Giorgio Bovo

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells

Summary Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Biological heterogeneity of putative bladder cancer stem-like cell populations from human bladder transitional cell carcinoma samples

Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells, the cancer stem‐like cells (CSCs) or tumor initiating cells. We report on the isolation and biological characterization of putative bladder CSC populations from primary TCCs. Isolated cells were induced to proliferate in stem cell culture conditions (serum‐free medium containing mitogenic growth factors). The proliferating cells formed spheroids (urospheres) and their abilities for extensive proliferation and self‐renewal were assayed. Their positivity for several stem cell markers (CD133, Oct‐3/4, nestin, and cytokeratins) was also assessed by immunofluorescence tests and they could have the potential to differentiate in the presence of serum. In stem cell culture conditions they gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might reflect their progressive acquisition of differentiative capacity and loss of self‐renewal ability. To evaluate if effective cell selection occurred after isolation, conventional cytogenetic studies on fresh chromosome spreads immediately after isolation and after culture were carried out. In addition, a molecular cytogenetic study by UroVysion assay was carried out on paraffin‐embedded tissue sections and on fresh and after culture nuclei preparations. The data collected indicated important karyotype changes and a positive selection for hypo‐ or near‐diploid cells, losing the complexity present in fresh tumors. (Cancer Sci 2009)

CORRELATION BETWEEN POSTOPERATIVE INFECTIONS AND LONG-TERM SURVIVAL AFTER COLORECTAL RESECTION FOR CANCER

Introduction Predicting long-term survival and cancer recurrence in patients with colorectal cancer is difficult because of the many factors that may affect the prognosis. This study investigated the prognostic significance of postoperative infections for patient outcome. Methods From an electronic database we selected 192 patients undergoing elective radical surgery for Dukes’ stage B and C colorectal adenocarcinoma. The five-year survival rates were analyzed by the Kaplan-Meier method. Univariate and multivariate analyses were carried out to evaluate the potential prognostic variables using the Cox proportional hazard model. Results Forty-three patients developed deep incisional or organ/space surgical site infections, while the remaining 149 were complication free. The two groups were comparable for baseline, surgical and histopathological characteristics. At univariate analysis, Dukes’ stage and infections were negative prognostic factors, while peritumoral infiltration of lymphocytes and eosinophils and fibrotic tissue appeared as protective variables. However, multivariate analysis showed that only Dukes’ stage (P = 0.048) and occurrence of postoperative infectious complications (P = 0.011) were independently associated with outcome. In patients with infectious complications, the survival rate was significantly lower than in patients without infections (log-rank = 0.0004). Conclusions The present results suggest the importance of evaluating other variables besides tumor stage in the prediction of long-term outcome. In prognostic studies more attention should be paid to postoperative infections.

Hepatocyte proliferation and risk of hepatocellular carcinoma in cirrhotic patients.

Abstract OBJECTIVES: High hepatocyte proliferation has been recently proposed as a risk factor for the development of hepatocellular carcinoma (HCC). The aim of this study was to assess whether hepatocyte proliferation is an independent risk factor for HCC when considered together with clinical and demographic characteristics. METHODS: We retrospectively evaluated 97 consecutive patients with a histological diagnosis of cirrhosis and preserved liver function, enrolled in a surveillance program for early diagnosis of HCC. Hepatocyte proliferation was evaluated by flow-cytometric analysis in liver samples collected at the time of histological diagnosis of cirrhosis. All patients were followed with abdominal US and serum α-fetoprotein (AFP) assays every 6 months. RESULTS: During a mean follow-up of 53 months (range, 12–120 months), 12 patients developed HCC, giving an annual incidence of 2.8%. The mean S-phase fraction was 2.5% ± 1.6 in patients who developed HCC and 0.9% ± 0.6 in those who did not ( p p p p p p CONCLUSIONS: Patients with high S-phase fraction and/or above-normal serum AFP are at higher risk of developing HCC and should be offered a close surveillance program.

Detection of high molecular weight proteins by MALDI imaging mass spectrometry

MALDI imaging mass spectrometry (IMS) is a unique technology to explore the spatial distribution of biomolecules directly on tissues. It allows the in situ investigation of a large number of small proteins and peptides. Detection of high molecular weight proteins through MALDI IMS still represents an important challenge, as it would allow the direct investigation of the distribution of more proteins involved in biological processes, such as cytokines, enzymes, neuropeptide precursors and receptors. In this work we compare the traditional method performed with sinapinic acid with a comparable protocol using ferulic acid as the matrix. Data show a remarkable increase of signal acquisition in the mass range of 20k to 150k Th. Moreover, we report molecular images of biomolecules above 70k Th, demonstrating the possibility of expanding the application of this technology both in clinical investigations and basic science.

Iron accumulation in chronic hepatitis C: relation of hepatic iron distribution, HFE genotype, and disease course.

The aim of the present study was to describe the histopathologic features of hepatic iron accumulation in patients with chronic hepatitis C (CH-C) infection, the relation between HFE mutations and hepatic iron location and among iron distribution, HFE, and hepatic damage. We studied 206 patients with CH-C infection. Of 101 patients with hemosiderin deposits, 90.1% had iron deposits in hepatocytes (alone or with sinusoidal and/or portal involvement). The hepatic iron score increased significantly as iron accumulation involved sinusoidal and portal tract compartments and according to HFE genotypes. Severe fibrosis and cirrhosis were associated more markedly with the presence of hemosiderin iron in the 3 hepatic compartments, HFE mutations, and high alcohol intake. We suggest that part of the iron accumulation in CH-C infection derives from increased iron absorption and release from storage cells and that the amount and distribution of hepatic iron deposits is related to hepatic damage. HFE mutations favor both processes, but other factors, genetic or acquired, are involved.

Melanoma metastatic to the gallbladder and small bowel: report of a case and review of the literature.

From post-mortem case records, the small bowel is the most frequent site of metastatic melanoma in the gastrointestinal (GI) tract, with gallbladder involvement occurring in 15% of cases. However, few cases have been documented in living patients and, when found, are associated with a poor prognosis. We report a case of a Caucasian man with metastatic gallbladder and small bowel melanoma from an unknown primary. He presented with diffuse abdominal pain, vomiting and progressive asthenia; subsequently, intestinal obstruction occurred. He had no past history of malignant melanoma and the primary lesion was not found. The multiple lesions, together with the absence of mucosal involvement in both the gallbladder and small bowel, led us to believe that the lesions were metastatic deposits from a probably regressed primary melanoma. It should be emphasized that surgical resection for melanoma metastatic to the GI tract is recommended for palliative reasons and can be performed safely. The clinical presentation, diagnosis, treatment and prognosis of previously reported cases of melanoma metastatic to the gallbladder and small bowel are reviewed. The differences between primary and secondary GI tract melanomas are also discussed.

Hepatic resection using a bipolar vessel sealing device: technical and histological analysis

INTRODUCTION Blood loss and bile leakage are well-known risk factors for morbidity and mortality during liver resection. Bleeding usually occurs during parenchymal transection, and surgical technique should be considered an important factor in preventing intraoperative and postoperative complications. OBJECTIVE Many approaches and devices have been developed to limit bleeding and bile leakage. The aim of the present study was to determine whether a bipolar vessel sealing device allows a safe and careful liver transection without routine inflow occlusion, achieving a satisfactory hemostasis and bile stasis, thus reducing blood loss and bile leak and related complications. PATIENTS AND METHODS A total of 50 consecutive patients (24 males, 26 females, with a mean age of 57 years) underwent major and minor hepatic resections using a bipolar vessel sealing device. A clamp crushing technique followed by energy application was used to perform the parenchymal transection. Inflow occlusion was used when necessary to control blood loss but not as a routine. No other devices were applied to achieve hemostasis. RESULTS The instrument was effective in 45 patients and failed to achieve hemostasis in 5 cases, all of whom had a cirrhotic liver. Median blood loss was 490 ml (range 100-2500 ml) and intraoperative blood transfusions were required in eight cases (16%). Mean operative time was 178 min (range 50-315 min). Inflow occlusion was necessary in 16 (32%) patients. The postoperative complication rate was 24%, with a postoperative hemorrhage in a cirrhotic patient. There was no clinical evidence of bile leak or procedure-related abdominal abscess. CONCLUSION We conclude that the device is a useful tool in standard liver resection, achieving good hemostasis and bile stasis in patients with normal liver parenchyma, but its use should be avoided in cirrhotic patients.

Fibrosis is associated with adiponectin resistance in chronic hepatitis C virus infection.

Eur J Clin Invest 2011; 41 (8): 898–905

PKHhigh cells within clonal human nephrospheres provide a purified adult renal stem cell population

The existence and identification of adult renal stem cells is a controversial issue. In this study, renal stem cells were identified from cultures of clonal human nephrospheres. The cultured nephrospheres exhibited the activation of stem cell pathways and contained cells at different levels of maturation. In each nephrosphere the presence of 1.12-1.25 cells mirroring stem cell properties was calculated. The nephrosphere cells were able to generate three-dimensional tubular structures in 3D cultures and in vivo. In clonal human nephrospheres a PKH(high) phenotype was isolated using PKH26 epifluorescence, which can identify quiescent cells within the nephrospheres. The PKH(high) cells, capable of self-renewal and of generating a differentiated epithelial, endothelial and podocytic progeny, can also survive in vivo maintaining the undifferentiated status. The PKH(high) status, together with a CD133(+)/CD24(-) phenotype, identified a homogeneous cell population displaying in vitro self-renewal and multipotency capacity. The resident adult renal stem cell population isolated from nephrospheres can be used for the study of mechanisms that regulate self-renewal and differentiation in adult renal tissue as well as in renal pathological conditions.