Tracking down of laryngo-pharyngeal metastasis.

BACKGROUND: In Romania, the most optimistic statistics give a 5 years survival rate in approximately 33% of laryngo-pharyngeal cancer patients. Considering that a cell carrying the viral DNA is originating from primary tumor, we have tested whether HPV DNA could be detected in the blood cell of patients with laryngeal cancer as a marker of disease progression and metastases. METHODS: The study was performed on 85 patients (59 +/- 8.7 age) with laryngo-pharyngeal cancer. HPV DNA was detected in tumor using nested PCR with consensus primers, and also in local lymph nodes and/or blood cells from patients HPV positive in primary tumor. RESULTS: HPV DNA was detected in 75.29% of analyzed tumours, and all HPV16 positive samples were confirmed by mRNA E6 expression. 56.3% of patients presented HPV DNA in peripheral circulation as confirmed by PCR with E6 HPV16 specific primers followed by Southern Blot. CONCLUSION: Our results sustain that the detection of HPV DNA in blood is a "surrogate marker" of metastasis when extension of metastasis cannot be estimated, this observation is very important for management of cancer patients with laryngopharyngeal localization.

In vitro hepatic differentiation of human bone marrow mesenchymal stem cells under differential exposure to liver-specific factors.

Recent findings demonstrated that stem cells could be harvested from a patient and used to repair his or her own damaged liver. Additionally, stem cells may be manipulated in vitro to induce hepatic differentiation. The current study aims to determine the differentiation efficacy of various liver-specific factors (hepatocyte growth factor, Insulin-Transferrin-Selenium, dexamethasone, and nicotinamide) used for stem cell differentiation into hepatocyte-like cells. Human mesenchymal stem cells were exposed to different media containing these compounds added individually or in various combinations. Hepatic differentiation was assessed via quantitative reverse transcription-polymerase chain reaction and immunocytochemical staining for stemness or liver-specific genes and proteins, including albumin, cytokeratins 18 and 19, HepPar-1, alpha-fetoprotein, and nestin. In addition, functional tests for glycogen storage, urea production, glucose, and albumin synthesis were also performed. The expression profiles of albumin, alpha-fetoprotein, and cytokeratin 19 demonstrated that when hepatocyte growth factor, nicotinamide, or dexamethasone were added individually, incomplete hepatocyte differentiation was achieved; the obtained cell populations contained progenitors that expressed both hepatic (albumin) and biliary (cytokeratin 19) markers, as well as alpha-fetoprotein. Hepatocyte growth factor and nicotinamide were the factors with the most hepatogenic potential. When all factors were added to the culture, cells exhibited features that closely resembled human adult hepatocytes as determined by their gene expression patterns (albumin, HepPar-1, and alpha-fetoprotein, but not cytokeratin 19) and functional testing. These cells with hepatic function may become important tools for liver transplant procedures, liver development studies, and pharmacologic research.

Transplantation of mesenchymal stem cells cultured on biomatrix support induces repairing of digestive tract defects, in animal model.

Transplanted mesenchymal stem cells (MSCs) appear to play a significant role in adult tissue repair. The aim of this research was to obtain MSCs enriched, three dimensional (3D) patches for transplant, and to test their ability to induce repair of iatrogenic digestive tract defects in rats. MSCs were obtained from human and rat bone marrow, cultured in vitro, and seeded in a collagen-agarose scaffold, where they showed enhanced viability and proliferation. The phenotype of the cultured cells was representative for MSCs (CD105+, CD90+, and CD34-, CD45-, CD3-, CD14-). The 3D patch was obtained by laying the MSCs enriched collagen-agarose scaffold on a human or swine aortic fragment. After excision of small portions of the rat digestive tract, the 3D patches were sutured at the edge of the defect using micro-surgical techniques. The rats were sacrificed at time-points and the regeneration of the digestive wall was investigated by immunofluorescence, light and electron microscopy. The MSCs enriched 3D patches were biocompatible, biodegradable, and prompted the regeneration of the four layers of the stomach and intestine wall in rats. Human cells were identified in the rat regenerated digestive wall as a hallmark of the transplanted MSCs. For the first time we constructed 3D patches made of cultured bone marrow MSCs, embedded into a collagen-rich biomatrix, on vascular bio-material support, and transplanted them in order to repair iatrogenic digestive tract defects. The result was a complete repair with preservation of the four layered structure of the digestive wall.

Immunohistochemical localization of caspase-3, caspase-9 and Bax in U87 glioblastoma xenografts.

Development of new therapies for glioblastoma requires animal models that mimic the biological characteristics of human brain tumors. On the other hand, potential antitumoral effects of a new therapeutic strategy are often established by evaluation of tumor cells apoptosis. Caspases are key mediators in the regulation and execution of apoptosis. Caspase-9 is activated during the intrinsic pathway downstream of mitochondria while caspase-3 is an effector caspase that initiates degradation of the cell in the final stages of apoptosis. Bax is a pro-apoptotic member of the Bcl-2 family that play key roles in the regulation of intrinsic apoptotic signaling. In the present study we investigated the immunohistochemical distribution of caspase 3, 9 and Bax in intracranial U87 glioblastoma xenograft. Immunohistochemistry showed that the glioblastoma xenografts contain cells positive for caspase-3, caspase-9, and Bax.

The development of xenograft glioblastoma implants in nude mice brain.

The inefficacity of the actual therapies for glioblastoma multiformis stimulates the researchers to search for new and innovative therapies. Therefore, the development of in vivo model for glioblastoma is an essential step during these researches, being a link between cells cultures studies and the first phases of clinical trials. In this paper, we present several procedures which have been performed for the first time in our country, such as: the cultivation and manipulation of U87MG line, the manipulation of athymic knock-out mice (NUDE Crl: CD-1 Foxn 1), the stereotactic inoculation of glioblastoma cells and finally the development of glioblastoma xenograft in the brain of inoculated nude mice. These results, which offer to the researchers from our country an in vivo model for glioblastoma, could be the start point for several projects oriented to the development of new therapies in glioblastoma, and could raise the performance of our scientific research to the European level.

The comparison of different protocols for expansion of umbilical-cord blood hematopoietic stem cells.

Hematopoiesis is maintained by the activity of multipotent stem cells, which have the dual capacity to self-renew and to differentiate into all of the blood cell lineages. The major challenge of stem cells based regenerative therapy is to expand ex vivo the primitive compartment to increase transplantable stem cells number. The present study was designed to evaluate several culture systems for in vitro maintenance of umbilical cord blood stem cells. The influences of different growth conditions such as stromal feeder layer, cytokines supplement and placental conditioned medium (PCM) have been evaluated over a relatively short period of time on CD34(+) cell expansion and maintenance of clonogenic progenitors. When cells were expanded on feeder layer in the presence of added cytokines and PCM on average a 2.96-fold increase of CD34(+)CD71(-) and a 3.13-fold increase of CD34(+)HLA-DR(-) was observed. The total number of colony forming cells (35 +/- 2.65) indicated also that the yield of clonogenic progenitors obtained with a combination of all factors was two folds higher than each of these factors alone and ten time above control (3.67 +/- 2.52). In conclusion, the results of our study clearly show that the ex vivo expansion of hematopoietic progenitor cells obtained from human umbilical cord blood is dependent on controlled experimental conditions, which might be helpful when designing culture systems for clinical applications.

The development of larger cells that spontaneously escape senescence--a step during the immortalization of a human cancer cell line.

There are few information concerning the changes associated with the transition interval when slow growing, primary explanted human cancer cells are displaced by new selected faster growing cells and became an immortal cell line. In a previous paper (J. Cell. Mol. Med., 5: 49-59, 2001) we described the TV cell line derived from a laryngeal tumor which harbors human papillomavirus (HPV) gene sequences throughout more than sixty in vitro passages. In this paper we analyze the modifications observed during the crisis interval when significant amount of cells senesce but occasional cells acquire some mutations that make them immortal. Confocal microscopy analysis revealed the heterogeneity of the cells in terms of their size and nucleus/cell ratio. Proliferation capacity was assessed by flow cytometry analyzing DNA content and expression of transferrin receptor (CD71). We discussed the possibility that HPV genome sequences alleviate a proliferation block during the crisis growth arrest of human larynx carcinoma cell line and the possibility that the cells monitor their size and growth by measuring the levels of some protein whose synthesis is coupled to cell development.

Ex vivo differentiation of umbilical cord blood progenitor cells in the presence of placental conditioned medium.

Hematopoetic stem cells (HSC) are the progenitors for the lympho-hematopoietic system, with long lifespan and high proliferation potential. Transplantation of HSC from bone marrow or peripheral blood represents a standard therapy in severe hematological conditions. A possible alternative source of HSC is the umbilical cord blood, prepared by various separation procedures followed by expansion in cultures supplemented with hematopoietic growth factors. In order to check the effects of placental conditioned medium (PCM) from placental cells culture upon viability of HSC, we added plasma, PCM, dimetil sulfoxyde or hemin in HSC cultures. Flow cytometry or direct scoring of solid cultures using CD45+, CD34+, CD71+ and CD14+ fluorescent-labeled monoclonal antibodies evaluated the effects upon cell proliferation and colony forming ability of HSC cultures, versus controls. PCM produced the highest proliferation, followed by plasma, DMSO and hemin. PCM improved the survival time and maintained a higher proportion of immature cells. PCM stimulates the differentiation towards myeloid lineage progenitor cells (>90% being CD45+), increasing the percentage of CD14+, granulocites /monocytes precursors. It is highly suggestive that PCM contains growth factors or cytokines, which regulate the development of HSC. Characterization of these factors is in progress.