ZAP-70 expression is associated with increased CD4 central memory T cells in chronic lymphocytic leukemia: cross-sectional study.

BACKGROUND: Although chronic lymphocytic leukemia is basically a B cell disease, its pathophysiology and evolution are thought to be significantly influenced by T cells, as these are probably the most important interaction partner of neoplastic B cells, participating in their expansion, differentiation and survival. Chronic lymphocytic leukemia B cells may also drive functional and phenotypic changes of non-malignant T cells. There are few data about the association between memory T cells and prognosis, especially related to ZAP-70, a common reliable surrogate of the gold standard chronic lymphocytic leukemia prognostic markers. OBJECTIVE: The aim of this study was to investigate whether the expression of ZAP-70 in chronic lymphocytic leukemia patients is associated with abnormal patterns of the distribution of naïve and memory T cells related to crosstalk between these cells. METHODS: In this cross-sectional, controlled study, patients with chronic lymphocytic leukemia were compared with healthy blood donors regarding the expression of ZAP-70 and the distribution of naïve and memory T cell subsets in peripheral blood as measured by flow cytometry. RESULTS: ZAP-70 positive patients presented an increased frequency and absolute number of central memory CD4+ T cells, but not CD8+ T cells, compared to ZAP-70 negative patients and age-matched apparently healthy donors. CONCLUSIONS: Because central memory CD4+ T cells are located in lymph nodes and express CD40L, we consider that malignant ZAP-70-positive B cells may receive beneficial signals from central memory CD4+ T cells as they accumulate, which could contribute to more aggressive disease.

ZAP-70 expression is associated with increased CD4 central memory T cells in chronic lymphocytic leukemia: cross-sectional study

ABSTRACT Background: Although chronic lymphocytic leukemia is basically a B cell disease, its pathophysiology and evolution are thought to be significantly influenced by T cells, as these are probably the most important interaction partner of neoplastic B cells, participating in their expansion, differentiation and survival. Chronic lymphocytic leukemia B cells may also drive functional and phenotypic changes of non-malignant T cells. There are few data about the association between memory T cells and prognosis, especially related to ZAP-70, a common reliable surrogate of the gold standard chronic lymphocytic leukemia prognostic markers. Objective: The aim of this study was to investigate whether the expression of ZAP-70 in chronic lymphocytic leukemia patients is associated with abnormal patterns of the distribution of naïve and memory T cells related to crosstalk between these cells. Methods: In this cross-sectional, controlled study, patients with chronic lymphocytic leukemia were compared with healthy blood donors regarding the expression of ZAP-70 and the distribution of naïve and memory T cell subsets in peripheral blood as measured by flow cytometry. Results: ZAP-70 positive patients presented an increased frequency and absolute number of central memory CD4+ T cells, but not CD8+ T cells, compared to ZAP-70 negative patients and age-matched apparently healthy donors. Conclusions: Because central memory CD4+ T cells are located in lymph nodes and express CD40L, we consider that malignant ZAP-70-positive B cells may receive beneficial signals from central memory CD4+ T cells as they accumulate, which could contribute to more aggressive disease.

CD40 ligand deficiency causes functional defects of peripheral neutrophils that are improved by exogenous IFN-γ.

BACKGROUND: Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches. OBJECTIVES: We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function. METHODS: We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function. RESULTS: Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients. CONCLUSION: Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases.

BACKGROUND: Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. AIMS: To evaluate expression of death receptors' family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. SUBJECTS AND METHODS: Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-ΔΔCt). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. RESULTS: In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. CONCLUSIONS: The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.

Melatonin protects CD4+ T cells from activation-induced cell death by blocking NFAT-mediated CD95 ligand upregulation.

Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4(+) T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation.

Pomolic acid may overcome multidrug resistance mediated by overexpression of anti-apoptotic Bcl-2 proteins.

Multidrug resistance (MDR) is multifactorial and may be mediated by overexpression of anti-apoptotic proteins. This paper investigated whether pomolic acid (PA) was able to overcome resistance mediated by overexpression of Bcl-2 or BcL-xL. The results obtained showed that overexpression of these proteins partially inhibited the PA-induced apoptosis, loss of mitochondrial membrane potential and caspase -3 and -9 activation observed in HL-60/neo. Since Bcl-2 transfected cell lines were shown to be quite resistant to a series of chemotherapeutic agents, the data presented call attention to the possible clinical significance of PA as an anti-MDR drug.

Involvement of DNA replication in ultraviolet-induced apoptosis of mammalian cells.

Exposure of cells to ultraviolet (UV) light damages the genome and the persistence of DNA lesions triggers apoptosis in mammalian cells. RNA transcription blockage by DNA damage is believed to be implicated in signaling for UV-induced apoptosis, but the role played by DNA replication in this process is still unclear. To address this point, we have employed the DNA polymerase inhibitor aphidicolin in UV-irradiated wild-type and XPB-mutated Chinese hamster ovary cells. The data obtained with synchronized cells indicate that induction of apoptosis by UV light is independent of the cell cycle phase. Nevertheless, cells treated with aphidicolin after UV exposure showed a significant prevention of apoptosis induction when compared to proliferating cells. These results were observed in both DNA-repair proficient and deficient cells, indicating that the prevention of apoptosis by aphidicolin is independent of the cells' ability to repair the photolesions caused by UV. Taken together, these data suggest that replication of damaged DNA also leads to critical events signaling for UV-induced cell death.

Pomolic acid triggers mitochondria-dependent apoptotic cell death in leukemia cell line.

One of the major goals in chemotherapy is to circumvent anti-apoptotic strategies developed by tumor cells. In a previous paper, we showed that pomolic acid (PA) is able to kill the leukemia cell line K562 and its MDR derivative, Lucena 1. Here, we demonstrated that PA-induced apoptosis of HL-60 cells is dependent on the activation of caspases-3 and -9 and dissipation of the mitochondrial transmembrane potential (Deltapsim). Disruption of Deltapsim precedes caspase activation and is not inhibited by zVAD-fmk indicating mitochondria as the main target of PA. Our data pointed to the potential use of PA to overcome apoptosis resistance.

Myriadenolide, a labdane diterpene isolated from Alomia myriadenia (asteraceae) induces depolarization of mitochondrial membranes and apoptosis associated with activation of caspases-8, -9, and -3 in Jurkat and THP-1 cells.

Myriadenolide is a diterpene that we have recently isolated from the extract of Alomia myriadenia (Asteraceae). Here we show for the first time that myriadenolide has caspase-dependent cytotoxic activity against human leukemia cells from both lymphocytic (Jurkat) and monocytic (THP-1) lineages, because preincubation of Jurkat or THP-1 cells with the broad-spectrum caspase inhibitor z-VAD-fmk completely abrogated cell death. Moreover, the mitochondrial pathway is implicated as mitochondrial depolarization and caspase-9 and caspase-3 activation were observed. Interestingly, caspase-8 and cleavage of the proapoptotic member of the Bcl-2 family BID was also observed during apoptosis induced by myriadenolide, suggesting a role for the caspase-8/BID pathway. However, interference with Fas or TNFR1 signaling did not interfere with apoptosis in our experimental system. Furthermore, pretreatment of cells with the caspase-3 inhibitor DEVD-fmk completely blocked the activation of caspase-8, suggesting that the activation of the caspase-8/BID pathway is part of an amplification loop initiated by caspase-3. Taken together, our results indicate myriadenolide as a novel candidate for the treatment of hematological malignancies.