Andrea Baiocchini

Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution

Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.

Patients with Tuberculosis Have a Dysfunctional Circulating B-Cell Compartment, Which Normalizes following Successful Treatment

B-cells not only produce immunoglobulins and present antigens to T-cells, but also additional key roles in the immune system. Current knowledge on the role of B-cells in infections caused by intracellular bacteria is fragmentary and contradictory. We therefore analysed the phenotypical and functional properties of B-cells during infection and disease caused by Mycobacterium tuberculosis (Mtb), the bacillus causing tuberculosis (TB), and included individuals with latent TB infection (LTBI), active TB, individuals treated successfully for TB, and healthy controls. Patients with active or treated TB disease had an increased proportion of antibodies reactive with mycobacteria. Patients with active TB had reduced circulating B-cell frequencies, whereas only minor increases in B-cells were detected in the lungs of individuals deceased from TB. Both active TB patients and individuals with LTBI had increased relative fractions of B-cells with an atypical phenotype. Importantly, these B-cells displayed impaired proliferation, immunoglobulin- and cytokine- production. These defects disappeared upon successful treatment. Moreover, T-cell activity was strongest in individuals successfully treated for TB, compared to active TB patients and LTBI subjects, and was dependent on the presence of functionally competent B-cells as shown by cellular depletion experiments. Thus, our results reveal that general B-cell function is impaired during active TB and LTBI, and that this B-cell dysfunction compromises cellular host immunity during Mtb infection. These new insights may provide novel strategies for correcting Mtb infection-induced immune dysfunction towards restored protective immunity.

Communication between Human Dendritic Cell Subsets in Tuberculosis: Requirements for Naive CD4(+) T Cell Stimulation.

Human primary dendritic cells (DCs) are heterogeneous by phenotype, function, and tissue localization and distinct from inflammatory monocyte-derived DCs. Current information regarding the susceptibility and functional role of primary human DC subsets to Mycobacterium tuberculosis (Mtb) infection is limited. Here, we dissect the response of different primary DC subsets to Mtb infection. Myeloid CD11c+ cells and pDCs (C-type lectin 4C+ cells) were located in human lymph nodes (LNs) of tuberculosis (TB) patients by histochemistry. Rare CD141hi DCs (C-type lectin 9A+ cells) were also identified. Infection with live Mtb revealed a higher responsiveness of myeloid CD1c+ DCs compared to CD141hi DCs and pDCs. CD1c+ DCs produced interleukin (IL)-6, tumor necrosis factor α, and IL-1β but not IL-12p70, a cytokine important for Th1 activation and host defenses against Mtb. Yet, CD1c+ DCs were able to activate autologous naïve CD4+ T cells. By combining cell purification with fluorescence-activated cell sorting and gene expression profiling on rare cell populations, we detected in responding CD4+ T cells, genes related to effector-cytolytic functions and transcription factors associated with Th1, Th17, and Treg polarization, suggesting multifunctional properties in our experimental conditions. Finally, immunohistologic analyses revealed contact between CD11c+ cells and pDCs in LNs of TB patients and in vitro data suggest that cooperation between Mtb-infected CD1c+ DCs and pDCs favors stimulation of CD4+ T cells.

Quasispecies tropism and compartmentalization in gut and peripheral blood during early and chronic phases of HIV-1 infection: possible correlation with immune activation markers

HIV quasispecies was analysed in plasma and proviral genomes hosted by duodenal mucosa and peripheral blood cells (PBMC) from patients with early or chronic infection, with respect to viral heterogeneity, tropism compartmentalization and extent of immune activation. Seventeen HIV-1-infected combined antiretroviral therapy naive patients were enrolled (11 early infection and six chronic infection). V3 and nef genomic regions were analysed by ultra-deep pyrosequencing. Sequences were used to infer co-receptor usage and to construct phylogenetic trees. As markers of immune activation, plasma sCD14 and soluble tumour necrosis factor receptor II (sTNFRII) levels were measured. Median diversity of HIV RNA was lower in patients with early infection versus chronic infection patients. Overall, direct correlation was observed between V3 diversity and X4 frequency; V3 diversity of HIV RNA was inversely correlated with CD4 T-cell count; median sCD14 and sTNFRII values were similar in early and chronic patients, but X4 frequency of HIV RNA was directly correlated with plasma sCD14. The proportion of patients harbouring X4 variants and median intra-patient X4 frequency of proviral genomes tended to be higher in chronic infection than early infection patients. More pronounced compartmentalization of proviral quasispecies in gut compared with PBMC samples was observed in patients with early infection compared with chronic patients. The loss of gut/PBMC compartmentalization in more advanced stages of HIV infection was confirmed by longitudinal observation. More studies are needed to understand the pathogenetic significance of early HIV quasispecies compartmentalization and progressive intermixing of viral variants in subsequent phases of the infection, as well as the role of immune activation in tropism switch.

Spike-in SILAC proteomic approach reveals the vitronectin as an early molecular signature of liver fibrosis in hepatitis C infections with hepatic iron overload

Hepatitis C virus (HCV)‐induced iron overload has been shown to promote liver fibrosis, steatosis, and hepatocellular carcinoma. The zonal‐restricted histological distribution of pathological iron deposits has hampered the attempt to perform large‐scale in vivo molecular investigations on the comorbidity between iron and HCV. Diagnostic and prognostic markers are not yet available to assess iron overload‐induced liver fibrogenesis and progression in HCV infections. Here, by means of Spike‐in SILAC proteomic approach, we first unveiled a specific membrane protein expression signature of HCV cell cultures in the presence of iron overload. Computational analysis of proteomic dataset highlighted the hepatocytic vitronectin expression as the most promising specific biomarker for iron‐associated fibrogenesis in HCV infections. Next, the robustness of our in vitro findings was challenged in human liver biopsies by immunohistochemistry and yielded two major results: (i) hepatocytic vitronectin expression is associated to liver fibrogenesis in HCV‐infected patients with iron overload; (ii) hepatic vitronectin expression was found to discriminate also the transition between mild to moderate fibrosis in HCV‐infected patients without iron overload.

Fulminant VZV infection in an adult AIDS patient treated with steroids: A case report

Varicella zoster virus (VZV) typically causes a benign disease in childhood. However, VZV can lead to severe complication in immunocompromised patients, involving skin and nearly every organ system, with significant morbidity and mortality. VZV infection occurs more frequently in patients treated with steroids. Herein, we describe a case of rapidly fatal disseminated VZV infection with cutaneous and visceral involvement in an adult AIDS patient treated with steroids.

Incomplete septal cirrhosis after high-dose methylprednisolone therapy and regression of liver injury.

To the Editor: High-dose pulsed intravenous methylprednisolone represents the standard therapy for relapsing Multiple Sclerosis (MS). Acute hepatitis related to this treatment has been described in the literature (1–9), rarely inducing fatal disease (4). Liver injury may be related to several mechanisms, including acute immuneallergic hepatitis and autoimmune hepatitis (AIH). In few cases, liver biopsy has been performed, showing a wide spectrum of lesions (1, 2, 4, 5, 8). We report a case of a 35-year-old woman treated with high-dose pulsed intravenous methylprednisolone between February 2002 and January 2005 after a diagnosis of optic neuritis and MS. On February 2005, she was admitted to our clinic for acute hepatitis. She had no history of alcohol abuse, herbal product use, abroad travelling, liver disease or family-related liver disease. She denied assumption of hepatotoxic drugs, except for methylprednisolone therapy (1 g per day for 5 days) 1 month before. Her physical examination revealed jaundice and liver function tests showed: aspartate aminotransferase (AST) 1104 mU/ml (normal level <40 mU/ml), alanine aminotransferase (ALT) 2000 mU/ml (normal level <40 mU/ml), c-glutamyl transferase (c GT) 232 mU/ml (normal level <64 mU/ ml), alkaline phosphatase (ALP) 114 U/L (normal level <129 U/L), total bilirubin 23.9 mg/dl (normal level <2 mg/dl), conjugated bilirubin 18 mg/dl and prothrombin time 41%. Serological tests for A, B, C, D and E hepatitis and serology for Toxoplasma, rubeola, Cytomegalovirus, Epstein Barr virus and syphilis were negative. Plasma HCV RNA and HBV DNA levels were undetectable. Antinuclear antibodies and autoantibodies against mitochondria, smooth muscle and liver-kidney microsome (LKM) were not detected. Antigen and antibody for Human Immunodeficiency Virus (HIV) 1 and 2 were negative. Abdominal ultrasound showed an intact liver. Liver tests gradually normalized and the patient was discharged after 1 month in good clinical conditions. On September 2009, 3 weeks after a further 5 days course of intravenous methylprednisolone (1 g per day) she developed weakness, fatigue, jaundice, nausea and lack of appetite. At readmission to our clinic laboratory tests showed: AST 972 mU/ml, ALT 1419 mU/ml, cGT 207 mU/ml, total bilirubin 22.5 mg/ dl, conjugated bilirubin 18.8 mg/dl, prothrombin time 51%, International Normalized Ratio (INR) 1.63, fibrinogen 158 mg/dl and colinestherasis 5685 mU/ml (normal range 3900–14 000 mU/ml). Serologic tests for A, B, C, D and E hepatitis were negative. CMV, EBV serology showed previous infection. Serology for Parvovirus, Toxoplasma, rubella, Herpes Simplex Virus (HSV) 1 and 2 and syphilis were negative. HCV RNA, HBV DNA and PCR for CMV, EBV, Parvovirus B19, HSV1, HSV2 and TTV, were all negative. Antinuclear antibodies and autoantibodies against mitochondria, smooth muscle and LKM were negative. Serum ferritin and ceruloplasmin were in the normal range. Abdominal ultrasound showed gallstones. Liver tests gradually improved and the patient was referred to the surgeons for cholecystectomy that was performed 1 month later. A wedge biopsy was performed during surgery. On gross examination, the liver showed an irregular surface with increased consistency; the specimen (dimensions 1.5 cm 9 1 cm 9 2 cm, deeper than 5 mm from the liver capsule) showed liver parenchyma septation, with an histological appearance of incomplete septal cirrhosis (Fig. 1A–C) as described in the literature (10, 11). The parenchyma was nodular and partially surrounded by fibrous septa with incomplete slender septa emerging from the portal tract at the Masson’s trichrome. Portal tracts contained a moderately dense mixed inflammatory infiltrate rich in lymphocytes which formed aggregates with the presence of plasma cells, eosinophils and neutrophils. Bridging and confluent necrosis were present. Deeper within the parenchyma, there were varying degrees of hepatocellular damage with ballooning, presence of apoptotic bodies and focal (spotty) necrosis; hepatic rosettes were observed. Fatty changes and Mallory-Denk bodies were not seen. There was not evidence of steatohepatitis. The Histological Activity Index according to ISHAK et al. (12) was 14. The clinical picture and liver injury were attributed to primary toxic damage of methylprednisolone and categorized according to the Roussel Uclaf Causality Assessment Method (RUCAM) scale (13). After 9 months from methylprednisolone withdrawal, the patient was in a stable clinical condition, and a needle liver biopsy was performed. The liver core

An anal cancer screening program for MSM in Italy: Prevalence of multiple HPV types and vaccine-targeted infections.

BACKGROUND Elevated HPV infection rates have been described in HIV-positive males, placing these subjects at high risk of anal neoplasia. Bivalent, quadrivalent, and nonavalent vaccines to prevent HPV infection have been developed, and recently proposed for gender-neutral immunization programs. OBJECTIVES In order to estimate the benefit that could be obtained by vaccination of HIV-positive men who have sex with men (MSM), we aimed at describing the frequency of multiple and vaccine-targeted HPV infections in MSM enrolled in an anal cancer screening program. STUDY DESIGN The anal cancer screening program was conducted between July 2009 and October 2012. Mucosal anal samples were tested for HPV DNA using MY09/MY11 PCR primers and, if positive, genotyped using the CLART2HPV Clinical Array (35HPV types). RESULTS A total of 220 MSM were screened and 88.6% were positive for HPV DNA: in 86.5% at least one high-risk (HR) type was found and in 13% only low-risk (LR) HPV were found. Multiple infections accounted for 84.5% of HPV DNA-positive cases and overall 160 different HPV genotype combinations were recognized (only three combinations were detected in more than one patient each). Based on strain coverage, at least one vaccine-targeted HPV type was found in 38.9%, 64%, and 78.4% of cases when considering bivalent, quadrivalent and nonavalent vaccines, respectively. At least one HR vaccine-targeted strain was found in 39% of MSM for bivalent and quadrivalent vaccines, and in 64% of cases for nonavalent prevention. CONCLUSIONS Anal HPV infections in unvaccinated mostly HIV-infected MSM are highly prevalent. The majority of this population has multiple infections with an extremely heterogeneous number of genotype combinations. The nonavalent vaccine could theoretically have prevented a minimum of one HR HPV type in two thirds of subjects.

Role and predictive strength of transglutaminase type 2 expression in premalignant lesions of the cervix

The demonstration that type 2 transglutaminase (TG2) can incorporate polyamine into the E7 oncoprotein of human papillomavirus (HPV) type 18 has led to the hypothesis that TG2 can have a role in the host cellular response to HPV infection. The aim of this study was to investigate whether HPV-related pathology, in infected human cervical epithelium, was associated with modulation of TG2 expression. Normal controls and HPV-infected cervical biopsies were analyzed for the expression of TG2, and the findings were compared with lesion grade. The correlation between TG2 expression and p16, a marker for HPV-induced dysplasia, and the retinoblastoma protein (Rb), a target of the E7 protein of HPV, was also investigated. Results obtained showed that TG2 was absent in normal squamous mucosa, whereas it was present in 100% CIN I lesions. Low-grade lesions showed significantly higher TG2 expression than high-grade lesions (P<0.0001). In 94% of CIN I more than 50% of the cells were positive for TG2, with a strong staining intensity (+3), whereas a decreased staining intensity and a low number of positive cells were found in CIN II/III. In CIN I cases, both nuclear and cytoplasmic staining were found in cells exhibiting classical morphological features of HPV infection. In addition, during progression from low-grade squamous intraepithelial lesions to severe dysplasia, TG2 expression was inversely correlated with p16 (Pearson: −0.930), whereas a positive correlation was observed between the expression of TG2 and pRb (Pearson: 0.997). TG2 is expressed in HPV infection as an early phenomenon, not restricted to high-risk genotypes. TG2 upregulation is probably part of host cell reaction against HPV-induced tissue modification. It may act as a cellular antioxidant defense factor, playing an important role in counteracting oxidative damage in neoplastic disease.

Fatal sclerosing peritonitis associated with primary effusion lymphoma after liver transplantation: A case report

Sclerosing peritonitis (SP) after liver transplantation has been described in 10 cases in the literature. The etiology is still unknown; however, SP is considered a consequence of chronic irritation and inflammation. It can be classified as primary (idiopathic) or secondary form. Although pathologically benign, it has a negative course, resulting in unrelenting abdominal pain, small bowel obstruction, malnutrition, and death. Posttransplantation lymphoproliferative disease (PTLD) is one of the leading causes of late death. Its development is related to complex interactions between immunosuppressive drugs and environmental agents. Primary effusion lymphoma (PEL) as an onset presentation of PTLD is relatively uncommon. Most examples of effusion-based PTLD have been secondary to widespread solid organ involvement and associated with Human herpes virus 8 (HHV-8) recurrence. Here in, we report a case of a 55-year-old man who rapidly developed refractory ascites and bacterial peritonitis at 1-year after orthotopic liver transplantation (OLT) with a fatal clinical course at the beginning of the second follow-up year after an uncomplicated liver transplantation due to cryptogenic cirrhosis. The diagnosis of HHV-8-positive lymphoma was established by postmortem examination with multiple solid localizations and massive dense fibrotic adhesions encompassing the small intestine, colon, liver, and porta hepatis without any involvement of body cavities.