Fourier Transform Infrared Imaging analysis of dental pulp inflammatory diseases.

OBJECTIVES: Fourier Transform Infrared microspectroscopy let characterize the macromolecular composition and distribution of tissues and cells, by studying the interaction between infrared radiation and matter. Therefore, we hypothesize to exploit this analytical tool in the analysis of inflamed pulps, to detect the different biochemical features related to various degrees of inflammation. MATERIALS AND METHODS: IR maps of 13 irreversible and 12 hyperplastic pulpitis, together with 10 normal pulps, were acquired, compared with histological findings and submitted to multivariate (HCA, PCA, SIMCA) and statistical (one-way ANOVA) analysis. The fit of convoluted bands let calculate meaningful band area ratios (means ± s.d., P < 0.05). RESULTS: The infrared imaging analysis pin-pointed higher amounts of water and lower quantities of type I collagen in all inflamed pulps. Specific vibrational markers were defined for irreversible pulpitis (Lipids/Total Biomass, PhII/Total Biomass, CH2 /CH3 , and Ty/AII) and hyperplastic ones (OH/Total Biomass, Collagen/Total Biomass, and CH3 Collagen/Total Biomass). CONCLUSION: The study confirmed that FTIR microspectroscopy let discriminate tissues' biological features. The infrared imaging analysis evidenced, in inflamed pulps, alterations in tissues' structure and composition. Changes in lipid metabolism, increasing amounts of tyrosine, and the occurrence of phosphorylative processes were highlighted in irreversible pulpitis, while high amounts of water and low quantities of type I collagen were detected in hyperplastic samples.

Study of oral cavity lesions by infrared spectroscopy.

Fourier transform infrared (FTIR) microspectroscopy is considered a useful tool in the biomedical field, for analysing in situ and at cellular level, very small areas of tissues and cells, with minimal sample preparation and without the use of stains or probes. This spectroscopic technique has been successfully applied to analyse biological samples from patients affected by tumoral pathologies, with particular attention to oral cavity lesions. In this study, we describe the application of FTIR microspectroscopy to characterize and discriminate the most recurrent benign and malignant diseases of oral cavity compartment. Infrared maps were acquired on tissues affected by the following pathologies: squamous cell carcinoma, adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma, squamous dysplasia, keratocystic odontogenic tumor, radicular cyst, residual cyst, unicystic ameloblastoma, and ameloblastic fibroma, together with healthy tissue samples (used as control group). The epithelial and connective components of all samples were distinguished and submitted to multivariate analysis. The results were in agreement with histological suggestions.

Pokemon proto-oncogene in oral cancer: potential role in the early phase of tumorigenesis.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) represents about 90% of all oral neoplasms with a poor clinical prognosis. To improve survival of OSCC patients, it is fundamental to understand the basic molecular mechanisms characterizing oral carcinogenesis. Dysregulation of oncogenes and tumor suppressor genes seems to play a central role in tumorigenesis, including malignant transformation of the oral cavity. MATERIALS AND METHODS: We analyzed the expression levels of the pro-oncogenic transcription factor Pokemon through real-time PCR, Western blot and immunohistochemistry in tumor, and normal oral tissue samples obtained from 22 patients with OSCC. The relationship between tumor characteristics and the level of Pokemon intratumor expression was also analyzed. RESULTS: Pokemon was significantly downregulated in OSCC. In particular, both mRNA and protein levels (tumor vs normal tissue) inversely correlated with histological grading, suggesting its potential role as a prognostic factor for OSCC. Moreover, a significant inverse correlation was found between Pokemon protein expression levels (OSCC vs normal oral mucosa) and tumor size, supporting the hypothesis that Pokemon could play an important role in the early phase of tumor expansion. CONCLUSION: This work shows that reduced expression of Pokemon is a peculiar feature of OSCC. Additional studies may establish the effective role of Pokemon in oral tumorigenesis.

Osteopontin, osteocalcin and OB-cadherin expression in Synthetic nanohydroxyapatite vs bovine hydroxyapatite cultured Osteoblastic-like cells.

Calcium phosphate ceramics have been applied in bone replacement for several decades due to their excellent biocompatibility, bioactivity, osteo-conductivity and mechanical strength. Several studies have demonstrated that porous hydroxyapatite (HA) is an excellent scaffold for osteogenic proliferation and differentiation of the osteoprogenitor cells. However, different methods of synthesis and production of HA ceramic-based materials may have considerable effect on the physical and biological properties. In the present work, two hydroxyapatite-based materials, a natural hydroxyapatite ceramic of bovine origin and a synthetic nano-cristalline hydroxyapatite were tested in vitro with MG63 cell line. The results displayed that both the materials demonstrated a good biocompatibility. The immunocytochemical stain revealed a different positivity of the osteogenic markers between the cultures with the biomaterials, and the control culture. Western blot data confirmed the immunocytochemical stain. Both the materials tested in the present study demonstrated a good biocompatibility with the osteoblastic cells allowing, at the same time, the osteogenic differentiation, and they may be useful in clinical use.

Periodontal status in the Down's syndrome subjects living in central-eastern Italy: the effects of place of living.

AIM: The purpose of this study was to determine the influence of the place of living on periodontal status of 62 Down's syndrome (DS) subjects resident at home (DSH) or in specialized institutes (DSI) in central-eastern Italy. METHODS: The demographic characteristics of the subjects and the periodontal variables were evaluated according to their living conditions. Descriptive analyses were conducted by stratifying subjects into three age groups (0-13; 14-22; >23 years), using medians and 25th-75th percentiles to summarized data. Comparisons between DSH and DSI subjects were performed using Wilcoxon rank sum test. The effect of demographic and clinical variables on periodontal status was evaluated by means of quantile regression analysis. RESULTS: No significant differences resulted between DSH and DSI patients, when compared for gender, age and mental retardation. No significant differences were found in the periodontal variables for the subjects with 0-13 years, while DSI subjects between 14 and 22 years of age presented higher levels of plaque index, probing depth, clinical attachment loss and a lower number of surviving teeth compared to DSH subjects. When DSI and DSH groups ≥ 23 years of age were compared, no differences were observed in the periodontal conditions except for PI and the number of surviving teeth. Age, body mass index and severe mental retardation were found to be significant predictors of periodontal conditions. CONCLUSIONS: Institutionalization has a negative effect on surviving teeth number of Down's syndrome subjects. Furthermore, the home care seems to produce benefits on the periodontal conditions of DSH 14-22 years of age.

Aurora B expression as a prognostic indicator and possible therapeutic target in oral squamous cell carcinoma.

The aim of this study is to investigate the expression of the chromosomal passenger protein Aurora B and its activated (phosphorylated) form in a large series of human oral squamous cell cancers (OSCC) and to evaluate its clinical and prognostic significance. Western blotting analysis revealed overexpression of both Aurora B and Thr-232 Phopsho-Aurora B in OSCC lines as compared to normal keratinocytes and bladder cancer cells. Furthermore, protein expression was analysed by immunohistochemistry in 101 OSCC of different site, stage and histological grade and in normal peritumoural areas. The intracellular localization of Aurora B in tumour cells was mainly nuclear, especially in proliferative areas, and significant overexpression was found in tumours in comparison to normal peritumoural areas (P=0.012). Staining results were correlated with clinicopathological parameters and long-term follow-up, and a significant association was found between protein expression and tumour stage (stage II, III and IV vs stage I, P=0.030) and size (<2cm vs >2cm, P=0.010). Cox regression analysis confirmed a poorer disease-free survival in cases with high expression of Aurora B protein. Kaplan-Meier curves showed shorter time to progression in patients with high levels of Aurora B expression (p<0.05). Moreover, the tumoral group with nuclear Aurora B immunolocalization had the worst prognosis (P=0.0364 in disease free survival). Our results suggest that assessing Aurora B expression might help in patients’ risk stratification and serve as a novel therapeutic target in advanced OSCCs.

Inhibiting proliferation in KB cancer cells by RNA interference-mediated knockdown of nicotinamide N-methyltransferase expression.

The enzyme Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide and other pyridines, playing a pivotal role in the biotransformation and detoxification of many drugs and xenobiotic compounds. Several tumours have been associated with abnormal NNMT expression, however its role in tumour development remains largely unknown. In this study we investigated expression levels of Nicotinamide N-methyltransferase in a cancer cell line and we evaluated the effect of shRNA-mediated silencing of NNMT on cell proliferation. Cancer cells were examined for NNMT expression by semiquantitative RT-PCR and Western blot analysis. A HPLC-based catalytic assay was performed to assess enzyme activity. Cells were transfected with four shRNA plasmids against NNMT and control cells were treated with transfection reagent only (mock). The efficiency of gene silencing was detected by Real-Time PCR and Western blot analysis. MTT cell proliferation assay and the soft agar colony formation assay were then applied to investigate the functional changes in cancerous cell. NNMT mRNA was detected in cancer cells, showing a very high expression level. In keeping with the results of RT-PCR analysis, the protein level and NNMT enzyme activity were particularly high in KB cells. ShRNA vectors targeted against NNMT efficiently suppressed gene expression, showing inhibition observed at both the mRNA and protein levels. Down-regulation of NNMT significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The present data support the hypothesis that the enzyme plays a role in tumour expansion and its inhibition could represent a possible molecular approach to the treatment of cancer.

Cloning and characterization of a senescence inducing and class II tumor suppressor gene in ovarian carcinoma at chromosome region 6q27.

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer.

The role of peroxisome proliferator-activated receptor gamma in bladder cancer in relation to angiogenesis and progression.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) immunohistochemical expression was analyzed in 75 human bladder tumor specimens, where the expression of some angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PDECGF), and tumor progression markers, such as epidermal growth factor receptor (EGFr), p16, mutated p53, and normal pRB, were also analyzed. The results were then compared to the clinical and pathological characteristics of the disease. PPAR gamma was expressed more significantly in papillary tumors than in solid cancers, and its presence was associated with statistical significance to low incidence of tumor recurrence or progression. This significant association was observed also when PPAR gamma was expressed in the presence of PDECGF, which resulted, when considered alone, to an angiogenic factor typical of solid cancers and appeared related to poor prognosis. In the presence of bFGF, on the contrary, PPAR gamma expression no longer resulted to a significant association with low incidence of tumor recurrence or progression, suggesting a possible worsening role of this angiogenic factor, typical of papillary cancers, in its interaction with PPAR gamma.

Antiangiogenic, antitumoural and antimetastatic effects of two distamycin A derivatives with anti-HIV-1 Tat activity in a Kaposi's sarcoma-like murine model.

The antiangiogenic, antitumoural and antimetastatic effects of two novel sulphonic derivatives of distamycin A, PNU145156E and PNU153429, were studied in a Kaposi's sarcoma-like tumour model obtained by injecting nude mice with cells releasing extracellular HIV-Tat protein, derived from a tumour which developed in a BK virus/tat transgenic mouse. Both PNU145156E and PNU153429 were administered intraperitoneally every fourth day for three weeks at doses of 100 or 50 mg/kg of body weight respectively, starting one day after injecting the tumour cells. Both drugs delayed tumour growth in nude mice, preventing neovascularization induced by the Tat protein. PNU153429 also significantly reduced the number and size of spontaneous tumour metastases. Both effects on tumour growth and metastases were augmented by treating simultaneously nude mice with 7.5 mg/kg of body weight of minocycline given per os daily for four weeks starting four days after injecting the tumour cells. Neither acute nor chronic toxic side-effects were observed during the life span of treated nude mice. Due to their antiangiogenic and anti-Tat effects, these drugs are promising for the treatment of Kaposi's sarcoma in AIDS patients.

Whole-cell bacterial peroxidase test with isoproterenol as the hydrogen donor.

The beta-adrenergic compound isoproterenol was used as oxidizable reagent in a whole-cell assay for the detection of bacterial peroxidase activities. Isoproterenol has been shown to constitute a useful reagent for detecting peroxidase activities in enzymatic tests, utilizing standard purified enzymes, and in the microbiological application proposed. The procedure developed is simple and rapid to perform. In contrast to currently used whole-cell tests for bacterial peroxidases, the assay described here does not need preliminary permeabilization; moreover, the compound utilized does not have related toxicological problems. Therefore, the isoproterenol assay may represent a low-cost safe additional peroxidase test in clinical bacteriology.

Isolation of lysozyme on chitosan.

Lysozyme has been immobilized on chitosan, a polyaminosaccharide, without using any intermediate reagent. The best pH conditions for operating the chitosan columns have been determined and the best eluting agent was found to be a 2% solution of propylamine. The lysozyme activity was determined after reacting lysozyme with the product of glycolchitin and Remazol Brilliant Blue R. The recovery of lysozyme from chicken egg white yields lysozyme with 55% activity.

Immobilized enzymes on chitosan columns: alpha-chymotrypsin and acid phosphatase.

alpha-Chymotrypsin and acid phosphatase have been immobilized on chitosan, a polyaminosaccharide, without using any intermediate reagent; the immobilized enzymes are active and their activity is much higher than for chitin-immobilized enzymes. The best pH conditions for operating chitosan columns have been determined and columns have been used to transform substrates in large amounts, with no decrease of activity or enzyme losses. Due to the nonconvalent interaction between chitosan and enzymes, the pure and active enzymes can be eventually recovered from the columns. The effects of metal ions, aldehydes, and salts are reported and discussed. Applications are foreseen in the food and biomedical sciences and industries.

Atomic-absorption determination of manganese, cobalt and copper in whole blood and serum, with a graphite atomizer.

A new and rapid method is described for the determination of Mn, Co and Cu by atomic absorption with a graphite atomizer and deuterium compensation, on very small samples of whole blood and serum, with no preliminary manipulations. The metal concentrations in blood serum from healthy donors have been found to be Mn 9 +/- 4.3 ng ml ; Co 7.7 +/- 1.9 ng ml and Cu 1.2 +/- 0.16 mug ml .

Enhanced capacity of chitosan for transition-metal ions in sulphate-sulphuric acid solutions.

Batch measurements have shown that the collection yields of chitosan for chromium(III), iron(III), nickel, copper(II), zinc and mercury(II) from sulphuric acid solutions are higher when the solutions contain ammonium sulphate, or when chitosan conditioned in ammonium sulphate is used, particularly at pH 3.0 and 5.0. The contrary is verified for the oxy-anions vanadate, chromate and molybdate. Manganese is never collected. At pH 1.0 no collection occurs. A procedure for recycling chromatographic columns includes fixation of Cu or Ni from a sulphate solution at pH 3-5 on sulphate-conditioned chitosan, and elution with 0.1M sulphuric acid/0.1M ammonium sulphate at pH 1.0; the presence of sulphate in the eluent obviates the detrimental effect of sulphuric acid on the next cycle. Sulphate is the favoured counter-ion of the chelated cations and its action produces shorter chromatographic bands. The interaction of sulphate with chitosan is discussed in terms of crystallinity and steric distribution of the protonated amino-groups in the polymer. Data on the new diethylaminohydroxypropylcellulose are included.