Natalia Ramirez

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes.In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor.Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Epigenetic regulation of microRNA expression in colorectal cancer

In the last years, microRNAs (miRNA) have emerged as new molecular players involved in carcinogenesis. Deregulation of miRNAs expression has been shown in different human cancer but the molecular mechanism underlying the alteration of miRNA expression is unknown. To identify tumor‐supressor miRNAs silenced through aberrant epigenetic events in colorectal cancer (CRC), we used a sequential approach. We first identified 5 miRNAs down‐regulated in patient with colorectal cancer samples and located around/on a CpG island. Treatment with a DNA methyltransferase inhibitor and a HDAC inhibitor restored expression of 3 of the 5 microRNAs (hsa‐miR‐9, hsa‐miR‐129 and hsa‐miR‐137) in 3 CRC cell lines. Expression of hsa‐miR‐9 was inversely correlated with methylation of their promoter regions as measure by MSP and bisulphate sequencing. Further, methylation of the hsa‐miR‐9‐1, hsa‐miR‐129‐2 and hsa‐miR‐137 CpG islands were frequently observed in CRC cell lines and in primary CRC tumors, but not in normal colonic mucosa. Finally, methylation of hsa‐miR‐9‐1 was associated with the presence of lymph node metastasis. In summary, our results aid in the understanding of miRNA gene regulation showing that aberrant DNA methylation and histone modifications work together to induce silencing of miRNAs in CRC.

microRNA-451 Regulates Macrophage Migration Inhibitory Factor Production and Proliferation of Gastrointestinal Cancer Cells

Purpose: microRNAs (miRNA) are small RNAs that function as post-transcriptional regulators of gene expression. Recent evidence has shown that some miRNAs can act as oncogenes or tumor suppressors. This study was conducted to evaluate the potential association of miRNA expression with clinical outcome in patients with gastric cancer. Experimental Design: Expression of 250 human mature miRNAs was measured by real-time PCR on paraffin-embedded tumor samples of 21 patients with gastric cancer stage III uniformly treated with surgical resection followed by chemoradiation. We identified the miRNAs correlated with disease-free and overall survival times, and the results were evaluated including 24 other patients. In vitro cell proliferation and radiosensitivity studies were done to support clinical data. Results: The results revealed that down-regulation of miR-451 was associated with worse prognosis. miR-451 was detected by in situ hybridization in epithelial cells and showed decreased expression in gastric and colorectal cancer versus nontumoral tissues. Overexpression of miR-451 in gastric and colorectal cancer cells reduced cell proliferation and increased sensitivity to radiotherapy. Microarray and bioinformatic analysis identified the novel oncogene macrophage migration inhibitory factor (MIF) as a potential target of miR-451. In fact, overexpression of miR-451 down-regulated mRNA and protein levels of MIF and decreased expression of reporter genes with MIF target sequences. Moreover, we found a significant inverse correlation between miR-451 and MIF expression in tumoral gastric biopsies. Conclusions: These findings support the role of miR-451 as a regulator of cancer proliferation and open new perspectives for the development of effective therapies for chemoradioresistant cancers.

Anti-Inflammatory Cytokines Induce Lipopolysaccharide Tolerance in Human Monocytes Without Modifying Toll-Like Receptor 4 Membrane Expression

Toll‐like receptor 4 (TLR4) participates in innate immunity by detecting lipopolysaccharides (LPS) of Gram‐negative bacterial cell walls. TLR4 macrophage expression in mice is modulated by LPS. This fact constitutes, at least partially, the molecular basis for LPS tolerance. Very recently, the effect of interferon‐γ (IFN‐γ), a pro‐inflammatory cytokine, has been described on TLR4 membrane expression of human monocytes. IFN‐γ up‐regulates TLR4 expression and antagonizes the LPS‐induced TLR4 down‐regulation. These data prompted us to study the expression of membrane TLR4 in human mono‐ cytes in which LPS tolerance was induced by LPS and by anti‐inflammatory cytokines [interleukin‐10 (IL‐10) and transforming growth factor β1 (TGFβ1)]. Data concerning this latter model, and more specifically, the effect of anti‐inflammatory cytokines over TLR4 expression, are not available at present. We show here that membrane TLR4 expression in human monocytes falls after LPS exposure. The effect was prolonged for 12 h, but then expression returned to normal levels. The incubation of human monocytes with IL‐10, TGFβ1 or a mixture of both induces no alterations in membrane TLR4 expression. However, these cytokines are able to substitute the tolerizing LPS exposure in order to induce LPS tolerance. Our data help to achieve a better understanding of the way cytokines control the cellular expression of TLR.

BY55/CD160 cannot be considered a cytotoxic marker in cytomegalovirus-specific human CD8 + T cells

CD160/BY55 is a glucosyl‐phosphatidylinositol (GPI)‐anchored cell membrane receptor that is expressed primarily in natural killer (NK) cells. Its presence in CD8+ T lymphocytes is considered to be a marker of cytotoxic activity, although there are few data in this regard. In the present work, we analysed the expression of CD160 in subpopulations of cytomegalovirus (CMV)‐specific CD8+ T cells. Subpopulations were defined by CD28 and CD57 expression and exhibited varying degrees of differentiation and cytotoxic potential, as evaluated by the expression of perforin, interferon (IFN)‐γ and interleukin (IL)‐7Rα/CD127. We included subjects with different intensities of anti‐viral immune response. Results showed that the terminally differentiated CD28– CD57+ subset displaying the highest level of perforin expressed CD160 at a level similar to that of memory CD28+ CD57–perforin– cells. A comparison of the expression of perforin in CD160+ cells versus CD160– cells showed that expression was significantly higher in the absence of CD160. Interestingly, the CMV‐specific CD8+ T cell subset from a patient with ongoing CMV reactivation did not begin to express CD160 until day +92 of the follow‐up period. Taken together, our data show that CD160 cannot be considered a cytotoxic marker in CMV‐specific CD8+ T cells.

Mobilization of hematopoietic progenitor cells from allogeneic healthy donors using a new biosimilar G‐CSF (Zarzio®)

Peripheral blood progenitor cells (PBPCs) have become the major source of hematopoietic progenitor cells for allogeneic transplantation. In February 2008, Zarzio® was approved by the European Medicine Agency for PBPCs mobilization, but this authorization was not based in trials analyzing safety and efficacy for PBPCs mobilization. Since August 2011, Zarzio® has been used at our institution for PBPCs mobilization. In total 36 healthy family donors underwent PBPCs mobilization, 18 with Neupogen® and 18 with Zarzio®. Donor characteristics were equivalent between groups, and no severe adverse effects were registered in the Zarzio® group. The number of CD34 cells collected/Kg recipient body weight was 6.7 × 106 (3.8–11.1) in the Zarzio® group versus 8.4 × 106 (5.6–16.6) in the Neupogen® group (P = 0.04). We collected the minimal target cell dose (2 × 106/kg) in all donors from each group and no significant differences were found in the collection of the optimal cell dose (5 × 106/kg) between groups, although 3/18 (16.6%) donors that received Zarzio® failed to mobilize the optimal cell dose compared with 0% in the Neupogen® group. A total of 35 patients proceeded to transplantation (17 in the Zarzio® and 18 in the Neupogen® groups, respectively). Platelet and neutrophil median time to engraftment was comparable between the two groups. Our retrospective study supports the conclusion that Zarzio® mobilization of PBPCs in healthy donors is safe but perhaps not as effective as the reference Neupogen. However, more prospective trials are required to definitively asses the safety and efficacy of G‐CSF biosimilars for PBPCs mobilization in healthy donors. J. Clin. Apheresis 31:48–52, 2016.

Streptamer technology allows accurate and specific detection of CMV-specific HLA-A*02 CD8(+) T cells by flow cytometry.

Multimer technology is widely used to screen antigen‐specific immune recovery after allogeneic hematopoietic stem cell transplantation (allo‐HSCT) as it enables identification, enumeration, phenotypic characterization and isolation of virus‐specific T‐cells. Novel approaches of multimerization might improve on classical tetramer staining; however, their use as standard monitoring technique to quantify antigen‐specific cells has not been validated yet. We have compared two of these available multimeric complexes: pentamer and streptamer to select the best strategy for the incorporation into clinical monitoring practice.

Impact of T cell selection methods in the success of clinical adoptive immunotherapy.

Chemotherapy and/or radiotherapy regular regimens used for conditioning of recipients of hematopoietic stem cell transplantation (SCT) induce a period of transient profound immunosuppression. The onset of a competent immunological response, such as the appearance of viral-specific T cells, is associated with a lower incidence of viral infections after haematopoietic transplantation. The rapid development of immunodominant peptide virus screening together with advances in the design of genetic and non-genetic viral- and tumoural-specific cellular selection strategies have opened new strategies for cellular immunotherapy in oncologic recipients who are highly sensitive to viral infections. However, the rapid development of cellular immunotherapy in SCT has disclosed the role of the T cell selection method in the modulation of functional cell activity and of in vivo secondary effects triggered following immunotherapy.

The abrogation of TCR-independent interactions with human serum ensures a selective capture of therapeutic virus-specific CD8+ T-cells by Multimer Technology in Adoptive Immunotherapy

Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8(+) T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes. Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8(+) T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.

Functional specific-T-cell expansion after first cytomegalovirus reactivation predicts viremia control in allogeneic hematopoietic stem cell transplant recipients

The use of preemptive antiviral therapy to prevent cytomegalovirus (CMV) disease in allogeneic hematopoietic stem cell transplantation (allo‐HSCT) recipients might result in over‐treatment, inducing drug‐related toxicity and viral resistance. A search for predictive markers is needed to determine requirement for antiviral therapy. Clinical follow‐up, in combination with the use of streptamers (STs) and cytokine‐intracellular staining, could help to identify patients at high risk for CMV reactivations. To study the immune response and reactivation control by CMV‐specific CD8+ T‐cell (CMV‐CTL) populations, we monitored 25 patients who have undergone allo‐HSCT by using ST multimer and intracellular cytokine staining. Our study has revealed that the presence of functional CMV‐specific T cells, determined by early interferon γ production or by significant T‐cell expansion after first CMV reactivation, correlated with short CMV viremia duration and low number of CMV reactivations. By contrast, the absence of functional CMV‐CTLs does correlate with CMV recurrence. These results support that behavior of CMV‐specific subpopulations after reactivation influences reactivations and can guide preemptive therapy.