Permissiveness of human biliary epithelial cells to infection by hepatitis C virus

The cellular tropism of hepatitis C virus (HCV) is an important but much debated issue. Permissivity to HCV of biliary cells has never been demonstrated. In this context, we used gallbladder epithelial cells (GBEC) as a model of the more proximal biliary epithelium. These cells were isolated from HCV-positive and -negative individuals and cultured for up to 40 days. Biliary cells from HCV-negative subjects were infected in vitro with various inocula. The retention of GBEC functional characteristics was assessed by the expression of cystic fibrosis transmembrane conductance regulator (CFTR). All 12 GBEC tested from HCV-negative patients were successfully infected by HCV. This was assessed by: 1) the detection of HCV-RNA positive and negative strands; 2) the detection of the viral capsid by immunofluorescence; and 3) the combination of single-strand conformation polymorphism (SSCP) and HVR1 sequence analysis demonstrating the distinct majoritary HCV genomes in serum and in GBEC. The level of HCV RNA in cell extracts and supernatants was low, but HCV infection was highly reproducible. Our results expand those showing the cellular tropism of HCV, and demonstrate the sensitivity of biliary cells to HCV infection. This might have an important impact in terms of pathogenesis and pathological features of HCV infection. In addition, given the easy access to these cells and the high reproducibility of in vitro infection, they should constitute an important tool for studies aimed at analyzing the issue of HCV penetration and neutralizing antibodies.     

Knee kinetics during functional electrical stimulation induced cycling in subjects with spinal cord injury: a preliminary study

The purpose of this preliminary study was to describe pedal effectiveness parameters and knee-joint reaction forces generated by subjects with chronic spinal cord injury (SCI) during functional electrical stimulation (FES)-induced bicycling. Three male subjects (age 33-36 years old), who were post-traumatic SCI (ASIA-modified level A, level T4-C5) and enrolled in an FES rehabilitation program, signed informed consent forms and participated in this study. Kinematic data and pedal forces during bicycling were collected and effective force, knee-joint reaction forces, knee generalized muscle moments, and knee-joint power and work were calculated. There were three critical findings of this study: 1) pedaling effectiveness was severely compromised in this subject population as indicated by a lack of overall positive crank work; 2) knee-joint kinetics were similar in magnitude to data reported for unimpaired individuals pedaling at higher rates and workloads, suggesting excessive knee-joint loading for subjects with SCI; and 3) shear reaction forces and muscle moments were opposite in direction to data reported for unimpaired individuals, revealing an energetically unfavorable knee stabilizing mechanism. The critical findings of this study suggest that knee-joint kinetics may be large enough to produce a fracture in the compromised lower limbs of individuals with SCI.     

Phase I study of AG 331, a novel thymidylate synthase inhibitor, in patients with refractory solid tumors

Purpose: This was a phase I study of AG 331 to determine systemic tolerance and pharmacokinetics following single and multiple escalating intravenous doses. Methods: The study was an open-label phase I trial that was divided into two components. In phase IA (single dose), six dose levels from 12.5 to 225 mg/m² were administered to 18 patients (3 at each dose level) and serial blood samples were collected for 72 h. Upon achieving satisfactory pharmacologic parameters, the multiple dosing component (phase IB) was initiated. Six dose levels from 50 to 800 mg/m² per day were administered for 5 consecutive days to 18 patients. Pre- and postdose blood samples were obtained on days 1–4 and serial blood samples were collected over 24 h following dose 5. Nonhematologic and hepatic toxicities were assessed, serum AG 331 concentrations were measured and pharmacokinetic parameters determined. Results: Other than fatigue, no severe toxicities were encountered in phase IA. Liver toxicity was manifested by elevations in transaminase first noted at multiple doses of 200 mg/m² per day for 5 days. Fever and malaise but no myelosuppression were noted. The mean terminal t_1/2 following single doses was significantly shorter than the t_1/2 following multiple dosing (6.8 vs 9.9 h) and clearance was significantly faster following single doses than following multiple dosing (81.7 vs 30.4 1/h), but no significant difference in V_d was noted. Conclusions: The dose-related toxicity profile precludes further clinical development at this time. The pharmacokinetics of AG 331 following single and multiple doses showed significant differences. Received: 11 July 1997 / Accepted: 18 September 1998     

Pathobiologic identification of two distinct breast carcinoma subsets with diverging clinical behaviors

Many different pathological and biological variables which characterize breast carcinomas have been found to be associated. The aim of this work was to analyze the complex relationship among these parameters. The pathologic, biologic, and clinical characteristics of a series of primary breast carcinomas from 676 patients were retrospectively investigated. Multiple correspondence analysis of 13 factors revealed clustering of eight pathobiologic variables, that is histologic grade, necrosis, lymphoid infiltration, number of mitoses, cerbB2 overexpression, p53, progesterone receptor, and bcl2 expression. An index for each tumor calculated on the basis of these eight factors served to distinguish two different tumor phenotypes, designated A and B. Phenotype A is represented by tumors sharing most of the biologic features of normal breast tissues: indeed, these tumors are characterized by a relatively high degree of differentiation, low proliferation, no necrosis or leukocyte infiltration, and no gene alterations. By contrast, phenotype B is quite divergent from the normal tissue because of its poor differentiation, high proliferation, frequent gene alterations and evidence of a host immune reaction. As regards the disease progression, these two subsets showed marked differences: phenotype A tumors had a low recurrence rate per year that remained constant over time and affected more frequently elderly patients, whereas group B tumors showed high aggressivity in the first years after surgery followed by a low longterm recurrence rate and were more frequently seen in younger patients. These data suggest that breast carcinoma consists of two different subsets that can be identified on the basis of pathobiologic features.     

Cis-dichlorodiammineplatinum (II) and VP 16–213 combination chemotherapy for non-small cell lung cancer

Twenty-three patients with non-small cell lung cancer were treated with a combination of cis-dichlorodiammineplatinum(II) 100 mg/m² IV on day 1 and VP 16–213 80 mg/m² IV on days 1–3. Eighteen patients are evaluable for response. Seven partial remissions with a median duration of 3 months (range, 1–13+) have been observed. Three patients exhibit stable disease, and eight patients show tumor progression. Overall survival was 5+ months (range, 1–13+); 7.5 months (range, 3–13+) for responders and 3+ months (range, 1–9+) for non-responders. Hematologic toxicity was acceptable, but poor subjective tolerance (nausea, vomiting, loss of appetite) was the main factor limiting treatment duration.     

Inhibition of metastases by a serum factor associated to concomitant resistance induced by unrelated murine tumors.

Murine lung metatases growing undisturbed by the primary tumor were significantly inhibited by the concomitant resistance induced by a secondary subcutaneous implant of two unrelated tumors. Such inhibition was T-independent since it was also observed in nude mice; its full expression was dependent on the presence of the secondary tumor implant and it was exerted on both macroscopic and microscopic established metastases and not on the process of tumor cell dissemination from the primary tumor. Direct and indirect mechanisms seemed to be involved, the former affecting the metastatic cells per se by causing a decrease in proliferation and an increase in apoptosis while the latter affected neo-vascularization. These antitumor and antiangiogenic effects could be attributed to a serum factor induced by the unrelated tumors generating concomitant resistance. This factor proved to be heat, acid and alkaline resistant and dialysable; it was recovered in an HPLC column with maximum absorption at 215 and 266 nm; it was anionic at neutral pH, exhibiting free carboxil groups and one or more molecules of tyrosine, with a molecular weight between 870 and 1300 Dalton. Intravenous administration of this factor significantly inhibited lung metastases, decreasing mitosis and increasing apoptosis similar to that observed in the presence of the unrelated tumors.     

Childhood non-Hodgkin malignant lymphomas: A clinicopathologic retrospective study

Between 1968 and 1975, 44 evaluable children under 16 years of age with the histologic diagnosis of non-Hodgkin malignant lymphoma (ML) were treated at the Istituto Nazionale Tumori of Milan. Histologic diagnoses were reclassified as follows: 13 lymphoblastic (others) ML, 15 convoluted cell type lymphoblastic ML, 9 Burkitt type ML, and 7 immunoblastic ML. Only 36% of the patients had stage I and II disease. At diagnosis 25% showed malignant cells in the bone marrow smears. Bone marrow infiltration was particularly frequent in the convoluted cell type lymphoblastic ML and in the lymphoblastic (others) ML subgroups. Burkitt type ML frequently was associated with abdominal lesions and subsequently a high incidence of central nervous system involvement. Patients with stage I and II ML were encountered mostly in the immunoblastic ML subgroup. After 1973 more intensive chemotherapy plus radiotherapy seems to have slightly improved the survival of the patients, except in the Burkitt type ML subgroup.     

Sensory neuronopathy and small cell lung cancer: antineuronal antibody reacting with neuroblastoma cells

The anti-Hu antibody is associated with a paraneoplastic subacute sensory neuronopathy (SSN) described in cases of small cell lung cancer (SCLC). The Hu antigen is a pan-neuronal nuclear antigen with a molecular weight of 35–40 kDa. In this study we demonstrated the presence of the paraneoplastic Hu antigen in different neuroblastoma cell lines. We showed that by indirect immunocytochemistry the serum of patients with SSN and SCLC reacts with the nuclei of neuroblastoma cell lines SKN-SH and LAN-1. Western blot analysis of nuclear extracts from neuroblastoma cell lines SKN-SH, IMR-32 and LAN-1 confirmed the presence of the Hu antigen in these neuroblastoma cell lines. By comparing the immunocytochemical method and the Western blot analysis we were able to determine that the Western blot analysis was a more sensitive test. Screening of the sera of a large population (a total of 122 patients with SCLC, 17 with paraneoplastic disorders as well as 121 controls with other neurological disorders) was performed and showed all 5 of the patients with SSN and SCLC to be positive for the anti-Hu antibody, whereas only 11 of the 122 SCLC patients and none of the controls were positive, thereby suggesting that this test has a very high degree of sensitivity.     

A Novel Model of Urinary Tract Differentiation,Tissue Regeneration,and Disease: Reprogramming Human Prostate and Bladder Cells into Induced Pluripotent Stem Cells

Background

Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.

Objective

To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.

Design, setting, and participants

Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.

Outcome measurements and statistical analysis

The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.

Results and limitations

Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.

Conclusions

IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.