Oxidative stress plays a key role in liver fibrosis. Both inflammatory cells and activated Kupffer cells produce H2O2, an oxidant involved in the activation of hepatic stellate cells (HSC). Increased production of reactive oxygen intermediates (ROIs) in fibrotic livers is associated in part with the up-regulation of transforming growth factor beta (TGF-beta), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF-beta induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H2O2 is a mediator of TGF-beta-elicited alpha1(I) collagen gene (col1a1) up-regulation. We demonstrated that TGF-beta induces the accumulation of H2O2, and that this oxidant is, in turn, directly involved in up-regulating the expression of the col1a1 gene. While the addition of H2O2 to HSC induced the expression of alpha1(I) procollagen mRNA, catalase, an H2O2 enzyme scavenger, abrogated TGF-beta-mediated col1a1 gene up-regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped a cis-acting element (-370 to -344) essential for TGF-beta responsiveness. We further showed that TGF-beta induced the activation and binding of a C/EBPbeta-containing transcriptional complex to this sequence, an effect that was also mimicked by the addition of H2O2. Taken together, these data demonstrate a direct connection between TGF-beta-mediated accumulation of H2O2 and the up-regulation of col1a1 gene in HSC.

Probiotics are live microorganisms that, when ingested in adequate amounts, provide health benefits to the host. The strains most frequently used as probiotics include lactic acid bacteria and bifidobacteria, which are isolated from traditional fermented products and the gut, faeces and breast milk of human subjects. The identification of microorganisms is the first step in the selection of potential probiotics. The present techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods, such as denaturing gradient gel electrophoresis/temperature gradient gel electrophoresis and fluorescence in situ hybridisation, are employed to identify and characterise probiotics. The ability to examine fully sequenced genomes has accelerated the application of genetic approaches to elucidate the functional roles of probiotics. One of the best-demonstrated clinical benefits of probiotics is the prevention and treatment of acute and antibioticassociated diarrhoea; however, there is mounting evidence for a potential role for probiotics in the treatment of allergies and intestinal, liver and metabolic diseases. These positive effects are generally attributed to the ability of probiotics to regulate intestinal permeability, normalise host intestinal microbiota, improve gut immune barrier function and equilibrate the balance between pro-inflammatory and antiinflammatory cytokines. However, the positive effects of probiotics are not always substantiated by findings from properly conducted clinical trials. Notably, even when the results from randomised, placebo-controlled trials support the beneficial effects of a particular probiotic for a specific indication, the benefits are generally not translatable to other probiotic formulations.

The microorganisms that live symbiotically in human beings are increasingly recognized as important players in health and disease. The largest collection of these microorganisms is found in the gastrointestinal tract. Microbial composition reflects both genetic and lifestyle variables of the host. This microbiota is in a dynamic balance with the host, exerting local and distant effects. Microbial perturbation (dysbiosis) could contribute to the risk of developing health problems. Various bacterial genes capable of producing estrogen-metabolizing enzymes have been identified. Accordingly, gut microbiota is capable of modulating estrogen serum levels. Conversely, estrogen-like compounds may promote the proliferation of certain species of bacteria. Therefore, a crosstalk between microbiota and both endogenous hormones and estrogen-like compounds might synergize to provide protection from disease but also to increase the risk of developing hormone-related diseases. Recent research suggests that the microbiota of women with breast cancer differs from that of healthy women, indicating that certain bacteria may be associated with cancer development and with different responses to therapy. In this review, we discuss recent knowledge about the microbiome and breast cancer, identifying specific characteristics of the human microbiome that may serve to develop novel approaches for risk assessment, prevention and treatment for this disease.

Maternal milk contains compounds that may affect newborn immunity. Among these are a group of oligosaccharides that are synthesized in the mammary gland from lactose; these oligosaccharides have been termed human milk oligosaccharides (HMOs). The amount of HMOs present in human milk is greater than the amount of protein. In fact, HMOs are the third-most abundant solid component in maternal milk after lactose and lipids, and are thus considered to be key components. The importance of HMOs may be explained by their inhibitory effects on the adhesion of microorganisms to the intestinal mucosa, the growth of pathogens through the production of bacteriocins and organic acids, and the expression of genes that are involved in inflammation. This review begins with short descriptions of the basic structures of HMOs and the gut immune system, continues with the beneficial effects of HMOs shown in cell and animal studies, and it ends with the observational and randomized controlled trials carried out in humans to date, with particular emphasis on their effect on immune system development. HMOs seem to protect breastfed infants against microbial infections. The protective effect has been found to be exerted through cell signaling and cell-to-cell recognition events, enrichment of the protective gut microbiota, the modulation of microbial adhesion, and the invasion of the infant intestinal mucosa. In addition, infants fed formula supplemented with selected HMOs exhibit a pattern of inflammatory cytokines closer to that of exclusively breastfed infants. Unfortunately, the positive effects found in preclinical studies have not been substantiated in the few randomized, double-blinded, multicenter, controlled trials that are available, perhaps partly because these studies focus on aspects other than the immune response (e.g., growth, tolerance, and stool microbiota).

Recent investigations have demonstrated a complex interrelationship between autophagy and cell death. A common mechanism of cell death in liver injury is tumor necrosis factor (TNF) cytotoxicity. To better delineate the in vivo function of autophagy in cell death, we examined the role of autophagy in TNF-induced hepatic injury. Atg7Dhep mice with a hepatocyte-specific knockout of the autophagy gene atg7 were generated and cotreated with D-galactosamine (GalN) and lipopolysaccharide (LPS). GalN/LPStreated Atg7Dhep mice had increased serum alanine aminotransferase levels, histological injury, numbers of TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling)-positive cells and mortality as compared with littermate controls. Loss of hepatocyte autophagy similarly sensitized to GalN/TNF liver injury. GalN/LPS injury in knockout animals did not result from altered production of TNF or other cytokines. Atg7Dhep mice had accelerated activation of the mitochondrial death pathway and caspase-3 and -7 cleavage. Increased cell death did not occur from direct mitochondrial toxicity or a lack of mitophagy, but rather from increased activation of initiator caspase-8 causing Bid cleavage. GalN blocked LPS induction of hepatic autophagy, and increased autophagy from beclin 1 overexpression prevented GalN/LPS injury. Autophagy, therefore, mediates cellular resistance to TNF toxicity in vivo by blocking activation of caspase-8 and the mitochondrial death pathway, suggesting that autophagy is a therapeutic target in TNF-dependent tissue injury. The relationship between the lysosomal, degradative pathway of macroautophagy and cell death remains unclear. Recent studies increasingly support the concept that autophagy functions to prevent rather than promote cell death.

The prevalence of the metabolic syndrome and nonalcoholic fatty liver disease (NAFLD) in humans increases with age. It is unknown whether this association is secondary to the increased incidence of risk factors for NAFLD that occurs with aging, reflects the culmination of years of exposure to lifestyle factors such as a high-fat diet (HFD), or results from physiological changes that characterize aging. To examine this question, the development of NAFLD in response to a fixed period of HFD feeding was examined in mice of different ages. Mice aged 2, 8 and 18 months were fed 16 weeks of a low-fat diet or HFD. Increased body mass and insulin insensitivity occurred in response to HFD feeding irrespective of the age of the mice. The amount of HFD-induced hepatic steatosis as determined biochemically and histologically was also equivalent among the three ages. Liver injury occurred exclusively in the two older ages as reflected by increased serum alanine aminotransferase levels, positive TUNEL staining and caspase activation. Older mice also had an elevated innate immune response with a more pronounced polarization of liver and adipose tissue macrophages into an M1 phenotype. Studies of cultured hepatocytes from young and old mice revealed that aged cells were selectively sensitized to the Fas death pathway. Conclusion Aging does not promote the development of hepatic steatosis but leads to increased hepatocellular injury and inflammation that may be due in part to sensitization to the Fas death pathway and increased M1 macrophage polarization.

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Tumor necrosis factor ␣ (TNF-␣) is one of the key cytokines of the acute phase response and of many inflammatory processes. This cytokine has several antifibrogenic actions and down-regulates the expression of the type I collagen genes and induces the expression of metalloproteinases. Because TNF-␣ directly antagonizes some fibrogenic actions of transforming growth factor ␤ 1 (TGF-␤ 1 ), we considered it important to map the cis-acting regulatory element of the ␣1(I) collagen (