T cell receptor excision circles (TREC) and recent thymic migrant cells in specific immunotherapy and respiratory allergy to Dermatophagoides pteronyssinus.

INTRODUCTION: T cell receptor excision circles (TREC) on CD31+ T cells are related to recent thymic emigrant cells (RTEs). The involvement of the functional thymic tissue occurs early in the IgE-mediated allergic reaction, and in response to specific immunotherapy (SIT). AIM: Evaluation of specific immunotherapy effects on TREC number in peripheral T cells in patients allergic to Dermatophagoides pteronyssinus (Dpt). METHOD: 85 respiratory allergic patients (both genders), 41 of them (Group II) under maintenance treatment to Dpt SIT (21 sublingual-SLIT, and 20 subcutaneous-SCIT), were selected. The allergic patients (Group I) without specific treatment were submitted to an allergen challenge test (22 nasal and 22 conjunctival). Peripheral cell analysis was performed immediately before treatment and 60 or 240 minutes after allergenic extract administration. TREC quantification was performed in CD4+CD31+ and CD8+CD31+. The results were expressed per 100.000 cells related to RTEs. Samples from 10 healthy individuals (Control - Group III) were obtained with the same method. RESULTS: The value of TRECs on RTEs was constant in control groups. For Group I patients (nasal or conjunctival test), TREC quantification in CD31+ T cells showed relevant individual changes, even in the patients tested earlier (60 minutes), and statistical significant at 240 minutes. Both SCIT and SLIT had also demonstrated enormous individual changes, particularly on TRECs/CD4+CD31+ cells assay. Basal values in Group III were significantly higher than those observed in active patients groups. CONCLUSION: Thymic functional activity is earlier involved in the allergic reaction and SIT IgE-mediated allergy is able to induce RTEs in the periphery, particularly TRECs/CD4+CD31+ cells. Both SLIT and SCIT showed reduced RETs in the periphery, probably due to maturation of regulatory T cells. Our results suggest a crucial role of the functional thymic tissue on the central mechanism of this therapy.

Expression patterns of HLA-DR+ or HLA-DR- on CD4+/CD25++/CD127low regulatory T cells in patients with allergy.

BACKGROUND: Allergic rhinoconjunctivitis induced by pollen is a highly prevalent chronic inflammatory disease in Europe. Parietariajudaica is a frequent trigger in the Mediterranean area. The function of regulatory T cells (Treg cells) in allergy has recently been investigated, but further data are necessary to better understand their role and to find new strategies to treat allergic diseases such as allergic rhinoconjunctivitis. OBJECTIVE: To characterize gene expression of HLA-DR+ or HLA-DR- on peripheral CD4+/CD25++/CD127low Treg cells in patients with allergy. METHODS: Peripheral Treg cells (CD4+/CD25++/CD127low) were quantified using flow cytometry and sorted according to HLA-DR expression during the pollen season in patients with allergic rhinoconjunctivitis caused by P. judaica. The results were compared with those of nonatopic controls. Expression of associated cytokines and their receptors was measured using quantitative reverse transcription-polymerase chain reaction after extraction of mRNA in sorted populations. RESULTS: During the pollen season, no significant differences were observed between allergic patients with rhinoconjunctivitis and healthy controls in terms of the absolute number or the percentage of Treg cells in peripheral blood. All patients had a higher number/percentage of HLA-DR- Treg cells than HLA-DR+ Treg cells. In both groups we found high levels of FOXP3 mRNA expression. Despite being lower in number, HLA-DR+ Treg cells presented higher expression of CD28, PRF1, GZMB, and FASL than HLA-DR-Treg cells. CONCLUSIONS: The most relevant results obtained suggest that HLA-DR+ Treg cells tend to present higher gene expression of molecules associated with contact-dependent cell activation and cytotoxicity.

Perioperative tumor cell dissemination in patients with primary or metastatic colorectal cancer.

INTRODUCTION: Although there is general correlation between the TNM stage of colorectal cancer (CRC) and its prognosis, there is often significant variability of tumor behaviour and individual patient outcome, which is unaccounted for by pathologic factors alone. Our aim was to estimate perioperative tumor cell dissemination in patients with primary or CRC liver metastases as a possible factor influencing the outcome. METHODS: Forty patients were prospectively enrolled in the study from the year 2007 to 2008. Eighteen patients had histologically proven CRC (50% rectal, 44% colonic, 6% colonic and rectal). Sixteen patients (47%) had CRC liver metastases only. The remaining six patients who underwent colon or liver resection for benign conditions, acted as the control group. All patients with malignant pathologies had R0 resections. Blood samples were taken before the surgical incision (T0), immediately after tumor resection (T1) and at the end of the surgical intervention (T2). Data acquisition was performed using a dual-laser FACSCalibur flow cytometer. Circulating malignant cells were identified as being CD45-/cytokeratin+. RESULTS: The analysis of patients overall (CRC resection subgroup and hepatectomy subgroup) revealed that there was no statistically significant difference of the tumoral cell count in the blood per million of hematopoietic cells at T0, T1 and T2. CONCLUSIONS: This study demonstrates no differences in the detected circulating numbers of tumor cells at different stages of surgical intervention.

Quantification and immunophenotypic characterization of bone marrow and umbilical cord blood mesenchymal stem cells by multicolor flow cytometry.

In recent years, mesenchymal stem cells (MSC) have been attracting the greatest interest in the regeneration of injured tissues, autoimmune diseases, and transplantation of hematopoietic progenitor cells. Bone marrow (BM) represents the major source of MSC; however, umbilical cord blood (UCB) MSC has some advantages over BM, such as the higher differentiation capability and noninvasive collection methods. We sought to establish a 7-color, single-tube flow cytometric assay to quantify MSC in fresh tissues, namely BM and UCB, based on phenotypic markers of these cells. Moreover, we evaluated the differential expression of these markers in BM and UCB MSC. We used 5 UCB samples and 5 BM samples obtained from individuals without hematologic disease. To characterize MSC we used the following combination of monoclonal antibodies: CD71-FITC; CD105-PE; CD184-PE-Cy5; CD34-PE-Cy7; CD133-APC; CD45-APC-H7; CD44-Pacific blue, acquiring at least 1 million nucleated cells. We observed a greater number of BM MSC when compared with UCB MSC as well as some differences in the expression of some MSC antigens, particularly CD105 and CD44. Based on our preliminary results, phenotypic identification of MSC by flow cytometry is possible using a 7-color, single-tube assay. However, culture assays after sorting of cells characterized in this study are required to prove that they correspond to MSC.

Functional aspects of cord blood lymphocytes response to polyclonal and allogeneic activation.

With the purpose of contributing to a better knowledge of the phenotypic characteristics and functional activity of umbilical cord blood (UCB) lymphocytes, we have carried out extensive immunophenotyping of these cells, evaluated their immune response to polyclonal and allogeneic activation and then compared these results with those obtained with peripheral blood lymphocytes of healthy donors (PBHD). Our results showed, in CD4+ UCB lymphocytes, an increase of CD38 and CD45RA and a decrease of CD11a (S6F1), CD54 and HLA-DR double positive cells. An increase of CD38, CD45RA and CD56, and a decrease of CD28, CD57 and HLA-DR were observed in CD8+ UCB lymphocytes. Most natural killer UCB cells are CD16+, CD56+, CD57-, and among the UCB cells there is a lower number of CD8+ and TCRgammadelta+ (either CD8- or CD8+), and higher number of CD4+ lymphocytes. After allogeneic stimulus the majority of these phenotypic differences disappeared, which seems to be in agreement with the normal allogeneic response (assessed through MLR, frequency of CTL and helper T lymphocytes precursors) presented by UCB lymphocytes. Regarding the response to polyclonal activation, among the mitogens used, only PHA induced a different result: a lower IFNgamma production by UCB cells.