Pectin nanocoating reduces proinflammatory fibroblast response to bacteria.

Implant failures are primarily related to bacterial infections and inflammation. Nanocoating of implant devices with organic molecules is a method used for improving their integration into host tissues and limiting inflammation. Bioengineered plant-derived rhamnogalacturonan-Is (RG-Is) from pectins improve tissue regeneration and exhibit anti-inflammatory properties. Therefore, the aim of this study is to evaluate the in vitro effect of RG-I nanocoating on human gingival primary fibroblast (HGF) activity and proinflammatory response following Porphyromonas gingivalis (P. gingivalis) infection. Infected HGFs were incubated on tissue culture polystyrene (TCPS) plates coated with unmodified RG-I isolated from potato pectin (PU) and dearabinanated RG-I (PA). HGF morphology, proliferation, metabolic activity, and expression of genes responsible for extracellular matrix (ECM) turnover and proinflammatory response were examined. Following the P. gingivalis infection, PU and PA significantly promoted HGF proliferation and metabolic activity. Moreover, gene expression levels of IL1B, IL8, TNFA, and MMP2 decreased in the infected cells cultured on PU and PA, whereas the expression of COL1A1, FN1, and FGFR1 was upregulated. The results indicate that RG-Is are promising candidates for nanocoating of an implant surface, can reduce inflammation, and enhance implant integration, particularly in medically compromised patients with chronic inflammatory diseases such as periodontitis and rheumatoid arthritis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3475-3481, 2017.

Gene expression profiles in three histologic types, clear-cell, endometrioid and serous ovarian carcinomas.

Ovarian carcinoma is the most lethal type of gynecologic malignancy in the Western world. Majority of early stage ovarian cancers are asymptomatic and this is the main reason that more than two-thirds of patients are diagnosed with advanced disease. Ovarian tumors are heterogeneous and the different histologic subtypes are further classified as benign, borderline (low-grade) and malignant (high-grade) to reflect their behavior. The aim of the study was to analyze gene expression profiles in three histologic types of ovarian carcinoma in an attempt to find the molecular differences among serous, endometrioid and clear cell subtypes. The analysis of gene expression was performed on 57 samples of ovarian carcinoma. RNA was isolated from the ovarian cancer tissues. The gene expression changes were determined by microarray analysis and quantitative real time polymerase chain reaction (qRT-PCR). Measurement of relative gene expression levels was used to identify molecular differences among three histologic types of ovarian carcinoma (clear-cell, endometrioid and serous). Unsupervised statistical analysis revealed four biological subtypes among three histotypes under study. The endometrioid ovarian carcinoma was divided into two molecular subtypes. The biggest molecular differences were observed between clear-cell and serous carcinomas (1070 genes, FDR 0.05), the smallest between endometrioid and serous carcinomas (81 genes, FDR 0.05). The biggest group of differentially expressed genes was involved in transport and metabolism. This finding can explain the differences in the response to chemotherapy observed among different histologic types of ovarian carcinomas. In conclusion, we found TCF2 (HNF1B) gene as a suitable marker for ovarian clear cell carcinoma. Gene expression profiling also shed light on the molecular mechanisms of different chemoresistance among the analyzed histotypes.

Ovarian small cell carcinoma of hypercalcemic type - evidence of germline origin and SMARCA4 gene inactivation. a pilot study.

Ovarian tumors from two patients, compatible by histological and immunohistochemical criteria with small cell carcinoma of hypercalcemic type (SCCHT) (WT1+, EMA dispersed+, synaptophysin+ or dispersed+), were extensively sampled in order to find clues to their histogenesis. Subsequently, small foci of immature teratoma were found in both of them (in 1/122 and in 3/80 tumor sections). In one case, microfoci of yolk sac tumor were also present within the teratoma area as well as in the background of the small cell tumor population - in the primary tumor and in omental metastasis. We found a resemblance of the microscopic patterns of SCCHT and atypical teratoid/rhabdoid tumor (AT/RT) of the central nervous system, and this prompted us to evaluate INI-1 and SMARCA4 immunohistochemical expression, because their alternative loss is regarded as a molecular hallmark of AT/RT. INI-1 expression was retained, while that of SMARCA4 was lost. We therefore analyzed tumor DNA by PCR amplification and sequencing for mutations in the SMARCA4 gene (NG_011556.1), which were identified in both tumors (c.2184_2206del; nonsense c.3277C>T - both in one tumor; nonsense c.3760G>T in another tumor). These data suggest that SCCHT is most likely an embryonal tumor originating from immature teratoma and related to malignant rhabdoid tumor. Further analyses are necessary to determine whether the tumors diagnosed as SCCHT constitute a homogeneous group or represent more than one entity.

Geographical variations in TP53 mutational spectrum in ovarian carcinomas.

The TP53 gene mutational spectrum in human tumours shows variations related to tissue of origin, carcinogen exposure or molecular background. We have compared TP53 mutations in ovarian carcinomas from different geographical regions; this study was based on data extracted and verified from the IARC database (R10, 2005), and on our results from 127 carcinomas. In total 873 mutations were evaluated. Tumours from Japan and Korea had a higher frequency of exon 7 mutations (38%vs 25%, p = 0.011) and lower frequency of exon 8 mutations (11%vs 29%, p = 0.0003) than those from Western countries; they were particularly different from Norwegian tumours which showed the lowest proportion of exon 7 (19%, p = 0.001) and highest proportion of exon 8 (37%, p < 0.0001) mutations. There were also differences in the profile of TP53 hotspots. The third hotspot in tumours from Poland was amino acid (AA) 176 (8.2% of substitutions vs 1.7% in other countries, p < 0.001), while in tumours from the UK it was AA 220 (8.9%vs 2.3%, p < 0.001). Codon 273 was the only apparent hotspot in the Norwegian tumours, while it was rarely mutated in Polish and Asian tumours. In contrast to other data tumours from Norway presented with 273(HIS) codon (82% of mutations at AA 273, p = 0.002), while tumours from the UK shared the 273(CYS) codon (80%, p < 0.001). Further analysis of TP53 gene mutations in ovarian cancer by geography could provide greater insights.

PTEN mutation, expression and LOH at its locus in ovarian carcinomas. Relation to TP53, K-RAS and BRCA1 mutations.

OBJECTIVE: We aimed to evaluate frequency of PTEN mutation, LOH and expression in ovarian tumors. In search for a molecular pathway, we confronted PTEN gene mutations with TP53, K-RAS and BRCA1 gene status in the same tumors. We also evaluated clinical significance of PTEN expression in a subgroup of patients uniformly treated with platinum-based regimens. METHODS: Molecular analysis was performed on 105 ovarian tumors (100 carcinomas) with the use of the SSCP and sequencing. Seventy-six tumors were analyzed for LOH at 10q23 locus with the use of six polymorphic markers. Immunohistochemical PTEN expression was done on paraffin-embedded material. Multivariate and univariate analysis was performed with the STATA program. RESULTS: PTEN mutations occurred in 5/100 (5%) of all carcinomas and in 3/15 (20%) of endometrioid carcinomas (EC). Low-grade EC that developed in borderline tumors had PTEN and/or K-RAS mutation (4/5, 80%), while high-grade EC had TP53 mutations only. There was a reverse association between PTEN and TP53 mutations (P = 0.005). LOH at PTEN locus was found in 60% of endometrioid and in 28% of serous and clear cell carcinomas. PTEN expression did not associate with PTEN mutations or LOH. Strong PTEN expression diminished risk of death in a TP53 positive group only (HR = 0.35, P = 0.029). CONCLUSION: Our results suggest that PTEN mutations may play a role in a development of low-grade endometrioid tumors. PTEN haploinsufficiency caused by LOH or epigenetic events may possibly contribute to development of other histological types and may be an adverse prognostic factor.

Nijmegen breakage syndrome gene (NBS1) alterations and its protein (nibrin) expression in human ovarian tumours.

We looked for NBS1 gene (602667) alterations and changes in nibrin expression in 162 human gynaecological tumours, mostly ovarian. Exons 6-8 and 10 of the NBS1 gene were evaluated by the SSCP and direct sequencing method. Nibrin expression was detected immunohistochemically with the use of the p95NBS1 (Ab-1) antibody. The 657del5 mutation (Slavic mutation) was found in two of 117 carcinomas studied (1.7%) - in both cases it was present in the germline; one of these tumours showed loss of heterozygosity (LOH) for the 657del5 mutation and loss of nibrin expression. We have found three types of novel germline intron variants: (1) two concomitant transitions (G to A) at bases 14009 and 14256; (2) C to T transition at base 13998; (3) G to C transversion at base 20035. Among the carcinomas studied, the intron variants were associated with a clear cell histological type (p = 0.004). Our results may suggest that NBS1 gene alterations contribute to the development of rare ovarian carcinomas. LOH for 657del5 in tumour tissue may support the hypothesis that the NBS1 gene functions as a tumour suppressor.

Spontaneous apoptosis in ovarian carcinomas: a positive association with p53 gene mutation is dependent on growth fraction.

Changes in cell survival contribute to tumour development, influence tumour biology and its response to chemotherapy. p53 gene alterations should negatively affect apoptosis by impaired p53-dependent apoptotic response. We looked for associations between spontaneous apoptosis, p53 gene mutation, p53 protein accumulation, growth fraction, bcl-2 expression and histological parameters in 64 ovarian, four tubal and three peritoneal carcinomas. Apoptotic cells were detected with the TUNEL method. p53 gene variants were detected by the single-strand conformation polymorphism and were sequenced directly. P53, Ki-67 and bcl-2 protein expressions were detected immunohistochemically. A weighed multiple logistic regression model was applied. Apoptotic index (AI) ranged 0.02-0.18 (mean 0.11); proliferation index (PI) ranged 3-90% (mean 54%). p53 gene mutations were present in 51, p53 protein accumulation in 46, and diffuse bcl-2 expression in 29 of 71 tumours. The AI was positively associated with the presence of p53 gene mutation (P = 0.011). However, the PI included into the analysis did positively influence the AI (P = 0.02) and diminished the association with p53 gene mutation (P = 0.082). The AI was negatively associated with good histological differentiation (P = 0.0006), the serous tumour type (P = 0.002), and diffuse bcl-2 expression (P = 0.025). Strong bcl-2 expression was associated with endometrioid tumour type (P = 0.002). FIGO stage and p53 protein accumulation were the only parameters that influenced overall survival time. Thus, our results suggest that histological tumour type and grade are major determinants of spontaneous apoptosis in ovarian carcinomas; p53 alterations do not adversely but rather positively affect spontaneous apoptosis by increasing growth fraction. This, in turn, suggests p53-independency of spontaneous apoptosis in ovarian carcinomas.