Gotowa bibliografia na temat „B3 receptors”

Brierley, Matthew J., Mark S. Yeoman, and Paul R. Benjamin. Glutamate is the transmitter for the N2v retraction phase interneurons of the Lymnaea feeding system. J. Neurophysiol. 78: 3408–3414, 1997. Electrophysiological and pharmacological methods were used to examine the role of glutamate in mediating the excitatory and inhibitory responses produced by the N2v rasp phase neurons on postsynaptic cells of the Lymnaea feeding network. The N2v → B3 motor neuron excitatory synaptic response could be mimicked by focal or bath application of l-glutamate at concentrations of ≥10−3 M. Quisqualate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were potent agonists for the B3 excitatory glutamate receptor (10−3 M), whereas kainate only produced very weak responses at the same concentration. This suggested that non- N-methyl-d-aspartate (NMDA), AMPA/quisqualate receptors were present on the B3 cell. The specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10−5 M) blocked 85% of the excitatory effects on the B3 cell produced by focal application of glutamate (10−3 M), confirming the presence of non-NMDA receptors. CNQX also blocked the major part of the excitatory postsynaptic potentials on the B3 cell produced by spontaneous or current-evoked bursts of spikes in the N2v cell. As with focal application of glutamate, a small delayed component remained that was CNQX insensitive. This provided direct evidence that glutamate acting via receptors of the non-NMDA, AMPA/quisqualate type were responsible for mediating the main N2v → B3 cell excitatory response. NMDA at 10−2 M also excited the B3 cell, but the effects were much more variable in size and absent in one-third of the 25 B3 cells tested. NMDA effects on B3 cells were not enhanced by bath application of glycine at 10−4 M or reduction of Mg2+ concentration in the saline to zero, suggesting the absence of typical NMDA receptors. The variability of the B3 cell responses to NMDA suggested these receptors were unlikely to be the main receptor type involved with N2v → B3 excitation. Quisqualate and AMPA at 10−3 M also mimicked N2v inhibitory effects on the B7 and B8 feeding motor neurons and the modulatory slow oscillator (SO) interneuron, providing further evidence for the role of AMPA/quisqualate receptors. Similar effects were seen with glutamate at the same concentration. However, CNQX could not block either glutamate or N2v inhibitory postsynaptic responses on the B7, B8, or SO cells, suggesting a different glutamate receptor subtype for inhibitory responses compared with those responsible for N2v → B3 excitation. We conclude that glutamate is a strong candidate transmitter for the N2v cells and that AMPA/quisquate receptors of different subtypes are likely to be responsible for the excitatory and inhibitory postsynaptic responses.

ABSTRACT Many entero-, parecho-, and rhinoviruses use immunoglobulin (Ig)-like receptors that bind into the viral canyon and are required to initiate viral uncoating during infection. However, some of these viruses use an alternative or additional receptor that binds outside the canyon. Both the coxsackievirus-adenovirus receptor (CAR), an Ig-like molecule that binds into the viral canyon, and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). A cryoelectron microscopy reconstruction of a variant of CVB3 complexed with DAF shows full occupancy of the DAF receptor in each of 60 binding sites. The DAF molecule bridges the canyon, blocking the CAR binding site and causing the two receptors to compete with one another. The binding site of DAF on CVB3 differs from the binding site of DAF on the surface of echoviruses, suggesting independent evolutionary processes.

The ephrins are a family of proteins known to bind the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinase family. In the present paper, we provide data showing that ephrin-B3 binds a sulfated cell-surface protein on HEK-293T (human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40) and HeLa cells, a binding that is nearly completely blocked by treatment of these cell lines with chlorate or heparinase, or by addition of the heavily sulfated glycosaminoglycan heparin. This indicates that heparan sulfate on these cells is essential for cell-surface binding of ephrin-B3. Heparin did not affect ephrin-B3 binding to EphB receptors expressed on transfected HEK-293T cells, indicating further that ephrin-B3 binds an alternative receptor which is a heparan sulfate proteoglycan. Site-directed mutagenesis analysis revealed that Arg178 and Lys179 are important for heparin binding of ephrin-B3 and also for ephrin-B3 binding to cells. These amino acids, when introduced in the non-heparin-binding ephrin-B1, conferred the heparin-binding property. Functional studies reveal that ephrin-B3 binding to cells induces cellular signalling and influences cell rounding and cell spreading. In conclusion, our data provide evidence for an unknown ephrin-B3-binding cell-surface proteoglycan involved in cellular signalling.

Abstract Acquired mutations truncating the C-terminal domain of the granulocyte colony-stimulating factor receptor (G-CSF-R) are found in about 20% of severe congenital neutropenia (SCN) patients, with this cohort of patients predisposed to acute myeloid leukemia (AML). In myeloid cells, such mutations act in a dominant-negative manner leading to hyperproliferation and lack of differentiation in response to G-CSF. However, why these truncated receptors are dominant in function over wild-type receptors has remained unclear. We report that ligand-induced internalization of truncated G-CSF-R is severely impaired compared with the wild-type receptor, which results in sustained activation of STAT proteins. Strikingly, in cells coexpressing both truncated and wild-type forms, the truncated receptors acted dominantly with regard to both internalization and sustained activation. Site-directed mutagenesis of the C-terminus showed that receptor tyrosines in this region were dispensable for internalization, whereas a di-leucine–containing motif in Box B3 played some role. However, loss of the di-leucine motif was not the critical determinant of the sustained activation status of truncated receptors. These data suggest that defective internalization, leading to extended receptor activation, is a major cause of the dominant hyperproliferative effect of truncated G-CSF receptors, which is only partially due to the loss of a di-leucine motif present in the Box B3 region of the full-length receptor.

Acquired mutations truncating the C-terminal domain of the granulocyte colony-stimulating factor receptor (G-CSF-R) are found in about 20% of severe congenital neutropenia (SCN) patients, with this cohort of patients predisposed to acute myeloid leukemia (AML). In myeloid cells, such mutations act in a dominant-negative manner leading to hyperproliferation and lack of differentiation in response to G-CSF. However, why these truncated receptors are dominant in function over wild-type receptors has remained unclear. We report that ligand-induced internalization of truncated G-CSF-R is severely impaired compared with the wild-type receptor, which results in sustained activation of STAT proteins. Strikingly, in cells coexpressing both truncated and wild-type forms, the truncated receptors acted dominantly with regard to both internalization and sustained activation. Site-directed mutagenesis of the C-terminus showed that receptor tyrosines in this region were dispensable for internalization, whereas a di-leucine–containing motif in Box B3 played some role. However, loss of the di-leucine motif was not the critical determinant of the sustained activation status of truncated receptors. These data suggest that defective internalization, leading to extended receptor activation, is a major cause of the dominant hyperproliferative effect of truncated G-CSF receptors, which is only partially due to the loss of a di-leucine motif present in the Box B3 region of the full-length receptor.

Kinins exert a variety of biological actions and have been implicated in the pathogenesis of inflammation, pain, asthma, and other diseases. Kinins act through specific receptors that are widespread and belong to two major categories, B1 and B2. B2 has been cloned and shown to be of the rhodopsin type, consisting of seven hydrophobic membrane domains connected by extracellular and intracellular loops. Recent pharmacological findings from various laboratories suggest the existence of new receptor types, which have been named B3, B4, and B5. These findings are analysed critically, especially with respect to the criteria that have been used for affirming the existence of new receptor entities. The analysis is restricted to data obtained in isolated organs, almost exclusively smooth muscle preparations. Criteria for receptor characterization and classification are the order of potency of agonists and the apparent affinities of antagonists. The analysis reveals that receptors for bradykinin and related kinins are of two types, B1 and B2. B1 mediates the rapid acute response (smooth muscle contraction or relaxation) as well as some effects occurring more slowly (e.g., collagen synthesis). B1 receptor functions have been shown to be modulated by interleukins. B2 receptors are responsible for most of the kinins' biological effects, including arterial vasodilatation, plasma extravasation, venoconstriction, activation of sensory fibers (e.g., fibers for pain), and stimulation of the release of prostaglandins, endothelium-dependent relaxing factor (from endothelia), noradrenaline (from nerve terminals and adrenals), and other endogenous agents. The pharmacological characteristics of the receptor sites (B2) mediating this array of biological effects show differences between species, and two B2 receptor subtypes are proposed, namely B2A (rabbit, dog, and possibly man) and B2B (guinea pig, hamster, rat). B2A and B2B receptor subtypes have been characterized by using fairly selective agonists and competitive antagonists (e.g., D-Arg[Hyp3,D-Phe7,Leu8]BK). Noncompetitive antagonists (non-equilibrium), such as HOE 140, do not discriminate between B2A and B2B subtypes. Species differences cannot account for the multiplicity of receptors that have been proposed for rat vas deferens, pre- and post-junctional sites, and rat uterus, guinea pig ileum, and rat blood pressure. The existence of hypothetical new receptor sites was based on data obtained with partial agonists and have not been substantiated by data obtained with potent pure antagonists. The B3 receptor, proposed to explain the unusual behaviour of the guinea pig tracheal response to kinins, has to be carefully reconsidered after the finding that HOE 140 acts as a pure antagonist on this tissue and shows a fairly high affinity for the tracheal site. B3, B4, and B5 receptors described in the esophagus of the opossum have not been sufficiently characterized either with agonists or with antagonists to be considered as new functional sites.Key words: kinins, smooth muscle, receptors, antagonists, action mechanisms.

Abstract Three cDNA clones encoding the guinea pig Fc receptor for IgG1 and IgG2 (Fc gamma 1/gamma 2 R) have been isolated by an expression cloning strategy using mAb directed against the receptor. When transfected into COS-7 cells, these cDNA induced cell surface expression of the receptor that bound IgG1 and IgG2 antibodies complexed with the Ag. The ligand-binding affinities of these receptors were indistinguishable. Nucleotide sequencing has indicated that one of these clones, Fc gamma 1/gamma 2R-B1, is identical to the previously isolated cDNA clone homologous to the b2 isoform of human Fc gamma RIIB and that of murine Fc gamma RII, encoding a transmembrane glycoprotein containing two Ig-like extracellular domains. Two other clones, Fc gamma 1/gamma 2R-B2 and -B3, are identical to Fc gamma 1/gamma 2R-B1 except for an inframe insertion in the cytoplasmic region. The 48-nucleotide insertion found in Fc gamma 1/gamma 2R-B2 is identical to the first 48 nucleotides of the B3 insert that comprises 132 bp. Based on the size and homology of the inserted sequence, Fc gamma 1/gamma 2R-B2 and -B3 are identified as the homologues of the b1 isoform of human Fc gamma RIIB and that of murine Fc gamma RII, respectively. Reverse transcription and polymerase chain reaction of RNA revealed that macrophages and polymorphonuclear leukocytes expressed preferentially Fc gamma 1/gamma 2R-B1. On the other hand, B lymphocytes expressed all three forms, among which Fc gamma 1/gamma 2R-B2 and -B3 were selectively expressed in LPS-activated B lymphocytes that showed a dramatic increase in the levels of cell surface expression of Fc gamma 1/gamma 2R. These results suggest that the inserted sequences of Fc gamma 1/gamma 2R-B2 and -B3 are important to generate responses specific for B lymphocytes, which may include regulation of cell activation.

Abstract Two variants of coxsackievirus group B, type 3 (CVB3) differ in ability to induce myocarditis in Balb/cCUM mice. Infection with the highly pathogenic variant (CVB3M) stimulates autoimmunity to normal cardiocyte antigens, and tissue injury results primarily from an autoreactive cytolytic T lymphocyte (ACTL). Animals infected with the less pathogenic CVB3o variant do not develop ACTL, although CVB3o replicates to high titers in the heart and polyclonal neutralizing antisera fail to distinguish between the two variant virions. The present study uses two IgM mAb derived by fusing spleen cells from CVB3M-infected mice with NS-1 cells. These mAb investigate important differences between the virus variants that may explain why only selected infections trigger autoimmunity. mAb 8A6 is a virus-neutralizing antibody that prevents infection of HeLa cells and cultured cardiocytes by attaching to the virus. mAb 10A1 also interferes with infection but presumably reacts to the virus receptor on the susceptible cells and shows little or no binding to the virions. While 8A6 is equally effective in neutralizing both CVB3o and CVB3M, suggesting that antigenic epitopes on both variants are either identical or highly cross-reactive, 10A1 distinguishes between the variants, suggesting that the pathogenic and less pathogenic viruses use distinct cell surface receptors. Competitive binding studies using radiolabeled CVB3M and either of the unlabeled variants confirm this hypothesis. Both mAb effectively prevent CVB3M-induced cardiac damage in vivo. mAb 10A1 also inhibits autoreactive ACTL lysis of cardiocytes, indicating that the autoimmune effectors may recognize the virus receptor, and that the receptor utilized by a virus may prove important in triggering auto-sensitization.

In viral myocarditis, adeno- and enteroviruses have most commonly been implicated as causes of infection. Both viruses require the human coxsackie-adenovirus receptor (CAR) to infect the myocardium. Due to its crucial role for viral entry, CAR-downregulation may lead to novel approaches for treatment for viral myocarditis. In this study, we report on pharmaceutical drug influences on CAR levels in human umbilical vein endothelial cells (HUVEC) and cervical carcinoma cells (HeLa) detected by immunoblotting, quantitative real time-PCR and cellular susceptibility to the cardiotropic coxsackie-B3 virus strain Nancy (CVB3). Our results indicate, for the first time, a dose-dependent CAR mRNA and protein downregulation upon Valsartan and Bosentan treatment. Most interestingly, drug-induced CAR diminution significantly reduced the viral load in CVB3-infected HUVEC. In order to assess the regulatory effects of both drugs in detail, we knocked down their protein targets, the G-protein coupled receptors angiotensin-II type-1 receptor (AT1R) and endothelin-1 type-A and -B receptors (ETAR/ETBR) in HUVEC. Receptor-specific gene silencing indicates that CAR gene expression is regulated by agonistic and antagonistic binding to ETBR, but not ETAR. In addition, neither stimulation nor inhibition of AT1R seemed to be involved in CAR gene regulatory processes. Our study indicates that Valsartan and Bosentan protected human endothelial cells from CVB3-infection. Therefore, besides their well-known anti-hypertensive effects these drugs may also protect the myocardium and other tissues from coxsackie- and adenoviral infection.

Flavonoids, a class of natural polyphenols abundantly present in our diet, have been shown to exert in vitro vasorelaxant activity. This was ascribed to a direct effect on the smooth muscle or factors released by the endothelium. Nowadays, perivascular adipose tissue (PVAT) is emerging as a fine regulator of blood vessel contractility. Therefore, it is conceivable to hypothesize that flavonoids vasoactivity may occur also through or is influenced, either positively or negatively, by PVATreleased factors. This hypothesis was assessed in vitro on rat aorta rings. Several flavonoids proved to display both antispasmodic and spasmolytic activity towards noradrenaline-induced contraction of rings deprived of PVAT (-PVAT). However, when PVAT was present (+PVAT), both activities of some flavonoids were lost and/or much decreased. In rings-PVAT, the superoxide donor pyrogallol mimicked the effect of PVAT, whereas in rings+PVAT the antioxidant mito-tempol restored both activities of the two most powerful flavonoids, namely apigenin and chrysin. The Rho-kinase inhibitor fasudil, or apigenin and chrysin concentration-dependently relaxed the vessel active tone induced by the Rho-kinase activator NaF; the presence of PVAT counteracted apigenin spasmolytic activity though only in the absence of mito-tempol. Similar results were obtained in rings pre-contracted with phenylephrine. Finally, when β3 receptors were blocked by SR59230A, the vasorelaxant activity of both flavonoids was no more affected by PVAT. These findings are coherent with the hypothesis that both noradrenaline and apigenin activated adipocyte β3 receptors with the ensuing release of mitochondrial superoxide anion, which once diffused toward myocytes counteracted flavonoid vasorelaxant activity, thus underlining the control of adipocytes upon the vascular tone. This phenomenon might limit the beneficial health effects of this class of natural compounds in patients affected by either obesity and/or other pathological conditions characterized by sympathetic nerve overactivity.

Introduction : Le cancer colorectal (CCR) est responsable de 500 000 morts par an dans le monde et représente la 2ème cause de mortalité par cancer dans les pays industrialisés. En dépit des progrès réalisés, il existe un réel besoin de développer de nouvelles thérapies pour améliorer la survie des patients. Un des principaux facteurs favorisant la survenue et la progression du CCR est le stress se traduisant notamment par la sécrétion de catécholamines activant les récepteurs β-adrénergiques (β1-, β2- et β3-AR) au niveau de la tumeur. Diverses études et observations ont démontré que l’activation des β-AR pouvait favoriser la prolifération tumorale de manière directe (via différents mécanismes comme la prolifération cellulaire) ou indirecte (via une action sur la composante immunitaire). Parmi les cellules immunitaires présentes au sein de la tumeur, les macrophages tumoraux (TAM) peuvent représenter jusqu’à 50% du volume tumoral. Ces derniers y sont retrouvés sous leurs différents phénotypes (M1-like antitumoral et M2-like pro-tumoral). Divers travaux ont fait état de la présence des β-AR à la surface des macrophages où leur effet semble être en faveur d’une polarisation vers un phénotype pro-tumoral. En outre, parmi les 3 sous-types de récepteurs, de nombreux arguments soulignent une implication majoritaire du β3-AR dans ces effets par rapport aux β1- et β2-AR, tandis que seule une surexpression du β3-AR a été observée dans les biopsies de tumeurs du côlon. Objectifs/Méthodes : Nous nous sommes donc attachés à mettre au point une stratégie méthodologique pour l’étude du β3-AR dans le macrophage en condition d’inflammation tissulaire présentant une importante composante macrophagique. Nous avons ensuite étudié les effets du β3-AR sur la prolifération de lignées de cancer colorectal puis sur la polarisation de macrophages humains ainsi que de TAM. Enfin, après avoir étudié la signalisation de ce récepteur chez les macrophages M1 et M2, nous avons observé les effets d’une inhibition pharmacologique du β3-AR sur la polarisation de TAM et sur la progression de tumeurs murines et humaines in vivo. Résultats : Nous avons confirmé que le β3-AR est présent et fonctionnel à la surface des macrophages où son activation résulte en un puissant effet antioxydant et anti-inflammatoire via l’inhibition de NOX2 et l’induction de l’expression de la catalase. Ces effets semblent passer par une signalisation Gs/PKA/Src/Erk1/2 induisant l’activation de PPARγ. Dans nos travaux, nous avons aussi pu voir que le β3-AR n’a pas d’effet prolifératif sur des lignées humaines de CCR. Nous avons également démontré que le β3-AR favorise la polarisation des macrophages vers un phénotype M2 et diminue la polarisation de ces derniers vers un phénotype M1. L’étude des signalisations de ce récepteur chez ces deux phénotypes a indiqué que les voies Gs/PKA/Src/ERK1/2 (M1) et Src/PI3K/ERK1/2 (M2) étaient impliquées. Enfin, l’inhibition du β3-AR a prévenu la progression de tumeurs murines (CT-26) et humaines (SW480) in vivo, via un effet anti-M2-like et pro-M1-like sur les TAM. En conclusion, ces résultats suggèrent que l’inhibition du β3-AR, à travers ses effets sur la polarisation des macrophages, pourrait être une stratégie prometteuse afin d’améliorer la prise en charge des patients souffrant de CCR
Background: Colorectal cancer (CRC) is responsible for 500.000 deaths per year worldwide and represents the 2nd cause of death by cancer in industrialized countries. Despite the progress made, there is a real need for new therapies to increase patients’ survival. Stress is one of the main factors, which contributes to the occurrence and the progression of CRC, by secreting catecholamines that activate β-adrenergic receptors (β1-, β2- and β3-AR) within the tumor. Several studies and observations have showed that the activation of β-ARs could directly increase tumor proliferation (via mechanisms such as cell proliferation), or indirectly (via an action on immune cells). Among immune cells within the tumor, tumor-associated macrophages (TAMs) represent up to 50% of the tumor mass where they exhibit their different phenotypes (M1-like anti-tumor and M2-like pro-tumor). Several studies report the presence of β-ARs in macrophages where they seem to favour a pro-tumor polarization. Furthermore, among the three subtypes of β-ARs, most of the studies seem to describe a major implication of the β3-AR compared to β1- and β2-AR. Moreover, only the β3-AR was found to be overexpressed in CRC biopsies. Objectives/Methods: We thus aimed to develop a model to study the β3-AR in macrophages within inflammatory macrophage-dependent conditions. Then, we studied the effects of the β3-AR on colorectal cancer cells’ proliferation and human macrophages and TAMs polarization. Finally, after the study of the signaling pathways of this receptor within M1 and M2 macrophages, we assessed the effects of a pharmacological inhibition of the β3-AR on TAM polarization and tumor progression. Results: We confirmed that the β3-AR is expressed and functional in human macrophages where its activation leads to potent antioxidant and anti-inflammatory effects through NOX2 inhibition and catalase expression. These effects appear to be subsequent to a Gs/PKA/Src/Erk1/2 signaling leading to the activation of PPARγ. In this work, we also saw that the β3-AR does not produce any effect on human CRC cell lines’ proliferation. We also showed that the β3-AR increases macrophage polarization towards the M2 phenotype while it decreases the M1 polarization. The study of β3-AR signaling in M1 and M2 macrophages exhibited Gs/PKA/Src/ERK1/2 and Src/PI3K/ERK1/2 pathways respectively. Finally, a pharmacological inhibition of the β3-AR prevented murine (CT-26) and human (SW480) tumors progression in vivo, through anti-M2-like and pro-M1-like effects on TAM polarization. In conclusion, these results suggest that the inhibition of the β3-AR, through its effects on macrophages polarization, could represent a promising strategy in order to improve CRC patient care

Orientadora : Profa. Dra. Neiva Leite
Co-orientadora: Profa. Dra. Lupe Furtado Alle
Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Educação Física. Defesa: Curitiba, 14/02/2013
Bibliografia: fls. 89-101
Área de concentraçao: Exercício e esporte
Resumo: O presente estudo teve dois objetivos, primeiro avaliar a frequência do polimorfismo Trp64Arg do gene _3-adrenérgico (ADBR3) e Arg16Gly e Gln27Glu no gene do receptor _2-adrenérgico (ADBR2) em crianças e adolescentes e relacioná-lo com as variáveis antropométricas, cardiorrespiratórias e metabólicas. O segundo investigar o papel do polimorfismo no gene ADBR3 (Trp64Arg) e ADBR2 (Arg16Gly e Gln27Glu) nas respostas das variáveis antropométricas, cardiorrespiratórias e metabólicas em crianças e adolescentes com excesso de peso, submetidas a tratamento multidisciplinar com 12 semanas de exercício físico e orientação nutricional. No primeiro estudo participaram 189 crianças e adolescentes, com excesso de peso, idade entre 10 e 16 anos, de ambos os sexos. Inicialmente avaliaram-se: estatura, peso, circunferência abdominal (CA), pressão arterial sistólica (PAS) e diastólica (PAD), consumo máximo de oxigênio (VO2max), glicemia basal, colesterol total (CT), lipoproteína de alta densidade (HDL-C), lipoproteína de baixa densidade (LDL-C) e triacilglicerol (TG). Foram calculados o índice de massa corporal (IMC) e o IMCESCORE Z. As mutações Trp64Arg (ADBR3) e Arg16Gly e Gln27Glu (ADBR2) foram avaliadas por genotipagem Taqman. No segundo estudo, participaram 83 crianças e adolescentes com excesso de peso em 12 semanas de exercício físico e orientação nutricional. Na fase inicial e após 12 semanas, avaliaram-se peso, estatura, CA, frequência cardíaca de repouso, PAS e PAD, taxa metabólica de repouso, VO2max, glicemia basal e após 120min, insulina basal e após 120min, CT, HDL-C, LDL-C e TG. O IMC, IMC-ESCORE Z, Homeostasis Metabolic Assessment (HOMA-IR) e Quantitative Insulin Sensitivity Check Index (QUICKI) foram calculados. O exercício físico consistiu em sessões de 110 min, três vezes por semana, 45 min de ciclismo indoor, 45 min de caminhada e 20 min de alongamento. A intensidade foi de 35 a 55% do VO2max nas primeiras quatro semanas e aumento até 55 a 75%. Utilizou-se teste t, Wilcoxon e teste de Qui-quadrado nos dados transversais. Nos dados longitudinais utilizou-se uma ANOVA com medidas repetidas e ANCOVA nas variáveis com dados inicias diferentes. Todos com nível de significância de p < 0,05. Os dados transversais revelaram que no polimorfismo Gln27Glu (ADBR2) o valor de TG foi superior no grupo portador da mutação quando comparado ao usual (p=0,01). Nas demais variáreis não houve diferenças significativas. Antes da intervenção, o HOMA-IR (p=0,001) e GLI120 (p=0,03) estavam mais elevados nos portadores do alelo 64Arg (ADBR3). Após as 12 semanas, o HOMA-IR apresentou maior redução no grupo com alelo 64Arg (p=0,01), as demais variáveis tiveram semelhanças das respostas. No polimorfismo Arg16Gly (ADBR2), a GLI120 inicial foi maior nos portadores do alelo 16Gly (p=0,01), as demais variáveis não diferenciaram. No polimorfismo Gln27Glu (ADBR2), a PAS (p=0,009), PAD (p=0,01) e TG (p=0,05) iniciais foram maiores nos portadores do alelo 27Glu, as demais variáveis foram semelhantes. Após a intervenção, houve tendência a maior redução nos valores de TG (p=0,06) e PAS (p=0,08) no grupo com alelo 27Glu. Conclui-se que as respostas antropométricas, de aptidão física e redução do peso foram semelhantes nos grupos com e sem polimorfismo ADBR2 e ADBR3. Entretanto, a terapêutica de 12 semanas de exercícios físicos aeróbios e orientação nutricional pode ser utilizada como estratégia na melhora da sensibilidade à insulina (polimorfismo Trp64Arg no receptor ADBR3) e na redução da PAS e TG (polimorfismo Gln27Glu do gene ADBR2). Sugere-se que, o incentivo às mudanças ambientais pode ser efetivo em crianças e adolescentes com excesso de peso, mesmo nos indivíduos com alterações metabólicas associadas ao componente genético.
Abstract: In this study we investigated associations between polymorphisms of the gene Trp64Arg _3-adrenergic receptor (ADRB3) and Arg16Gly and Gln27Glu gene _2- adrenergic receptor (ADRB2) in children and adolescents with anthropometric, cardiorespiratory and metabolic. The study included 189 children and adolescents aged between 10 and 16 years, of both sexes. Initially evaluated: height, weight, waist circumference (WC), systolic blood pressure (SBP) and diastolic (DBP), maximal oxygen consumption (VO2max), basal glucose, total cholesterol (TC), high density lipoprotein (HDL -C), low density lipoprotein (LDL-C) and triglyceride (TG). We calculated the body mass index (BMI) and BMI Z-SCORE Mutations Trp64Arg (ADRB3), and Arg16Gly Gln27Glu (ADRB2) was evaluated by PCR with Taqman. We also assessed the role of these variants (Trp64Arg, and Arg16Gly Gln27Glu) in the responses of anthropometric, cardiorespiratory and metabolic diseases in children and adolescents with overweight undergo multidisciplinary treatment with 12 weeks of exercise and nutritional counseling. Participated 83 children and adolescents with overweight at 12 weeks of exercise and nutritional guidance. In the initial stage and after 12 weeks were evaluated for weight, height, WC, resting heart rate, SBP and DBP, resting metabolic rate, VO2max, and after 120min basal glucose, basal insulin and after 120min, TC, HDL-C , LDL-C and TG. BMI, BMI ZSCORE, Metabolic Homeostasis Assessment (HOMA-IR) and Quantitative Insulin Sensitivity Check Index (QUICKI) were calculated. Exercise sessions consisted of 110 minutes, three times per week, 45 minutes of indoor cycling, 45 min and 20 min walk stretching for 12 weeks. The intensity was between 35 to 55% VO2max in the first four weeks and gradually increasing until 55 to 75%. In cross-sectional data t test was used for normal data and Wilcoxon test for nonparametric data. In longitudinal data was used an ANOVA with repeated measures ANCOVA and the variables with different initial data, the non-normal data were transformed into logarithms. Data analysis showed that only the transverse Gln27Glu polymorphism (ADBR2) the value of TG was higher in the group carrying the mutation compared to usual. The remaining variables were not significant differences. Before the intervention, and HOMA-IR were higher in GLI120 allele 64Arg (ADRB3), after 12 weeks, only HOMAIR showed a greater reduction in the group with allele 64Arg, other variables had similar responses. In Arg16Gly polymorphism (ADRB2) just GLI120 was higher in initial allele 16Gly and after intervention responses on lipid profile, metabolic, physical fitness and weight reduction between the groups with and without the mutation were similar. In Gln27Glu polymorphism (ADRB2) SBP, DBP, and TG were higher in initial allele 27Glu and other variables did not differ. After intervention responses on metabolic profile, anthropometric and physical fitness were similar, only the values of TG and SBP showed a trend toward greater reduction in the group with allele 26Glu. It is suggested that the relationship between gene ADRB3 with insulin resistance may be affected by physical activity and nutritional counseling, as well as SBP, DBP and TG mutation in Gln27Glu (ADRB2).

Una posible mutación patogénica en el gen del receptor β3-adrenérgico (Trp64Arg) ha sido reportada por estar asociada con Diabetes tipo 2 en sujetos de diferentes grupos étnicos. Desde entonces, diversos genes candidatos mutados han sido reportados asociados con Diabetes tipo 2 en peruanos y en otras nacionalidades. Se investigó la frecuencia del polimorfismo Trp64Arg en el gen del receptor β3-adrenérgico, parámetros bioquímicos y medidas antropométricas de 72 sujetos diabéticos tipo 2, y 21 controles, de ambos géneros, con edades entre 30 y 70 años, de la provincia de Sullana (Norte del Perú), por ser de alta prevalencia en Diabetes tipo 2. El estudio de laboratorio se realizó en: Instituto Peruano de Biología Molecular (IPBM), Laboratorio de Biología Molecular de la Facultad de Medicina Veterinaria y en la Facultad de Farmacia y Bioquímica de la UNMSM. Del análisis de los resultados utilizando el programa SPSS v 15 se encontró que las frecuencias de los genotipos del gen del receptor β3-adrenérgico en la población de estudio fue de: 61.1% (44) Trp64Trp, 27.8% (20) Trp64Arg y 11.1% (8) Arg64Arg para diabéticos; y de: 57.1% (12) Trp64Trp, 14.3% (3) Trp64Arg y 28.6% (6) Arg64Arg para controles. Asimismo, se halló las siguientes frecuencias alélicas en el gen receptor β3-adrenérgico en diabéticos y controles, para el alelo Trp: 75% y 64.3% y para Arg: 25.0% y 35.7%, respectivamente, de lo que se deduce que la frecuencia alélica de la mutación (Arg) fue un poco menor pero estadísticamente no significativa (p igual a 0.088). Los diabéticos se encontraron con mal control metabólico glucémico e hiperglicemia. Al aplicar el test t de Student en diabéticos y controles se pudo observar que solo la Hemoglobina glicosilada (Hb-G) tuvo significancia estadística (p igual a 0.039), que podría estar asociado al polimorfismo Trp64Arg. En el análisis de los genotipos vs IMC, Dislipidemia y Control Metabólico glucémico en controles se encontró diferencia significativa para el parámetro IMC en los diferentes genotipos, pero no en diabéticos tipo 2. Del análisis del riesgo Odds ratio se halló que, en el total de sujetos de estudio (diabéticos más controles) el genotipo Trp64Arg alcanzó cierto grado de factor de riesgo a sobrepeso, dislipidemia, y mal control metabólico glucémico, y que el genotipo Arg64Arg es un factor de riesgo a sobrepeso y a padecer diabetes, pero en todos los casos no tuvo diferencia estadística significativa. Se concluye que, el polimorfismo en estudio es importante, pero no es el único, ni el mayor factor determinante a desarrollar Diabetes tipo 2.
A possible pathogenic mutation in the β3-adrenergic receptor gene (Trp64Arg) has been reported to be associated with Diabetes type 2, Non-insulin-dependent diabetes mellitus (NIDDM), in different group ethnic subjects. Since that, several mutations of candidate genes have been reported associated with Diabetes type 2 in Peruvians and subjects from other nationalities. It was investigated the frequency of polymorphism Trp64Arg in the β3-adrenergic receptor gene, biochemical and anthropometric measurements of 72 type 2 diabetic subjects, and 21 as control group, both genders, aged between 30 and 70 years all of them of the province Sullana (North of Perú) as this city as high prevalence of Type 2 Diabetes in the country. The laboratory study was conducted in: Peruvian Institute of Molecular Biology (IPBM), the Laboratory of Molecular Biology of Faculty of Veterinary Medicine and Faculty of Pharmacy and Biochemistry – Universidad Nacional Mayor de San Marcos. The analysis of the results was performed using the SPSS 15 version found that the frequencies of the genotypes of β3-adrenergic receptor gene in the study population was: 61.1% (44) Trp64Trp, 27.8% (20) Trp64Arg and 11.1% (8) Arg64Arg for diabetics, and: 57.1% (12) Trp64Trp, 14.3% (3) Trp64Arg and 28.6% (6) Arg64Arg to controls. Furthermore, we found the following allelic frequencies in the β3-adrenergic receptor gene in diabetic patients and controls for the Trp allele, 75% and 64.3% for Arg: 25.0% and 35.7% respectively, which suggests that the frequency allelic to the mutation (Arg) was slightly lower but not statistically significant (p same to 0.088). Diabetics were found with bad glycemic metabolic control and hyperglycemia. In applying the Student t test in diabetics and controls was observed that only the glycosylated Hemoglobin (Hb-G) had statistical significance (p same to 0.039), which could be associated with the Trp64Arg polymorphism. In the analysis of genotypes versus BMI, dyslipidemia and glycemic metabolic control in subject controls was significant difference for the BMI parameter in the different genotypes, but not in type 2 Diabetes. Of risk analysis found that Odds ratios in the total study subjects (diabetic controls more) Trp64Arg genotype achieved a degree of risk factor for overweight, dyslipidemia, and bad glycemic metabolic control, and that Arg64Arg genotype is a risk factor related with overweight and diabetes, but none had significant statistical difference. We conclude that the polymorphism under this study is important, but neither the only, nor the most decisive factor to develop type 2 Diabetes.
Tesis

碩士
國立成功大學
微生物及免疫學研究所
97
Group B coxsackieviruses (CVB) belong to members of Human Enterovirus B (HEV-B) and contain six serotypes (CVB1-6). They are associated with some severe illness in neonates, including meningoencephalitis, hepatitis, myocarditis, pneumonitis and coagulopathy. There are several epidemics of CVB-associated fulminant hepatitis in Taiwan between 1994 and 2008. Cellular receptors play an important role in the tissue tropism of viral infection. CVB was found to use the coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF, CD55) as a primary receptor and coreceptor to infect permissive cells. The previous studies suggested that CVB infection may influence the expressions of CAR and DAF on host cells. To investigate the change of viral receptor expressions of CVB3 infection on Huh7 cells, the in vitro infection model was established. Flow cytometry analysis showed that CAR and DAF are highly expressed on Huh7 cells. By antibody blocking assay, anti-CAR mAb was shown to block CVB3 infection on Huh7 cells, while anti-DAF mAb has synergistic inhibitory effect. At 8 hours of CVB3 infection, cellular expression of CAR was markedly reduced on Huh7 cells. Reduction of CAR expression was also observed among different CVB serotypes. CVB3 infection reduced CAR expression in protein level, but not in transcription level and not associated with converting to soluble form on Huh7 cells. Confocal microscopy analysis illustrated that internalization of CAR is associated with endosomal/lysosomal marker, Lamp1 at 8 hours postinfection. Moreover, low-pH mediated endocytosis may be involved in the mechanism of CAR internalization and digestion in dose-dependent manner. In conclusion, CVB infection could enhance CAR internalization and downregulate cellular CAR expression.

碩士
國立成功大學
微生物及免疫學研究所
100
Coxsackievirus B3 is a member of the human enterovirus B. CVB3 infection can cause a series of severe diseases in neonates such as myocarditis, hepatitis, meningitis and encephalitis. Antibody-dependent enhancement (ADE) of CVB3 infection has been shown to associate with disease severity in myocarditis. A hepatotropic CVB3 strain, CVB3/2630, can increase liver inflammation and damage through ADE mechanism, which was reported previously. Both coxsackievirus and adenovirus receptor (CAR) and Fcγ receptors (FcγRs) on immune cells are involved in the homologous ADE mechanisms of CVB3 infection. To study the role of CAR or FcγRs in ADE of CVB3 infection in hepatocytes, diluted mouse anti-CVB3 IgG, human intravenous immunoglobulin (IVIG) and anti-CVB3 IgG from human IVIG each infected with CVB3 directly to hepatocytes or hepatocytes pretreated with anti-CAR and anti-DAF antibodies were conducted. We found that diluted mouse anti-CVB3 IgG enhanced CVB3 infection at the concentration 0.02-0.18 μg/ml in AML12 (mouse hepatocytes); IVIG and anti-CVB3 IgG from IVIG enhanced CVB3 infection at the concentration 3-7 μg/ml and 0.012-2 μg/ml respectively in Huh7 (human hepatocytes). Blocking CAR on AML12 and Huh7 before infection, the viral titer showed no difference between with and without mouse anti-CVB3 IgG. In addition, the cytokines expression pattern did not change significantly after infection for 10 hours in Huh7 via ADE. On the other hand, blocking CAR on Huh7 before CVB3 infection resulted in a dose-dependent inhibition of viral titer, while blocking DAF didn’t inhibit viral titer obviously. However, blocking both CAR and DAF showed synergistic inhibition on viral titer. To further investigate the F(ab’)2 portion in ADE mechanisms of CVB3 in Huh7, we remove the Fc fragment from human IVIG and then infected Huh7 with CVB3 and F(ab’)2 fragment. The viral titer didn’t enhance as IgG lost its Fc fragment, which indicated the CAR-dependent ADE of CVB3 infection in Huh7 is not via F(ab’)2 fragment. As heparan sulfate is a CAR-independent mediator of the CVB3 infection, we hypothesized that heparan sulfate might participate the ADE. The expression level of heparan sulfate on AML12 and Huh7 was analyzed, then removed the heparan sulfate by heparinase I on AML12. Flow cytometry analysis showed that the infection rate of CVB3 on AML12 cells have no difference with heparan sulfate-free AML12 cells. Heparan sulfate may not involve in ADE of CVB3 infection in AML12. In conclusion, we demonstrated that CVB3 can induce ADE via CAR in hepatocyte, and the ADE of CVB3 infection is not through F(ab’)2 fragment or the involvement of heparan sulfate.

The lesions of uncertain malignant potential of the breast, classified as B3, besides increasing the relative risk for breast cancer, have very heterogeneous abnormalities and raise a big question when defining conduct. A good multidisciplinary evaluation is necessary, comparing biopsy and imaging examination results. This study reports the case of a 54-year-old patient, without other risk factors for breast cancer, who was referred to MAMARJ, a mastology clinic, from a gynecology service, in November 2019 for evaluation of category 4 mammography, due to alterations in the right breast: linear and heterogeneous calcifications in the upper outer quadrant (UOQ) and punctiform and grouped calcifications lower inner quadrant (LIQ). Mammotomies were indicated, and histopathological reports were compatible with columnar cell hyperplasia with a focus on planar atypia — in the UOQ — and adenomyoepithelioma and columnar cell hyperplasia without atypia — in the LIQ. She was taken to surgery to remove the lesion from the UOQ (histopathology without malignancy). In July 2020, she underwent a mammography with a category 2 (BIRADS) report due to parenchymal distortion from previous surgery, and a ultrasonography with sparse cysts and bilateral ductal ectasia (category 3). One year later, in July 2021, she presented mammography — amorphous calcifications in the upper quadrants and punctate calcifications in the LIQ, near the clip from previous mammotomy. A mammotomy of the calcifications in the upper quadrants was performed. The diagnosis of the vacuum-guided biopsy was columnar cell changes with minimal architectural atypia in the upper quadrants. Removal of the lesion from the upper quadrants and the LIQ (target of the previous mammotomy) was indicated. The histopathological diagnosis was ductal carcinoma in situ (LIQ), associated with an atypical ductal hyperplasia, microcalcifications, and flat epithelial atypia. Immunohistochemical panel: estrogen receptor (ER) was positive, progesterone receptor (PR) was positive, and human epidermal growth factor receptor type 2 (HER2) was negative. The upper quadrant lesion was compatible with a focus on intraductal proliferation with discrete atypia. A simple mastectomy was performed with immediate reconstruction in the right breast. The mastectomy was indicated mainly because it was the patient’s choice. As suggested, since the first diagnosis of B3 lesion and after that of ductal carcinoma in situ, the patient did not accept chemoprevention. It should be noted that risk-reducing mastectomy is cited only rarely for the prevention in cases of even recurrent and multicentric premalignant lesions, as in this case.