Influx of kynurenine into the brain is involved in the reduction of ethanol consumption induced by Ro 61-8048 after chronic intermittent ethanol in mice.

BACKGROUND AND PURPOSE: The kynurenine pathway has been proposed as a target for modulating drug abuse. We previously demonstrated that inhibition of kynurenine 3-monooxygenase (KMO), using Ro 61-8048, reduces ethanol consumption in a binge drinking model. Here, we investigate the effect of the kynurenine pathway modulation in ethanol-dependent mice. EXPERIMENTAL APPROACH: Adult male and female mice were subjected to a Chronic Intermittent Ethanol (CIE) paradigm. On the last day of CIE, mice were treated with Ro 61-8048, Ro 61-8048 + PNU-120596, a positive allosteric modulator of α7nAChR, and Ro 61-8048 + L-leucine or probenecid, which blocks the influx or efflux of kynurenine from the brain, respectively. Ethanol, water consumption and preference were measured and kynurenine levels in plasma and limbic forebrain were determined. KEY RESULTS: Ro 61-8048 decreases consumption and preference for ethanol in both sexes exposed to the CIE model, an effect that was prevented by PNU-120596. The Ro 61-8048-induced decrease in ethanol consumption depends on the influx of kynurenine into the brain. CONCLUSION AND IMPLICATIONS: Inhibition of KMO reduces ethanol consumption and preference in both male and female mice subjected to CIE model by a mechanism involving α7nAChR. Moreover, this centrally-mediated effect depends on the influx of peripheral kynurenine to the brain and can be prolonged by blocking the efflux of kynurenine from the brain. Here, for the first time, we demonstrate that the modulation of the kynurenine pathway is an effective strategy for the treatment of ethanol dependence in both sexes.

Addiction and the kynurenine pathway: A new dancing couple?

Drug use poses a serious threat to health systems throughout the world and the number of consumers rises relentlessly every year. The kynurenine pathway, main pathway of tryptophan degradation, has drawn interest in this field due to its relationship with addictive behaviour. Recently it has been confirmed that modulation of kynurenine metabolism at certain stages of the pathway can reduce, prevent or abolish drug seeking-like behaviours in studies with several different drugs. In this review, we present an up-to-date summary of the evidences of a relationship between drug use and the kynurenine pathway, both the alterations of the pathway due to drug use as well as modulation of the pathway as a potential approach to treat drug addiction. The review discusses ethanol, nicotine, cannabis, amphetamines, cocaine and opioids and new prospects in the drug research field are proposed.

Changes in brain kynurenine levels via gut microbiota and gut-barrier disruption induced by chronic ethanol exposure in mice.

Inflammatory processes have been shown to modify tryptophan (Trp) metabolism. Gut microbiota appears to play a significant role in the induction of peripheral and central inflammation. Ethanol (EtOH) exposure alters gut permeability, but its effects on Trp metabolism and the involvement of gut microbiota have not been studied. We analyzed several parameters of gut-barrier and of peripheral and central Trp metabolism following 2 different EtOH consumption patterns in mice, the binge model, drinking in the dark (DID), and the chronic intermittent (CI) consumption paradigm. Antibiotic treatment was used to evaluate gut microbiota involvement in the CI model. Mice exposed to CI EtOH intake, but not DID, show bacterial translocation and increased plasma LPS immediately after EtOH removal. Gut-barrier permeability to FITC-dextran is increased by CI, and, furthermore, intestinal epithelial tight-junction (TJ) disruption is observed (decreased expression of zonula occludens 1 and occludin) associated with increased matrix metalloproteinase (MMP)-9 activity and iNOS expression. CI EtOH, but not DID, increases kynurenine (Kyn) levels in plasma and limbic forebrain. Intestinal bacterial decontamination prevents the LPS increase but not the permeability to FITC-dextran, TJ disruption, or the increase in MMP-9 activity and iNOS expression. Although plasma Kyn levels are not affected by antibiotic treatment, the elevation of Kyn in brain is prevented, pointing to an involvement of microbiota in CI EtOH-induced changes in brain Trp metabolism. Additionally, CI EtOH produces depressive-like symptoms of anhedonia, which are prevented by the antibiotic treatment thus pointing to an association between anhedonia and the increase in brain Kyn and to the involvement of gut microbiota.-Giménez-Gómez, P., Pérez-Hernández, M., O'Shea, E., Caso, J. R., Martín-Hernández, D., Cervera, L. A., Centelles. M. L. G.-L., Gutiérrez-Lopez, M. D., Colado, M. I. Changes in brain kynurenine levels via gut microbiota and gut-barrier disruption induced by chronic ethanol exposure in mice.

Increasing kynurenine brain levels reduces ethanol consumption in mice by inhibiting dopamine release in nucleus accumbens.

Recent research suggests that ethanol (EtOH) consumption behaviour can be regulated by modifying the kynurenine (KYN) pathway, although the mechanisms involved have not yet been well elucidated. To further explore the implication of the kynurenine pathway in EtOH consumption we inhibited kynurenine 3-monooxygenase (KMO) activity with Ro 61-8048 (100 mg/kg, i.p.), which shifts the KYN metabolic pathway towards kynurenic acid (KYNA) production. KMO inhibition decreases voluntary binge EtOH consumption and EtOH preference in mice subjected to "drinking in the dark" (DID) and "two-bottle choice" paradigms, respectively. This effect seems to be a consequence of increased KYN concentration, since systemic KYN administration (100 mg/kg, i.p.) similarly deters binge EtOH consumption in the DID model. Despite KYN and KYNA being well-established ligands of the aryl hydrocarbon receptor (AhR), administration of AhR antagonists (TMF 5 mg/kg and CH-223191 20 mg/kg, i.p.) and of an agonist (TCDD 50 µg/kg, intragastric) demonstrates that signalling through this receptor is not involved in EtOH consumption behaviour. Ro 61-8048 did not alter plasma acetaldehyde concentration, but prevented EtOH-induced dopamine release in the nucleus accumbens shell. These results point to a critical involvement of the reward circuitry in the reduction of EtOH consumption induced by KYN and KYNA increments. PNU-120596 (3 mg/kg, i.p.), a positive allosteric modulator of α7-nicotinic acetylcholine receptors, partially prevented the Ro 61-8048-induced decrease in EtOH consumption. Overall, our results highlight the usefulness of manipulating the KYN pathway as a pharmacological tool for modifying EtOH consumption and point to a possible modulator of alcohol drinking behaviour.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) produces edema due to BBB disruption induced by MMP-9 activation in rat hippocampus.

The recreational drug of abuse, 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier (BBB) integrity in rats through an early P2X7 receptor-mediated event which induces MMP-9 activity. Increased BBB permeability often causes plasma proteins and water to access cerebral tissue leading to vasogenic edema formation. The current study was performed to examine the effect of a single neurotoxic dose of MDMA (12.5 mg/kg, i.p.) on in vivo edema development associated with changes in the expression of the perivascular astrocytic water channel, AQP4, as well as in the expression of the tight-junction (TJ) protein, claudin-5 and Evans Blue dye extravasation in the hippocampus of adult male Dark Agouti rats. We also evaluated the ability of the MMP-9 inhibitor, SB-3CT (25 mg/kg, i.p.), to prevent these changes in order to validate the involvement of MMP-9 activation in MDMA-induced BBB disruption. The results show that MDMA produces edema of short duration temporally associated with changes in AQP4 expression and a reduction in claudin-5 expression, changes which are prevented by SB-3CT. In addition, MDMA induces a short-term increase in both tPA activity and expression, a serine-protease which is involved in BBB disruption and upregulation of MMP-9 expression. In conclusion, this study provides evidence enough to conclude that MDMA induces edema of short duration due to BBB disruption mediated by MMP-9 activation.

Disruption of blood-brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge-like drinking model.

Inflammatory cytokines and reactive oxygen species are reported to be involved in blood-brain barrier (BBB) disruption. Because there is evidence that ethanol (EtOH) induces release of free radicals, cytokines and inflammatory mediators we examined BBB integrity and matrix metalloproteinase (MMP) activity in postmortem human alcoholic brain and investigated the role of TLR4 signaling in BBB permeability in TLR4-knockout mice under a binge-like EtOH drinking protocol. Immunohistochemical studies showed reduced immunoreactivity of the basal lamina protein, collagen-IV and of the tight junction protein, claudin-5 in dorsolateral prefrontal cortex of alcoholics. There was also increased MMP-9 activity and expression of phosphorylated ERK1/2 and p-38. Greater number of CD45+ IR cells were observed associated with an enhanced neuroinflammatory response reflected by increased GFAP and Iba-1 immunostaining. To further explore effects of high EtOH consumption on BBB integrity we studied TLR4-knockout mice exposed to the drinking in the dark paradigm. Repetitive EtOH exposure in wild-type mice decreased hippocampal expression of laminin and collagen-IV and increased IgG immunoreactivity, indicating IgG extravasation. Western blot analysis also revealed increased MyD88 and p-ERK1/2 levels. None of these changes was observed in TLR4-knockout mice. Collectively, these findings indicate that chronic EtOH increases degradation of tight junctions and extracellular matrix in postmortem human brain and induces a neuroinflammatory response associated with activation of ERK1/2 and p-38 and greater MMP-9 activity. The EtOH-induced effects on BBB impairment are not evident in the hippocampus of TLR4-knockout mice, suggesting the involvement of TLR4 signaling in the underlying mechanism leading to BBB disruption in mice.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier integrity through a mechanism involving P2X7 receptors.

The recreational drug 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') produces a neuro-inflammatory response in rats characterized by an increase in microglial activation and IL-1ß levels. The integrity of the blood-brain barrier (BBB) is important in preserving the homeostasis of the brain and has been shown to be affected by neuro-inflammatory processes. We aimed to study the effect of a single dose of MDMA on the activity of metalloproteinases (MMPs), expression of extracellular matrix proteins, BBB leakage and the role of the ionotropic purinergic receptor P2X7 (P2X7R) in the changes induced by the drug. Adult male Dark Agouti rats were treated with MDMA (10 mg/kg, i.p.) and killed at several time-points in order to evaluate MMP-9 and MMP-3 activity in the hippocampus and laminin and collagen-IV expression and IgG extravasation in the dentate gyrus. Microglial activation, P2X7R expression and localization were also determined in the dentate gyrus. Separate groups were treated with MDMA and the P2X7R antagonists Brilliant Blue G (BBG; 50 mg/kg, i.p.) or A-438079 (30 mg/kg, i.p.). MDMA increased MMP-3 and MMP-9 activity, reduced laminin and collagen-IV expression and increased IgG immunoreactivity. In addition, MDMA increased microglial activation and P2X7R immunoreactivity in these cells. BBG suppressed the increase in MMP-9 and MMP-3 activity, prevented basal lamina degradation and IgG extravasation into the brain parenchyma. A-438079 also prevented the MDMA-induced reduction in laminin and collagen-IV immunoreactivity. These results indicate that MDMA alters BBB permeability through an early P2X7R-mediated event, which in turn leads to enhancement of MMP-9 and MMP-3 activity and degradation of extracellular matrix.

The JNK inhibitor, SP600125, potentiates the glial response and cell death induced by methamphetamine in the mouse striatum.

This study investigates the effect of the selective Jun NH2-terminal kinase 1/2 (JNK1/2) inhibitor, (SP600125) on the striatal dopamine nerve terminal loss and on the increased interleukin-15 (IL-15) expression and glial response induced by methamphetamine (METH). Mice were given repeated low doses of METH (4 mg/kg, i.p., three times separated by 3 h) and killed 24 h or 7 d after the last dose. SP600125 (30 mg/kg, i.p) was administered 30 min before the last METH injection. Results indicate that METH produced dopaminergic axonal neurotoxicity reflected as a marked decrease in the striatal density of tyrosine hydroxylase-immunoreactive (TH-ir) fibres and dopamine transporter-immunoreactivity (DAT-ir) 24 h after dosing. These effects were not modified by SP600125. This compound also failed to prevent the long-term loss of dopamine levels and DAT observed 7 d following METH injection. Nevertheless, SP600125 potentiated METH-induced striatal cell loss reflected by an increase in Fluoro-Jade immunostaining, cleaved capase-3 immunoreactivity and the number of terminal deoxyncleotidyl transferase-mediated dUTP nick end labelling (TUNEL) positive cells. In line with a deleterious effect of JNK1/2 inhibition, SP600125 increased the astroglial and microglial response induced by METH and interfered with drug-induced IL-15 expression. Together these data indicate that, not only does SP600125 fail to protect against the dopaminergic damage induced by METH but also, in fact, it potentiates the glial response and the non-dopaminergic striatal cell loss caused by the drug.

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9.

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

A study on the effect of JNK inhibitor, SP600125, on the disruption of blood-brain barrier induced by methamphetamine.

Methamphetamine (METH) is a widely consumed drug with high abuse potential. Studies in animals have shown that the drug produces dopaminergic neurotoxicity following both single high-dose and repeated low-dose administration. In addition, METH produces an increase in matrix metalloproteinase expression and loss of BBB integrity. We have examined the effect of repeated low-dose METH on MMP-9/2 expression and activity and laminin expression and the role of MMPs and JNK 1/2 phosphorylation on the changes induced by the drug in BBB integrity. Mice were given METH (4 mg/kg, i.p., three times separated by 3 h) and killed at different times after the last dose. Striatal MMP-9/2 activity was determined by zymography and expression of MMPs, laminin and phosphorylated JNK 1/2 was determined by western blot. BBB integrity was determined by IgG immunoreactivity. SP600125 and BB-94 were used to inhibit JNK and MMPs respectively. METH increased striatal MMP-9 expression and activity, IgG immunoreactivity and p-JNK 1/2 expression and decreased laminin expression. Increased IgG immunoreactivity colocalized with areas of greater MMP-9 activity. JNK inhibition prevented METH-induced changes in MMP-9 activity, laminin degradation and BBB leakage. BB-94 also prevented laminin degradation and BBB leakage. The decrease in BBB integrity induced by METH is mediated by the JNK pathway which activates MMP-9 causing degradation of laminin and BBB leakage.

Functional interplay between tetraspanins and proteases.

Several recent publications have described examples of physical and functional interations between tetraspanins and specific membrane proteases belonging to the TM-MMP and α-(ADAMs) and γ-secretases families. Collectively, these examples constitute an emerging body of evidence supporting the notion that tetraspanin-enriched microdomains (TEMs) represent functional platforms for the regulation of key cellular processes including the release of surface protein ectodomains ("shedding"), regulated intramembrane proteolysis ("RIPing") and matrix degradation and assembly. These cellular processes in turn play a crucial role in an array of physiological and pathological phenomena. Thus, TEMs may represent new therapeutical targets that may simultaneously affect the proteolytic activity of different enzymes and their substrates. Agonistic or antagonistic antibodies and blocking soluble peptides corresponding to tetraspanin functional regions may offer new opportunities in the treatment of pathologies such as chronic inflammation, cancer, or Alzheimer's disease. In this review article, we will discuss all these aspects of functional regulation of protease activities by tetraspanins.

The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9.

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-α and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-α and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17.

Evidence that MDMA ('ecstasy') increases cannabinoid CB2 receptor expression in microglial cells: role in the neuroinflammatory response in rat brain.

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') produces selective long-lasting serotonergic neurotoxicity in rats. The drug also produces acute hyperthermia which modulates the severity of the neurotoxic response. In addition, MDMA produces signs of neuroinflammation reflected as microglial activation and an increase in the release of interleukin-1beta, the latter of which appears to be a consequence of the hyperthermic response and to be implicated in the neurotoxicity induced by the drug. Over-expression of the cannabinoid CB2 receptor in microglia during non-immune and immune pathological conditions is thought to be aimed at controlling the production of neurotoxic factors such as proinflammatory cytokines. Our objective was to study the pattern of CB2 receptor expression following MDMA and to examine the effect of JWH-015 (a CB2 agonist) on the MDMA-induced neuroinflammatory response as well as 5-hydroxytryptamine (5-HT) neurotoxicity. Adult Dark Agouti rats were given MDMA (12.5 mg/kg, i.p.) and killed 3 h or 24 h later for the determination of CB2 receptor expression. JWH-015 was given 48 h, 24 h and 0.5 h before MDMA and 1 h and/or 6 h later and animals were killed for the determination of microglial activation (3 h and 24 h) and 5-HT neurotoxicity (7 days). MDMA increased CB2 receptor expression shortly after administration and these receptors were found in microglia. JWH-015 decreased MDMA-induced microglial activation and interleukin-1beta release and slightly decreased MDMA-induced 5-HT neurotoxicity. In conclusion, CB2 receptor activation reduces the neuroinflammatory response following MDMA and provides partial neuroprotection against the drug.

Administration of neurotoxic doses of MDMA reduces sensitivity to ethanol and increases GAT-1 immunoreactivity in mice striatum.

RATIONALE: Mice with reduced dopamine activity following neurotoxic doses of 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') consume more ethanol (EtOH) and show greater preference for EtOH. In keeping with human studies and other animal models where alcohol consumption and preference are also high, MDMA treatment will reduce sensitivity to certain physiological effects of EtOH. OBJECTIVE: We have examined the sensitivity to the acute effects of EtOH in MDMA-lesioned mice and the effects of EtOH on striatal gamma-aminobutyric acid (GABA) accumulation and expression of GABA subtype-1 transporter (GAT-1). METHODS: C57BL/6J mice were injected with neurotoxic MDMA (30 mg/kg, three times, every 3 h, i.p.). Seven days later, mice were given EtOH (3 g/kg, i.p.) to determine the loss of righting response and the development of rapid tolerance to the hypothermic effect of EtOH. The effect of EtOH on the striatal accumulation of GABA after inhibiting GABA transaminase and on GAT-1 immunoreactivity was also determined. RESULTS: Mice pre-treated with a neurotoxic dose of MDMA were less sensitive to the sedative-hypnotic effect of acute EtOH and exhibited alterations in the development of rapid tolerance to the hypothermic effect of EtOH. These animals showed an increase in striatal GAT-1 immunoreactivity. EtOH reduced GABA concentration in the striatum of non-lesioned mice, an effect not observed in MDMA-lesioned mice. CONCLUSION: These findings indicate that mice with a MDMA-induced dopaminergic lesion show increased expression of striatal GAT-1 that may contribute to the lower sensitivity to EtOH-induced sedative effects and the resistance to the development of rapid tolerance to hypothermia produced by EtOH.

Immunological synapse formation inhibits, via NF-kappaB and FOXO1, the apoptosis of dendritic cells.

The immunological synapse (IS) is a cell-cell junction formed between CD4(+) T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-kappaB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.

The tetraspanin CD9 inhibits the proliferation and tumorigenicity of human colon carcinoma cells.

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well-established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin-dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti-CD9-specific antibodies resulted in (i) increased beta1 integrin-mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF-alpha, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF-alpha, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.

Endothelial tetraspanin microdomains regulate leukocyte firm adhesion during extravasation.

Tetraspanins associate with several transmembrane proteins forming microdomains involved in intercellular adhesion and migration. Here, we show that endothelial tetraspanins relocalize to the contact site with transmigrating leukocytes and associate laterally with both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Alteration of endothelial tetraspanin microdomains by CD9-large extracellular loop (LEL)-glutathione S-transferase (GST) peptides or CD9/CD151 siRNA oligonucleotides interfered with ICAM-1 and VCAM-1 function, preventing lymphocyte transendothelial migration and increasing lymphocyte detachment under shear flow. Heterotypic intercellular adhesion mediated by VCAM-1 or ICAM-1 was augmented when expressed exogenously in the appropriate tetraspanin environment. Therefore, tetraspanin microdomains have a crucial role in the proper adhesive function of ICAM-1 and VCAM-1 during leukocyte adhesion and transendothelial migration.

A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin.

Tetraspanins associate on the cell membrane with several transmembrane proteins, including members of the integrin superfamily. The tetraspanin CD9 has been implicated in cell motility, metastasis, and sperm-egg fusion. In this study we characterize the first CD9 conformation-dependent epitope (detected by monoclonal antibody (mAb) PAINS-13) whose expression depends on changes in the activation state of associated beta(1) integrins. MAb PAINS-13 precipitates CD9 under conditions that preserve the association of this tetraspanin with integrins, but not under conditions that disrupt these interactions. Induction of activation of beta(1) integrins by temperature, divalent cation Mn(2+), or mAb TS2/16 correlated with enhanced expression of the PAINS-13 epitope on a variety of cells. Through the use of different K562 myeloid leukemia transfectant cells expressing specific members of the beta(1) integrin subfamily we show that the expression of the PAINS-13 epitope depends on CD9 association with alpha(6)beta(1) integrin. The mAb PAINS-13 reactivity has been mapped to the CD9 region comprising residues 112-154 in the NH(2) half of the large extracellular loop. Also, we show that the CD9 conformation recognized by mAb PAINS-13 is functionally relevant in beta(1) integrin-mediated cellular processes including wound healing migration, tubular morphogenesis, cell adhesion and spreading and in signal transduction involving phosphatidylinositol 3-kinase activation.

Predisposición genética para desarrollar diabetes mellitus insulino-dependiente: estudio en población de Santiago y Temuco, Chile / Genetic predisposition for developing insulin-dependent diabetes mellitus: study in population from Santiago and Temuco, Chile

Insulin dependent diabetes mellitus (IDDM) is strongly associated with particular HLA-DQ alpha/beta markers in white population. The heterodimers confirmation composed of a DQ alpha chain with an arginine at residue 52 (Arg52) combined to a DQ beta chain lacking an aspartic acid at residue 57 (non asp57) increase markedly the risk to develop IDDM. To confirm this association, 63 IDDM patients from Santiago de Chile registry, 20 IDDM patients from Temuco registry and 74 unrelated helathy non diabetic control subjects were studied. With polymerase chain reaction (PCR) and sequence specific oligonucleotide probes the individuals were typed for their HLA-DQA1 and DQB1 alleles, their DQA1/DQB1 genotype and heterodimers conformation were compared. In diabetic population both markers Arg52 homocygote and non Asp57 homocygote were increased regard to control subjects (R/R: 0.76 and 0.85 vs 0.33; ND/ND: 0.78 and 0.75 vs 0.50, p<0.05). A high relative risk (RR) was determined for both homocygote markers in IDDM groups.compared. Arg52 DQ alpha (R)/non Asp57 DQ beta (ND) heterodimers were strongly associated with susceptibility to IDDM. A high RR was observed in patients with four susceptibility DQ heterodimers (RR1: 13.7 in IDDM-Santiago and RR2: 18.6 in IDDM-Temuco, p<0.00003). The HLA-DQ alpha/beta markers and their risk heterodimers are increased in our diabetic population and could be considered as susceptibility markers to develop IDDM

HLA y diabetes mellitus insulino-dependiente / HLA and insulin-dependent diabetes mellitus

The propensity of an individual to develop type I (insulin dependent) diabetes mellitus is directly related to specipic HLA clase II proteins, specially those from DR and DQ regions. Genetic susceptibility to insulin dependent diabetes arises from a preestablished conformation of alpha and ß chains of DQ and ß chain of DR. Since the classic demonstration by McDevitt and colleagues that DQ ß chain aspartate at position 57 was protective against the development of the disease, many populations have been surveyed to study the association between the incidence Type I diabetes and determined frequencies of DR and DQ haplotypes. The assocation between these markers and susceptibility to Type I diabetes is well established in caucasians at the present time. However, little information is available for Latin American populations, that share a mixture of european, african and native genes. Our group is studying genetic markers of three Latin American populations (Argentina, Perú and Chile) and their possible association to the different incidence of Type I diabetes mellitus in each country