Increased T regulatory cells are associated with adverse clinical features and predict progression in multiple myeloma.

Background: Regulatory T (Treg) cells play an important role in the maintenance of immune system homeostasis. Multiple myeloma (MM) is a plasma cell disorder frequently associated with impaired immune cell numbers and functions. Methods: We analyzed Treg cells in peripheral blood (n = 207) and bone marrow (n = 202) of pre-malignant and malignant MM patients using flow cytometry. Treg cells and their subsets from MM patients and healthy volunteers were functionally evaluated for their suppressive property. A cohort of 25 patients was analyzed for lymphocytes, CD4 T cells and Treg cells before and after treatment with cyclophosphamide, thalidomide plus dexamethasone (CTD). Results: We found elevated frequencies of Treg cells in newly diagnosed (P<0.01) and relapsed MM patients (P<0.0001) compared to healthy volunteers. Also, Treg subsets including naive (P = 0.015) and activated (P = 0.036) Treg cells were significantly increased in MM patients compared to healthy volunteers. Functional studies showed that Treg cells and their subsets from both MM and healthy volunteers were similar in their inhibitory function. Significantly increased frequencies of Treg cells were found in MM patients with adverse clinical features such as hypercalcemia (.10 mg/dL), decreased normal plasma cell (<5%) count and IgA myeloma subtype. We also showed that MM patients with >5% of Treg cells had inferior time to progression (TTP) (13 months vs. median not reached; P = 0.013). Furthermore, we demonstrated the prognostic value of Treg cells in prediction of TTP by Cox regression analysis (P = 0.045). CTD treatment significantly reduced frequencies of CD4 T cells (P = 0.001) and Treg cells (P = 0.018) but not Treg cells/CD4 T cells ratio compared to pretreatment. Conclusions: Our study showed immune deregulation in MM patients which is evidenced by elevated level of functionally active Treg cells and patients with increased Treg cells have higher risk of progression

Konsensuální doporučení ČNHP (Českého národního hemofilického programu) pro diagnostiku a léčbu pacientů s vrozenou hemofilií a s inhibitorem FVIII/FIX

Cílem standardu je stanovit základní diagnostické a především terapeutické postupy v péči o pacienty s vrozenou hemofilií a s inhibitorem koagulačního faktoru VIII, resp. faktoru IX.The aim of the guidelines is to set-up and standardize diagnostic as well as therapeutic approaches to care about patients with congenital haemophilia and inhibitors to coagulation factor VIII or factor I

Phospholipid profiling enables to discriminate tumor- and non-tumor-derived human colon epithelial cells: Phospholipidome similarities and differences in colon cancer cell lines and in patient-derived cell samples.

Identification of changes of phospholipid (PL) composition occurring during colorectal cancer (CRC) development may help us to better understand their roles in CRC cells. Here, we used LC-MS/MS-based PL profiling of cell lines derived from normal colon mucosa, or isolated at distinct stages of CRC development, in order to study alterations of PL species potentially linked with cell transformation. We found that a detailed evaluation of phosphatidylinositol (PI) and phosphatidylserine (PS) classes allowed us to cluster the studied epithelial cell lines according to their origin: i) cells originally derived from normal colon tissue (NCM460, FHC); ii) cell lines derived from colon adenoma or less advanced differentiating adenocarcinoma cells (AA/C1, HT-29); or, iii) cells obtained by in vitro transformation of adenoma cells and advanced colon adenocarcinoma cells (HCT-116, AA/C1/SB10, SW480, SW620). Although we tentatively identified several PS and PI species contributing to cell line clustering, full PI and PS profiles appeared to be a key to the successful cell line discrimination. In parallel, we compared PL composition of primary epithelial (EpCAM-positive) cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients, with PL profiles of cell lines derived from normal colon mucosa (NCM460) and from colon adenocarcinoma (HCT-116, SW480) cells, respectively. In general, higher total levels of all PL classes were observed in tumor cells. The overall PL profiles of the cell lines, when compared with the respective patient-derived cells, exhibited similarities. Nevertheless, there were also some notable differences in levels of individual PL species. This indicated that epithelial cell lines, derived either from normal colon tissue or from CRC cells, could be employed as models for functional lipidomic analyses of colon cells, albeit with some caution. The biological significance of the observed PL deregulation, or their potential links with specific CRC stages, deserve further investigation

Inhibitory function of naïve and activated T regulatory cells.

<p>Similarly, we assessed the suppressive function of naïve, activated and non-Treg cells by CFSE based proliferation assay. Naïve CD4 T cells (CD4⁺CD25⁻CD45RA⁺) were CFSE labeled and co-cultured with naïve, activated and non-Treg cells at a ratio of 1∶1 in the presence of anti-CD3/CD28 beads and accessory cells. (A) Represents proliferation assay from a MM patient. In the presence of naïve and activated Treg cells in the proliferation assay, naïve CD4 T cell proliferation was inhibited but not in their absence/presence of non-Treg cells. This was clearly shown by dilution of CFSE in the FITC channel. (B) In a head to head analysis, proliferation of naïve CD4 T cells between MM patients and HVs did not differ significantly either in the presence or absence of naïve and activated Treg cells in the proliferation assay (proliferation/division of naïve CD4 T cells is expressed in %). (C) Similarly, level of IFN-γ secretion by naïve CD4 T cells did not differ significantly between MM and HV cohorts in the presence or absence of naïve and activated Treg cells (IFN-γ concentration is expressed in pg/ml). Mann-Whitney U test was used to assess the statistical difference between MM and HV cohorts. Statistical difference between MM and HV cohorts is indicated by P value. Median is represented by horizontal line, and raw data from each experiment are represented by small dots and squares. CFSE, carboxyfluorescein succinimidyl ester; MM, multiple myeloma; HV, healthy volunteer.</p

Phenotypic feature of T regulatory cells in peripheral blood and bone marrow.

<p>(A) Illustrates isotype-matched control for FoxP3 expression and phenotype of Treg cells (CD4⁺CD25hi⁺FoxP3⁺) from peripheral blood and bone marrow of a MM patient. (B) Histogram shows negative/dim expression for CD127 by Treg cells compared to CD4⁺CD25⁻ cells. (C) Phenotype of naïve (CD4⁺CD45RA⁺FoxP3dim⁺), activated (CD4⁺CD45RA⁺FoxP3hi⁺) and non-Treg cells (CD4⁺CD45RA^-FoxP3dim⁺) from peripheral blood of a MM patient.</p