RESUMO
BACKGROUND: Obesity-induced hypogonadism (OIH) is a prevalent, but often neglected condition in men, which aggravates the metabolic complications of overweight. While hypothalamic suppression of Kiss1-encoded kisspeptin has been suggested to contribute to OIH, the molecular mechanisms for such repression in obesity, and the therapeutic implications thereof, remain unknown. METHODS: A combination of bioinformatic, expression and functional analyses was implemented, assessing the role of the evolutionary-conserved miRNAs, miR-137 and miR-325, in mediating obesity-induced suppression of hypothalamic kisspeptin, as putative mechanism of central hypogonadism and metabolic comorbidities. The implications of such miR-137/325-kisspeptin interplay for therapeutic intervention in obesity were also explored using preclinical OIH models. RESULTS: MiR-137/325 repressed human KISS1 3'-UTR in-vitro and inhibited hypothalamic kisspeptin content in male rats, while miR-137/325 expression was up-regulated, and Kiss1/kisspeptin decreased, in the medio-basal hypothalamus of obese rats. Selective over-expression of miR-137 in Kiss1 neurons reduced Kiss1/ kisspeptin and partially replicated reproductive and metabolic alterations of OIH in lean mice. Conversely, interference of the repressive actions of miR-137/325 selectively on Kiss1 3'-UTR in vivo, using target-site blockers (TSB), enhanced kisspeptin content and reversed central hypogonadism in obese rats, together with improvement of glucose intolerance, insulin resistance and cardiovascular and inflammatory markers, despite persistent exposure to obesogenic diet. Reversal of OIH by TSB miR-137/325 was more effective than chronic kisspeptin or testosterone treatments in obese rats. CONCLUSIONS: Our data disclose that the miR-137/325-Kisspeptin repressive interaction is a major player in the pathogenesis of obesity-induced hypogonadism and a putative druggable target for improved management of this condition and its metabolic comorbidities in men suffering obesity. SIGNIFICANCE STATEMENT: Up to half of the men suffering obesity display also central hypogonadism, an often neglected complication of overweight that can aggravate the clinical course of obesity and its complications. The mechanisms for such obesity-induced hypogonadism remain poorly defined. We show here that the evolutionary conserved miR137/miR325 tandem centrally mediates obesity-induced hypogonadism via repression of the reproductive-stimulatory signal, kisspeptin; this may represent an amenable druggable target for improved management of hypogonadism and other metabolic complications of obesity.
Assuntos
Hipogonadismo , Hipotálamo , Kisspeptinas , MicroRNAs , Obesidade , MicroRNAs/genética , MicroRNAs/metabolismo , Hipogonadismo/genética , Hipogonadismo/metabolismo , Hipogonadismo/complicações , Kisspeptinas/genética , Kisspeptinas/metabolismo , Animais , Obesidade/metabolismo , Obesidade/complicações , Obesidade/genética , Masculino , Ratos , Hipotálamo/metabolismo , Humanos , Camundongos , Ratos Wistar , ComorbidadeRESUMO
Macrophages (MΦ) infected with human immunodeficiency virus (HIV)-1 or activated by its envelope protein gp120 exert neurotoxicity. We found previously that signaling via p38 mitogen-activated protein kinase (p38 MAPK) is essential to the neurotoxicity of HIVgp120-stimulated MΦ. However, the associated downstream pathways remained elusive. Here we show that cysteinyl-leukotrienes (CysLT) released by HIV-infected or HIVgp120 stimulated MΦ downstream of p38 MAPK critically contribute to neurotoxicity. SiRNA-mediated or pharmacological inhibition of p38 MAPK deprives MΦ of CysLT synthase (LTC4S) and, pharmacological inhibition of the cysteinyl-leukotriene receptor 1 (CYSLTR1) protects cerebrocortical neurons against toxicity of both gp120-stimulated and HIV-infected MΦ. Components of the CysLT pathway are differentially regulated in brains of HIV-infected individuals and a transgenic mouse model of NeuroHIV (HIVgp120tg). Moreover, genetic ablation of LTC4S or CysLTR1 prevents neuronal damage and impairment of spatial memory in HIVgp120tg mice. Altogether, our findings suggest a novel critical role for cysteinyl-leukotrienes in HIV-associated brain injury.
Assuntos
Cisteína , Infecções por HIV , HIV-1 , Camundongos , Humanos , Animais , HIV-1/metabolismo , Macrófagos/metabolismo , Leucotrienos/metabolismo , Neurônios/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Camundongos Transgênicos , Infecções por HIV/metabolismoRESUMO
This study evaluated the in vivo therapeutic efficacy of oncolytic serotype 5 adenovirus TAV255 in CAR-deficient tumors. In vitro experiments were performed with cell lines that expressed different levels of CAR (HEK293, A549, CT26, 4T1, and MCF-7). Low CAR cells, such as CT26, were poorly transduced by Ad in vitro unless the adenovirus was encapsulated in liposomes. However, the CT26 tumor in an immune-competent mouse model responded to the unencapsulated TAV255; 33% of the tumors were induced into complete remission, and mice with complete remission rejected the rechallenge with cancer cell injection. Encapsulation of TAV255 improves its therapeutic efficacy by transducing more CT26 cells, as expected from in vitro results. In a bilateral tumor model, nonencapsulated TAV255 reduced the growth rate of the locally treated tumors but had no effect on the growth rate of the distant tumor site. Conversely, encapsulated TAV255-infected CT26 induced a delayed growth rate of both the primary injected tumor and the distant tumor, consistent with a robust immune response. In vivo, intratumorally injected unencapsulated adenoviruses infect CAR-negative cells with only limited efficiency. However, unencapsulated adenoviruses robustly inhibit the growth of CAR-deficient tumors, an effect that constitutes an 'in situ vaccination' by stimulating cytotoxic T cells.
RESUMO
Adenovirus (Ad) is a widely studied viral vector for cancer therapy as it can be engineered to cause selective lysis of cancer cells. However, Ad delivery is limited in treating cancers that do not have coxsackievirus and adenovirus receptors (CAR). To overcome this challenge, Ad-encapsulated liposomes were developed that enhance the delivery of Ads and increase therapeutic efficacy. Cationic empty liposomes were manufactured first, to which an anionic Ad were added, which resulted in encapsulated Ad liposomes through charge interaction. Optimization of the liposome formula was carried out with series of formulation variables experiments using an extrusion process, which is ideal for laboratory-scale small batches. Later, the optimized formulation was manufactured with a homogenization technique-A high shear rotor-stator blending, that is ideal for large-scale manufacturing and is in compliance with Good Manufacturing Practices (GMP). Comparative in vitro transduction, physicochemical characterization, long-term storage stability at different temperature conditions, and in vivo animal studies were performed. Ad encapsulated liposomes transduced CAR deficient cells 100-fold more efficiently than the unencapsulated Ad (p ≤ 0.0001) in vitro, and 4-fold higher in tumors injected in nude mice in vivo. Both extrusion and homogenization performed similarly-with equivalent in vitro and in vivo transduction efficiencies, physicochemical characterization, and long-term storage stability. Thus, two Ad encapsulated liposomes preparation methods used herein, i.e., extrusion vs. homogenization were equivalent in terms of enhanced Ad performance and long-term storage stability; this will, hopefully, facilitate translation to the clinic.
RESUMO
Adenovirus (Ad)-based vectors have shown considerable promise for gene therapy. However, Ad requires the coxsackievirus and adenovirus receptor (CAR) to enter cells efficiently and low CAR expression is found in many human cancers, which hinder adenoviral gene therapies. Here, cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-folate liposomes (Df) encapsulating replication-deficient Ad were synthesized, which showed improved transfection efficiency in various CAR-deficient cell lines, including epithelial and hematopoietic cell types. When encapsulating replication-competent oncolytic Ad (TAV255) in DOTAP-folate liposome (TAV255-Df), the adenoviral structural protein, hexon, was readily produced in CAR-deficient cells, and the tumor cell killing ability was 5× higher than that of the non-encapsulated Ad. In CAR-deficient CT26 colon carcinoma murine models, replication-competent TAV255-Df treatment of subcutaneous tumors by intratumoral injection resulted in 67% full tumor remission, prolonged survival, and anti-cancer immunity when mice were rechallenged with cancer cells with no further treatment. The preclinical data shows that DOTAP-folate liposomes could significantly enhance the transfection efficiency of Ad in CAR-deficient cells and, therefore, could be a feasible strategy for applications in cancer treatment.
Assuntos
Adenoviridae , Neoplasias , Camundongos , Humanos , Animais , Adenoviridae/genética , Adenoviridae/metabolismo , Lipossomos/metabolismo , Propano , Ácido Fólico/metabolismoRESUMO
In this work, we present the synthesis of a novel Zn-Salphen complex containing an allyl group, which was used as building block in the further preparation of a new family of functional terpolymers. These polymers were obtained through radical co-polymerization with methyl metacrylate (MMA) and n-butyl acrylate (nBuA) in different ratios. The supramolecular recognition behavior of each polymer was evaluated via potentiometric measurements against selected anions in aqueous media. Interestingly, this proof of concept study shows that these systems were selective against only fluoride (F-) or both, fluoride and acetate (OAc-), by tailoring the relative content of Zn-Salphen monomer, thus making them a promising starting point for modular design of chemical sensors through straightforward synthetic approaches.
Assuntos
Resinas Acrílicas/química , Fenilenodiaminas/química , Polímeros/química , Zinco/química , Estrutura Molecular , Polimerização , Polímeros/síntese química , Polimetil Metacrilato/químicaRESUMO
The neuropathogenesis of HIV associated neurocognitive disorders (HAND) involves disruption of mitochondrial homeostasis and increased neuroinflammation. However, it is unknown if alterations in mitochondrial biogenesis in the brain underlie the neuropathogenesis of HAND. In this study, neuropathological and molecular analyses of mitochondrial biogenesis and inflammatory pathways were performed in brain specimens from a well-characterized cohort of HIV+ cases that were on antiretroviral regimens. In vitro investigations using primary human astroglia and neurons were used to probe the underlying mechanisms of mitochondrial alterations. In frontal cortices from HAND brains compared to cognitive normal brains, total levels of transcription factors that regulate mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were decreased. Immunohistochemical analyses revealed that TFAM was decreased in neurons and increased in astroglia. These changes were accompanied by decreased total mitochondrial DNA per cell and increased levels of messenger RNA for the proinflammatory cytokine interleukin (IL)-1ß. To determine how IL-1ß affects astroglial bioenergetic processes and mitochondrial activity, human astroglial cultures were exposed to recombinant IL-1ß. IL-1ß induced mitochondrial activity within 30â¯min of treatment, altered mitochondrial related gene expression, altered mitochondrial morphology, enhanced adenoside triphosphate (ATP) utilization and increased the expression of inflammatory cytokines. WIN55,212-2 (WIN), an aminoalkylindole derivative and cannabinoid receptor agonist, blocked IL-1ß-induced bioenergetic fluctuations and inflammatory gene expression in astroglia independent of cannabinoid receptor (CB)1 and peroxisome proliferator-activated receptor (PPAR) γ. A PPARα antagonist reversed the anti-inflammatory effects of WIN in human astroglia. These results show that mitochondrial biogenesis is differentially regulated in neurons and astroglia in HAND brains and that targeting astroglial bioenergetic processes may be a strategy to modulate neuroinflammation.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Astrócitos/metabolismo , Encéfalo/metabolismo , Soropositividade para HIV/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Fármacos Anti-HIV/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Soropositividade para HIV/tratamento farmacológico , Soropositividade para HIV/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
HIV-1 infection causes injury to the central nervous system (CNS) and is often associated with neurocognitive disorders. One model for brain damage seen in AIDS patients is the transgenic (tg) mouse expressing a soluble envelope protein gp120 of HIV-1 LAV in the brain in astrocytes under the control of the promoter of glial fibrillary acidic protein. These GFAP-gp120tg mice manifest several key neuropathological features observed in AIDS brains, such as decreased synaptic and dendritic density, increased numbers of activated microglia, and pronounced astrocytosis. Several recent studies show that brains of GFAP-gp120tg mice and neurocognitively impaired HIV patients share also a significant number of differentially regulated genes, activation of innate immunity and other cellular signaling pathways, disturbed neurogenesis, and learning deficits. These findings support the continued relevance of the GFAP-gp120tg mouse as a useful model to investigate neurodegenerative mechanisms and develop therapeutic strategies to mitigate the consequences associated with HIV infection of the CNS, neuroAIDS, and HAND.
Assuntos
Complexo AIDS Demência/genética , Disfunção Cognitiva/genética , Modelos Animais de Doenças , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Complexo AIDS Demência/imunologia , Complexo AIDS Demência/fisiopatologia , Animais , Astrócitos/imunologia , Astrócitos/patologia , Encéfalo/imunologia , Encéfalo/patologia , Disfunção Cognitiva/imunologia , Disfunção Cognitiva/fisiopatologia , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/química , Imunidade Inata , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Sinapses/imunologia , Sinapses/patologiaRESUMO
Infection with human immunodeficiency virus-1 (HIV-1) causes brain injury. Type I interferons (IFNα/ß) are critical mediators of any anti-viral immune response and IFNß has been implicated in the temporary control of lentiviral infection in the brain. Here we show that transgenic mice expressing HIV-1 envelope glycoprotein 120 in their central nervous system (HIVgp120tg) mount a transient IFNß response and provide evidence that IFNß confers neuronal protection against HIVgp120 toxicity. In cerebrocortical cell cultures, neuroprotection by IFNß against gp120 toxicity is dependent on IFNα receptor 1 (IFNAR1) and the ß-chemokine CCL4, as IFNAR1 deficiency and neutralizing antibodies against CCL4, respectively, abolish the neuroprotective effects. We find in vivo that IFNß mRNA is significantly increased in HIVgp120tg brains at 1.5, but not 3 or 6 months of age. However, a four-week intranasal IFNß treatment of HIVgp120tg mice starting at 3.5 months of age increases expression of CCL4 and concomitantly protects neuronal dendrites and pre-synaptic terminals in cortex and hippocampus from gp120-induced damage. Moreover, in vivo and in vitro data suggests astrocytes are a major source of IFNß-induced CCL4. Altogether, our results suggest exogenous IFNß as a neuroprotective factor that has potential to ameliorate in vivo HIVgp120-induced brain injury.
Assuntos
Lesões Encefálicas/prevenção & controle , Modelos Animais de Doenças , Interferon beta/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Lesões Encefálicas/virologia , Células Cultivadas , Quimiocina CCL4/genética , Quimiocina CCL4/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Humanos , Interferon beta/genética , Interferon beta/metabolismo , Camundongos Transgênicos , Neurônios/patologia , Neurônios/virologia , Ratos , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismoRESUMO
Multiple mechanisms appear to contribute to neuronal stress and injury underlying HIV-associated neurocognitive disorders (HAND), which occur despite the successful introduction of combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can itself be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine (METH), seems to compromise antiretroviral therapy and aggravate HAND. However, the combined effect of virus and recreational and therapeutic drugs on the brain is still incompletely understood. However, several lines of evidence suggest a shared critical role of oxidative stress, compromised neuronal energy homeostasis and autophagy in promotion and prevention of neuronal dysfunction associated with HIV-1 infection, cART and psychostimulant use. In this review, we present a synopsis of recent work related to neuronal stress and injury induced by HIV infection, antiretrovirals (ARVs) and the highly addictive psychostimulant METH.
RESUMO
BACKGROUND: The chemokine receptor CXCR4 (CD184) and its natural ligand CXCL12 contribute to many physiological processes, including decisions about cell death and survival in the central nervous system. In addition, CXCR4 is a co-receptor for human immunodeficiency virus (HIV)-1 and mediates the neurotoxicity of the viral envelope protein gp120. However, we previously observed that CXCL12 also causes toxicity in cerebrocortical neurons but the cellular mechanism remained incompletely defined. METHODS: Primary neuronal-glial cerebrocortical cell cultures from rat were exposed to a neurotoxicity-inducing CXCL12 concentration for different times and the activity of the stress-associated mitogen-activated protein kinase p38 (p38 MAPK) was assessed using an in vitro kinase assay. Neurotoxicity of CXCL12 and cellular localization of p38 MAPK was analyzed by immunofluorescence microscopy. Pharmacological inhibition of NMDA-type glutamate receptor-gated ion channels (NMDAR) of L-type Ca2+ channels was employed during 12- and 24-h exposure to neurotoxic amounts of CXCL12 to study the effects on active p38 MAPK and neuronal survival by Western blotting and microscopy, respectively. Neurotoxicity of CXCL12 was also assessed during pharmacological inhibition of p38 MAPK. RESULTS: Here, we show that a neurotoxic amount of CXCL12 triggers a significant increase of endogenous p38 MAPK activity in cerebrocortical cells. Immunofluorescence and Western blotting experiments with mixed neuronal-glial and neuron-depleted glial cerebrocortical cells revealed that the majority of active/phosphorylated p38 MAPK was located in neurons. Blockade of NMDAR-gated ion channels or L-type Ca2+ channels both abrogated an increase of active p38 MAPK and toxicity of CXCL12 in cerebrocortical neurons. Inhibition of L-type Ca2+ channels with nimodipine kept the active kinase at levels not significantly different from baseline while blocking NMDAR with MK-801 strongly reduced phosphorylated p38 MAPK below baseline. Finally, we confirmed that directly blocking p38 MAPK also abrogated neurotoxicity of CXCL12. CONCLUSIONS: Our findings link CXCL12-induced neuronal death to the regulation of NMDAR-gated ion channels and L-type Ca2+ channels upstream of p38 MAPK activation.
RESUMO
INTRODUCTION: Pharmacotherapy for the management of obesity is primarily aimed at weight loss, weight loss maintenance and risk reduction (reduction in body fat, risk factors for cardiovascular disease and the incidence of diabetes mellitus). Among drugs that have been evaluated for weight loss include antidepressants (fluoxetine) and antiepileptic (topiramate). MATERIAL AND METHODS: We analyzed eating behavior and weight loss in a sample of morbid obesity patients before bariatric surgery. The patients suffering eating disturbances symptoms were grouped into three groups: one group received 40 mg of flouxetine/day (Group A); another group received topiramate 200 mg/day (Group B); and the third group of patients were treated with fluoxetine 40 mg and 200 mg of topiramate/day (Group C). RESULTS: Patients treated with fluoxetine plus topiramate lost more weight at 3 and 6 months before surgery. CONCLUSIONS: The use of the psychopharmaceutical drug (fluoxetine and topiramate) in morbid obese patients with eating disorders could represent a new approach to the management of eating behavior before bariatric surgery.
Assuntos
Fármacos Antiobesidade/administração & dosagem , Cirurgia Bariátrica , Comportamento Alimentar/efeitos dos fármacos , Fluoxetina/administração & dosagem , Frutose/análogos & derivados , Obesidade Mórbida/tratamento farmacológico , Obesidade Mórbida/cirurgia , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Adulto , Quimioterapia Combinada , Feminino , Frutose/administração & dosagem , Humanos , Masculino , Cuidados Pré-Operatórios , TopiramatoRESUMO
HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine.
Assuntos
Proteína gp120 do Envelope de HIV/farmacologia , Inibidores de Integrase de HIV/farmacologia , Metanfetamina/farmacologia , Neurônios/efeitos dos fármacos , Nevirapina/farmacologia , Saquinavir/farmacologia , Zidovudina/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Autofagia/efeitos dos fármacos , Técnicas de Cultura de Células , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Combinação de Medicamentos , Embrião de Mamíferos , Homeostase/efeitos dos fármacos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Sirolimo/farmacologiaRESUMO
This study aims to identify the profile of immunohistochemical (IHC) parameters, copy number aberrations (CNAs) and epigenetic alterations [promoter methylation (PM) and miR expression] related to hereditary (H) and triple negative (TN) breast cancer (BC). This profile could be of relevance for guiding tumor response to treatment with targeting therapy. The study comprises 278 formalin fixed paraffin-embedded BCs divided into two groups: H group, including 88 hereditary BC (HBC) and 190 non hereditary (NHBC), and TN group, containing 79 TNBC and 187 non TNBC (NTNBC). We assessed IHC parameters (Ki67, ER, PR, HER2, CK5/6, CK18 and Cadherin-E), CNA of 20 BC related genes, and PM of 24 tumor suppressor genes employing MLPA/MS-MLPA (MRC Holland, Amsterdam). MiR-4417, miR-423-3p, miR-590-5p and miR-187-3p expression was assessed by quantitative RT-PCR (Applied Biosystems). Binary logistic regression was applied to select the parameters that better differentiate the HBC or TN groups. For HBC we found that, ER expression, ERBB2 CNA and PM in RASSF1 and TIMP3 were associated with NHBC whereas; MYC and AURKA CNA were linked to HBC. For TNBC, we found that CDC6 CNA, GSTP1 and RASSF1 PM and miR-423-3p hyperexpression were characteristic of NTNBC, while MYC aberrations, BRCA1 hypermethylation and miR-590-5p and miR-4417 hyperexpression were more indicative of TNBC. The selected markers allow establishing BC subtypes, which are characterized by showing similar etiopathogenetic mechanisms, some of them being molecular targets for known drugs or possible molecular targets. These results could be the basis to implement a personalized therapy.
RESUMO
INTRODUCTION. The challenge of early detection can be tackled from an evolutionary perspective. Early intervention treatments have shown themselves to be effective provided that they are applied systematically as part of the strategic planning of the treatment. AIMS. The aim of this study is to provide an updated review in response to the criticism targeted towards early detection and to offer some considerations on the intervention strategy. Our research is based on a review of the early care techniques that are commonly used within the field of autism and it intends to reflect the most significant aspects that can be deduced from the experiments and studies carried out to date. CONCLUSIONS. From the findings of the review it can be concluded that early detection may be more efficient if carried out within the framework of developmental surveillance, which also offers the opportunity to provide guidance on the child's development. Early care is an effective resource for attending to the needs of children with autism. Professionals have the duty to assess the work they do on available treatments with a reflexive, judicious attitude, taking into account the values and preferences of the families. Programmes must focus on the core symptoms and apply the active ingredients of the treatment.
TITLE: De la deteccion precoz a la atencion temprana: estrategias de intervencion a partir del cribado prospectivo.Introduccion. El reto de la deteccion precoz puede realizarse desde una perspectiva evolutiva. Los tratamientos de intervencion temprana han demostrado su eficacia siempre y cuando se apliquen sistematicamente en el marco de una planificacion estrategica del tratamiento. Objetivos. Proporcionar una revision actualizada para responder a las criticas a la deteccion precoz y aportar una reflexion sobre la estrategia de intervencion, basada en la revision de las tecnicas de atencion temprana usuales en el campo del autismo, reflejando los aspectos mas relevantes que se deducen de las experiencias y estudios llevados a cabo hasta el momento. Conclusiones. Tras la revision realizada se concluye que la deteccion precoz puede ser mas eficiente si se lleva a cabo en el marco de la vigilancia del desarrollo, que ademas ofrece la oportunidad de proporcionar orientacion sobre el desarrollo del menor. La atencion temprana constituye un recurso eficaz para atender las necesidades del menor con autismo. Los profesionales tienen la responsabilidad de evaluar el trabajo que hacen con una actitud reflexiva y critica sobre los tratamientos disponibles tomando en consideracion los valores y las preferencias de las familias. Los programas deben centrarse en los sintomas nucleares aplicando ingredientes activos del tratamiento.
Assuntos
Transtorno Autístico/diagnóstico , Transtorno Autístico/terapia , Criança , Diagnóstico Precoce , Intervenção Médica Precoce , HumanosRESUMO
This study investigates the relationship of promoter methylation in tumor suppressor genes with copy-number aberrations (CNA) and with tumor markers in breast cancer (BCs). The study includes 98 formalin fixed paraffin-embedded BCs in which promoter methylation of 24 tumour suppressor genes were assessed by Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA), CNA of 20 BC related genes by MLPA and ER, PR, HER2, CK5/6, CK18, EGFR, Cadherin-E, P53, Ki-67 and PARP expression by immunohistochemistry (IHC). Cluster analysis classed BCs in two groups according to promoter methylation percentage: the highly-methylated group (16 BCs), containing mostly hyper-methylated genes, and the sparsely-methylated group (82 BCs) with hypo-methylated genes. ATM, CDKN2A, VHL, CHFR and CDKN2B showed the greatest differences in the mean methylation percentage between these groups. We found no relationship of the IHC parameters or pathological features with methylation status, except for Catherin-E (p = 0.008). However the highly methylated BCs showed higher CNA proportion than the sparsely methylated BCs (p < 0.001, OR = 1.62; IC 95% [1.26, 2.07]). CDC6, MAPT, MED1, PRMD14 and AURKA showed the major differences in the CNA percentage between the two groups, exceeding the 22%. Methylation in RASSF1, CASP8, DAPK1 and GSTP1 conferred the highest probability of harboring CNA. Our results show a new link between promoter methylation and CNA giving support to the importance of methylation events to establish new BCs subtypes. Our findings may be also of relevance in personalized therapy assessment, which could benefit the hyper methylated BC patients group.
RESUMO
Methamphetamine (METH) abuse is frequent in individuals infected with human immunodeficiency virus type-1 (HIV-1) and is suspected to aggravate HIV-associated neurocognitive disorders (HAND). METH is a psychostimulant that compromises several neurotransmitter systems and HIV proteins trigger neuronal injury but the combined effects of viral infection and METH abuse are incompletely understood. In this study we treated transgenic mice expressing the HIV envelope protein gp120 in the brain (HIV-1 gp120tg) at 3-4 months of age with an escalating-dose, multiple-binge METH regimen. The long-term effects were analyzed after 6-7 months of drug abstinence employing behavioral tests and analysis of neuropathology, electrophysiology and gene expression. Behavioral testing showed that both HIV-1 gp120tg and WT animals treated with METH displayed impaired learning and memory. Neuropathological analysis revealed that METH similar to HIV-1 gp120 caused a significant loss of neuronal dendrites and pre-synaptic terminals in hippocampus and cerebral cortex of WT animals. Electrophysiological studies in hippocampal slices showed that METH exposed HIV-1 gp120tg animals displayed reduced post-tetanic potentiation, whereas both gp120 expression and METH lead to reduced long-term potentiation. A quantitative reverse transcription-polymerase chain reaction array showed that gp120 expression, METH and their combination each caused a significant dysregulation of specific components of GABAergic and glutamatergic neurotransmission systems, providing a possible mechanism for synaptic dysfunction and behavioral impairment. In conclusion, both HIV-1 gp120 and METH caused lasting behavioral impairment in association with neuropathology and altered gene expression. However, combined METH exposure and HIV-1 gp120 expression resulted in the most pronounced, long lasting pre- and post-synaptic alterations coinciding with impaired learning and memory.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Infecções por HIV/complicações , Metanfetamina/efeitos adversos , Transtornos Relacionados ao Uso de Substâncias/complicações , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/virologia , Estimulantes do Sistema Nervoso Central/efeitos adversos , Proteína gp120 do Envelope de HIV , HIV-1 , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/efeitos dos fármacosRESUMO
The innate immune system has been implicated in several neurodegenerative diseases, including HIV-1-associated dementia. In this study, we show that genetic ablation of CCR5 prevents microglial activation and neuronal damage in a transgenic model of HIV-associated brain injury induced by a CXCR4-using viral envelope gp120. The CCR5 knockout (KO) also rescues spatial learning and memory in gp120-transgenic mice. However, the CCR5KO does not abrogate astrocytosis, indicating it can occur independently from neuronal injury and behavioral impairment. To characterize further the neuroprotective effect of CCR5 deficiency we performed a genome-wide gene expression analysis of brains from HIVgp120tg mice expressing or lacking CCR5 and nontransgenic controls. A comparison with a human brain microarray study reveals that brains of HIVgp120tg mice and HIV patients with neurocognitive impairment share numerous differentially regulated genes. Furthermore, brains of CCR5 wild-type and CCR5KO gp120tg mice express markers of an innate immune response. One of the most significantly upregulated factors is the acute phase protein lipocalin-2 (LCN2). Using cerebrocortical cell cultures, we find that LCN2 is neurotoxic in a CCR5-dependent fashion, whereas inhibition of CCR5 alone is not sufficient to abrogate neurotoxicity of a CXCR4-using gp120. However, the combination of pharmacologic CCR5 blockade and LCN2 protects neurons from toxicity of a CXCR4-using gp120, thus recapitulating the finding in CCR5-deficient gp120tg mouse brain. Our study provides evidence for an indirect pathologic role of CCR5 and a novel protective effect of LCN2 in combination with inhibition of CCR5 in HIV-associated brain injury.
Assuntos
Complexo AIDS Demência/genética , Proteínas de Fase Aguda/metabolismo , Proteína gp120 do Envelope de HIV/genética , HIV-1 , Lipocalinas/metabolismo , Proteínas Oncogênicas/metabolismo , Receptores CCR5/genética , Proteínas de Fase Aguda/biossíntese , Animais , Antagonistas dos Receptores CCR5 , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Gliose , Lipocalina-2 , Lipocalinas/biossíntese , Aprendizagem em Labirinto , Memória , Camundongos , Camundongos Knockout , Microglia/patologia , Proteínas Oncogênicas/biossíntese , Receptores CCR5/biossíntese , Receptores CXCR4/metabolismo , Transdução de Sinais/genéticaRESUMO
Autoantibodies against p53 have been observed in many cancers, often linked with abnormalities in the TP53 gene. Since p53 mutations and deletions at chromosome 17p are known to occur in CLL, we measured anti-p53 autoantibodies by ELISA in plasma samples from patients with normal cytogenetics as well as those with 13q, 11q, and 17p deletions as well as trisomy 12. Anti-p53 autoantibodies were detected in over half of the patients with a 17p deletion but in very few of the others. There was no correlation between the levels of anti-p53 antibodies and the percentage of cells with 17p abnormalities. The levels of the anti-p53 autoantibodies remained stable for most patients with serial samples. Increased levels of antibodies that bound to two peptide fragments of p53 were also seen in patients with 17p deletions. At least on case with high levels of anti-p53 autoantibodies had a heterozygotic mutation known to result in a dominant negative phenotype, suggesting that aberrant expression of p53 may contribute to the development of autoantibodies and suggests that these autoantibodies may reflect biological features relevant to prognosis.
Assuntos
Autoanticorpos/imunologia , Deleção Cromossômica , Cromossomos Humanos Par 17/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Western Blotting , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Heterozigoto , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Linfocítica Crônica de Células B/sangue , Mutação/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Prognóstico , Taxa de SobrevidaRESUMO
PURPOSE: Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations. METHODS: Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay. RESULTS: Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 microg/ml, and the assay for alemtuzumab had a limit of detection of 1 microg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner. CONCLUSION: Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies.