Adhesion and proliferation of human dermal fibroblasts on collagen matrix

The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon. Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy). By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous. By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel. By transmission electron microscopy, collagen fibrils showed the typical banding. Human dermal fibroblasts were seeded on the collagen matrix, previously equilibrated in growth medium. Fibroblast proliferation stopped in the second week and was always significantly lower than that of the same cell strain seeded on plastic and cultured in parallel. By light microscopy, after six days culture, cells formed a confluent multilayer on the surface of the gel. By scanning and transmission electron microscopy, fibroblasts appeared flat and adherent to the matrix. Contacts of cells among themselves and with the collagen fibrils were observed. Fibroblasts never moved into the collagen gel. In conclusion, human dermal fibroblasts can be grown in a three-dimensional matrix made by horse tendon that, on the other hand, seems to condition their proliferation rate.     

Testicular cancer and Hodgkin's disease: evaluation of semen quality

BACKGROUND: The aim of our study was to establish whether there is a difference in semen quality between patients with testicular cancer (TC) and Hodgkin's disease (HD). METHODS: We evaluated 342 patients affected by TC (n = 232) or HD (n = 110) who cryobanked sperm before initiating chemo- or radiotherapy. All TC patients were evaluated approximately 1 month after orchidectomy. RESULTS: A total of 14 patients were azoospermic or cryptozoospermic. In the TC group (n = 222) the mean of the semen parameters was normal according to the World Health Organization (1992). However, dividing the cases into total sperm count > or =40x10(6)/ejaculate and <40x10(6)/ejaculate, 35.5% of the patients showed an impaired semen quality. The quality of sperm parameters was higher in seminoma patients than for the other histological groups. A significant difference for all semen variables was observed between patients with serum betahCG levels classified as pathological (>5 mIU/ml) and those with normal serum betahCG. Comparison of semen parameters between TC stages I and II showed no significant differences. In the HD group (n = 106), we found that by and large they showed normal spermatogenesis, with only 24.5% having a total sperm count <40x10(6)/ejaculate. There was a significant decrease in semen quality in stages III and IV of HD. CONCLUSIONS: Better semen quality was observed in patients with HD than in those with TC. The semen quality observed in our TC and HD groups seems better than previous results reported in the literature.     

Novel alginate-acrylic polymers as a platform for drug delivery

The aim of the present work was to develop a family of novel materials based on a combination of sodium alginate and acrylic polymers and to evaluate their potential in drug delivery applications. In the presence of sodium alginate, acrylic chains with acidic as well as basic moieties were polymerized to create an interpolymer complex based on electrostatic interactions that are able to modulate the release rate of low molecular weight drugs. The synthesized materials were used to prepare hydrophilic matrices for drug delivery and tested for their adhesion properties to glass, used as a model substrate for mucoadhesion.     

Placing mitochondrial DNA mutations within the progression model of type I endometrial carcinoma

Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.     

Widespread proliferation impairment and hypocellularity in the cerebellum of fetuses with down syndrome

Evidence in mouse models for Down syndrome (DS) and human fetuses with DS clearly shows severe neurogenesis impairment in various telencephalic regions, suggesting that this defect may underlie the cognitive abnormalities of DS. As cerebellar hypotrophy and motor disturbances are part of the clinical features of DS, the goal of our study was to establish whether these defects may be related to neurogenesis impairment during cerebellar development. We found that in fetuses with DS (17-21 weeks of gestation) the cerebellum had an immature pattern, a reduced volume and notably fewer cells (-25%/-50%) in all cerebellar layers. Immunohistochemistry for Ki-67, a marker of cycling cells, showed impaired proliferation (-17%/-50%) of precursors from both cerebellar neurogenic regions (external granular layer and ventricular zone). No differences in apoptotic cell death were found in DS vs. control fetuses. The current study provides novel evidence that in the cerebellum of DS fetuses there is a generalized hypocellularity and that this defect is due to proliferation impairment, rather than to an increased cell death. The reduced proliferation potency found in the DS fetal cerebellum, in conjunction with previous evidence, strengthens the idea that the trisomic brain is characterized by widespread neurogenesis disruption.     

Villin,intestinal brush border hydrolases and keratin polypeptides in intestinal metaplasia and gastric cancer; an immunohistologic study emphasizing the different degrees of intestinal and gastric differentiation in signet ring cell carcinomas

Summary Gastric carcinomas have been assayed for the presence of villin and for the small intestinal hydrolases aminopeptidase N and sucrase isomaltase. These proteins seem not to be present in normal stomach epithelium. However intestinal metaplasia in stomach, and tumour cells in the glandular patterns of gastric carcinoma were positive for all three markers, showing characteristic apical positivity. In contrast, in diffuse gastric carcinomas the percentage of signet ring cells positive for these markers varied from 10–100% with each marker showing a similar percentage of positive cells. Testing of gastric carcinomas with antibodies specific for different keratin polypeptides showed that while all 7 tumours were positive for keratins 8 and 18,2 were also positive for keratin 7. In the keratin 7 positive tumours all tumour cells were keratin 7 positive. The keratin 8 antibody also reacted on routinely fixed specimens. Thus gastric carcinomas reveal different degrees of gastric and intestinal differentiation     

Complete replication cycle and acquisition of tegument in nucleus of human herpesvirus 6A in astrocytes and in T-cells

The ultrastructural replication cycle of human herpesvirus 6A and 6B, both T-lymphotropic viruses, with tropism for the central nervous system, was compared by electron microscopy in the same cells, that is, in the T-lymphoblastoid cell line SupT-1 and in human astrocytes. Both HHV-6A and HHV-6B replicated efficiently in SupT-1 and formed viral particles. The tegument is the least characterized structure of the herpesviral particle and both variants were able to form intranuclear membrane compartments called tegusomes in SupT-1 where tegumentation occurred. Also, tegumentation occurred in HHV-6A infected cells in the nucleoplasm without the presence of a tegusome. This suggests that there is more than one possible route of tegumentation. Differences in the replication cycles between HHV-6A and HHV-6B were also observed in the cytoplasm. One such difference was that prominent annulate lamellae were only found in the cytoplasm of HHV-6A infected cells. In astrocytes a successful formation of viral particles was only seen with the HHV-6A variant. The HHV-6A virus life cycle in astrocytes resembled the life cycle in the T-cell line SupT-1, except that no annulate lamellae were found. Complete viral particles were found extracellularly around the astrocytes and the supernatant of infected astrocytes were able to re-infect SupT-1 cells. This suggests that HHV-6A infection in astrocytes can generate complete, viable, and infectious viral particles. The HHV-6 variants behave differently in the same type of cells and have different tropisms for astrocytes, supporting the notion that the variants might induce different diseases.     

In-situ sol-gel synthesis and characterization of bioactive pHEMA/SiO2 blend hybrids

A novel procedure to synthesize poly(2-hydroxyethylmethacrylate)-silica blend hybrids is presented. Methacrylate monomers bearing an alkoxysilyl unit, prepared by Michael addition of 2-hydroxyethylmethacrylate (HEMA) to 3-Aminopropyltriethoxysilane (APTS) were employed. By (13)C NMR and mass analysis it was possible to establish the formation of coupling hybrid species. Hybrid materials, with final concentration ranging from 10% to 30% w/w of silica gel to the mass of polymer, were obtained through basic catalyzed sol-gel process of tetraethoxysilane (TEOS) and the alkoxysilyl unit of the hybrid monomer, followed by in-situ free-radical polymerization. The hybrids were characterized as far as concerns their thermal properties (glass transition temperature, decomposition temperature), their sorption behavior in water, and in-vitro bioactivity. Optical transparency, higher glass transition temperature, and higher decomposition temperature than pHEMA suggest an increase in either density or intensity of cross-links between the organic and the inorganic phases. The swelling ratio of the 30% hybrids is comparable to pHEMA, whereas it is lower for the other compositions. In-vitro bioactivity of the hybrids, due to the inorganic phase, was ascertained. Soaking time required for apatite deposition on the samples surface decreases as the content of silica gel increases. Therefore, the obtained bioactive hybrids can be used to make bioactive scaffolds for bone engineering.