Enhanced metabolic activation of chemical hepatocarcinogens in woodchucks infected with hepatitis B virus

The metabolism of chemical carcinogens was investigated in liverpreparations from 28 captive woodchucks (Marmotamonax). Of these,23 were naturally infected with the wood-chuck hepatitis virus(WHV), and eight also had primary hepatocellular carcinoma (PHC).Twenty-nine parameters were investigated in liver subcelhilarfractions, including cross-reactivity with HBsAg, and biochemicalparameters, such as -glutamyl transpeptidase, cytochrome P-450and mirosomal monooxygenases (aryl hydrocarbon hydroxylase,ethodycoumarin and ethoxyresorufin deethylases, amino-pyrineand dimethylnitrosamine demethylases, and testosterone 7-and16ß- and 6ß-hydroxylases), uridine 5'-diphos-phoglucuronosyltransferase, GSH and related enzymes (peroxidase, reductaseand S-transferase), as well as other cytosolic enzyme activities(glucose 6-phosphate and 6-phosphogluconate dehydrogenases,NADPH- and NADH-dependent diaphorases, and DT diaphorase). Inaddition, liver preparations were used in order to quantifythe metabolic activation into bacterial mutagens of five procarcinogens(aflatoxin B¹, the pyrolysis products Trp-P-2 and MelQ, 2-aminofluoreneand dimethylnitrosamine) and the decrease of potency of threedirect-acting mutagens (sodium di-chromate, ICR 191 and 4-nitroquinoline1-oxide). WHV infection produced a significant stimulation ofcarcinogen metabolism, as shown by the simultaneous change indetoxification parameters (GSH depletion) and activation indices(enhancement of microsomal monooxygenases and of pro-carcinogenactivation into mutagenic metabolites). There were no significantdifferences between WHV-positive samples from animals, withoutPHC and the noncancerous tissue of PHC-bearing animals, whereasa decrease of both activation and detoxification indices wasrecorded in the turmorous tissue. There was a considerable interindividualvariability among WHV carriers, which was tentatively ascribedto genetic factors. Pregnancy was the only known factor influencingthe results in WHV carriers. However, even by excluding pregnantanimals, the effects on carcinogen metabolism produced by WHVinfection were still statistically significant. These results,together with previous data obtained in humans, revealed thatmetabolic factors may play a role in the synergism between viralhepatitis and chemical hepato-carcinogens in the etiopathogenesisof PHC.     

Portal hypertension in Williams syndrome: report of two patients

Williams or Williams-Beuren syndrome (WBS) is a developmental disorder with multisystemic manifestations characterized by distinctive facial features, mental disability with unique cognitive and personality profiles, vascular stenoses, growth retardation, and occasional infantile hypercalcemia, caused by haploinsufficiency for genes deleted in chromosome band 7q11.23. However, with the exception of arterial stenoses caused by haploinsufficiency for the elastin gene (ELN), no specific implication of any other gene in the phenotype has been established. We present two patients with portal hypertension leading to splenomegaly and pancytopenia carrying the common 1.5 Mb WBS deletion. We propose this is an additional severe vascular complication of ELN deficiency and discuss the specific characteristics of the portal venous tract that could explain the impact of ELN deficiency in that venous territory. This complication is potentially lethal and should thus be considered in any patient with WBS and splenomegaly.     

Single step enrichment of blood dendritic cells by positive immunoselection

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44(+) cells, including up to 67% CMRF-44(+) CD14(-) CD19(-) DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44(+) cells (range 19-99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.     

Crystallization and thermal behaviour of blends of isotactic polypropylene and hydrogenated oligo(cyclopentadiene)

The crystallization and the thermal behaviour of thin films of isotactic polypropylene/hydrogenated oligo(cyclopentadiene) of low molecular weight (iPP/HOCP) are studied using optical microscopy and differential scanning calorimetry (DSC). The spherulite growth rate, the overall crystallization rate and the equilibrium melting temperature of iPP are decreased by the addition of HOCP to iPP. This leads to the hypothesis that iPP and HOCP form a miscible blend in the amorphous phase. This hypothesis is also supported by the detection of a single blend-composition-dependent glass transition temperature of each blend as determined by DSC.     

Qualitative difference between the cytotoxic T lymphocyte responses to melanocyte antigens in melanoma and vitiligo

Vitiligo is a skin disorder characterized by depigmented macules secondary to melanocyte loss. An unusual facet is its relation to melanoma: cytotoxic T lymphocytes directed to melanocyte antigens are found in both conditions and imply a breakdown of tolerance, yet the resulting immune reaction is the opposite. The mechanisms at the basis of these opposite effects are not known. Here, we performed a direct comparison of whole melanocyte-specific T cell populations in the two diseases. We demonstrate that neither precursor frequencies of Melan-A/MART-1-specific T lymphocytes nor their status of activation differ significantly. However, by using a tetramer-based T cell receptor down-regulation assay, we documented a higher affinity of vitiligo T cells. We calculated that the peptide concentration required for 50% of maximal receptor down-regulation differed by 6.5-fold between the two diseases. Moreover, only vitiligo T cells were capable of efficient receptor down-regulation and IFN-gamma production in response to HLA-matched melanoma cells, suggesting that this difference in receptor affinity is physiologically relevant. The differences in receptor affinity and tumor reactivity were confirmed by analyzing Melan-A/MART-1-specific clones established from the two diseases. Our results suggest that the quality, and not the quantity, of the melanocyte-specific cytotoxic responses differs between the two pathologies.     

Search for poliovirus long-term excretors among patients affected by agammaglobulinemia

Patients with agammaglobulinemia may excrete enteroviruses, including vaccine-derived poliovirus, for prolonged periods of time. This poses a risk to the patients but it also may pose a risk to the population after eradication of poliovirus and the cessation of routine vaccination. To assess this risk, a pilot study was performed to identify potential poliovirus long-term excretors in a cohort of 38 patients with a definite/presumptive diagnosis of X-linked agammaglobulinemia (XLA). Stool samples were analyzed to detect any polio or other enteroviruses replicating in the gut and neutralizing antibodies against polioviruses were measured in the sera. No viruses were isolated from the stool samples and most sera had neutralizing antibody levels against all three poliovirus serotypes considered by the WHO to be protective in immunocompetent individuals. This suggests that long-term excretion of enteroviruses in patients with agammaglobulinemia is relatively uncommon.     

Efficacy and tolerability of estraderm MX, a new estradiol matrix patch

Objectives: To assess the efficacy and tolerability of a new matrix patch delivering 0.05 mg estradiol per day (Estraderm MX 50) in postmenopausal women with moderate to severe postmenopausal symptoms. Methods: A multicenter, double-blin, randomized, between-patient, placebo controlled trial in 109 postmenopausal women was carried out. Patches were applied twice weekly for 12 weeks. Patients were assessed at 4, 8 and 12 weeks of treatment. The primary efficacy variable was change from baseline in mean number of moderate to severe hot flushes (including night sweats) per 24 h during the last 2 weeks of treatment. Other variables included Kupperman Index, local and systemic tolerability. Plasma concentrations of estradiol (E2), estrone (E1) and estrone sulfate (E1S) were determined before and after treatment. Results: Estraderm MX was significantly superior to placebo (P < 0.001) in reducing mean number of moderate to severe hot flushes (including night sweats) per 24 h after 4, 8 and 12 weeks of treatment. The estimate of treatment group differences after 12 weeks was 4.2 hot flushes (95% confidence interval: 2.6–5.5). Estraderm MX also significantly reduced Kupperman Index at all time points compared to placebo (P < 0.001). Estraderm MX induced increases in mean E2, E1 and E1S plasma levels as expected (E2: baseline 2.7 pg/ml, 12 weeks 38.9 pg/ml; E1: baseline 18.8 pg/ml, 12 weeks 41.6 pg/ml; E1S: baseline 235.6 pg/ml, 12 weeks 765.1 pg/ml). Overall rates of adverse experiences were similar for Estraderm MX and placebo. The number of patients reporting skin irritation was low and similar in both groups. Conclusions: Estraderm MX 50, a new matrix patch, offers an effective and well tolerated dosage form for transdermal delivery of 0.05 mg E2 per day.     

Evaluation of stromal metalloproteinases and vascular endothelial growth factors in a spontaneous metastasis model

This study aims to investigate MMP2 and MT1-MMP protein as well as VEGF-C and VEGF-D mRNA expression in tumor cells and distant organs considered to be targets for metastasis in a tumor spontaneous metastasis model previously described. Cultured tumor cells, able to express pro-MMP2, MMP2, pro-MMP9, and MT1-MMP, develop tumor growth and metastasis, mainly in the liver and spleen, when they are injected in the mammary pad gland of Wistar rats. Immunohistochemical studies of tumor masses showed small groups of tumor cells staining for MT1-MMP but not for MMP2. In the liver, tumor metastatic foci and a stromal positive staining for both MMP2 and MT1-MMP were shown. The spleen and lymph nodes, with only scattered metastatic cells, did not show MMPs immunostaining. Using RT-PCR, a significantly higher VEGF-C and VEGF-D gene expression was shown in the liver of tumor-bearing rats respect to normal rats, whereas spleen and lymph nodes did not show significant differences in mRNA VEGF-C/D levels. Taken together, our results suggest that the stroma microenvironment of target organs for metastasis has the ability to produce MMPs and VEGFs that facilitate the anchorage of tumor cells and promote tumor cell growth and angiogenesis.     

Lymphoepithelioma-like carcinoma of the bladder: three cases with clinicopathological and p53 protein expression study

Lymphoepithelioma-like carcinoma of the bladder is an uncommon neoplasm, of which 49 cases have been described in the English literature, none of which has been studied for p53 protein expression. We studied three muscle-infiltrating cases of this tumor using immunohistochemical, in situ hybridization and polymerase chain reaction (PCR) methods. The three cases were positive for epithelial markers and negative for lymphoid antigens in the tumoral syncytial areas. The intensive infiltrate of small cells was negative for epithelial and positive for lymphoid markers. This population was mainly made up of cytotoxic T-lymphocytes, positive for TIA-1. p53 protein was intensely positive in more than 90% of the epithelial component nuclei, being negative in the lymphoid cells. PCR study did not show mutations on p53. Both lymphocytes and epithelium were negative for Epstein–Barr virus markers, such as the latent membrane protein and EBER (Epstein–Barr-encoded RNA). The prognosis was very good after radiotherapy and chemotherapy treatment, preserving the bladder despite the muscle infiltration. The presence of an intense cytotoxic T-lymphocyte population may be related to this good prognosis. Both aspects, p53 protein status and T-lymphoid population, had never been studied before in bladder lymphoepithelioma-like carcinoma.