Apoptotic Epitope-Specific CD8+ T Cells and Interferon Signaling Intersect in Chronic Hepatitis C Virus Infection.

CD8(+) T cells specific to caspase-cleaved antigens derived from apoptotic T cells represent a principal player in chronic immune activation. Here, we found that both apoptotic epitope-specific and hepatitis C virus (HCV)-specific CD8(+) T cells were mostly confined within the effector memory (EM) or terminally differentiated EM CD45RA(+) cell subsets expressing a dysfunctional T-helper 1-like signature program in chronic HCV infection. However, apoptotic epitope-specific CD8(+) T cells produced tumor necrosis factor α and interleukin 2 at the intrahepatic level significantly more than HCV-specific CD8(+) T cells, despite both populations expressing high levels of programmed death 1 receptor. Contextually, only apoptotic epitope-specific CD8(+) T cells correlated with both interferon-stimulated gene levels in T cells and hepatic fibrosis score. Together, these data suggest that, compared with HCV-specific CD8(+) T cells, apoptotic epitope-specific CD8(+) T cells can better sustain chronic immune activation, owing to their capacity to produce tumor necrosis factor α, and exhibit greater resistance to inhibitory signals during chronic HCV infection.

Proteomic analysis reveals a major role for contact inhibition in the terminal differentiation of hepatocytes.

BACKGROUND & AIMS: Hepatocytes are considered an exception of the paradigmatic inverse correlation between cell proliferation and terminal differentiation. In fact, hepatic vital functions are guaranteed by proliferating parenchymal cells during liver regeneration. However, a fine molecular characterization of the relationship between proliferation and differentiation in hepatocytes has been hampered by the lack of reliable in vivo or in vitro models. METHODS: The hepatocyte terminal differentiation program was characterized in the immortalized, untransformed and differentiated hepatocytic cell line MMH, using several techniques. Particularly, two-dimensional difference gel electrophoresis combined to tandem mass spectrometry proteomic approach was used. Cell cycle and cell adhesion properties of MMH have been altered using either myc-overexpression and MEK1/2 inhibition or a constitutive active beta-catenin mutant, respectively. RESULTS: The hepatocyte terminal differentiation program is stimulated by the exit from the cell cycle induced by cell-cell contact. Comparative proteomic analysis of proliferating versus quiescent hepatocytes validated the importance of contact inhibition, identifying 68 differently expressed gene products, representing 49 unique proteins. Notably, enzymes involved in important liver functions such as detoxification processes, lipid metabolism, iron and vitamin A storage and secretion, anti-inflammatory response and exocytosis were found significantly up-regulated in quiescent hepatocytes. Finally, we found that: (i) cell cycle arrest induced by MEK1/2 inhibition is not sufficient to induce hepatic product expression; (ii) constitutive activation of beta-catenin counteracts the contact inhibition-induced terminal differentiation. CONCLUSION: The hepatocyte terminal differentiation program requires a quiescent state maintained by cell-cell contact through the E-cadherin/beta-catenin pathway, rather than the inhibition of proliferation.

IP-10 detection in urine is associated with lung diseases.

BACKGROUND: Blood cytokines and chemokines have been proposed as biomarkers for tuberculosis (TB). Recently, some immune mediators found in the urine of patients with renal dysfunctions have also been suggested as potential biomarkers. Finding biomarkers for TB in urine would present several advantages over blood in terms of collection and safety. The objective of this study was to investigate the presence of cytokines and chemokines in the urine of patients with pulmonary TB at the time of diagnosis. In a subgroup, the evaluation was also performed during TB treatment and at therapy completion. Patients with lung diseases other than TB, and healthy subjects were also enrolled. METHODS: Urine samples from 138 individuals, after exclusion of renal dysfunctions, were collected during an 18 month-period. Among them, 58 received a diagnosis of pulmonary TB, 28 resulted having lung diseases other than TB, and 34 were healthy subjects. Moreover, 18 TB patients, 9 of whom were tested 2 months after AFB smear sputum reversion and 9 of whom were cured of TB were also included. Cytokines and chemokines in urine were evaluated using a Cytometric-Bead-Array-Flex-Set. IP-10 detection in 49 subjects was also carried out in parallel by using an Enzyme Linked ImmunoSorbent Assay (ELISA). RESULTS: IFN-γ, TNF-α, IL-2, IL-8, MIP-1α, MIP-1ß and RANTES were poorly detected in all urine samples. Conversely, IP-10 was consistently detected in urine and its level was significantly increased in patients with lung disease compared to healthy subjects (p < 0.001). Increased IP-10 levels were found in both pulmonary TB and lung diseases other than TB. Moreover lower IP-10 levels were found in cured-TB patients compared to the levels at the time of diagnosis, and this difference was close to significance (p = 0.06). Interestingly, we demonstrated a significant correlation between the data obtained by flow cytometry and ELISA (r² 0.82, p < 0.0001). CONCLUSIONS: IP-10, in contrast to IFN-γ, TNF-α, IL-2, IL-8, MIP-1α, MIP-1ß and RANTES, is detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunctions. Moreover, the IP-10 level in cured-TB patients is comparable to that found in healthy subjects. More studies are needed to further investigate the clinical utility of these findings.

Characterization of black patina from the Tiber River embankments using Next-Generation Sequencing.

Black patinas are very common biological deterioration phenomena on lapideous artworks in outdoor environments. These substrates, exposed to sunlight, and atmospheric and environmental agents (i.e. wind and temperature changes), represent extreme environments that can only be colonized by highly versatile and adaptable microorganisms. Black patinas comprise a wide variety of microorganisms, but the morphological plasticity of most of these microorganisms hinders their identification by optical microscopy. This study used Next-Generation Sequencing (NGS) (including shotgun and amplicon sequencing) to characterize the black patina of the travertine embankments (muraglioni) of the Tiber River in Rome (Italy). Overall, the sequencing highlighted the rich diversity of bacterial and fungal communities and allowed the identification of more than one hundred taxa. NGS confirmed the relevance of coccoid and filamentous cyanobacteria observed by optical microscopy and revealed an informative landscape of the fungal community underlining the presence of microcolonial fungi and phylloplane yeasts. For the first time high-throughput sequencing allowed the exploration of the expansive diversity of bacteria in black patina, which has so far been overlooked in routine analyses. Furthermore, the identification of euendolithic microorganisms and weathering agents underlines the biodegradative role of black patina, which has often been underestimated. Therefore, the use of NGS to characterize black patinas could be useful in choosing appropriate conservation treatments and in the monitoring of stone colonization after the restoration interventions.

Structural Variations of Vaginal and Endometrial Microbiota: Hints on Female Infertility.

Microbiota are microorganismal communities colonizing human tissues exposed to the external environment, including the urogenital tract. The bacterial composition of the vaginal microbiota has been established and is partially related to obstetric outcome, while the uterine microbiota, considered to be a sterile environment for years, is now the focus of more extensive studies and debates. The characterization of the microbiota contained in the reproductive tract (RT) of asymptomatic and infertile women, could define a specific RT microbiota associated with implantation failure. In this pilot study, 34 women undergoing personalized hormonal stimulation were recruited and the biological samples of each patient, vaginal fluid, and endometrial biopsy, were collected immediately prior to oocyte-pick up, and sequenced. Women were subsequently divided into groups according to fertilization outcome. Analysis of the 16s rRNA V4-V5 region revealed a significant difference between vaginal and endometrial microbiota. The vaginal microbiota of pregnant women corroborated previous data, exhibiting a lactobacilli-dominant habitat compared to non-pregnant cases, while the endometrial bacterial colonization was characterized by a polymicrobial ecosystem in which lactobacilli were exclusively detected in the group that displayed unsuccessful in vitro fertilization. Overall, these preliminary results revisit our knowledge of the genitourinary microbiota, and highlight a putative relationship between vaginal/endometrial microbiota and reproductive success.

IFN-α promotes rapid human Treg contraction and late Th1-like Treg decrease.

Type I IFNs are pleiotropic cytokines that exert concerted activities in the development of antiviral responses. Regulatory T cells represent a physiologic checkpoint in the balance between immunity and tolerance, requiring fine and rapid controls. Here, we show that human regulatory T cells are particularly sensitive to the sequential effects of IFN-α. First, IFN-α exerts a rapid, antiproliferative and proapoptotic effect in vitro and in vivo, as early as after 2 d of pegylated IFN/ribavirin therapy in patients with chronic hepatitis C. Such activities result in the decline, at d 2, in circulating regulatory T cell frequency and specifically of the activated regulatory T cell subset. Later, IFN-based therapy restrains the fraction of regulatory T cells that can be polarized into IFN-γ-producing Th1-like regulatory T cells known to contribute to chronic immune activation in type 1 inflammation. Indeed, Th1-like regulatory T cell frequency significantly declines after 30 d of therapy in vivo in relation to the persistent decline of relevant IL-12 sources, namely, myeloid and 6-sulfo LacNAc-expressing dendritic cells. This event is recapitulated by experiments in vitro, providing evidence that it may be attributable to the inhibitory effect of IFN-α on IL-12-induced, Th1-like regulatory T cell polarization. In summary, our results suggest that IFN-α-driven, early regulatory T cell depletion contributes to the development of antiviral immunity, ultimately resulting in the resolution of type 1 inflammation.

Synergy between truncated c-Met (cyto-Met) and c-Myc in liver oncogenesis: importance of TGF-beta signalling in the control of liver homeostasis and transformation.

The c-Met tyrosine kinase receptor and its ligand, Hepatocyte Growth Factor/ Scatter Factor, have been implicated in human cancer. We have previously described that the transgenic expression of a truncated form of human c-Met (cyto-Met) in the liver confers resistance to several apoptotic stimuli. Here we show the impact of cyto-Met expression on liver proliferation and transformation. Despite a sixfold increase of hepatocyte proliferation, adult transgenic livers displayed normal size and architecture. We present evidence showing that activation of TGF-beta1 signalling controls the liver mass in cyto-Met mice. The oncogenic potential of cyto-Met was further assessed in the context of c-Myc-induced hepatocarcinogenesis, using WHV/c-Myc transgenic mice. Co-expression of cyto-Met and c-Myc further enhanced hepatocyte proliferation and caused a dramatic acceleration of the Myc-induced tumorigenesis, leading to the emergence of hepatocarcinomas in 3-4-month-old animals. Importantly, the TGF-beta receptor type II expression was strongly downregulated in most tumours, indicating that impairment of TGF-beta1-mediated growth inhibition plays a major role in accelerated neoplastic development. The strong potential of cyto-Met for oncogenic cooperation without direct transforming activity designates cyto-Met mice as an ideal tool for studying the early steps of multistage hepatocarcinogenesis and for identification of prognostic markers of transformation.

IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome.

The HBV covalently closed circular DNA (cccDNA) is organized as a mini-chromosome in the nuclei of infected hepatocytes by histone and non-histone proteins. Transcription from the cccDNA of the RNA replicative intermediate termed pre-genome (pgRNA), is the critical step for genome amplification and ultimately determines the rate of HBV replication. Multiple evidences suggest that cccDNA epigenetic modifications, such as histone modifications and DNA methylation, participate in regulating the transcriptional activity of the HBV cccDNA. Inflammatory cytokines (TNFα, LTß) and the pleiotropic cytokine interleukin-6 (IL6) inhibit hepatitis B virus (HBV) replication and transcription. Here we show, in HepG2 cells transfected with linear HBV monomers and HBV-infected NTCP-HepG2 cells, that IL6 treatment leads to a reduction of cccDNA-bound histone acetylation paralleled by a rapid decrease in 3.5kb/pgRNA and subgenomic HBV RNAs transcription without affecting cccDNA chromatinization or cccDNA levels. IL6 repressive effect on HBV replication is mediated by a loss of HNF1α and HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes.

Implications of storing urinary DNA from different populations for molecular analyses.

BACKGROUND: Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days. METHODOLOGY: Urine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4 degrees C, -20 degrees C & -80 degrees C) with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J) and single copy (TLR2) targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy. CONCLUSION: Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.