Impaired LAIR-1-mediated immune control due to collagen degradation in fibrosis.

Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.

Programmed Cell Death-1 (PD-1) anchoring to the GPI-linked co-receptor CD48 reveals a novel mechanism to modulate PD-1-dependent inhibition of human T cells.

Activation of PD-1 by anchoring it to Antigen Receptor (AR) components or associated co-receptors represents an attractive approach to treat autoimmune conditions. In this study, we provide evidence that CD48, a common lipid raft and Src kinase-associated coreceptor, induces significant Src kinase-dependent activation of PD-1 upon crosslinking, while CD71, a receptor excluded from these compartments, does not. Functionally, using bead-conjugated antibodies we demonstrate that CD48-dependent activation of PD-1 inhibits proliferation of AR-induced primary human T cells, and similarly, PD-1 activation using PD-1/CD48 bispecific antibodies inhibits IL-2, enhances IL-10 secretion, and reduces NFAT activation in primary human and Jurkat T cells, respectively. As a whole, CD48-dependent activation of PD-1 represents a novel mechanism to fine tune T cell activation, and by functionally anchoring PD-1 with receptors other than AR, this study provides a conceptual framework for rational development of novel therapies that activate inhibitory checkpoint receptors for treatment of immune-mediated diseases.

Metformin and simvastatin exert additive antitumour effects in glioblastoma via senescence-state: clinical and translational evidence.

BACKGROUND: Glioblastoma is one of the most devastating and incurable cancers due to its aggressive behaviour and lack of available therapies, being its overall-survival from diagnosis ∼14-months. Thus, identification of new therapeutic tools is urgently needed. Interestingly, metabolism-related drugs (e.g., metformin/statins) are emerging as efficient antitumour agents for several cancers. Herein, we evaluated the in vitro/in vivo effects of metformin and/or statins on key clinical/functional/molecular/signalling parameters in glioblastoma patients/cells. METHODS: An exploratory-observational-randomized retrospective glioblastoma patient cohort (n = 85), human glioblastoma/non-tumour brain human cells (cell lines/patient-derived cell cultures), mouse astrocytes progenitor cell cultures, and a preclinical xenograft glioblastoma mouse model were used to measure key functional parameters, signalling-pathways and/or antitumour progression in response to metformin and/or simvastatin. FINDINGS: Metformin and simvastatin exerted strong antitumour actions in glioblastoma cell cultures (i.e., proliferation/migration/tumoursphere/colony-formation/VEGF-secretion inhibition and apoptosis/senescence induction). Notably, their combination additively altered these functional parameters vs. individual treatments. These actions were mediated by the modulation of key oncogenic signalling-pathways (i.e., AKT/JAK-STAT/NF-κB/TGFß-pathways). Interestingly, an enrichment analysis uncovered a TGFß-pathway activation, together with AKT inactivation, in response to metformin + simvastatin combination, which might be linked to an induction of the senescence-state, the associated secretory-phenotype, and to the dysregulation of spliceosome components. Remarkably, the antitumour actions of metformin + simvastatin combination were also observed in vivo [i.e., association with longer overall-survival in human, and reduction in tumour-progression in a mouse model (reduced tumour-size/weight/mitosis-number, and increased apoptosis)]. INTERPRETATION: Altogether, metformin and simvastatin reduce aggressiveness features in glioblastomas, being this effect significantly more effective (in vitro/in vivo) when both drugs are combined, offering a clinically relevant opportunity that should be tested for their use in humans. FUNDING: Spanish Ministry of Science, Innovation and Universities; Junta de Andalucía; CIBERobn (CIBER is an initiative of Instituto de Salud Carlos III, Spanish Ministry of Health, Social Services and Equality).

Somatostatin Receptor Splicing Variant sst5TMD4 Overexpression in Glioblastoma Is Associated with Poor Survival, Increased Aggressiveness Features, and Somatostatin Analogs Resistance.

Glioblastoma (GBM) is the most malignant and lethal brain tumor. Current standard treatment consists of surgery followed by radiotherapy/chemotherapy; however, this is only a palliative approach with a mean post-operative survival of scarcely ~12-15 months. Thus, the identification of novel therapeutic targets to treat this devastating pathology is urgently needed. In this context, the truncated splicing variant of the somatostatin receptor subtype 5 (sst5TMD4), which is produced by aberrant alternative splicing, has been demonstrated to be overexpressed and associated with increased aggressiveness features in several tumors. However, the presence, functional role, and associated molecular mechanisms of sst5TMD4 in GBM have not been yet explored. Therefore, we performed a comprehensive analysis to characterize the expression and pathophysiological role of sst5TMD4 in human GBM. sst5TMD4 was significantly overexpressed (at mRNA and protein levels) in human GBM tissue compared to non-tumor (control) brain tissue. Remarkably, sst5TMD4 expression was significantly associated with poor overall survival and recurrent tumors in GBM patients. Moreover, in vitro sst5TMD4 overexpression (by specific plasmid) increased, whereas sst5TMD4 silencing (by specific siRNA) decreased, key malignant features (i.e., proliferation and migration capacity) of GBM cells (U-87 MG/U-118 MG models). Furthermore, sst5TMD4 overexpression in GBM cells altered the activity of multiple key signaling pathways associated with tumor aggressiveness/progression (AKT/JAK-STAT/NF-κB/TGF-ß), and its silencing sensitized GBM cells to the antitumor effect of pasireotide (a somatostatin analog). Altogether, these results demonstrate that sst5TMD4 is overexpressed and associated with enhanced malignancy features in human GBMs and reveal its potential utility as a novel diagnostic/prognostic biomarker and putative therapeutic target in GBMs.

SF3B1 inhibition disrupts malignancy and prolongs survival in glioblastoma patients through BCL2L1 splicing and mTOR/ß-catenin pathways imbalances.

BACKGROUND: Glioblastoma is one of the most devastating cancer worldwide based on its locally aggressive behavior and because it cannot be cured by current therapies. Defects in alternative splicing process are frequent in cancer. Recently, we demonstrated that dysregulation of the spliceosome is directly associated with glioma development, progression, and aggressiveness. METHODS: Different human cohorts and a dataset from different glioma mouse models were analyzed to determine the mutation frequency as well as the gene and protein expression levels between tumor and control samples of the splicing-factor-3B-subunit-1 (SF3B1), an essential and druggable spliceosome component. SF3B1 expression was also explored at the single-cell level across all cell subpopulations and transcriptomic programs. The association of SF3B1 expression with relevant clinical data (e.g., overall survival) in different human cohorts was also analyzed. Different functional (proliferation/migration/tumorspheres and colonies formation/VEGF secretion/apoptosis) and mechanistic (gene expression/signaling pathways) assays were performed in three different glioblastomas cell models (human primary cultures and cell lines) in response to SF3B1 blockade (using pladienolide B treatment). Moreover, tumor progression and formation were monitored in response to SF3B1 blockade in two preclinical xenograft glioblastoma mouse models. RESULTS: Our data provide novel evidence demonstrating that the splicing-factor-3B-subunit-1 (SF3B1, an essential and druggable spliceosome component) is low-frequency mutated in human gliomas (~ 1 %) but widely overexpressed in glioblastoma compared with control samples from the different human cohorts and mouse models included in the present study, wherein SF3B1 levels are associated with key molecular and clinical features (e.g., overall survival, poor prognosis and/or drug resistance). Remarkably, in vitro and in vivo blockade of SF3B1 activity with pladienolide B drastically altered multiple glioblastoma pathophysiological processes (i.e., reduction in proliferation, migration, tumorspheres formation, VEGF secretion, tumor initiation and increased apoptosis) likely by suppressing AKT/mTOR/ß-catenin pathways, and an imbalance of BCL2L1 splicing. CONCLUSIONS: Together, we highlight SF3B1 as a potential diagnostic and prognostic biomarker and an efficient pharmacological target in glioblastoma, offering a clinically relevant opportunity worth to be explored in humans.

Clinical Characteristics of Hospitalized Infants With Laboratory-Confirmed Pertussis in Guatemala.

BACKGROUND: Pertussis is an important cause of hospitalization and death in infants too young to be vaccinated (aged <2 months). Limited data on infant pertussis have been reported from Central America. The aim of this study was to characterize acute respiratory illnesses (ARIs) attributable to Bordetella pertussis among infants enrolled in an ongoing surveillance study in Guatemala. METHODS: As part of a population-based surveillance study in Guatemala, infants aged <2 months who presented with ARI and required hospitalization were enrolled, and nasopharyngeal and oropharyngeal swab specimens were obtained. For this study, these specimens were tested for B pertussis using real-time polymerase chain reaction (PCR). RESULTS: Among 301 infants hospitalized with ARI, we found 11 with pertussis confirmed by PCR (pertussis-positive infants). Compared to pertussis-negative infants, pertussis-positive infants had a higher mean admission white blood cell count (20900 vs 12579 cells/µl, respectively; P = .024), absolute lymphocyte count (11517 vs 5591 cells/µl, respectively; P < .001), rate of admission to the intensive care unit (64% vs 35%, respectively; P = .054), and case fatality rate (18% vs 3%, respectively; P = .014). Ten of the 11 pertussis-positive infants had cough at presentation; the majority (80%) of them had a cough duration of <7 days, and only 1 had a cough duration of >14 days. Fever (temperature ≥ 38°C) was documented in nearly half (45%) of the pertussis-positive infants (range, 38.0-38.4°C). CONCLUSIONS: In this study of infants <2 months of age hospitalized with ARI in Guatemala, pertussis-positive infants had a high rate of intensive care unit admission and a higher case fatality rate than pertussis-negative infants.

Situación del parasitismo intestinal en preescolares de un hogar infantil estatal en Popayán, Colombia / Situation of intestinal parasitism in preschools of a state child's home in Popayan, Colombia

deficientes. Se describen prevalencias a nivel nacional de este mayores al 80%, con predominio de protozoos. Objetivo: describir la situación de parasitismo intestinal en preescolares de un hogar infantil estatal de Popayán (Colombia) y su relación con variables sociodemográficas. Materiales y métodos: se realizó un estudio descriptivo de corte transversal, entre mayo y septiembre de 2013, en 187 niños de 1 a 5 años de edad, matriculados en un hogar infantil estatal de Popayán (Colombia). Se realizó una encuesta sociodemográfica y análisis parasitológico de muestras de materia fecal mediante examen directo y método de concentración modificado de Ritchie-Frick. Se calcularon prevalencias, distribuciones de frecuencia y asociaciones de factores sociodemográficos con la presencia de parasitismo intestinal utilizando regresiones logísticas. Resultados: se identificó una prevalencia de parasitismo intestinal de 43,3%. La especie encontrada con mayor frecuencia fue Blastocystis spp. (24,6%), seguida por Entamoeba coli (13,4%) y Giardia intestinalis (11,8%). En ninguna de las muestras se observaron helmintos. Conclusiones: la prevalencia encontrada de parasitismo intestinal se aproxima al promedio nacional, de acuerdo con reportes para población preescolar en otros municipios del país. La ausencia de helmintos y el predominio de especies de protozoos respaldan la necesidad de futuras investigaciones que permitan conocer la epidemiología local. Además, se identificaron condiciones sociodemográficas de riesgo para infecciones en la población estudiada, algunas de ellas asociadas a la presencia de protozoos intestinales

Intestinal parasitism, generally asymptomatic problem but with important repercussions at socioeconomic and health field, mainly affects children with poor sanitary conditions. Their nationwide prevalence is higher than 80% with predominance of protozoa. Objective: To describe the situation of intestinal parasitism in preschools of a statechild's home in Popayan (Colombia) and its relationship with socio-demographic variables. Materials and methods: A cross - sectional descriptive study was carried out, between May and September 2013, with 187 children between 1 and 5 years of age, enrolled in a statechild's home in Popayan (Colombia). A sociodemographic survey was applied and parasitological analysis of stool samples was performed by direct examination and by the modified Ritchie-Frick concentration method. Prevalence and frequency distributions were calculated as well as logistic regression of associations of sociodemographic factors with the presence of intestinal parasitism. Results: The prevalence of intestinal parasites was 43.3%. The most frequently species was Blastocystis spp. (24.6%), followed by Entamoeba coli (13.4%) and Giardia instestinalis (11.8%). Helminths were not observed in any of the samples. Conclusions: The identified prevalence of intestinal parasites is close to the national average, according with previous reports for pre-school children in other municipalities in the country. The absence of helminths and the predominance of the protozoan species support the need for future research that allows knowing the local epidemiology. In addition, it was identified that the studied population is continuously exposed to different sociodemographic risk conditions that increase the possibility of acquiring enteroparasitosis

Kinase-independent feedback of the TAK1/TAB1 complex on BCL10 turnover and NF-κB activation.

Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.

G alpha q-containing G proteins regulate B cell selection and survival and are required to prevent B cell-dependent autoimmunity.

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G(alphaq) subunit of trimeric G proteins (Gnaq(-/-) mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq(-/-) B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq(-/-) chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq(-/-) B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.

MAGUK-controlled ubiquitination of CARMA1 modulates lymphocyte NF-kappaB activity.

The adaptor protein CARMA1 is required for antigen receptor-triggered activation of IKK and JNK in lymphocytes. Once activated, the events that subsequently turn off the CARMA1 signalosome are unknown. In this study, we found that antigen receptor-activated CARMA1 underwent lysine 48 (K48) polyubiquitination and proteasome-dependent degradation. The MAGUK region of CARMA1 was an essential player in this event; the SH3 and GUK domains contained the main ubiquitin acceptor sites, and deletion of a Hook domain (an important structure for maintaining inactive MAGUK proteins) between SH3 and GUK was sufficient to induce constitutive ubiquitination of CARMA1. A similar deletion promoted the ubiquitination of PSD-95 and Dlgh1, suggesting that a conserved mechanism may control the turnover of other MAGUK family protein complexes. Functionally, we demonstrated that elimination of MAGUK ubiquitination sites in CARMA1 resulted in elevated basal and inducible NF-kappaB and JNK activation as a result of defective K48 ubiquitination and increased persistence of this ubiquitination-deficient CARMA1 protein in activated lymphocytes. The coordination of degradation with the full activation of the CARMA1 molecule likely provides an intrinsic feedback control mechanism to balance lymphocyte activation upon antigenic stimulation.

Serine 649 phosphorylation within the protein kinase C-regulated domain down-regulates CARMA1 activity in lymphocytes.

Phosphorylation of CARMA1 is a crucial event initiating the assembly of IkappaB kinase and JNK signaling complexes downstream of activated Ag receptors. We previously mapped three protein kinase C (PKC) target sites in murine CARMA1 in vitro, and demonstrated that mutation of two of these serines (S564 and S657) resulted in reduced NF-kappaB activation, whereas mutation of the third serine (S649) had no clear effect. In this study, we report that when low concentrations of Ag receptor activators are used, loss of S649 (by mutation to alanine) promotes enhanced IkappaB kinase and JNK activation in both B and T cell lines. Reconstitution of CARMA1(-/-) DT40 B cells with CARMA1 S649A leads to increased cell death and reduced cell growth in comparison to wild-type CARMA1, likely a result of enhanced JNK activation. To directly determine whether S649 is modified in vivo, we generated phospho-specific Abs recognizing phospho-S649, and phospho-S657 as a positive control. Although phospho-S657 peaked and declined rapidly after Ag receptor stimulation, phospho-S649 occurred later and was maintained for a significantly longer period poststimulation in both B and T cells. Interestingly, phospho-S657 was completely abolished in PKCbeta-deficient B cells, whereas delayed phosphorylation at S649 was partially intact and depended, in part, upon novel PKC activity. Thus, distinct PKC-mediated CARMA1 phosphorylation events exert opposing effects on the activation status of CARMA1. We propose that early phosphorylation events at S657 and S564 promote the initial assembly of the CARMA1 signalosome, whereas later phosphorylation at S649 triggers CARMA1 down-regulation.

CD38 induces differentiation of immature transitional 2 B lymphocytes in the spleen.

CD38 is a surface receptor able to induce activation, proliferation, and survival of human and mouse lymphocytes; this molecule is expressed on the surface of both mature and immature B cells. In this work, the function of CD38 in the maturation of murine B lymphocytes in the spleen was analyzed. The results showed that CD38 is highly expressed on Transitional 2 (T2) B lymphocytes with an intermediate expression on Transitional 1 (T1) and mature follicular B cells (M). Correlating with a high expression of CD38, T2 cells are also larger and more granular than T1 or M B cells. T2 cells also showed high levels of other molecules, which indicate an activated phenotype. CD38 crosslinking induced proliferation and maturation of T2 B lymphocytes; in contrast, T1 subset died by apoptosis. Finally, CD38 stimulation of T2 B lymphocytes obtained from Btk-, Lyn-, or Fyn-deficient mice showed a defective differentiation; similarly, drugs interfering with PI3K or ERK decreased the proliferation or differentiation of this subset. This suggests that these molecules participate in the CD38 signaling pathway. As a whole, the results indicate that CD38 plays an important role in the regulation of B-cell maturation in the spleen.

CD38 cross-linking enhances TLR-induced B cell proliferation but decreases IgM plasma cell differentiation.

It is becoming increasingly clear that the regulation of proliferation and differentiation of B cells to plasma cells involves the integration of a variety of intracellular signals provided by receptors of both the adaptive and innate immune system. The cross-linking of the surface molecule CD38 induces calcium mobilization, protein phosphorylation and NF-kappaB translocation into the nucleus, ultimately leading to proliferation and isotype switching toward IgG1. Here we describe (a) the effect on B cell activation of stimulating through both CD38 and Toll-like receptors 4, 7 and 9; and (b) that CD38 cross-linking increases the number of proliferating cells and the rate of proliferation in LPS-stimulated B cells by a Bruton's tyrosine kinase- and protein kinase C-dependent mechanism. In contrast, CD38 cross-linking reduces the number of cells committed to IgM plasma cell differentiation as measured by the number of CD138+ cells, antibody secretion, and the expression of PAX5, Bcl6 and Blimp-1. Since a putative ligand for CD38 is expressed by germinal center follicular dendritic cells, and CD38 expression is down-regulated in germinal center B cells, we speculate that CD38 might participate in the outcome of post-germinal center antibody responses.

The CARMA1 signalosome links the signalling machinery of adaptive and innate immunity in lymphocytes.

The recognition of antigen by B- or T-cell receptors initiates an intracellular signalling cascade that results in the nuclear translocation and activation of the transcription factor nuclear factor-kappaB (NF-kappaB). NF-kappaB is an important regulator of lymphocyte development and function, and its dysregulation is associated with many immune disorders. Defining the mechanisms that transmit signals from the antigen receptor to NF-kappaB is therefore an important goal for immunologists. In this Review, we merge information gleaned from research of the innate immune system with what we know about antigen-receptor signals in the adaptive immune system, to propose a cohesive model of how antigen receptors activate NF-kappaB.

CD38 induces apoptosis of a murine pro-B leukemic cell line by a tyrosine kinase-dependent but ADP-ribosyl cyclase- and NAD glycohydrolase-independent mechanism.

Cross-linking of CD38 on hematopoietic cells induces activation, proliferation and differentiation of mature T and B cells and mediates apoptosis of myeloid and lymphoid progenitor cells. In addition to acting as a signaling receptor, CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites, including cyclic adenosine diphosphate ribose (cADPR). It has been previously postulated that the calcium-mobilizing metabolites produced by CD38 may regulate its receptor-based activities. To test this hypothesis, we examined whether the enzyme activity of CD38 controls the apoptosis of an anti-CD38-stimulated leukemic B cell. We show that anti-CD38-induced apoptosis of Ba/F3 cells, a murine pro-B cell line, is not affected by blocking the calcium-mobilizing activity of cADPR or by inhibiting intracellular or extracellular calcium mobilization. In addition, we demonstrate that blocking CD38 enzyme activity with 2'-deoxy-2'-fluoro-nicotinamide arabinoside adenine dinucleotide has no effect on apoptosis and that Ba/F3 cells expressing catalytically inactive mutant forms of CD38 still undergo apoptosis upon CD38 cross-linking. Instead, we find that anti-CD38-induced apoptosis is dependent on tyrosine kinase and caspase activation, and that this process appears to be potentiated by the presence of membrane microdomains. Thus, the receptor-mediated functions of CD38 can be separated from its enzyme activity in a murine leukemic cell line, suggesting that CD38 plays multiple, but independent, biologic roles.

Phosphorylation of the CARMA1 linker controls NF-kappaB activation.

PKC isoforms and CARMA1 play crucial roles in immunoreceptor-dependent NF-kappaB activation. We tested whether PKC-dependent phosphorylation of CARMA1 directly regulates this signaling cascade. B cell antigen receptor (BCR) engagement led to the progressive recruitment of CARMA1 into lipid rafts and to the association of CARMA1 with, and phosphorylation by, PKCbeta. Furthermore, PKCbeta interacted with the serine-rich CARMA1 linker, and both PKCbeta and PKCtheta phosphorylated identical serine residues (S564, S649, and S657) within this linker. Mutation of two of these sites ablated the functional activity of CARMA1. In contrast, deletion of the linker resulted in constitutive, receptor- and PKC-independent NF-kappaB activation. Together, our data support a model whereby CARMA1 phosphorylation controls NF-kappaB activation by triggering a shift from an inactive to an active CARMA1 conformer. This PKC-dependent switch regulates accessibility of the CARD and CC domains and controls assembly and full activation of the membrane-associated IkappaB kinase (IKK) signalosome.

Cas IIgly induces apoptosis in glioma C6 cells in vitro and in vivo through caspase-dependent and caspase-independent mechanisms.

In this work, we investigated the effects of Casiopeina II-gly (Cas IIgly)--a new copper compound exhibiting antineoplastic activity--on glioma C6 cells under both in vitro and in vivo conditions, as an approach to identify potential therapeutic agents against malignant glioma. The exposure of C6 cells to Cas IIgly significantly inhibited cell proliferation, increased reactive oxygen species (ROS) formation, and induced apoptosis in a dose-dependent manner. In cultured C6 cells, Cas IIgly caused mitochondrio-nuclear translocation of apoptosis induction factor (AIF) and endonuclease G at all concentrations tested; in contrast, fragmentation of nucleosomal DNA, cytochrome c release, and caspase-3 activation were observed at high concentrations. Administration of N-acetyl-L-cystein, an antioxidant, resulted in significant inhibition of AIF translocation, nucleosomal DNA fragmentation, and caspase-3 activation induced by Cas IIgly. These results suggest that caspase-dependent and caspase-independent pathways both participate in apoptotic events elicited by Cas IIgly. ROS formation induced by Cas IIgly might also be involved in the mitochondrio-nuclear translocation of AIF and apoptosis. In addition, treatment of glioma C6-positive rats with Cas IIgly reduced tumor volume and mitotic and cell proliferation indexes, and increased apoptotic index. Our findings support the use of Cas IIgly for the treatment of malignant gliomas.

Localization of CD38 in murine B lymphocytes to plasma but not intracellular membranes.

CD38 has been widely characterized both as an ecto-enzyme and as a receptor. As an enzyme, CD38 catalyzes the conversion of NAD(+) and NADP to several metabolites including cADPR and NAADP, which mediate Ca(2+) release from separate intracellular stores, and ADPR, which activates the TRPM2 plasma membrane Ca(2+) channel. Since the catalytic domain of CD38 is exposed to the extracellular milieu, several mechanistic and topological studies have been performed to explain how CD38 gains access to its substrates, which are found at highest concentration in the cytosol of cells, and how the non-permeant metabolites produced by ecto-CD38 arrive at their intracellular site(s) of action. Accordingly, several studies have reported that CD38 is not only expressed on the plasma membrane but is also found in various sub-cellular compartments, including the nucleus where it is localized to the inner nuclear membrane. In this work, we employed a protocol of mild membrane solubilization to cleanly separate plasma membranes from other intracellular membranes and then analyzed the sub-cellular expression of murine CD38 in purified primary B lymphocytes. After immunoprecipitation, CD38 was exclusively detected in the plasma membrane protein containing soluble fraction and not in the insoluble fraction which was highly enriched for nuclear, endoplasmic reticulum and mitochondrial proteins. Likewise, NAD(+) glycohydrolase measurements and confocal microscopy analysis corroborated that CD38 was not localized in nuclear membranes and indicated that CD38 is primarily, if not exclusively, localized to the plasma membrane of murine B lymphocytes.

CD38 signaling regulates B lymphocyte activation via a phospholipase C (PLC)-gamma 2-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-gamma2 (PLC-gamma2), 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-gamma2 in the CD38 signaling pathway. Accordingly, we demonstrate that CD38 cross-linking does not result in the functional phosphorylation of PLC-gamma2 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) beta-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-kappaB 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, IkappaB-alpha 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-gamma2-independent, B lymphocyte activation.