Effect of mTORC1/mTORC2 inhibition on T cell function: potential role in graft-versus-host disease control

Producción CientíficaThe mechanistic target of rapamycin (mTOR) pathway is crucial for the activation and function of T cells, which play an essential role in the development of graft-versus-host disease (GvHD). Despite its partial ability to block mTOR pathway, the mTORC1 inhibitor rapamycin has shown encouraging results in the control of GvHD. Therefore, we considered that simultaneous targeting of both mTORC1 and mTORC2 complexes could exert a more potent inhibition of T cell activation and, thus, could have utility in GvHD control. To assess this assumption, we have used the dual mTORC1/mTORC2 inhibitors CC214-1 and CC214-2. In vitro studies confirmed the superior ability of CC214-1 versus rapamycin to block mTORC1 and mTORC2 activity and to reduce T cell proliferation. Both drugs induced a similar decrease in Th1/Th2 cytokine secretion, but CC214-1 was more efficient in inhibiting na€ıve T cell activation and the expression of Tcell activation markers. In addition, CC214-1 induced specific tolerance against alloantigens, while preserving anti-cytomegalovirus response. Finally, in a mouse model of GvHD, the administration of CC214-2 significantly improved mice survival and decreased GvHD-induced damages. In conclusion, the current study shows, for the first time, the immunosuppressive ability of CC214-1 on T lymphocytes and illustrates the role of CC214-2 in the allogeneic transplantation setting as a possible GvHD prophylaxis agent.Gerencia Regional de Salud de Castilla y León (Proyecto GRS 726/A13

Host virus and pneumococcus-specific immune responses in high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia: implications for disease progression

[EN]Patients diagnosed with chronic lymphocytic leukemia (CLL) display a high incidence of infections due to an associated immunodeficiency that includes hypogammaglobulinemia. A higher risk of infections has also been recently reported for high-count monoclonal B-cell lymphocytosis, while no information is available in low-count monoclonal B-cell lymphocytosis. Here, we evaluated the status of the humoral immune system in patients with chronic lymphocytic leukemia (n=58), as well as in low- (n=71) and high- (n=29) count monoclonal B-cell lymphocytosis versus healthy donors (n=91). Total free plasma immunoglobulin titers and specific levels of antibodies against cytomegalovirus, Epstein-Barr virus, influenza and S.pneumoniae were measured by nephelometry and ELISA-based techniques, respectively. Overall, our results show that both CLL and high-count monoclonal B-cell lymphocytosis patients, but not low-count monoclonal B-cell lymphocytosis subjects, present with relatively high levels of antibodies specific for the latent viruses investigated, associated with progressively lower levels of S.pneumoniae-specific immunoglobulins

Low-count monoclonal B-cell lymphocytosis persists after seven years of follow up and is associated with a poorer outcome

[EN]Low-count monoclonal B-cell lymphocytosis is defined by the presence of very low numbers of circulating clonal B cells, usually phenotypically similar to chronic lymphocytic leukemia cells, whose biological and clinical significance remains elusive. Herein, we re-evaluated 65/91 low-count monoclonal B-cell lymphocytosis cases (54 chronic lymphocytic leukemia-like and 11 non-chronic lymphocytic leukemialike) followed-up for a median of seven years, using high-sensitivity flow cytometry and interphase fluorescence in situ hybridization. Overall, the clone size significantly increased in 69% of low-count monoclonal B-cell lymphocytosis cases, but only one subject progressed to high-count monoclonal B-cell lymphocytosis. In parallel, the frequency of cytogenetic alterations increased over time (32% vs. 61% of cases, respectively). The absolute number of the major T-cell and natural killer cell populations also increased, but only among chronic lymphocytic leukemia-like cases with increased clone size vs. age- and sex-matched controls. Although progression to chronic lymphocytic leukemia was not observed, the overall survival of low-count monoclonal B-cell lymphocytosis individuals was significantly reduced vs. non-monoclonal Bcell lymphocytosis controls (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer were the main causes of death in low-count monoclonal B-cell lymphocytosis. In summary, despite the fact that mid-term progression from low-count monoclonal B-cell lymphocytosis to high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia appears to be unlikely, these clones persist at increased numbers, usually carrying more genetic alterations, and might thus be a marker of an impaired immune system indirectly associated with a poorer outcome, particularly among females

Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality

[EN]Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56(low) NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56(low) NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94(hi)/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality.This work has been partially supported by the following grants: FIS 02/1244-FEDER, DTS 15/00119-FEDER, RTICC RD06/0020/0035-FEDER and RTICC RD12/0036/0048-FEDER from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain; SA103/03 and SA079U14 from the Consejería de Educación, Junta de Castilla y León, Valladolid, Spain. The research activities of the EuroFlow Consortium were supported by the European Commission (grant STREP EU-FP6, LSHB-CT-2006–018708, entitled ‘Flow cytometry for fast and sensitive diagnosis and follow-up of hematological malignancies’)

Fish Oil Enriched Intravenous Lipid Emulsions Reduce Triglyceride Levels in Non-Critically Ill Patients with TPN and Type 2 Diabetes. A Post-Hoc Analysis of the INSUPAR Study

There are no studies that have specifically assessed the role of intravenous lipid emulsions (ILE) enriched with fish oil in people with diabetes receiving total parenteral nutrition (TPN). The objective of this study was to assess the metabolic control (glycemic and lipid) and in-hospital complications that occurred in non-critically ill inpatients with TPN and type 2 diabetes with regard to the use of fish oil emulsions compared with other ILEs. We performed a post-hoc analysis of the Insulin in Parenteral Nutrition (INSUPAR) trial that included patients who started with TPN for any cause and that would predictably continue with TPN for at least five days. The study included 161 patients who started with TPN for any cause. There were 80 patients (49.7%) on fish oil enriched ILEs and 81 patients (50.3%) on other ILEs. We found significant decreases in triglyceride levels in the fish oil group compared to the other patients. We did not find any differences in glucose metabolic control: mean capillary glucose, glycemic variability, and insulin dose, except in the number of mild hypoglycemic events that was significantly higher in the fish oil group. We did not observe any differences in other metabolic, liver or infectious complications, in-hospital length of stay or mortality

Regular insulin added to total parenteral nutrition vs subcutaneous glargine in non-critically ill diabetic inpatients, a multicenter randomized clinical trial: INSUPAR trial

Background: There is no established insulin regimen in T2DM patients receiving parenteral nutrition. Aims: To compare the effectiveness (metabolic control) and safety of two insulin regimens in patients with diabetes receiving TPN. Design: Prospective, open-label, multicenter, clinical trial on adult inpatients with type 2 diabetes on a non-critical setting with indication for TPN. Patients were randomized on one of these two regimens: 100% of RI on TPN or 50% of Regular insulin added to TPN bag and 50% subcutaneous Gl. Data were analyzed according to intention-to-treat principle. Results: 81 patients were on RI and 80 on GI. No differences were observed in neither average total daily dose of insulin, programmed or correction, nor in capillary mean blood glucose during TPN infusion (165.3 +/- 35.4 in RI vs 172.5 +/- 43.6 mg/dL in GI; p = 0.25). Mean capillary glucose was significantly lower in the GI group within two days after TPN interruption (160.3 +/- 45.1 in RI vs 141.7 +/- 43.8 mg/dL in GI; p = 0.024). The percentage of capillary glucose above 180 mg/dL was similar in both groups. The rate of capillary glucose <= 70 mg/dL, the number of hypoglycemic episodes per 100 days of TPN, and the percentage of patients with non-severe hypoglycemia were significantly higher on GI group. No severe hypoglycemia was detected. No differences were observed in length of stay, infectious complications, or hospital mortality. Conclusion: Effectiveness of both regimens was similar. GI group achieved better metabolic control after TPN interruption but non-severe hypoglycemia rate was higher in the GI group. (C) 2019 The Author(s). Published by Elsevier Ltd

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO

CLL-like B-lymphocytes are systematically present at very low numbers in peripheral blood of healthy adults

[EN] Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the Western world. The disease is typically diagnosed in adults >40 years old, who show an expansion (>5 × 109 cells per l) of clonal B-cells with a unique CD5+, CD23+, B-cell receptor (BCR)low immunophenotype in peripheral blood (PB) and bone marrow, frequently in association with involvement of other lymphoid tissues, disease symptoms and a heterogeneous clinical outcome. In parallel, monoclonal B-cell lymphocytosis (MBL), typically characterized by an expansion of clonal CLL-like B-cells (<5 × 109 cells per l), has been also frequently reported in otherwise asymptomatic subjects.1, 2 Increasing evidence suggests that this could represent a pre-leukemic condition, as CLL frequently develops in individuals with previous history of MBL, and MBL cases progress to CLL at a rate of 1% per year

Methylation is an inactivating mechanism of the p16 gene in multiple myeloma associated with high plasma cell proliferation and short survival

[EN]In order to gain further insights into the role of the p16 gene in cell cycle regulation and the prognostic implications of its inactivation, we investigated the methylation status of the p16 gene in 98 untreated patients using a polymerase chain reaction assay based on the inability of some restriction enzymes to digest methylated sequences. Forty-one patients showed a p16 methylated gene (42%). The percentage of S-phase plasma cells (PC) in these patients was almost three times higher than in those with an unmethylated p16 gene (4Æ16% ± 3Æ37% vs 1Æ5% ± 1Æ41%, P < 0Æ001). The presence of p16 methylation also correlated with both elevated b2-microglobulin serum levels and high C-reactive protein values. Patients with a p16 methylated gene had shorter overall and progression-free survival than those patients without p16 methylation. However, this feature did not retain independent prognostic influence on multivariate analysis, probably due to its association with the S-phase PC, which had more potent statistical significance in the Cox model. These findings showed methylation of the p16 gene was a frequent event in MM patients at diagnosis, and was associated with an increased proliferative rate of plasma cells and a poor prognosis, indicating an important role for p16 gene in the cell cycle regulation of multiple myeloma tumour cells, and thus in the clinical outcome of the disease