Plasticity of regulatory T cells under cytokine pressure.

CD4+ T helper (Th) cells have been divided into different subsets as defined by their cytokine products and functions after their activation. CD4+ T cell subsets are continuously discovered and until now Th1, Th2, Th9, Th17, and regulatory T (Treg) cells have been almost unanimously recognized but yet not completely characterized. The selective production of cytokines by each of the subsets is probably the master key of the mechanisms of immune regulation. The cytokine milieu is extremely important on deciding the fate of T cells. Generally, more than one cytokine is needed for differentiating to a particular lineage and just recently it was shown that this status quo of commitment could be challenged. It is well known that cytokines bind to Type I/II cytokine receptors signaling via Janus kinases (JAKs) followed by activation of Signal Transducer and Activator of Transcription (STAT). STAT molecules work together with other transcription factors (Foxp3, RORgammat and RORalpha, T-bet, GATA3, Runx 1, NFAT, etc.) also controlled by cytokines, in modulating the Th phenotype and functions. In this review, we analyze the plasticity of Treg population focusing on the most recent discoveries on how microenvironmental cytokines refine/modify Treg phenotype and function, thus changing their fate.

Gastrointestinal cancer stem cells as targets for innovative immunotherapy.

The role of cancer stem cells in gastrointestinal cancer-associated death has been widely recognized. Gastrointestinal cancer stem cells (GCSCs) are considered to be responsible for tumor initiation, growth, resistance to cytotoxic therapies, recurrence and metastasis due to their unique properties. These properties make the current therapeutic trials against GCSCs ineffective. Moreover, recent studies have shown that targeting stem cell surface markers or stemness associated pathways might have an additional off-target effect on the immune system. Recent advances in oncology and precision medicine have opened alternative therapeutic strategies in the form of cancer immunotherapy. This approach differs from classical anti-cancer therapy through its mechanism of action involving the activation and use of a functional immune system against tumor cells, instead of aiming physically destruction of cancer cells through radio- or chemotherapy. New immunological approaches for GCSCs targeting involve the use of different immune cells and various immune mechanisms like targeting specific surface antigens, using innate immune cells like the natural killer and T cells, T-cell chimeric antigen receptor technology, dendritic cell vaccine, or immune checkpoint inhibitors. In this respect, better understandings of immune regulatory mechanisms that govern anti-tumor response bring new hope in obtaining long-term remission for cancer therapy.

Comparative methods for genotyping hepatitis C virus isolates from Romania.

Accurate genotyping of hepatitis C virus (HCV) has clinical implications for treatment orientation and epidemiological impact in tracing the contamination sources. The aim of the study was to compare a genotyping assay by restriction fragment length polymorphism (RFLP) in the HCV 5'untranslated region (5'UTR) with sequencing in the 5'untranslated and NS5B regions. One hundred and three samples, collected between 2004 and 2006 from chronically infected patients with HCV, were tested with the 5'UTR and NS5B protocols. Of the total number of the samples tested by the 5'UTR-RFLP assay (n=103) the HCV subtype could be inferred by this method for 92 samples, by 5'UTR sequencing for 16 samples out of 23 tested (n=23) and by using the NS5B sequencing for all the samples tested (n=34). Our results showed that the HCV genotype distribution in Romania is: 1b--86.4%, 1a--10.7% and 4a--2.9%. In conclusion, RFLP screening in the 5'UTR is a convenient method for HCV genotyping and discrimination between 1b and non-1b genotypes but has a poor resolving power for subtyping and evaluation of the transmission routes. Sequencing in NS5B region is more adapted than RFLP and sequencing in 5'UTR for subtyping and epidemiological investigation.

IL-6 and IL-11 as markers for tumor aggressiveness and prognosis in gastric adenocarcinoma patients without mutations in Gp130 subunits.

BACKGROUND AND AIMS: A point mutation (gp130Y757F/Y759F) was identified as being responsible for aberrant activation of gp130 in mice and associated with gastric adenocarcinoma induction. As a result, we investigated the possible role of key point mutations in Tyr from IL6ST exon 17 that encode for the catalytic domain of gp130, and of its respective activators (IL-6 family member cytokines) in human gastric cancer initiation and development. METHOD: DNA, protein and plasma from 51 patients with gastric adenocarcinoma have been used in exploring gp130 status. We used sequencing analysis of IL6ST exon 17 in order to identify possible mutations that would lead to constitutive active forms of the receptor. The levels of gp130 activators (IL-6, IL-11, LIF) were analyzed by ELISA in plasma and mucosa of patients with gastric adenocarcinoma. RESULTS: Sequencing analysis did not identify mutations in gp130 key positions (Y759, Y767, Y814, Y905 and Y915). An increased IL-6 and IL-11 level in gastric mucosa was observed, correlated with staging, indicating these cytokines as gp130 activators in tumor epithelial cell. Those variations were consistent with increased IL-6 level in plasma. Furthermore, IL-6, but not IL-11 showed a significant correlation with patient's survival time, suggesting that tissue and plasma concentration of IL-6 might be a marker of tumor aggressiveness with prognostic value. CONCLUSIONS: According to our results, no mutations were detected in gp130 key positions in human gastric adenocarcinoma samples. However, gp130 activation may occur due to the increased level of IL-6 and IL-11 cytokines detected that can become valuable biomarkers.

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.

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.

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.