rNDV-P05 is mediated, at least partially, through its immune-stimulatory effect on the upregulation of TNF-α, TRAIL, IFN-α, and IFN-γ, and its ability to recruit CD8+ T cells into tumor tissue. CONCLUSION: Systemic treatment with rNDV-P05 decreases the tumoral parameters in the breast cancer murine model.