Alejandro Zentella

CD8 Cells of Patients with Diffuse Cutaneous Leishmaniasis Display Functional Exhaustion: The Latter Is Reversed, In Vitro, by TLR2 Agonists

Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular “exhaustion” described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.

Dexamethasone protection from TNF-alpha-induced cell death in MCF-7 cells requires NF-kappaB and is independent from AKT

BackgroundThe biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex) dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death.ResultsCellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs) expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected.ConclusionNF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1.

CD38 Signaling Regulates B Lymphocyte Activation via a Phospholipase C (PLC)-γ2-Independent, Protein Kinase C, Phosphatidylcholine-PLC, and Phospholipase D-Dependent Signaling Cascade

The CD38 cell surface receptor is a potent activator for splenic, B lymphocytes. The molecular mechanisms regulating this response, however, remain incompletely characterized. Activation of the nonreceptor tyrosine kinase, Btk, is essential for CD38 downstream signaling function. The major Btk-dependent substrate in B cells, phospholipase C-γ2 (PLC-γ2), functions to generate the key secondary messengers, inositol-1,4,5 trisphosphate and diacylglycerol. Surprisingly, CD38 ligation results in no detectable increase in phosphoinositide metabolism and only a minimal increase in cytosolic calcium. We hypothesized that Btk functioned independently of PLC-γ2 in the CD38 signaling pathway. Accordingly, we demonstrate that CD38 cross-linking does not result in the functional phosphorylation of PLC-γ2 nor an increase in inositol-1,4,5 trisphosphate production. Furthermore, splenic B cells exhibit a normal CD38-mediated, proliferative response in the presence of the phosphoinositide-PLC inhibitor, U73122. Conversely, protein kinase C (PKC) β-deficient mice, or PKC inhibitors, indicated the requirement for diacylglycerol-dependent PKC isoforms in this pathway. Loss of PKC activity blocked CD38-dependent, B cell proliferation, NF-κB activation, and subsequent expression of cyclin-D2. These results suggested that an alternate diacylglycerol-producing phospholipase must participate in CD38 signaling. Consistent with this idea, CD38 increased the enzymatic activity of the phosphatidylcholine (PC)-metabolizing enzymes, PC-PLC and phospholipase D. The PC-PLC inhibitor, D609, completely blocked CD38-dependent B cell proliferation, IκB-α degradation, and cyclin-D2 expression. Analysis of Btk mutant B cells demonstrated a partial requirement for Btk in the activation of both enzymes. Taken together, these data demonstrate that CD38 initiates a novel signaling cascade leading to Btk-, PC-PLC-, and phospholipase D-dependent, PLC-γ2-independent, B lymphocyte activation.

Ceramide promotes the death of human cervical tumor cells in the absence of biochemical and morphological markers of apoptosis.

C8-ceramide, a synthetic cell-permeable analog of endogenous ceramides, interfered with cell proliferation, and was cytotoxic to papilloma virus-containing human cervix carcinoma cells, CALO, INBL, and HeLa, that match two clinical stages of tumor progression. C8-ceramide (3 microM) markedly reduced the tumor cell number after 48 h of treatment, an effect that endured even after the removal of C8-ceramide. The carcinoma cells showed morphologic changes, characteristic of necrosis and released lactate dehydrogenase (LDH). A biologically inactive analog C8-dihydro-ceramide had no effect on cell viability in any of the cell lines tested. Seventy-two hours after C8-ceramide treatment none of the biochemical and morphological markers characteristic of apoptosis: (a) nuclear chromatin condensation, (b) DNA fragmentation, (c) proteolysis of the caspase-3 substrate poly-(ADP-ribose)-polymerase (PARP), and (d) appearance of phosphatidylserine on the external cell membrane, were observed. C8-ceramide had no effect on human cervix fibroblasts and induced a mild reduction (30%) in the proliferation of normal human cervix epithelia and HeLa cells (IV-B metastatic stage). The cytotoxicity of C8-ceramide was restricted to CALO (early II-B) and INBL (IV-A non-metastatic) carcinoma cells. The possible application of ceramide in the treatment of early stages of cervical cancer is discussed.

Synthesis and cytotoxic activity of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidine derivatives

A series of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidines derivatives were synthesized using a convenient synthetic route. We evaluate the isosteric replacement of methyl groups in 4-(2-methylimidazo[1,2-a]pyridin-3-yl)-N-p-tolylpyrimidin-2-amine (compound 1) by trifluoromethyl groups and the isosteric substitution of the 2-methylimidazo[1,2-a]pyridin-3-yl scaffold by quinolin-4-yl or quinolin-3-yl moieties. The replacement of hydrogen by fluorine does not affect notably the cytotoxic activity and CDK inhibitor activity in this series. Quinolin-4-yl-substituted compound, 8, presents cytotoxic activity and is most effective and selective against CDK1/CycA than against CDK2/CycB. Compound 11, which has a quinolin-3-yl moiety is CDK inhibitor but presents null cytotoxic activity. Quinolin-4-yl-substituted compounds constitute a new lead of cytotoxic and CDK inhibitor compounds from which more compelling and selective inhibitors can be designed.

Opposite effect of Hsp90α and Hsp90β on eNOS ability to produce nitric oxide or superoxide anion in human embryonic kidney cells.

Heat shock protein 90 subfamily is composed by two cytosolic isoforms known as Hsp90α and Hsp90β. Endothelial nitric oxide synthase (eNOS) is regulated by Hsp90, however the specific role of each Hsp90 isoform on NO production has not been established. This study was designed to evaluate the effect of Hsp90α and Hsp90β over-expression on eNOS/NO pathway. Rat Hsp90α and Hsp90β were cloned into pcDNA3.1(+) and transfected in human embryonic kidney cells (HEK-293). Hsp90α and Hsp90β transfection was corroborated by Western blot analysis and their effect on NO production (NO2/NO3), eNOS protein and its phosphorylation at Ser1177 and Thr495, as well as Akt/PKB Ser473 phosphorylation was determined. The interaction of Hsp90α and Hsp90β with eNOS and the dimer/monomer ratio of Hsp90, as well as O2- generation were also assessed. After transfection, Hsp90α and Hsp90β levels were significantly increased in HEK-293 cells. The Hsp90α over-expression induced a significant increase in NO2/NO3 levels, an effect that was associated with increased phosphorylation of eNOS Ser 1177 and Akt/PKB Ser473, as well as with a greater Hsp90α dimerization. Noteworthy, pcHsp90β transfection reduced significantly NO2/NO3 and increased O2- generation. These effects were associated with a reduction of eNOS dimeric conformation, increased eNOS Thr495 phosphorylation, reduced Akt/PKB phosphorylation, and by a greater amount of monomeric Hsp90β conformation. These data show for first time that Hsp90α and Hsp90β differentially modulate NO and O2- generation by eNOS through promoting changes in eNOS conformation and phosphorylation state.

Leishmania mexicana: Participation of NF-κB in the differential production of IL-12 in dendritic cells and monocytes induced by lipophosphoglycan (LPG)

Dendritic cells (DC) and macrophages (Mphi) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mphi IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-alpha, and IL-10 and nuclear translocation of NF-kappaB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-kappaB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-kappaB in monocytes with the subsequent decrease in IL-12 production.

A noncanonical NF-κB pathway through the p50 subunit regulates Bcl-2 overexpression during an oxidative-conditioning hormesis response

Cells can respond to damage and stress by activating various repair and survival pathways. One of these responses can be induced by preconditioning the cells with sublethal stress to provoke a prosurvival response that will prevent damage and death, and which is known as hormesis. Bcl-2, an antiapoptotic protein recognized by its antioxidant and prosurvival functions, has been documented to play an important role during oxidative-conditioning hormesis. Using an oxidative-hormetic model, which was previously established in the L929 cell line by subjecting the cells to a mild oxidative stress of 50 μM H₂O₂ for 9 h, we identified two different transductional mechanisms that participate in the regulation of Bcl-2 expression during the hormetic response. These mechanisms converge in activating the nuclear transcription factor NF-κB. Interestingly, the noncanonical p50 subunit of the NF-κB family is apparently the subunit that participates during the oxidative-hormetic response.

Glycosylated VCAM-1 isoforms revealed in 2D western blots of HUVECs treated with tumoral soluble factors of breast cancer cells

Background Several common aspects of endothelial phenotype, such as the expression of cell adhesion molecules, are shared between metastasis and inflammation. Here, we analyzed VCAM-1 variants as biological markers of these two types of endothelial cell activation. With the combination of 2-DE and western blot techniques and the aid of tunicamycin, we analyzed N-glycosylation variants of VCAM-1 in primary human endothelial cells stimulated with either TNF or tumoral soluble factors (TSFs) derived from the human breast cancer cell line ZR75.30. Results Treatments induced a pro-adhesive endothelial phenotype. 2D western blots analysis of cells subjected to both treatments revealed the expression of the two known VCAM-1 isoforms and of previously unknown isoforms. In particular TSFZR75.30 induced an isoform with a relative molecular mass (Mr) and isoelectric point (pI) of 75-77 kDa and 5.0, respectively. Conclusion The unknown isoforms of VCAM-1 that were found to be overexpressed after treatment with TSFs compared with TNF, could serve as biomarkers to discriminate between inflammation and metastasis. 2D western blots revealed three new VCAM-1 isoforms expressed in primary human endothelial cells in response to TSF stimulation. Each of these isoforms varies in Mr and pI and could be the result of differential glycosylation states.

Bcl-2 sustains hormetic response by inducing Nrf-2 nuclear translocation in L929 mouse fibroblasts

Hormesis is the process whereby exposure to a low dose of a chemical agent induces an adaptive effect on the cell or organism. This response evokes the expression of cytoprotective and antioxidant proteins, allowing pro-oxidants to emerge as important hormetic agents. The antiapoptotic protein Bcl-2 is known to protect cells against death induced by oxidants; it has been suggested that Bcl-2 might also modulate steady-state reactive oxygen species levels. The aim of this work was to find out if Bcl-2 might play a role during the hormetic response and in Nrf-2 activation. We have established a model to study the oxidative conditioning hormesis response (OCH) by conditioning the cell line L929 with 50muM H(2)O(2) for 9h. This condition did not induce oxidative damage nor oxidative imbalance, and OCH cells maintained a 70-80% survival rate after severe H(2)O(2) treatment compared to nonconditioned cells. When cells were pretreated with the Bcl-2 inhibitor HA14-1 or were silenced with Bcl-2-siRNA, both the hormetic effect and the Nrf-2 nuclear translocation previously observed were abrogated. Our results suggest a sequence of causal events related to increase in Bcl-2 expression, induction of Nrf-2 activation, and sustained expression of cytoprotective proteins such as GST and gammaGCS.