Performance of a polymer-based DNA chip platform in detection and genotyping of human papillomavirus in clinical samples.

Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods.

Granulocyte colony-stimulating factor (G-CSF) receptor gene expression of ovarian carcinoma does not correlate with G-CSF caused cell proliferation.

BACKGROUND: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a growth factor commonly used to avoid leukopenia after chemotherapy. Endogenous G-CSF is produced by macrophages and granulocytes that infiltrate tumors. It has been reported that rhG-CSF stimulates the proliferation of several cell lines as well as bladder carcinoma cells. Conversely, in some hematopoietic cell lines such as U-937, WEHI-3B, and K-562 no effect or in some cases a differentiation pattern was found. Moreover, the role of rhG-CSF on the proliferation of solid tumors is not well understood. METHODS: In this study, 10 ovarian carcinoma biopsies were characterized for the presence of G-CSF and G-CSF receptor by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. Proliferation was analyzed by ATP viability assays. RESULTS: Performing RT-PCR, these biopsies and four ovarian carcinoma cell lines were analyzed for endogenous G-CSF production, which was found in some biopsies and in all cell lines. Despite the presence of the G-CSF receptor in all biopsies and cell lines, no proliferation was found after rhG-CSF incubation of the cell lines or the tumor samples for 3 and for 6 days, respectively. CONCLUSIONS: Summarizing the authors' in vitro studies, rhG-CSF does not affect the proliferation of ovarian carcinoma cells in vitro.

Evaluation of methods to detect p53 mutations in ovarian cancer.

OBJECTIVE: The p53 status is increasingly regarded as a marker predictive of response to particular cancer therapies, but for this approach it is self-evident that the p53 status must be determined correctly. METHODS: We have tested ovarian cancers with single-strand conformation polymorphism analysis (SSCP), immunohistochemical staining with DO-1 anti-p53 antibody (IHC), and yeast p53 functional assay (FASAY). RESULTS: These techniques commonly used to detect p53 mutations showed important differences in their sensitivity. Of 53 tumors tested with three indirect techniques, 27 (50%), 33 (62%) and 41 (77%) were positive by SSCP, IHC, and FASAY, respectively. In a subset of 32 tumors strongly suspected of containing mutations, 25 (78%), 26 (81%), 29 (91%) and 30 (94%) were positive by SSCP, immunostaining, DNA sequencing and yeast assay, respectively. CONCLUSIONS: Under comparable routine conditions, the FASAY reached the highest sensitivity. Since no single technique detected all mutations, we recommend the use of at least two different techniques in situations where the p53 status will affect patient management.

G-CSF receptor expression in ovarian cancer.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is clinically used to overcome neutropenic periods during chemotherapy. In vitro studies using cell lines as a model system have recently suggested that G-CSF can promote ovarian cancer growth. The objective of this work is to determine whether tumor cells express G-CSF-receptors (G-CSFR). A set of ovarian tumor biopsies and ovarian cancer cell lines was analyzed by RT-PCR, immunohistochemistry and immunofluorescence. The presence of a 276 bp-amplicon (exon 8-10) obtained by RT-PCR showed that 12 out of 16 ovarian tumor biopsies and two out of four ovarian cancer cell lines expressed G-CSFR-mRNA. G-CSFR-protein was detected in tumor cells of the 12 biopsies that also contained G-CSFR-mRNA. A second 409 bp-amplicon (exon 17) obtained by RT-PCR from the variable C-terminal cytoplasmic region of G-CSFR could be amplified only in four out of 16 biopsies and in none of the ovarian cancer cell lines studied. The results presented here indicate that G-CSFR is frequently expressed in ovarian cancer cells. Moreover, the failure of RT-PCR amplification of the 409 bp-amplicon in samples that express G-CSFR-mRNA suggests that C-terminal truncated receptor forms are also expressed.

Expression of interleukin-6 and interleukin-6 receptors in human granulosa lutein cells.

Cytokines are important regulators of reproductive functions. Significant amounts of interleukin-6 (IL-6) have been detected in the serum and ascites of patients with ovarian hyperstimulation syndrome (OHSS). These findings suggest the involvement of IL-6 as a mediator in the pathogenesis of OHSS. This study was performed to analyse IL-6 and IL-6 receptor (IL-6-R) expression in human granulosa lutein cells (GC). GC were cultured after isolation from follicular fluid. IL-6 concentrations in follicular fluid and serum from individual patients and GC supernatants were measured by enzyme-linked immunosorbent assay. We found detectable concentrations of IL-6 in serum and follicular fluid of all patients. Expression of IL-6 in GC was shown immunocytochemically. IL-6 mRNA was detected in GC by in-situ hybridization. Gene expression for IL-6 and IL-6-R in GC was demonstrated using reverse transcription-polymerase chain reaction. IL-6 significantly inhibited human chorionic gonadotrophin (HCG)-induced progesterone secretion of GC. The results of our study suggest that IL-6 is expressed in HGC and that this cytokine is able to modulate GC function via its specific receptor. This is the first report that describes the precence of IL-6-R in human granulosa lutein cells.

rhG-CSF affects genes involved in mitogen signalling and early gene expression in the ovarian cancer cell line HEY.

The ovarian adenocarcinoma cell line HEY was used as an in vitro model to study the influence of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on epithelial tumours such as ovarian cancer. Serum-starved cells were treated with rhG-CSF in a time- and dose-dependent manner. Cell proliferation, measured as cell division and DNA synthesis, was stimulated about 40% by rhG-CSF. After harvesting, cells were examined for the presence of G-CSF receptor (FACS analysis and RT-PCR), as well as for expression of genes involved in mitogen signalling (ERKs, JNKs) and early gene expression (c-jun). rhG-CSF affected mitogen-activated pathways and was receptor-mediated if the G-CSF receptor was present. After rhG-CSF induction, Janus N-terminal kinases (JNK 1 and 2) were simultaneously increased in the cytosol, up to 30-fold as measured by Western blotting), whereas ERK 1 and 2 accumulated maximally by 2.5-fold 1 hr after rhG-CSF induction. c-Jun was up-regulated strongly by this cytokine at the translational level. Our data suggest that rhG-CSF affects genes involved in mitogen signalling and early gene expression in solid tumours. We also noted the presence of G-CSF receptor on ovarian cancer cell lines.

Influencia del G-CSF en el crecimiento celular en el adenocarcinoma ovarico / Influence of G-CSP on the proliferation an ovarian adenocarcinoma cell line

El factor de crecimiento hematopoyético G-CSF (factor estimulante de las colonias de granulocitos) es clínicamente usado para superar períodos de neutropenia y para aumentar los niveles endógenos de neutrófilos durante la quimioterapia. Sin embargo poco es lo conocido acerca de los aspectos sobre células tumorales. Por el otro lado, los cánceres de ovario son frecuentemente infiltrados con glóbulos blancos que producen localmente G-CSF, por lo tanto esta cytoquina puede en forma sistémica o parácrina jugar un importante rol en el desarrollo tumoral en tumores que presentan receptores de G-CSF. Las cytoquinas pueden presentar ambos efectos, positivo o negativo, en las células que presentan cancerogénesis, signos incipientes de proliferación, diferenciación o apoptosis. Ellas juegan un rol mayor en la determinación del microambiente tumoral por las interacciones entre el huésped y las células tumorales en los estadios precoces del desarrollo tumoral y el crecimiento celular

Influencia del G-CSF en el crecimiento celular en el adenocarcinoma ovarico / Influence of G-CSP on the proliferation an ovarian adenocarcinoma cell line

El factor de crecimiento hematopoyético G-CSF (factor estimulante de las colonias de granulocitos) es clínicamente usado para superar períodos de neutropenia y para aumentar los niveles endógenos de neutrófilos durante la quimioterapia. Sin embargo poco es lo conocido acerca de los aspectos sobre células tumorales. Por el otro lado, los cánceres de ovario son frecuentemente infiltrados con glóbulos blancos que producen localmente G-CSF, por lo tanto esta cytoquina puede en forma sistémica o parácrina jugar un importante rol en el desarrollo tumoral en tumores que presentan receptores de G-CSF. Las cytoquinas pueden presentar ambos efectos, positivo o negativo, en las células que presentan cancerogénesis, signos incipientes de proliferación, diferenciación o apoptosis. Ellas juegan un rol mayor en la determinación del microambiente tumoral por las interacciones entre el huésped y las células tumorales en los estadios precoces del desarrollo tumoral y el crecimiento celular (AU)

c-jun expression and growth stimulation in human ovarian carcinoma cell lines following exposure to cytokines.

We evaluated the effects of recombinant human G-CSF, rhGM-CSF, rhM-CSF and rhIL-1 alpha on proliferation and regulation of c-jun gene expression in 4 human ovarian-carcinoma (HOC) cell lines, NIH:OVCAR-3, SK-OV-3, HEY and BG-1, and in one primary ovarian tumor in vitro. The cytokines were administered in concentrations of 0.1 U/ml to 1000 U/ml. Cell growth was measured by crystal-violet- and thiazolyl-blue(MTT)-based cell counts. c-jun transcripts were measured by the solution hybridization/RNAse protection assay. RhM-CSF and rhGM-CSF showed no growth stimulation of any of the 5 cell lines tested. Results from exposure to rhG-CSF were different. The cell lines NIH:OVCAR-3, SK-OV-3, BG-1 and the primary ovarian tumor showed no proliferative response. A 2- to 3-fold increase in proliferation was observed in the HEY HOC cell line. rhIL-1 alpha led to growth stimulation in the BG-1 cell line, but showed an inhibitory effect in the NIH:OVCAR-3 cells. No effects of rhIL-1 alpha were observed in the remaining 2 cell lines nor in the primary ovarian tumor. Growth stimulation was accompanied by an increase in c-jun expression in the HEY cell line, and the BG-1 cell line. No alterations in c-jun expression were observed in the remaining 3 cell lines. Our results indicate that rhG-CSF or rhIL-1 alpha influence cell proliferation in 2 out of 5 human ovarian-tumor cell lines, accompanied by an increase in c-jun expression.

[Deciduous dentition and orthognathodontic prevention. 2]. / Dentatura decidua e prevenzione ortognatodontica. Parte II.

One of the main factors in the prevention of malocclusion is the preservation of healthy primary dentition until the time of normal shedding. By this contribution the Authors describe the restorative procedures and the pulp and canal treatments to be applied in deciduous dentition as well as the clinical and therapeutic problems connected with the early loss and the abnormal persistence of deciduous teeth. These therapeutic measures, in addition to caries prevention, have a very important role in the promotion of the normal development of occlusion.