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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

Aim Intestinal dysfunction in cirrhosis patients is linked to death by bacterial infections. Currently, there is no effective therapy for this complication. This study aims to evaluate butyrate, a novel postbiotic, on the intestinal inflammatory response, tight junction proteins, and the microbiota in the cholestasis model. Methods and results Wistar rats underwent 15 days of bile duct ligation (BDL). We administered butyrate at a concentration of 1%. The BDL group did not receive treatment. The results showed that butyrate could significantly reduce pro-inflammatory cytokines (IL-17A, IFN-γ, TNF- α) in the ileum and colon while promoting IL-10 expression in the colon. Moreover, it significantly promotes tight junction protein (cld-1, occludin, and ZO-1) expression in the ileum. A similar effect was observed in the colon except for ZO-1. Additionally, butyrate limited taxa diversity loss and promoted probiotic genera expansion such as Lachnospira, Prevotella, and Lactobacillus. The increase of Turicibacter and Clostridiaceae distinguished the BDL group. Conclusions Butyrate is effective in regulating the inflammatory response, tight junction proteins, and limits bacterial diversity loss. Significance and impact of study This research reveals that butyrate could represent an interesting postbiotic metabolomic intervention for intestinal epithelium dysfunction in liver disease.

Summary Immune checkpoint therapy to reverse natural killer (NK) and T cell exhaustion has emerged as a promising treatment in various cancers. While anti-programmed cell death 1 (PD-1) pembrolizumab has recently gained Food and Drug Administration (FDA) approval for use in recurrent or metastatic cervical cancer, other checkpoint molecules, such as T cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) and T cell immunoglobulin and mucin-domain containing-3 (Tim-3), have yet to be fully explored in this disease. We report expression of TIGIT, Tim-3 and PD-1 on subsets of peripheral blood NK (CD56dim/negCD16bright/dim/neg and CD56brightCD16dim/neg) and T cells. The percentages of these cells were increased in women with cervical cancer and pre-malignant lesions. PD-1+ NK and T cells were likely to co-express TIGIT and/or Tim-3. These cells, with an apparently ‘exhausted’ phenotype, were augmented in patients. A subset of cells were also natural killer group 2 member D (NKG2D)- and DNAX accessory molecule 1 (DNAM-1)-positive. PD-1int and PD-1high T cells were notably increased in cervical cancer. Soluble programmed cell death ligand 1 (PD-L1) was higher in cancer patient blood versus healthy donors and we observed a positive correlation between sPD-L1 and PD-1+ T cells in women with low-grade lesions. Within the cancer group, there were no significant correlations between sPD-L1 levels and cervical cancer stage. However, when comparing cancer versus healthy donors, we observed an inverse association between sPD-L1 and total T cells and a correlation between sPD-L1 and CD56dim NK cells. Our results may show an overview of the immune response towards pre-cancerous lesions and cervical cancer, perhaps giving an early clue as to whom to administer blocking therapies. The increase of multiple checkpoint markers may aid in identifying patients uniquely responsive to combined antibody therapies.

Background HIV/HCV coinfection is associated with a rapid progression to liver damage. Specifically, the dysregulation of NK cell populations is of special interest, as NK cells have been shown to effectively block HCV replication as well as an anti-fibrogenic activity. The NKp30 receptor has been linked to tumor cell lysis and has a crucial role during viral infections. In the present study, we determine the subpopulations of NK cells and the expression of NKp30 receptor and cytotoxicity molecules in patients with HIV/HCV. Results HIV/HCV coinfected patients present greater liver damage than the HIV control group, using the APRI and FIB-4 indices. NK cells frequency was decreased in the HIV/HCV group compared to the HIV group. CD56brigth cell frequency was increased and CD56dim was decreased in HIV/HCV patients. NKp30 expression was higher in CD56brigth subpopulation and lower in CD56dim subpopulation in the HIV/HCV group. However, the expression of the ζ chain (p dim NKp30 + cells from HIV/HCV patients. Conclusions The expansion of the highly dysfunctional CD56brigth subpopulation and the low expression of cytotoxicity markers in CD56dim cells reflects an altered activity in NK cells of coinfected patients, which could contribute to the accelerated progression of liver damage.

Neutrophils represent the first line of host cellular defense against various pathogens. The most recently described microbicidal mechanism of these cells is the release of neutrophil extracellular traps (NET). Currently, a wide range of chemical and biological stimuli are known to induce this response; however, the effect of short-chain fatty acids (SCFAs) on the induction of NET is still unknown. SCFAs are produced mainly by bacterial fermentation of dietary fiber and are found in host tissues and blood. This study aimed to determine whether physiological levels of SCFAs can induce the formation of NET. Previously reported concentrations of SCFAs (as found in the colonic lumen and peripheral blood in postprandial and basal states) were used to stimulate the neutrophils. In order to determine the signaling pathway utilized by SCFAs, we tested the inhibition of the Free Fatty Acid 2 Receptor (FFA2R) expressed in neutrophils using CATPB, the inhibitor of FFA2R, genistein, an inhibitor of the downstream Gα/q11 proteins and DPI, an inhibitor of the NADPH oxidase complex. The SCFAs at colonic intestinal lumen concentrations were able to induce the formation of NET, and when tested at concentrations found in the peripheral blood, only acetic acid at 100 μM (fasting equivalent) and 700 μM (postprandial equivalent) was found to induce the formation of NET. The administration of the competitive inhibitor against the receptor or blockade of relevant G protein signaling and the inhibition of NADPH oxidase complex decreased NET release. SCFAs stimulate NET formation in vitro and this effect is mediated, in part, by the FFA2R.

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.

Introduction. The present study aimed to elucidate the potential antifibrotic effects of pinocembrin (PIN), a flavanone found abundantly in honey and propolis, by studying its effect on different oxidative stress, inflammatory and fibrosis markers in an experimental model of CCl4-induced liver fibrosis.Material and methods. PIN (20 mg/kg) was given orally 3 times/week for 6 consecutive weeks alternating with CCl4 (0.5 mL/kg, 1:1 mixture with corn oil, i. p.) twice weekly. Different hepatotoxicity indices, oxidative stress, inflammatory and liver fibrosis markers were assessed.Results. PIN significantly restored liver transaminases and total cholesterol to normal levels. Also, PIN ameliorated oxidative stress injury evoked by CCl4 as evidenced by inhibition of reduced glutathione depletion and lipid peroxidation as well as elevation of antioxidant enzyme superoxide dismutase (SOD). Further, PIN up-regulated the nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), thereby inducing the expression and activity of the cytoprotective enzyme hemeoxygenase-1 (HO-1). Moreover, PIN alleviated pro-inflammatory cytokines such as TNF-α via inhibiting nuclear factor-KB (nF-kB) activation. As markers of fibrosis, collagen and α-SMA expression increased markedly in the CCl4 group and PIN prevented these alterations. In addition, PIN down-regulated TGFβ1 and p-Smad2/3, thereby inhibiting TGFβ1/Smad signaling pathway.Conclusion. These results suggest that PIN possess potent antifibrotic effects that can be explained on its antioxidant properties. It ameliorates oxidative stress and inflammation during induction of fibrogenesis via its ability to augment cellular antioxidant defenses, activating Nrf2-mediated HO-1 expression and modulating NF-KB and TGF-β1/Smad signaling pathway.

Excess of visceral adipose tissue (VAT) characteristic of obesity leads to a proinflammatory state disrupting the insulin signaling pathway, triggering insulin resistance (IR) and inflammation, the main processes contributing to obesity comorbidities. Ursolic acid (UA), a pentacyclic triterpenoid occurring in a variety of plant foods, exhibits anti-inflammatory properties. The aim of this study was to evaluate UA effects on IR, hyperinsulinemia, and inflammation in experimental diet-induced obesity. Forty male Wistar rats were randomly assigned to eight groups (

ADAM10 has been implicated in the progression of various solid tumors. ADAM10 regulates the cleavage of the FasL ectodomain from the plasma membrane of different cell types, generating the soluble FasL fragment (sFasL). Currently, there are few studies in oral squamous cell carcinoma (OSCC) that correlate levels of ADAM10 and FasL in the tumor microenvironment with clinical parameters of the disease. To determine the expression of ADAM10, Fas, FasL and sFasL in patients with OSCC and its association with TNM stage. Twenty-five patients with OSCC and 25 healthy controls were included. Biopsies of tumor tissue from patients with OSCC and buccal mucosa in controls were obtained. ADAM10, Fas, and FasL were analyzed by Western blotting. sFasL was quantified by ELISA. ADAM10 and Fas decreased significantly in OSCC compared with controls. Relatedly, within the OSCC group, Fas and ADAM10 decreased in accordance with tumor disease stage; in stages I/II, as well as in tumors of smaller diameter (T1-T2), ADAM10 showed higher levels when compared to patients with T3-T4 tumors and in stage III-IV. FasL in the tumor microenvironment and serum FasL showed no significant differences between both groups. Levels of complete FasL and cleaved FasL were positively correlated in controls; this correlation is preserved in patients with tumors in early stages (I-II), but is lost in later stage (III-IV). The dysregulation of ADAM10, Fas and FasL could be useful indicators of the progression and severity of OSCC.

Background and rationale for the study. IL-17, TGF-β1/2 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-β1, and TGF-β2 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. TGFβ1 and TGFβ2 gene expression significantly increased throughout the whole fibrotic process. At the protein level in liver homogenates, IL-17, TGF-β1, and RORγt significantly increased at 8 and 30 days after BDL. Interestingly, a significant increase in the protein levels of TGF-β2 and decrease of NKp46 was observed only 30 days after BDL. Unexpectedly, TGF-β2 exhibited stronger signals than TGF-β1 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-β1 and, strikingly, TGF-β2 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.

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-β, 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-β, IL-6, TNF-α, IL-8, IL-12, and IL-10. However, the PRL costimulation considerably decreased IL1-β, 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.

BACKGROUND 3,3'-Diindolylmethane (DIM) is a condensation product of indole-3-carbinol, a glucosinolate naturally occurring in Brassica genus vegetables. The antiinflammatory properties of DIM through the inhibition of NF-κB, as well as its ameliorating effects on glucose tolerance and hyperglicemic states, have been described. A subclinical proinflammatory profile resultant from the interaction of adipocytes and macrophages has been reported in obesity, affecting the insulin signaling pathway, contributing to insulin resistance. OBJECTIVE The aim of this study was to evaluate the effect of DIM on proinflammatory cytokines and phosphorylation of IRS-1 pY612 and Akt-1/PKB pT308 in an obesity-induced inflammation model. METHODS Differentiated 3T3-L1 adipocytes were co-cultured with RAW 264.7 macrophages and exposed to 20 µM, 40 µM and 60 µM DIM for 24 h followed by 100 nM insulin for 20 min. MCP-1, IL-6 and TNFα were quantified in the supernatant through individual ELISAs. Adipocyte lysates were used to determine the relative expression of the proinflammatory mediators by qPCR, and the phosphorylation of IRS-1 pY612 and Akt-1/PKB pT308 proteins by western blot analysis. RESULTS DIM significantly (p

B7H6, an endogenous ligand expressed on tumor cell surfaces, triggers NKp30-mediated activation of human NK cells. In contrast, the release of soluble B7H6 has been proposed as a novel mechanism by which tumors might evade NK cell-mediated recognition. Since NK cells are critical for the maintenance of early pregnancy, it is not illogical that soluble B7H6 might also be an important factor in directing NK cell activity during normal pregnancy. Thus, this study was focused on the characterization of soluble B7H6 during the development of normal pregnancy. Serum samples were obtained from healthy pregnant women who were experiencing their second pregnancies (n=36). Additionally, 17 of these pregnant participants were longitudinally studied for the presence of B7H6 during their second and third trimesters. Age-matched healthy non-pregnant women served as controls (n=30). The presence of soluble B7H6 was revealed by Western blotting. A further characterization was performed using an immunoproteomic approach based on 2DE-Western blotting combined with MALDI-MS. The results show that sera from all pregnant women were characterized by the presence of two novel isoforms of B7H6, both with lower MW than the reported of 51kDa. These isoforms were either a heavy (∼37kDa) or a light isoform (∼30kDa) and were mutually exclusive. N-glycosylation did not completely explain the different molecular weights exhibited by the two isoforms, as was demonstrated by enzymatic deglycosylation with PNGase F. The confirmation of the identity and molecular mass of each isoform indicates that B7H6, while maintaining the C- and N-termini, is most likely released during pregnancy by a mechanism distinct from proteolytic cleavage. We found that both isoforms, but mainly the heavier B7H6, were released via exosomes; and that the lighter isoform was also released in an exosome-free manner that was not observed in the heavy isoform samples. In conclusion, we find that soluble B7H6 is constitutively expressed during pregnancy and that, moreover, the soluble B7H6 is present in two new isoforms, which are released by exosomal and exosome-free mechanisms.

Prolactin (PRL) is a hormone–cytokine that has been involved in autoimmunity due to its immunoregulatory and lymphoproliferative effects. It is produced by various extrapituitary sites including immune cells, under control of a superdistal promoter that contains a single nucleotide polymorphism − 1149 G/T previously associated with rheumatoid arthritis (RA) susceptibility in European population. The aim of this study was to investigate the association of the extrapituitary PRL − 1149 G/T promoter polymorphism with clinical parameters, clinical activity and disability indices in RA patients from Western Mexico and to analyze the PRL mRNA expression according to the PRL − 1149 G/T promoter polymorphism in total leucocytes from RA patients and controls. We conducted a case–control study that included 258 RA patients and 333 control subjects (CS). The DNA samples were genotyped using the PCR–RFLP method and the PRL mRNA expression was determined by quantitative real time PCR. PRL serum levels and antibodies to cyclic citrullinated peptides (anti-CCP) were measured with ELISA. We found significant differences in the genotype (p = 0.022) and allelic (p = 0.046) distribution of the polymorphism between RA patients and control subjects. According to the dominant genetic model, there is an association between the T allele (GT + TT genotypes) and decreased RA susceptibility in comparison to the G allele carriers (GG genotype) (OR 0.64, 95% CI 0.45–0.92; p = 0.011). The T allele carriers (GT + TT genotypes) had lower titers of anti-CCP antibodies in comparison to the G allele carriers (GG genotype) (median, 66 U/mL vs. 125 U/mL; p = 0.03). Furthermore, the GG homozygotes had higher PRL mRNA expression in comparison to the GT heterozygotes, and this latter with respect to the TT homozygotes, in both groups (RA: 1 > 0.72 > 0.19; CS: 1 > 0.54 > 0.28). However, PRL serum levels were similar in both groups. Our results suggest that the PRL − 1149 T allele is a genetic marker for decreased RA susceptibility and is associated with lower titers of anti-CCP antibodies in Mexican population. We also suggest influence of genotype upon PRL mRNA expression.

CD28 is well characterized as an essential co-stimulatory receptor critical for activation, proliferation and survival processes in CD4

Background and Aim Development of hepatic cirrhosis involves oxidative stress, inflammation, hepatic stellate cells (HSC)s activation and fibrosis. On the other hand, quercetin, a natural flavonoid is a potent antioxidant and activator of superoxide dismutase and catalase. The aim was to determinate the effect of quercetin on HSCs and development of hepatic fibrosis. Methods Wistar male rats were chronically intoxicated with CCl4 for 8 weeks and concomitantly treated with 100 mg/kg per day of quercetin. Oxidative state, inflammation and fibrosis were evaluated. Effect of quercetin on apoptosis of HSC was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling reaction. Results Sixty percent of reduction in fibrosis index was observed with quercetin treatment compared with control animals. Considerable reduction on hepatic enzymes was detected in the quercetin group. Expression of pro-fibrotic genes (transforming growth factor-β [TGF-β], Collagen 1α [Col-1α] and connective tissue growth factor [CTGF]) were decreased by quercetin. Quercetin increased gene expression and functional activity of antioxidant enzymes superoxide dismutase and catalase. Inflammatory index was highly reduced as determined by H-E staining and pro-inflammatory cytokines expression and nuclear factor-κB activation were also inhibited. A significant reduction of 65% on activated HSC number was detected when rats were treated with quercetin. Quercetin also induced activation of matrix metalloproteinases MMP2 and MMP9 contributing to decreased index of fibrosis. Conclusions Treatment with quercetin reduces oxidation and inflammation and also prevents liver fibrosis, through induction of HSC apoptosis and activation of MMPs.

Capillarization of the sinusoid impedes the clearance of neurotoxic substances in liver fibrosis. These events may result in hepatic encephalopathy. Neurological and hepatic features of rats after bile duct ligation (BDL) supplemented with Manganese (BDL+Mn(2+)) were examined. The 4-week-old BDL rats had elevated levels of ammonia and were concomitantly fed with 1 mg ml(-1) of MnCl(2) in drinking water (BDL/Mn(+2)). Five out of fifteen rats were killed and the serum, liver and brain tissue (striatum and substantia nigra) were recovered. Of the remaining BDL/Mn(+2)-cirrhotic animals (n=10), five were injected with a combination of Adenovirus-human plasminogen activator (Ad-huPA) and Adenovirus-matrix metalloproteinase-8 (Ad-MMP-8) (3 × 10(11)+1.5 × 10(11) vector particles per kg), and five with 4.5 × 10(11) vector particles per kg of Adenovirus-β-galactosidase (Ad-β-Gal). This treatment was carried on for 10 days. The BDL/Mn(+2) rats displayed tremor, rigidity and gait abnormalities, which improved notably with combinatorial gene therapy, as well as motor coordination. Liver fibrosis was evidently less after treatment with Ad-huPA+Ad-MMP-8 (25%). In the brain (striatum), Ad-huPA+Ad-MMP-8 treatment rendered higher concentrations of dopamine compared with Ad-β-Gal-treated encephalopathic rats (210 and 162 ng g(-1) of tissue, respectively). The BDL/Mn(+2) animals and controls treated with Ad-β-Gal showed abnormal morphology in astrocytes (gliosis) in striatum and substantia nigra, in which expressions of green fibrillar acidic protein and tyrosine hydroxylase were altered. These abnormalities decreased with Ad-huPA+Ad-MMP-8 treatment. Importantly, the latter animals showed an increment in sprouting of nervous fibers in substantia nigra. Combinatorial gene therapy improves neuroanatomical and neurochemical characteristics similar to human hepatic encephalopathy.

BACKGROUND Plasmin role in transforming growth factor-beta (TGF-beta)-responsive gene regulation remains to be elucidated. Also, plasmin action on co-repressor Ski-related novel protein N (SnoN) and differential activation of matrix metalloproteinases (MMPs) are unknown. Thus, the role of plasmin on profibrogenic molecule expression, SnoN transcriptional kinetics and gelatinase activation was investigated. METHODS Hepatic stellate cells (HSC) were transduced with adenovirus-mediated human urokinase plasminogen activator (Ad-huPA) (4 x 10(9) viral particles/ml). Overexpression of urokinase plasminogen activator and therefore of plasmin, was blocked by tranexamic acid (TA) in transduced HSC. Gene expression was monitored by reverse transcriptase polymerase chain reaction. HSC-free supernatants were used to evaluate MMP-2 and MMP-9 by zymography. SnoN, TGF-beta and tissue inhibitor of metalloproteinase (TIMP)-1 were analysed by Western blot. Plasmin and SnoN expression kinetics were evaluated in bile duct-ligated (BDL) rats. RESULTS Plasmin overexpression in Ad-huPA-transduced HSC significantly decreased gene expression of profibrogenic molecules [alpha1(I)collagen 66%, TIMP-1 59%, alpha-smooth muscle actin 90% and TGF-beta 55%]. Interestingly, both SnoN gene and protein expression increased prominently. Plasmin inhibition by TA upregulated the profibrogenic genes, which respond to TGF-beta-intracellular signalling. In contrast, SnoN mRNA and protein dropped importantly. Plasmin-activated MMP-9 and MMP-2 in HSC supernatants. Taken together, these findings indicate that MMP-9 activation is totally plasmin dependent. SnoN levels significantly decreased in cholestatic-BDL rats (82%) as compared with control animals. Interestingly, hepatic plasmin levels dropped 46% in BDL rats as compared with control. CONCLUSION Plasmin plays a key role in regulating TGF-beta-responding genes. In particular, regulation of TGF-beta-co-repressor (SnoN) is greatly affected, which suggests SnoN as a cardinal player in cholestasis-induced fibrogenesis.

Background The authors' previous data support the notion that adenoviral-driven urokinase plasminogen activator (u-PA) expression results in reversion of experimental liver cirrhosis. The specific aim of the present study was to decipher the mechanisms involved in the regulation by endogenous/gene-delivered u-PA of matrix metalloproteinases (MMP) and related proteins engaged in degradation of excessive hepatic connective tissue. Methods Tissue slices from cirrhotic rat livers were incubated with u-PA-rich supernatants from 24-h-cultured hepatic stellate cells (HSC). Matrix metalloproteinase-2, -9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) were detected by western blot and biologic activity. The HSC that discontinued u-PA production were transfected with the adenovector Adu-PA and serum-free supernatants evaluated for proteolytic activity by MMP-3, MMP-2 and MMP-9. Collagen I, transforming growth factor-beta1 (TGF-beta1), plasminogen activator inhibitor-1 (PAI-1) and TIMP-1 mRNA levels were also evaluated. Results and conclusion Endogenous u-PA from cultured HSC significantly induced the active forms of MMP-2 (68 kDa) and MMP-9 (78 kDa) in cirrhotic tissue slices. The TIMP-1 molecular forms demonstrated that u-PA pushed the presence of 'free' TIMP-1 (not complexed with MMP; 71%) in cirrhotic tissue. When non-producing u-PA-HSC were transfected with adenoviral vector coding for the functional human protein u-PA (Adhu-PA), an overactivation of MMP-3, MMP-2 and MMP-9 (800%, 48% and 100%, respectively) was found as compared with HSC transfected with control adenovirus encoding green fluorescent protein (Ad-GFP). Finally, gene expression of collagen I, TGF-beta1, PAI-1 and TIMP-1 were downregulated by Adhu-PA action as well.

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.

Background Experimental liver fibrosis induced by carbon tetrachloride (CCl(4)) is associated with oxidative stress, lipid peroxidation, and inflammation. This work was focused on elucidating the anti-inflammatory and antioxidant effects of ethylenediaminetetraacetic acid (EDTA) in this model of hepatotoxicity Methods Wistar male rats were treated with CCl(4) and EDTA (60, 120, or 240 mg/kg). Morphometric analyses were carried out in Masson's stained liver sections to determine fibrosis index. Coagulation tests prothrombin time (PT) and partial thromboplastin time (PTT) were also determined. Gene expression for transforming growth factor beta (TGF-beta1), alpha1(I) procollagen gene (alpha1 Col I), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and superoxide dismutase (SOD) was monitored by real-time PCR. Antioxidant effect of EDTA was measured by its effects on lipid peroxidation; biological activity of ceruloplasmin (Cp), SOD, and catalase (Cat) were analyzed by zymography assays. Results Animals with CCl(4)-hepatic injury that received EDTA showed a decrement in fibrosis (20%) and lipid peroxidation (22%). The mRNA expression for TNF-alpha (55%), TGF-beta1 (50%), IL-6 (52%), and alpha1 Col I (60%) was also decreased. This group of animals showed increased Cp (62%) and SOD (25%) biological activities. Coagulation blood tests, Cat activity, and gene expression for SOD were not modified by EDTA treatment. Conclusion This study demonstrates that EDTA treatment induces the activity of antioxidant enzymes, decreases lipid peroxidation, hepatic inflammation, and fibrosis in experimental liver fibrosis induced by CCl(4).

Dominant-negative transforming growth factor beta type II receptor (TbetaRIIDeltacyt) is a protein that blocks transforming growth factor (TGF-beta) signaling. Because the consequences of blocking TGF-beta have not been completely elucidated in liver fibrosis, we analysed the effects of adenoviral delivery of TbetaRIIDeltacyt on profibrogenic genes and matrix metalloproteinase (MMP) proteins, as well as on TGF-beta signal repressor SKI-like oncogene (SnoN), in cultured hepatic stellate cells (HSCs) and in a rat model of liver fibrosis.To induce liver fibrosis, rats were treated with thioacetamide for 7 weeks and administrated once with Ad-TbetaRIIDeltacyt or Ad-betagal through the iliac vein. Fibrosis was measured by morphometric analysis. We evaluated SnoN by western blot, immunocytochemistry and immunohistochemistry; MMP activity was determined by zymography and profibrogenic gene expression by the real-time reverse transcriptase-polymerase chain reaction in cultured HSCs and liver tissue.Profibrogenic gene expression of collagen alpha1 (I), TGF-beta1, platelet-derived growth factor-B, plasminogen activator inhibitor (PAI)-1, tissue inhibitor of matrix metalloproteinase-1 and MMP-2 was down-regulated; whereas MMP-3 was over-expressed in response to Ad-TbetaRIIDeltacyt in HSCs. Moreover, zymography assays corroborated MMP-2 and MMP-3 changes in activity. Surprisingly, anti-TGF-beta molecular intervention increased nuclear SnoN in HSCs. In vivo, Ad-TbetaRIIDeltacyt reduced liver fibrosis, increased nuclear SnoN in sinusoidal cells, and also produced significant suppression in collagen alpha1 (I), TGF-beta1, PAI-1, MMP-2 and over-expression in MMP-3 in thioacetamide-intoxicated animals.The results obtained in the present study suggest that the molecular mechanism for the blocking effects of Ad-TbetaRIIDeltacyt in TGF-beta signaling acts via up-regulation of the transcriptional repressor SnoN, which antagonizes TGF-beta signaling (TGF-beta/Smad-pathway inhibitor). Consequently, profibrogenic genes are down-regulated.

P0092 IL-17 A AND F ISOFORMS AND THEIR RECEPTORS MEDIATE LIVER DAMAGE IN EXPERIMENTAL CHOLESTASIS AND THE IL17 A/F HETERODIMER INDUCES A PROFIBROGENIC PROFILE IN HEPATIC STELLATE CELLS IN VITRO S. Zepeda-Morales, S. Del Toro-Arreola, L. Sanchez-Orozco, M. Fafutis-Morris, L. Garcia-Benavides, A.L. Pereira-Suarez, M.R. Bueno-Topete. Instituto de Enfermedades Cronico Degenerativas, Bioloǵia Molecular y Genomica, Laboratorio de Inmunoloǵia, Instituto de Terapeutica Experimental y Cĺinica, CUCS, U de G., Guadalajara, jal, Mexico E-mail: ruthmyriamtop@hotmail.com

A 19 moth-old child who presented seizures secondary to intractable hypoglycemia, fulfilling the clinical and biochemical criteria for hyperinsulinism was studied. Histopathological findings of the pancreas showed the presence of small clusters of b cell islets throughout acinar tissue near ducts, in both the head and the proximal third of the body. Proximal pancreatectomy (60%) and distal pancreatic-jejunostomy (Roux-in-Y) were performed. This procedure was effective in reverting hypoglycemia and constitutes the first successful alternative treatment.

1. The association of ethanol and phenformin during 6 months in the Sprague-Dawley rat produces an alteration in lactate homeostasis. A basal blood lactate value of 54.13 +/- 15.43 mg/dl was found compared to 23.65 +/- 7.4 mg/dl in control rats. 2. Lactic acid levels increased to 28.8 +/- 7.42 mg/dl and 22.01 +/- 8.08 mg/dl after chronic administration of ethanol or phenformin in Sprague-Dawley rats, respectively. Nevertheless, these were not statistically significant with respect to those of control group. 3. The total hepatic collagen content after chronic administration of phenformin and ethanol was moderately elevated 7.12 +/- 1.85 mg/g of wet tissue, and statistically significant with respect to the control group, 4.77 +/- 1.17 mg/g. Collagen content values in phenformin and ethanol rats did not reveal statistically significant differences. 4. Liver showed histological degenerative changes but not any sign of fibrosis after chronic administration of ethanol and phenformin together.

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.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.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.

BackgroundThe 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.ResultsHigh 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.ConclusionsOur 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.