Julio Sánchez Avalos

Forskolin induces U937 cell line differentiation as a result of a sustained cAMP elevation

The present study examines the effects of forskolin on U937 cell differentiation. We recently reported that dibutyryl cAMP (dbcAMP), but not cAMP-elevating agents such as histamine, promotes U937 cell differentiation. cAMP production elicited by stimulation of histamine H2 receptors showed a rapid, homologous desensitization, which might explain the dissimilar responses to histamine and dbcAMP. Forskolin induced an increase in cAMP levels in a concentration-dependent manner (EC50=30 microM) for an extended period of at least 24 h. Forskolin but not histamine (up to 100 microM), also inhibited cell growth in a dose-dependent fashion (EC50=22 microM). After 3 days of incubation, 75 microM forskolin induced U937 cell differentiation as judged by an increased rate of reduction of nitrobluetetrazolium (mean+/-S.E.M.: 21.3+/-6.6% in treated cells vs. 3.2+/-1.9% in the control group, P < 0.001) and an augmented chemotactic response to complement 5a (C5a) (33.2+/-5.9% in forskolin-treated vs. 0.34+/-0.12% in control cells, P < 0.01). Furthermore, c-Myc levels decreased following forskolin treatment, while the histamine H2 receptor agonist dimaprit had no effect. We conclude that forskolin induces U937 cell differentiation through a sustained rise in cAMP levels.

Biallelic deletion 13q14.3 in patients with chronic lymphocytic leukemia: cytogenetic, FISH and clinical studies

Background and objective:  Monoallelic deletion of 13q14.3 (13q14x1) is the most common abnormality in chronic lymphocytic leukemia (CLL). As a sole alteration, it predicts a favorable outcome. Biallelic 13q14.3 (13q14x2) deletion or concomitant 13q14x1/13q14x2 has been scarcely evaluated in the literature. We present the clinical, cytogenetic and fluorescence in situ hybridization (FISH) analysis of six CLL patients with normal karyotypes and 13q14x2 and their comparison to cases with 13q14x1 as a single abnormality.

Erythrocytapheresis with Recombinant Human Erythropoietin in Hereditary Hemochromatosis Therapy: A New Alternative

Background and Objectives: The purposes of this study were to evaluate the tolerance, efficacy and safety of isovolemic erythrocytapheresis (EA) in nonanemic patients with hereditary hemochromatosis (HH), and to assess the usefulness of recombinant human erythropoietin (rHuEPO) associated with EA to reduce treatment duration. Materials and Methods: In 10 asymptomatic patients with serum ferritin >400 μg/l, transferrin saturation >50%, and GPT elevation, EA with rHuEPO and folic acid was performed. Results: Red cell indices, serum ferritin, and other iron metabolism parameters (serum iron, transferrin, and transferrin saturation); GPT and other laboratory data were considerably improved. Conclusion: This method offers better results in less time than traditional phlebotomy. EA with rHuEPO is an effective therapeutic alternative for patients with HH.

CXCL12-induced chemotaxis is impaired in T cells from patients with ZAP-70-negative chronic lymphocytic leukemia

Background T cells from patients with chronic lymphocytic leukemia may play an important role in contributing to the onset, sustenance, and exacerbation of the disease by providing survival and proliferative signals to the leukemic clone within lymph nodes and bone marrow. Design and Methods By performing chemotaxis assays towards CXCL12, CCL21 and CCL19, we sought to evaluate the migratory potential of T cells from chronic lymphocytic leukemia patients. We next analyzed the chemokine-induced migration of T cells, dividing the chronic lymphocytic leukemia samples according to their expression of the poor prognostic factors CD38 and ZAP-70 in leukemic cells determined by flow cytometry. Results We found that T cells from patients with chronic lymphocytic leukemia are less responsive to CXCL12, CCL21 and CCL19 than T cells from healthy adults despite similar CXCR4 and CCR7 expression. Following separation of the patients into two groups according to ZAP-70 expression, we found that T cells from ZAP-70-negative samples showed significantly less migration towards CXCL12 compared to T cells from ZAP-70-positive samples and that this was not due to defective CXCR4 down-regulation, F-actin polymerization or to a lesser expression of ZAP-70, CD3, CD45, CD38 or CXCR7 on these cells. Interestingly, we found that leukemic cells from ZAP-70-negative samples seem to be responsible for the defective CXCR4 migratory response observed in their T cells. Conclusions Impaired migration towards CXCL12 may reduce the access of T cells from ZAP-70-negative patients to lymphoid organs, creating a less favorable microenvironment for leukemic cell survival and proliferation.

SHIP‐1 protein level and phosphorylation status differs between CLL cells segregated by ZAP‐70 expression

The clinical course of B-cell chronic lymphocytic leukaemia (CLL) is very heterogeneous, with some patients that present an indolent disease and others that succumb rapidly despite therapy. Leukaemic cells from aggressive CLL patients typically display B-cell receptors (BCR) encoded by unmutated immunoglobulin variable heavy-chain genes (IGHV) and express the protein tyrosine kinase ZAP-70 (Crespo et al, 2003). In contrast, mutated IGHV genes and the absence of ZAP-70 are mostly found in patients with indolent disease (Crespo et al, 2003). It has been proposed that survival differences between these two subgroups are related to the differential ability of the BCR to respond to stimulation (Chen et al, 2002; Stevenson & Caligaris-Cappio, 2004). Thus, ZAP-70 patients display a more effective BCR signal transduction that could contribute to their relatively aggressive clinical behaviour (Chen et al, 2002). Inhibitory phosphatases SHP-1 (SH2 domain-containing tyrosine phosphatase-1), SHIP-1 (SH2 domain-containing phosphatidylinositol 5-phosphatase-1) and SHIP-2 are involved in the complex and organized machinery aimed at counterbalancing B-cell activation upon BCR engagement by restricting the duration and/or intensity of the signalling (Zhang et al, 2000; March & Ravichandran, 2002). As ZAP-70 patients present an impaired BCR signalling capacity, we have explored the possibility that these phosphatases were preferentially expressed in CLL cells from this subgroup. Following informed consent and ethical approval, Western blotting analysis was performed on 49 purified CLL samples. SHP-1 and SHIP-1 proteins were found to be expressed in B cells from ZAP-70 and ZAP-70 CLL patients, while SHIP-2 could not be detected in any of the samples analysed (Fig 1A), even when the amount of protein loaded in each lane was duplicated (data not shown). Normal tonsillar B lymphocytes (TBL), used as controls, showed specific bands at 68, 145 and 150 kDa corresponding to SHP-1, SHIP-1 and SHIP-2 respectively (Fig 1A). There were no significant differences in SHP-1 expression between CLL subgroups (data not shown). By contrast, ZAP-70 CLL cells not only displayed a higher SHIP-1 protein expression compared with ZAP-70 subgroup (Fig 1B), but also it was constitutively tyrosine phosphorylated to a greater extent (Fig 1C and D). As SHIP-1 phosphorylation may correlate with its inhibitory capacity by enhancing the interaction with other adapter proteins (March & Ravichandran, 2002), ZAP-70 CLL cells might be more prone to inhibition by SHIP-1 than ZAP-70 ones. Finally, we evaluated SHIP-1 phosphorylation status upon BCR cross-linking with goat anti-human IgM (anti-l) and rabbit anti-goat IgG (RAG). We found that, in ZAP-70 patients, who present an impaired IgM signalling capacity (Chen et al, 2002), BCR cross-linking led to a time-dependent increase in reactivity of the anti-P-SHIP-1 antibody, which was maximal at 15 min and then decreased back towards baseline by 30 min (Fig 1E and F). By contrast, ZAP-70 samples did not modify SHIP-1 phosphorylation status upon BCR engagement (Fig 1E and F) although they expressed similar levels of surface IgM compared with ZAP-70 samples (data not shown). In conclusion, we found that the inhibitory phosphatase SHIP-1 exclusively participated in BCR signal transduction in ZAP-70 CLL cells, wherein it is expressed and constitutively tyrosine phosphorylated to a greater extent compared with ZAP-70 samples. Taken together, our data suggest that SHIP1 might be involved in the impaired BCR signalling commonly found in the former subgroup of patients. Moreover, given that SHIP-1 can negatively modulate not only BCR but also cytokine and chemokine receptor signalling (March & Ravichandran, 2002), the possibility exist that ZAP-70 CLL cells, by expressing higher SHIP-1 levels, hold higher signalling thresholds to different microenvironment stimuli. Experiments are in progress to determine whether SHIP-1 can regulate CLL cell responsiveness.

Consumption Coagulopathy in Acute Renal Failure Induced by Hypolipotropic Diets

Weanling male rats fed on a hypolipotropic diet develop acute renal failure whose morphological features vary from focal tubular necrosis to cortical necrosis. We have sequentially studied the hemostatic mechanism in correlation with the morphology of various tissues, mainly renal and hepatic, in choline-deficient rats as well as in three control groups. No important changes were observed in the hemostatic mechanisms before the development of tubular necrosis. Along with tubular necrosis a consumption coagulopathy was found, evidenced mainly by a decrease in the activity of factors V and VIII as well as a prolongation in PTTK and Quicks time and a decrease in platelets. Fibrin degradation products were found in serum and urine and soluble fibrin monomer complexes in the former. Following tubular necrosis thrombi were found in the renal microvasculature. It is possible to speculate that the tubular necrosis induced by choline deficiency could produce an activation of the coagulation system which in turn would lead to thrombosis of the renal microcirculation and cortical necrosis.

Expression of Fcγ receptors type II (FcγRII) in chronic lymphocytic leukemia B cells

Fil: Gamberale, Romina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Interferon DNA polymorphism in chronic leukemia.

The interferon (IFN) system (alpha, beta and gamma IFNs) is closely related to the first line of defenses against viral and tumoral diseases. Chronic leukemic and chronic lymphoproliferative patients respond in variable degrees to therapy with exogenous IFN. Remission after treatment with IFN-alpha in hairy cell leukemia (HCL) and in chronic myelogenous leukemia (CML) have been reported by other authors. In order to determine whether there are differences in IFN-alpha and beta genes between healthy and chronic leukemic individuals and among the different chronic leukemic patients, restriction fragment length polymorphism (RFLP) analyses was performed in a panel of patients with HCL, CML and chronic lymphocytic leukemia (CLL), and in a sample of healthy individuals. A significant difference in the allelic frequencies for the IFN-beta and Sst I enzyme in Chronic leukemias, mainly of myeloid origin, compared with the healthy individuals, was found.

Evaluation of the primitive fraction by functional in vitro assays at the RNA and DNA level represents a novel tool for complementing molecular monitoring in chronic myeloid leukemia

Quantification of BCR-ABL1 mRNA levels in peripheral blood of chronic myeloid leukemia patients is a strong indicator of response to tyrosine-kinase inhibitors (TKI) treatment. However, additional prognostic markers are needed in order to better classify patients. The hypothesis of leukemic stem cells (LSCs) heterogeneity and persistence, suggests that their functional evaluation could be of clinical interest. In this work, we assessed the primitive and progenitor fractions in patients at diagnosis and during TKI treatment using functional in vitro assays, defining a “functional leukemic burden” (FLB). We observed that the FLB was reduced in vivo in both fractions upon treatment. However, different FLB levels were observed among patients according to their response to treatment, suggesting that quantification of the FLB could complement early molecular monitoring. Given that FLB assessment is limited by BCR-ABL1 mRNA expression levels, we developed a novel detection method of primitive cells at the DNA level, using patient-specific primers and direct nested PCR in colonies obtained from functional in vitro assays. We believe that this method could be useful in the context of discontinuation trials, given that it is unknown whether the persistent leukemic clone represents LSCs, able to resume the leukemia upon TKI removal.