Immunopathological characterization of cryptoglandular anal fistula: a pilot study investigating its pathogenesis.

AIM: The pathogenesis of cryptoglandular anal fistula (AF) is still under debate. Tissue inflammation could play a primary role. The pathological process of epithelial mesenchymal transition (EMT) might be involved but has never been investigated. METHOD: In a prospective pilot study, 12 patients with an AF had a fistulectomy. The excised track was divided into proximal (intrasphincteric) and distal (extrasphincteric) parts which were subjected to standard histopathological examination. The cytokines IL-8 and IL-1beta were analysed as markers of inflammation, while EMT was evaluated by expression of TGF-beta, Vimentin, Zeb-1, Snail and E-cadherin. The mRNA and protein expression of these molecules was investigated by real-time PCR (RT-PCR), Western blot analysis and immunohistochemistry and was compared with that of the normal adjacent tissue. RESULTS: Chronic inflammation and granulation tissue and a stratified epithelium were evident on standard histopathological examination. The cytokine IL-8 was more expressed in the proximal than the distal part of the track (fold increase 4.34 vs 3.60), while the reverse was found for IL-1beta (fold increase 1.33 vs 2.01); both were more intensely expressed compared with the normal anal mucosa. EMT was demonstrated, in both proximal and distal parts of the track, with an increase of TGF-beta, Vimentin, Zeb-1 and Snail and a mean decrease of E-cadherin. Western blot analysis and immunohistochemistry confirmed the protein expression. CONCLUSION: The study suggests that chronic inflammation is present in cryptoglandular fistulas. The inflammatory pattern might be different in the proximal than in the distal part of the fistula track. The cytokines IL-1beta and IL-8 could play a possible role in fistula formation. The study demonstrates for the first time the potential importance of EMT in the pathogenesis of cryptoglandular AF.

Post-translational modulation of CD133 expression during sodium butyrate-induced differentiation of HT29 human colon cancer cells: implications for its detection.

The CD133 molecule has been proposed as a surface marker of cancer stem cells in several human malignancies, including colon cancers. The function and the mechanisms regulating CD133 expression remain unknown. The HT29 human colon cancer cells undergo differentiation following treatment with various agents and represent a useful in vitro model of colon differentiation. This study evaluated the behavior of CD133 during sodium butyrate-induced differentiation of HT29 cells. Treatment with sodium butyrate induced a progressive decrease of CD133 expression, as assessed by flow cytometry using the AC133 monoclonal antibody. Indeed, expression of CD133, which was about 47% in untreated control cells, gradually decreased down to about 3% after 72 h in a time- and dose-dependent manner. No relationship was observed between CD133 protein evaluated by flow cytometry and mRNA expression level, and no changes were detected in the methylation status of the CD133 gene promoter during HT29 differentiation. Moreover, the expression of the CD133 protein, evaluated by Western blot analysis using a specific anti-CD133 antibody directed against the C-terminal intracytoplasmic region of human CD133 protein, did not correlate with flow cytometry results. Different results were also obtained using the two antibodies to analyze the expression of the CD133 molecule in human colon cancers. These findings demonstrate that membrane expression of the CD133 stem cell marker might undergo a complex regulation during differentiation of colon cells and suggest that HT29 cells are a useful in vitro model to study the mechanisms involved in this regulation which likely occurs at a post-transcriptional level.

Isolation and characterization of CD133+ cell population within human primary and metastatic colon cancer.

BACKGROUND: "Cancer stem cells" (CSC) have been identified as a minority of cancer cells responsible for tumor initiation, maintenance and spreading. Although a universal marker for CSC has not yet been identified, CD133 has been proposed as the hallmark of CSC in colon cancer. The aim of our study was to assess the presence of a CD133+ cell fraction in samples of colon cancer and liver metastasis from colon cancer and evaluate their potential as tumor-initiating cells. METHODS: Tissue samples from 17 colon cancers and 8 liver metastasis were fragmented and digested using collagenase. Cell suspensions were characterized by flow cytometry using anti-CD133, CD45 and CD31 antibodies. CD133+ cells were also isolated by magnetic cell sorting and their tumor-initiating potential was assessed versus the remaining CD133- fraction by soft-agar assay. RESULTS: Our results confirmed the existence of a subset of CD133+ tumor cells within human colon cancers. Interestingly, CD133+ cells were detectable in liver metastasis at a higher percentage when compared to primary tumors. Soft-agar assay showed that CD133+ cell fraction was able to induce larger and more numerous colonies than CD133-cells. CONCLUSION: Our findings data that the CD133+ colon cancer cells might play an important role in both primary tumors as well as in metastatic lesions thus warranting further studies on the role(s) of this subset of cells in the metastatic process.

IFN-γ and other serum cytokines in head and neck squamous cell carcinomas.

SUMMARY: Altered immune responses have been reported in head and neck cancer, and some of these responses have been associated with poor clinical outcomes. A multiple-array technology platform was used to simultaneously evaluate the levels of 25 cytokines. Pre-treatment serum levels were evaluated in 31 HNSCC patients and 6 healthy controls. The levels of 8 cytokines, specifically IL-1ra, IL-2, IL-5, IL-6, IL-8, IL-17, IFN-γ and IP-10, were significantly higher in patients than in controls. Among cancer patients we observed lower levels of IFN-γ and IL-7 in cases with nodal metastases compared to those with cN0 disease. We observed increases in the levels of some serum cytokines in HNSCC patients, as well as reductions in selected cytokines associated with regional progression. These findings provide an intriguing perspective on the development and validation of novel markers for follow-up evaluations and predictions of regional spreading in HNSCC patients.

In vitro evaluation of the potential toxic effects of palladium nanoparticles on fibroblasts and lung epithelial cells.

Palladium nanoparticles have been increasingly used in catalytic processes, wastewater treatment, electronics, and biomedicine. However, recent evidence proved that these nanoparticles are able to induce adverse effects both in in vitro and in vivo models. Nevertheless, molecular mechanisms underlying the toxic effects are still poorly understood. Therefore, this study aimed to investigate the potential toxicological mechanisms of palladium nanoparticles assessing their effects on normal diploid rat fibroblast and lung carcinoma human epithelial cell lines. Several endpoints such as cell growth, cell cycle progression, DNA damage, induction of apoptosis, reactive oxygen species production and expression of cell cycle regulatory proteins were evaluated. Results showed that palladium nanoparticles inhibited cell growth in a dose- and time-dependent manner in both cell lines, although with a more evident action on fibroblasts. Interestingly, inhibition of cell growth was not associated with the induction of apoptosis. Cell cycle progression was arrested in the G0/G1 phase and DNA damage was evident in both cell lines even if only a slight increase in the intracellular reactive oxygen species levels was detected. These findings provide valuable insight into understanding the molecular mechanisms responsible of palladium nanoparticles toxicity whose identification is essential to define an adequate risk assessment process.

Redox regulation of cell proliferation by pyrrolidine dithiocarbamate in murine thymoma cells transplanted in vivo.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic compound largely used in cell biological studies and known to exert either antioxidant or pro-oxidant effects. Recently, its antitumoral activity has been proposed on the basis of its antioxidant and proapoptotic effects. In the present study, we evaluated the effect of increasing i.p. doses of PDTC on the growth of a strain of highly malignant thymoma cells inoculated in the peritoneum of inbred Balb/c mice. PDTC treatment increased the number of thymoma cells in a dose-dependent manner, enhancing the percentage of proliferating tumor cells. PDTC exerted regulatory effects on cell cycle distribution, decreasing the expression of cell cycle inhibitors. Alterations in the production of intracellular reactive oxygen species, levels of oxidized glutathione, and intracellular levels of the redox-active metals iron and copper were also observed. The above results represent the first evidence that PDTC may induce in vivo cell proliferation in a murine thymoma cell model. In addition, we suggest that the ability of PDTC to bind and transport metals inside the cell and its pro-oxidant property may be factors underlying its effects on thymoma cell proliferation and cell cycle distribution.

Evaluation of polycyclic aromatic hydrocarbon-DNA adducts in exfoliated oral cells by an immunohistochemical assay.

Polycyclic aromatic hydrocarbon-DNA adducts were evaluated in oral cells from 98 healthy volunteers by an immunohistochemical method using a specific antiserum against benzo(a)pyrene-DNA adducts revealed by the immunoperoxidase reaction. Mean adduct content, determined as relative staining intensity by absorbance image analyzer, was significantly higher in the cells from tobacco smokers compared with nonsmokers (330 +/- 98, n = 33 versus 286 +/- 83, n = 64, respectively) with a P = 0.013 obtained by two-sample t test with equal variances. We found that in the smoker group, the PAH-DNA adduct content increases with the number of cigarettes. Thus, the relative staining intensity was 305 +/- 105 in the group smoking 1-10 cigarettes/day (n = 16), 347 +/- 77 in the 11-20 group (n = 14), and 386 +/- 112 in the group smoking more than 20 cigarettes/day (n = 3; P = 0.03 by nonparametric test for trend). No significant association was detected between PAH-DNA adducts in oral cells and variables such as residential area, oral infections, alcohol or vitamin intake, grilled food consumption, and professional activity. This work confirms and extends previous data suggesting that this immunohistochemical method might be used as a valuable dosimeter of genotoxic damage in a carcinogen-exposed population, although further studies are needed to verify the applicability of the test in high-risk populations other than smokers.

Prognostic significance of cytoplasmic p53 overexpression in colorectal cancer. An immunohistochemical analysis.

p53 overexpression was studied by immunohistochemistry in 96 consecutive colorectal cancer patients, subdividing positive specimens according to two staining patterns: cytoplasmic or nuclear. Forty-seven per cent of the cases were p53 positive, a significant correlation being found with Dukes' stage (P = 0.0036). A prevalence of nuclear staining was observed in Dukes' B and cytoplasmic in Dukes' D stages. After 36 months, 23% of the patients had a recurrence, and 45% were p53 positive, all Dukes' C-D stage with cytoplasmic staining. The Kaplan-Meier curve showed a significant correlation between p53 cytoplasmic staining and disease-free survival period (P = 0.002). With respect to disease-free survival, the Cox proportional hazard regression test, comparing p53 positivity with Dukes' stage, showed the latter to be the most significant variable. In our series of patients, advanced Dukes' stage tumours were localised in the right colon, where a higher percentage of p53 positivity (67% versus 40% of the left side), as well as a higher frequency of cytoplasmic staining was observed. In conclusion, from the data obtained, a strong correlation between p53 cytoplasmic staining and patient prognosis is clearly indicated.

Immunohistochemical analysis of p53 protein in transplant recipients with Kaposi's sarcoma.

PURPOSE: Kaposi's sarcoma (KS) is a proliferative process of suspected viral aetiology associated with immune deficiency. In transplanted patients, lesions regress on discontinuation of immunosuppressive therapy. The purpose of this work was to analyse the expression of the p53 oncosuppressor gene product, a proliferation regulator overexpressed in both malignant and non-malignant conditions, with the aim of better qualifying KS proliferation characteristics. METHODS: We analysed p53 expression in a group of transplanted, cyclosporin A-treated, KS patients by immunohistochemistry, utilizing the DO-7 (with and without the antigen retrieval pretreatment), and the PAb 240 monoclonal anti-p53 antibodies, the latter of which is able to detect a mutated epitope, and evaluating staining intensity and localization, whether cytoplasmic or nuclear. RESULTS: Seventy five percent of KS lesions from transplanted patients presented both nuclear and cytoplasmic positive p53 immunostaining with DO-7 antibody, thus demonstrating a presumably functional inactivation; one case also presented immunoreactivity with the PAb 240 antibody. CONCLUSIONS: On the basis of the results obtained and in the presence of lesion regression upon immunosuppression withdrawal, it may be concluded that KS in transplanted patients can be considered a non-malignant proliferative process, and that the cytoplasmic expression of p53 may stand for a functional inactivation pattern.

Effect of portacaval shunt on circulating free fatty acids and ketone bodies in rats.

To ascertain whether portal diversion affects ketone body (KB) metabolism, fasting circulating levels of free fatty acids (FFA), acetoacetate (AcAc) and B-hydroxybutyrate (3-OH-B) were measured in portacaval shunted (PCS), sham-operated (S-O) and unoperated control (C) rats. In PCS animals blood KB concentration was clearly reduced when compared with S-O and C rats. Beta-hydroxybutyrate level was significantly lower in PCS rats, whereas AcAc concentration did not appear significantly modified in these animals. The hypothesis is proposed that hypoketonemia induced by portal diversion is due to reduced hepatic availability of fatty acids.

Evaluation of 8-hydroxydeoxyguanosine in human oral cells: the importance of tobacco smoke and urban environment.

The DNA adduct 8-hydroxydeoxyguanosine (8-OHdG) has been widely used as a sensitive biomarker for oxidative damage. To investigate the role of environmental factors on oxidative DNA damage formation, the level of 8-OHdG was determined in oral cells from 109 healthy volunteers by an immunohistochemical method. A statistically significantly higher content of 8-OHdG was detected in oral cells from smokers (111 +/- 55, n = 38) compared with non smokers (78 +/- 48, n = 71), (p < 0.01). Moreover, subjects living in an urban area showed a higher level of oxidative damage with respect to those living in a countryside-suburban area (99 +/- 53, n = 58 vs. 78 +/- 51, n = 51), (p = 0.03). No significant association was detected between 8-OHdG in oral cells and other variables such as passive smoke, oral infections, alcohol or vitamin intake and grilled food consumption. This work suggests that tobacco smoke and environmental exposure to pollutants lead to a measurable increase of oxidative damage in oral cells and confirms that the immunoperoxidase method is an appropriate approach for epidemiological analyses.

Immunohistochemical analysis of 4-aminobiphenyl-DNA adducts in oral mucosal cells of smokers and nonsmokers.

BACKGROUND: The "biologically effective dose markers", DNA and protein adducts, are a direct index of carcinogen induced cell damage and an indirect one of genetic susceptibility. This study aimed to examine the dose-response relationship for 4-Aminobiphenyl-DNA adducts in oral cells of smokers and non smokers. MATERIALS AND METHODS: An immunoperoxidase method with the monoclonal 3C8 antibody, which recognizes 4-Aminobiphenyl-DNA, has been used for detecting DNA damage in oral cells of 12 smokers and 12 non smokers. RESULTS: Higher staining for 4-Aminobiphenyl-DNA was detected in the cells of smokers (187 +/- 42) vs. non smokers (135 +/- 35) (p = 0.004), with a twofold range in relative staining for both groups, suggesting individual differences relevance in metabolizing carcinogens and/or repairing DNA damage. CONCLUSIONS: This non invasive method requiring small cell amounts is a tool for monitoring large groups of subjects at risk in primary prevention programs.

Biological characterization of central and peripheral primary non small cell lung cancers (NSCLC).

BACKGROUND: Non Small Cell Lung Carcinomas (NSCLC) comprise 90% of all lung carcinomas. Studies have demonstrated a preferential central (bronchus-derived) localization for squamous cells, whereas adenocarcinomas are frequently peripheral (bronchiolo-alveolus derived). It has been suggested that exposure to carcinogenic insults including cigarette smoke, may induce different types of tumors in different locations. MATERIALS AND METHODS: Forty one NSCLC patients staged according to WHO and TNM were considered for localization and biological parameters (p53 expression, cell ploidy and S-phase). RESULTS: p53 overexpression was found more frequently in central than in peripheral tumors (69% vs 39%) (p = 0.074). Central tumors were more aneuploid (69%) than peripheral ones (46%) (p = 0.03) No difference in smoking habit was observed in the two groups. CONCLUSIONS: Our results suggest that there is no apparent biological difference between these two groups of NSCLCs, and that the smoking does not play a role in either histotype determination or biological behavior.

Analysis of 4-ABP-DNA adducts and p53 alterations in urinary bladder carcinoma.

BACKGROUND: Activated intermediates of 4-aminobiphenyl (4-ABP) are able to covalently interact with DNA to form adducts. There is a large body of evidence indicating that carcinogen-DNA adduct formation can be one of the cancer initiating mechanisms. MATERIALS AND METHODS: (4-ABP)-induced DNA damage in association with p53 overexpression and mutations were evaluated in specimens of urothelial bladder cancers from 106 patients. RESULTS: 4-ABP-DNA adduct levels resulted higher in smokers compared to non smokers, with a borderline statistical value. p53 nuclear overexpression was related to tumor grading, while no significant correlation with stage, 4-ABP-DNA adducts, smoking habit, and disease recurrence could be observed. Concerning molecular analysis, p53 point mutations were found in 17 of 106 cases (16%) and mutational pattern was significantly associated both with higher grade and stage, but no correlation was found with disease recurrence. CONCLUSIONS: These results suggest that other sources, in addition to tobacco smoke, may contribute to 4-ABP-DNA adducts formation in bladder tissue and that p53 expression/mutation cannot be considered a prognostic factor in bladder cancer.

Resveratrol, a natural phenolic compound, inhibits cell proliferation and prevents oxidative DNA damage.

Resveratrol (3,4',5-trihydroxystilbene) is a naturally occurring phenolic compound which is present at high levels in wine and has been recently proposed as a potential cancer chemopreventive and chemoterapeutic agent. In this study, we evaluated the antiproliferative activity of resveratrol on a panel of cell lines of various histogenetic origin, including normal rat fibroblasts and mouse mammary epithelial cells compared to human breast, colon and prostate cancer cells. The concentration of resveratrol inhibiting cell growth by 50% (IC(50)) ranged from about 20 to 100 microM. At such concentration, we were unable to detect a significant increase in the apoptotic index in most of the cell lines analyzed. We also studied the effects of resveratrol on cell cycle distribution. The most striking effect was a reduction in the percentage of cells in the G2/M phase which was most frequently associated with an increase of cells in the S phase of the cell cycle. We also found that resveratrol is able to prevent the increase in reactive oxygen species (ROS) following exposure to oxidative agents (i.e. tobacco-smoke condensate (TAR) and H(2)O(2)). Resveratrol also reduced nuclear DNA fragmentation, as assessed by single cell gel electrophoresis (comet test). Taken together our results suggest that resveratrol can act as an antimutagenic/anticarcinogenic agent by preventing oxidative DNA damage which plays a pivotal role in the carcinogenic activity of many genotoxic agents.

A processing system for elaboration of data obtained by the oxygraph.

A Data Processing System is presented to resolve some problems usually present in the oxygraph research. The possibility to obtain a file of data in which each experiment is arranged as completely as possible in the description of experimental conditions and the corresponding data permits the execution of some operations such as calculations, statistical elaborations, kinetic studies, research and comparation of experiments and plotting. This system may result very helpful in the programmation of the experiments and in the elaboration of the data obtained by the oxygraph technique.

[Osteopathies and vitamin D. New concepts and prospects in the light of recent advances]. / Osteopatie e vitamina D. Nuovi concetti e prospettive da recenti acquisizioni.

Recognition over the last ten years of the fact that vitamin D does not act as such, but must be converted into a hormonal form, has filled in the picture physiological endocrine regulation of calcium and phosphate homeostasis. While vitamin D has thus lost the dietetic significance associated with it for over 50 years. Nevertheless, new interpretations of the aetiopathogenesis of many demineralizing bone diseases are of much greater utility. Nor is it futuristic to suppose that all the biochemical parameters establishing one of the metabolisms that are under strict homeostatic control in the body, such as that of calcium and that of phosphate, are understood.

[Beneficial effect of swimming in thermal waters on muscle glycogen depletion]. / Benefico effetto del nuoto in acqua termale sulla deplezione del glicogeno muscolare.

The effect of swimming in the termal water on muscle glycogen stores was studied. After 30 min the muscle glycogen results in a diminution, but it is not depleted. On the contrary, 30 min of swimming in normal water results in a depletion of muscle glycogene stores. The glycemic homeostasis is well maintained in thermal water, and hypoglicemia occurs only after swimming in normal water.

[Lipolysis and ketosis during swimming in thermal water]. / Lipolisi e chetosi nel nuoto in acqua termale.

Plasma free fatty acids, blood glucose, beta-hydroxybutyrate and acetoacetate variations were studied in rats during swimming. Rats were forced to swim for 30 min in thermal water (source of Abano Terme) at 35 degrees and in normal water at 25 degrees. During swimming in thermal water plasma free fatty acids were increased, the glycemia remained unaffected, the beta-hydroxybutyrate and acetoacetate decreased. The swim in normal water induced a sharp increase of plasma free fatty acids, a decrease of blood glucose, an increase of blood beta-hydroxybutyrate and a marked decrease of acetoacetate. From these data, some indications of clinical interest are presented and discussed.