CDH1 POLYMORPHISMS AND LOW EXPRESSION OF E-CADHERIN AND ß-CATENIN IN COLORECTAL CANCER PATIENTS.

Epithelial-mesenchymal transition (EMT) process has a central role in tumor progression and metastases. Loss of cell-to-cell adhesiveness is a key step in EMT. In particular, E-cadherin and ß-catenin, components of the adherens junctions, play a strategic role. Accumulation of ß-catenin at cytoplasmic level following adherens junctions disruption, induces its translocation into the nucleus, where it binds to members of the TCF/LEF family of transcription factors. In particular, Lymphoid Enhancer-Binding factor 1 (LEF1) product can target genes involved in EMT. The aim of the present study was to evaluate the influence of CDH1 and CTNNB1 genes, coding for E-cadherin and ß-catenin respectively and LEF1 in a sample study of 140 Italian patients affected by colorectal cancer. An association study between four single nucleotide polymorphisms (rs11865026, rs11642413, rs13689, and rs10431923) of CDH1 and the disease did not provide statistically significant results. The gene expression analysis carried out for CDH1, CTNNB1 and LEF1 in 54 paired specimens from 27 patients provided evidence of a reduced expression of the first two in cancer tissues. We believe there may be a sort of cross regulation between the products of these two genes which closely interact in EMT activation and that such hypothesis should be further investigated in a greater number of cases.

Lung regions differently modulate bronchial branching development and extracellular matrix plays a role in regulating the development of chick embryo whole lung.

Normal branching development is dependent on the correlation between cells and extracellular matrix. In this interaction glycosaminoglycans, cytokines and growth factors play a fundamental role. In order to verify the distribution and influence of extracellular matrix and related enzymes on chick embryo lung development, 6 day-old whole lungs were maintained in vitro with testicular hyaluronidase, beta-N-acetyl-D-glucosaminidase and chondrotinase ABC or in linkage with apical, medial and caudal lung regions of 6-day development before and after enzyme treatment. In a separate lung region beta-N-acetyl-D-glucosaminidase and hyaluronidase were determined. Our data show that the whole lung cultures increase bronchial branching development when the medial region is admixed separately, while the separate apical or caudal regions or apical combined with caudal region do not affect bronchial branching development. The enzyme treatment of medial region prevents the branching development in associated whole lung. The bronchial branching development of whole lung cultured in medium containing the enzymes related to glycosaminoglycans turnover is significantly altered. In conclusion, these data show that the different influence of separate apical, medial, caudal lung regions on bronchial branching development is related to the extracellular matrix composition.

Interactions of fibroblasts with soldered and laser-welded joints.

Relatively little is known about the biocompatibility of the soldered or laser-welded joints of dental appliances. We investigated the reaction of human gingival fibroblasts cultured in vitro in direct contact with samples of soldered and laser-welded joints from orthodontic lingual arches. Contrast phase light microscopy was used to evaluate cell adhesion, morphology and proliferation after 6 and 24h and after 7 and 16 days. Scanning electron microscopy (SEM) was performed at 16 days. Our in vitro findings provide evidence that laser-welded orthodontic appliances have superior fibroblast biocompatibility.

Expression profile of epidermal differentiation complex genes in normal and anal cancer cells.

Anal cancer originates from a peculiar histological region and provides a useful model for investigating alterations in proliferation and/or differentiation of neoplastic keratinocytes. Epidermal differentiation complex (EDC) genes, which form one of the major gene clusters in the human genome, are involved in the terminal differentiation of epithelial cells and in many instances have been implicated in epithelial tumours. We constructed a DNA macroarray capable of characterising the expression profiles of the entire EDC gene complex in normal mucosa and anal cancer biopsies of seven unrelated patients. Brain tissue and cultured keratinocytes were used as controls. All anal cancer samples showed expression profiles in which none of the EDC genes was silent, as evaluated by phosphor-imager analysis. Variance analysis showed significantly lower expression of SPRR2 with respect to SPRR1 or SPRR3, and significantly higher expression of S100A8 than of other S100A subfamily members. At hierarchical clustering analysis, the four basaloid anal cancer cases conglomerated in the top five positions. The macroarray method used by us provides the first demonstration of the expression profile of the EDC gene family in anal cancer, and is capable of producing significant information on the subgrouping of epithelial tumours such as anal cancer.

Expression analysis and mutational screening of the epithelium-specific ets gene-1 (ESE-1) in patients with squamous anal cancer.

To investigate whether ESE-1 gene abnormalities are involved in alterations of epithelial cell differentiation in squamous anal cancer ESE-1 expression and structure were screened in six patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and automated sequence analysis. The complete cDNA of isoform ESE-1b was always expressed and correctly spliced, with single nucleotide polymorphism being observed in two cases. Presence of ESE-1b point mutations was excluded. Expression of SPRR2A and ENDOA/CK8, two epithelium-specific ESE-1 target genes, were revealed by RT-PCR in all cases. This first report of expression of ESE-1, and of SPRR2A and ENDOA/CK8 (both related to terminal differentiation in different types of epithelia lining) in anal cancer excludes the hypothesis that these genes influenced carcinogenesis in our patients. Despite selecting of patients without clinical evidence of HPV infection, PCR consistently revealed HPV-16 DNA, highlighting the importance of HPV infection in anal cancer.

Qualitative assessment of natural apatite in vitro and in vivo.

Among the natural and synthetic materials investigated as bone graft substitutes, much interest has been focused on natural apatite obtained from low temperature heat-deproteinated compact bone. Previous research demonstrates that, when treated at a temperature below 500 degrees C, this material maintains its characteristic ultrastructural features, with a high surface/volume ratio, while as an implant material, it offers the host tissue a large surface of interaction. In vitro and in vivo tests showed that natural apatite is well tolerated and is a good osteoconducing material. The present in vivo study in rabbits was carried out to first investigate the behavior and capacity of natural apatite implants to stimulate bone ingrowth, and then to analyze the cells located at the bone/material interface. Synthetic hydroxyapatite was used as a control material. In a parallel in vitro study, we investigated the activity of differentiated osteoblasts and periosteal cells obtained from rats and new-born rabbits, incubated with natural apatite and synthetic hydroxyapatite. The in vivo study showed that natural apatite allows osteoblasts to form new bone tissue, adhering to the implant with ingrowth into the implant structure. In the presence of synthetic hydroxyapatite, a less pronounced osteoblastic activity was observed. In agreement with these observations, the in vitro study showed that natural apatite is more effective in attracting cells, favoring their proliferation and stimulating alkaline phosphatase activity. These findings suggest that natural apatite is more suitable for bone filling or bone regeneration than synthetic hydroxyapatite.

Cell growth on cordierite: an approach to the identification of reliable supports for continuous-flow solid-bed reactors.

This study aimed to assess the biocompatibility of two cordierite ceramics (DF and Cord 1014), with similar chemical composition and different porosity, as a potential support for cell growth in a continuous-flow, solid-bed reactor. The Chinese hamster ovary (CHO) cell line transfected with HBV-DHFR recombinant plasmid was seeded on cordierite or polystyrene dishes and evaluated for cell growth and production of recombinant hepatitis B surface antigen. Proliferation of the CHO cells, in terms of cell number, was generally similar in polystyrene and Cord 1014 and always lower in DF. Flow cytometric analysis showed no difference in cell cycle distribution for cells grown on different supports, and showed a two-fold increase in percentage of debris for cells grown on DF than for those grown on Cord 1014 and polystyrene culture dishes. Moreover, the morphology of cells grown on Cord 1014 did not change during the experiment, and cells were well spread and organized. Finally, total recombinant hepatitis B surface antigen production was higher on Cord 1014 than on polystyrene and DF samples. Such evidence suggests that Cord 1014 could be a promising support for growing cells in a continuous-flow, solid-bed reactor.

An experimental pilot study of tacalcitol activities during modulation of parakeratotic skin features.

Establishing guidelines and experimental models preclinical and clinical evaluations of new agents for treatment, and/or prevention of human diseases has become a task of crucial importance. Psoriasis is such one disease holding great interest for dermatology owing to its high rate of incidence and complexity of treatment. However the absence of psoriatic lesions in animals and the inability to induce them, calls for experimental techniques both in vitro and in vivo. The purpose of this study was to evaluate experimentally the effects of tacalcitol on cell proliferation and differentiation process. Thereafter a human pilot study on psoriatic patients has been developed.

In vitro study of gingival fibroblasts from normal and inflamed tissue: age-related responsiveness.

The aim of this study was to characterize some phenotypic expressions of fibroblasts from the human oral mucosa. Gingival and lower forearm fibroblasts from young (20-30 years) and elderly (> 60 years) subjects were analyzed. Gingival fibroblasts were taken from donors with (P) and without (NP) periodontal disease, while skin biopsies were taken from healthy subjects. Cell proliferation was assessed by evaluating the cell multiplication coefficient (C.M.C.). The proliferation potential of gingival fibroblasts from elderly individuals with and without periodontopathy did not differ from that of young subjects in the same condition but differed significantly in the skin samples. Enzyme neutral endopeptidase (EC 3.4.24.11) (NEP) activity, studied as a possible marker of cell ageing, showed an age-related increase in human skin fibroblasts but not consistently in gingival fibroblasts from individuals with or without periodontal disease. Cell area and substrate adhesion were evaluated by morphometric analysis. There were no significant differences between elderly P and NP subjects, while significant differences were observed between young and elderly P subjects. In conclusion, proliferative capacity and NEP activity in gingival fibroblasts did not appear to be age-related, probably because their microenvironment is continually moistened by saliva, which continues to contain growth factors, notably EGF, even into senescence. Tissue reaction and repair are important clinical and therapeutic implications.

Human skeletal muscle mitochondria in aging: lack of detectable morphological and enzymic defects.

We have investigated structural and functional properties of skeletal muscle mitochondria obtained from biopsies from young and old individuals. The morphometric analysis of muscle sections revealed a tendency to an increase of total area, numerical density and volume density of mitochondria in the aged. The enzymatic activities of NADH-Coenzyme Q reductase, succinate cytochrome c reductase, ubiquinol-cytochrome c reductase exhibited a high variability of specific activities without any correlation with age. Expression of the values as enzyme turnovers reduced the variability but was unable to reveal any age-dependent modification.

Fibroblast proliferation over dialysis membrane: an experimental model for "tissue" biocompatibility evaluation.

The present study reports on a biological model based on fibroblast proliferation applied to 3 different types of flat-plate dialysis membrane, in order to ascertain whether the artificial materials currently used in hemodialysis cause in vitro cellular proliferation. The study plan we followed involved plate membrane isolation from non-used dialyzers and used dialyzers, observed through scanning electron microscopy (SEM) both before and after testing with human fibroblasts by means of cell culture. Fibroblast growth was assessed by phase contrast light microscopy examination and cytometric DNA content evaluation. Our investigations proved that the artificial materials we considered interact with fibroblast cultures. Noticeable proliferative response was observed both after contact with unused material and on mediation by the protein layer absorbed on the membrane surface at the end of dialysis sessions. In this last case fibroblast proliferative activity appeared higher than that observed with unused membranes, showing that the soluble molecules entrapped in the protein layer appeared able to exert a biological activity even in vitro tests.

Lack of major mitochondrial bioenergetic changes in cultured skin fibroblasts from aged individuals.

Human skin fibroblasts from young and old donors were cultured in vitro and compared in their mitochondrial morphology and function. A decreased numerical density of mitochondria in the fibroblasts from old individuals was balanced by the increased size of individual mitochondria. The mitochondrial membrane potential, estimated in the intact cells by the difference between the total accumulation ratio of the lipophilic cation tetraphenylphosphonium and the accumulation ratio in presence of uncoupler, was unchanged, as were some mitochondrial enzymatic activities tested in the homogenates. The results point out that the decreased proliferating capacity observed in the fibroblasts from the old subjects was accompanied by a likely decrease of mitochondrial duplication; the decreased energy utilization for cell division balances a possible energetic decline in such way that the steady-state energy status is unchanged.

Gingival fibroblasts "in vitro" and Down's syndrome.

Gingival fibroblast cultures from four patients with Down's Syndrome (DS) and periodontal disease were compared with four in vitro age-matched fibroblast cultures of handicapped subjects (ND) also affected by periodontitis. The extra copy of chromosome 21 could alter growth regulation and biochemical mechanisms, so we examined quantitatively some DS phenotypical aspects to detect possible differences from those of controls. The growth properties of gingival fibroblast cultures from DS patients were more elevated than their ND age-matched controls. There were no differences in plasma membrane polarization and in neutral endopeptidase activity. The succinate-cytochrome C reductase activity decreases in DS fibroblasts compared with ND. Our results outline the difficulties to in using fibroblast cultures as an in vitro system to study premature ageing Down's Syndrome.

Gingival fibroblasts "in vitro" and Down's Syndrome.

Gingival fibroblast cultures from four patients with Down's Syndrome (DS) and periodontal disease were compared with four in vitro age-matched fibroblast cultures of handicapped subjects (ND) also affected by periodontitis. The extra copy of cromosome 21 could alter growth regulation and biochemical mechanisms, so we examined quantitatively some DS phenotypical aspects to detect possible differences from those of controls. The growth properties of gingival fibroblast cultures from DS patients were more elevated than their ND age-matched controls. There were no differences in plasma membrane polarization and in neutral endopeptidase activity. The succinate-cytochrome C reductase activity decreases in DS fibroblasts compared with ND. Our results outline the difficulties to inusing fibroblast cultures as an in vitro system to study premature ageing Down's Syndrome.

In vitro growth of periodontal fibroblasts on treated cementum.

The aim of the present study was to assess the ability in vitro of phosphoric and citric acids, applied on human root cementum, to neutralize noxious plaque and calculus and to allow the growth of human gingival fibroblasts. Fibroblasts grown on cementum treated with phosphoric acid appeared typically elongated and aligned parallel to the root surface. Fibroblasts grown on cementum treated with citric acid, in both normal and periodontally diseased teeth, lost their elongated shape, acquiring polygonal borders with irregular cytoplasmic extrusions, and the cell density was significantly lower. These findings suggest that phosphoric acid cleaning of both normal and diseased root surfaces may result in an oriented, high rate of fibroblastic growth with more effective periodontal cellular proliferation than that observed after citric acid treatment.

[In vitro structural aspects of the human trophoblastic cell]. / Aspetti strutturali "in vitro" delle cellule trofoblastiche umane.

Maternal- fetal exchanges are mainly regulated by trophoblast, which displays an active role during embryo growth. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and better elucidated by "in vitro" studies. In light of this, we have carried out morphological and biochemical analyses in order to evaluate 1) the syncytiotrophoblast formation in culture (48 h, 5-30 days) the Na+/K+ATPase activity and 3) the plasmalemmal microviscosity changes occurring during "in vitro" trophoblast production. Morphological and biochemical modulations have been pointed out.

[Intrinsic potential of the gingival interdental epithelium and its "therapeutic" induction using brushing. Clinico-morphologic aspects]. / Potenzialità intrinseche dell'epitelio interdentale gengivale e loro induzione "terapeutica" mediante spazzolamento. Aspetti clinico-morfologici.

Toothbrushing technique may represent an important tool to improve gingival keratinization. Our experience evidenced a close relationship between this endoral therapy and interdental epithelial recovery of gingiva, after two months of treatment. Mechanical or microenvironmental stimuli and genetically determined potentialities are the main factors involved in this clinical-therapeutical recovery to modulate structural epithelial behaviour.