Towards the Development of a Female Animal Model of T1DM Using Hyaluronic Acid Nanocoated Cell Transplantation: Refinements and Considerations for Future Protocols.

Female mice (Black 6 strain) (C57BL/6) aged 6 weeks were subject to low dose streptozotocin (STZ) treatment for five consecutive days to mimic type 1 diabetes mellitus (T1DM) with insulitis. At two weeks after STZ injections, evaluation of the elevated glucose levels was used to confirm diabetes. The diabetic mice were then subject to the transplantation of pancreatic ß-cells (MIN-6 line). Four groups of mice were studied. The first group was injected with saline-only acting as the placebo surgery control, also known as SHAM group, the second and third groups were injected with MIN-6 single cells and polyethylene glycol-modified dipalmitoyl-glycerol-phosphatidyl ethanolamine (PEG-DPPE) modified MIN-6 single cells (500 µg per 1.106 cells), respectively, while the fourth group was injected with hyaluronic acid (HA)-coated MIN-6 single cells (5 bilayers). At seven- and fourteen-days following transplantation, the mice were euthanised. The renal and pancreatic tissues were then collected and histologically analysed. The induction of diabetes in female mice, through five-consecutive daily STZ injections resulted in inconsistent glycaemic levels. Interestingly, this shows an incomplete diabetes induction in female mice, of which we attribute to sex dimorphism and hormonal interferences. Transplantation failure of free-floating encapsulated cells was unable to decrease blood glucose hyperglycaemia to physiological ranges. The result is attributed to deprived cell-cell interactions, leading to decreased ß-cells functionality. Overall, we highlight the necessity of refining T1DM disease models in female subjects when using multiple low-dose STZ injections together with transplantation protocols. Considerations need to be made regarding the different developmental stages of female mice and oestrogen load interfering with pancreatic ß-cells susceptibility to STZ. The use of pseudo islets, cell aggregates and spheroids are sought to improve transplantation outcome in comparison to free-floating single cells.

Targeting Glucose Transporters for Breast Cancer Therapy: The Effect of Natural and Synthetic Compounds.

Reprogramming of cellular energy metabolism is widely accepted to be a cancer hallmark. The deviant energetic metabolism of cancer cells-known as the Warburg effect-consists in much higher rates of glucose uptake and glycolytic oxidation coupled with the production of lactic acid, even in the presence of oxygen. Consequently, cancer cells have higher glucose needs and thus display a higher sensitivity to glucose deprivation-induced death than normal cells. So, inhibitors of glucose uptake are potential therapeutic targets in cancer. Breast cancer is the most commonly diagnosed cancer and a leading cause of cancer death in women worldwide. Overexpression of facilitative glucose transporters (GLUT), mainly GLUT1, in breast cancer cells is firmly established, and the consequences of GLUT inhibition and/or knockout are under investigation. Herein we review the compounds, both of natural and synthetic origin, found to interfere with uptake of glucose by breast cancer cells, and the consequences of interference with that mechanism on breast cancer cell biology. We will also present data where the interaction with GLUT is exploited in order to increase the efficiency or selectivity of anticancer agents, in breast cancer cells.

Competencia clínica de médicos de seguridad social guatemaltecos para manejar hepatitis virales en atención primaria / Clinical competences for human viral hepatitis treatment of primary healthcare physicians from the Guatemalan social security system

RESUMEN Objetivo Medir la competencia clínica para el diagnóstico y manejo de hepatitis virales en médicos de primer nivel de atención a la salud. Metodología Se efectuó un estudio transversal en el que usando un instrumento previamente validado se midió la competencia y posteriormente se comparó entre médicos adscritos a diversas unidades médicas de atención primaria a la salud (UMAPS) del Instituto Guatemalteco de Seguridad Social (IGSS). La información fue analizada mediante estadística descriptiva e inferencial no paramétrica. Se evaluaron 104 médicos de 5 UMAPS del IGSS. Resultados Se encontró un nivel muy bajo de competencia clínica para el diagnóstico y tratamiento de las hepatitis virales, dentro de un intervalo de 9 a 62 puntos obtenidos en el instrumento que tiene un valor máximo teórico de 88, sin encontrar diferencias estadísticamente significativas entre UMAPS. Conclusiones: Se requiere educación continua en los médicos de las UMAPS del IGSS para mejorar sus competencias en hepatitis virales.(AU)

ABSTRACT Objective To measure the clinical competence for diagnosis and treatment of human viral hepatitis in primary health care physicians. Methodology Cross-sectional study in which a previously validated instrument to measure competences was used, and subsequent comparison between physicians at various primary health care units (PHCT) from the Guatemalan Institute of Social Security (GISS). This information was analyzed using descriptive and non-parametrical statistics. 104 physicians, from 5 PHCT ascribed to GISS were analyzed. Results A low level of clinical competence for diagnosis and treatment of human viral hepatitis in this physicians group was found, within a range of 9 to 62 points obtained through an instrument with a maximum theoretical value of 88; no significant statistical difference between PHCT was found. Conclusions PHCT physicians from require continuing education to improve their clinical competence on human viral hepatitis.(AU)

[Clinical competences for human viral hepatitis treatment of primary healthcare physicians from the Guatemalan social security system]. / Competencia clínica de médicos de seguridad social guatemaltecos para manejar hepatitis virales en atención primaria.

OBJECTIVE: To measure the clinical competence for diagnosis and treatment of human viral hepatitis in primary health care physicians. METHODOLOGY: Cross-sectional study in which a previously validated instrument to measure competences was used, and subsequent comparison between physicians at various primary health care units (PHCT) from the Guatemalan Institute of Social Security (GISS). This information was analyzed using descriptive and non-parametrical statistics. 104 physicians, from 5 PHCT ascribed to GISS were analyzed. RESULTS: A low level of clinical competence for diagnosis and treatment of human viral hepatitis in this physicians group was found, within a range of 9 to 62 points obtained through an instrument with a maximum theoretical value of 88; no significant statistical difference between PHCT was found. CONCLUSIONS: PHCT physicians from require continuing education to improve their clinical competence on human viral hepatitis.

OBJETIVO: Medir la competencia clínica para el diagnóstico y manejo de hepatitis virales en médicos de primer nivel de atención a la salud. METODOLOGÍA: Se efectuó un estudio transversal en el que usando un instrumento previamente validado se midió la competencia y posteriormente se comparó entre médicos adscritos a diversas unidades médicas de atención primaria a la salud (UMAPS) del Instituto Guatemalteco de Seguridad Social (IGSS). La información fue analizada mediante estadística descriptiva e inferencial no paramétrica. Se evaluaron 104 médicos de 5 UMAPS del IGSS. RESULTADOS: Se encontró un nivel muy bajo de competencia clínica para el diagnóstico y tratamiento de las hepatitis virales, dentro de un intervalo de 9 a 62 puntos obtenidos en el instrumento que tiene un valor máximo teórico de 88, sin encontrar diferencias estadísticamente significativas entre UMAPS. Conclusiones: Se requiere educación continua en los médicos de las UMAPS del IGSS para mejorar sus competencias en hepatitis virales.

Neuromuscular activity of Bothrops fonsecai snake venom in vertebrate preparations.

The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160µg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160µg/ml, respectively) and attenuated the contractures to 110µM ACh (78-100% inhibition) and 40mM KCl (45-90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200µg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200µg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K(+) were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom.

Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B1 receptor gene.

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ (10 microM). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 microM), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice.

Kinin B1 receptor deficiency leads to leptin hypersensitivity and resistance to obesity.

OBJECTIVE: Kinins mediate pathophysiological processes related to hypertension, pain, and inflammation through the activation of two G-protein-coupled receptors, named B(1) and B(2). Although these peptides have been related to glucose homeostasis, their effects on energy balance are still unknown. RESEARCH DESIGN AND METHODS: Using genetic and pharmacological strategies to abrogate the kinin B(1) receptor in different animal models of obesity, here we present evidence of a novel role for kinins in the regulation of satiety and adiposity. RESULTS: Kinin B(1) receptor deficiency in mice (B(1)(-/-)) resulted in less fat content, hypoleptinemia, increased leptin sensitivity, and robust protection against high-fat diet-induced weight gain. Under high-fat diet, B(1)(-/-) also exhibited reduced food intake, improved lipid oxidation, and increased energy expenditure. Surprisingly, B(1) receptor deficiency was not able to decrease food intake and adiposity in obese mice lacking leptin (ob/ob-B(1)(-/-)). However, ob/ob-B(1)(-/-) mice were more responsive to the effects of exogenous leptin on body weight and food intake, suggesting that B(1) receptors may be dependent on leptin to display their metabolic roles. Finally, inhibition of weight gain and food intake by B(1) receptor ablation was pharmacologically confirmed by long-term administration of the kinin B(1) receptor antagonist SSR240612 to mice under high-fat diet. CONCLUSIONS: Our data suggest that kinin B(1) receptors participate in the regulation of the energy balance via a mechanism that could involve the modulation of leptin sensitivity.

Disruption of the kinin B1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus.

A transgenic mouse model, deficient in kinin B(1) receptor (B(1)(-/-)) was used to evaluate the role of B(2) receptor in the smooth muscle stomach fundus. The results showed that the potency of bradykinin (BK) to induce contraction in the gastric tissue was maintained whereas the efficacy was markedly reduced. The angiotensin converting enzyme (ACE) inhibitor captopril potentiated BK-induced effect in wild type (WT) but not in B(1)(-/-) fundus. However, ACE activity detected by the convertion of Ang I to Ang II was inhibited by captopril in both types of gastric tissues. Taking into account the hypothesis that captopril and ACE bind to the B(2) receptor, we suggest that this complex was not formed in the stomach deficient in B(1) receptor. Therefore, our finding strongly support the hypothesis that in smooth muscles that constitutively express the kinin B(1) and B(2) receptors, an interaction between captopril and ACE, B(1) and B(2) receptors should occur forming a complex protein interaction for the potentiating effect of ACE on kinin receptors.

Pharmacological characterization of mouse hind paw oedema induced by Bothrops insularis (jararaca ilhoa) snake venom.

Bothrops snake venoms produce marked local effects, including oedema, haemorrhage and necrosis. The ability of Bothrops insularis venom to induce oedema in mice was investigated. Venom was injected into hind paws and the change in volume over time was measured by plethysmometry. B. insularis venom (0.01-2.5 microg/paw) induced paw oedema which, at high doses (>/=0.5 microg/paw), was accompanied by haemorrhage. The peak oedematogenic response occurred 3 h after venom injection with all doses and decreased gradually thereafter, but was still elevated with high doses after 24 h. Pretreating the mice with cyproheptadine (histamine H(1) and serotonin 5-HT(2) receptor antagonist), mepyramine (histamine H(1) receptor antagonist), L-NAME (inhibitor of nitric oxide synthase), indomethacin and rofecoxib (inhibitors of cyclooxygenases), and dexamethasone (indirect inhibitor of PLA(2)) significantly attenuated venom-induced oedema, whereas methysergide, a serotonin 5-HT(1)/5-HT(2) receptor antagonist, had no effect. The administration of antivenom 30 min before or immediately after venom injection also significantly inhibited venom-induced oedema. These results show that B. insularis venom causes oedema in the mouse hind paw and that this response is mediated by histamine, nitric oxide, and arachidonic acid metabolites formed by cyclooxygenases 1 and 2. The neutralization by commercial antivenom indicates that the venom components responsible for oedema are recognized by the antivenom and share immunological identity with their counterparts in the venoms of mainland Bothrops species.

Evidence for changes in the tachyphylactic property of recombinant angiotensin II AT(1) receptor expressed in CHO cells.

The manifestation of tachyphylaxis to angiotensin II in Chinese hamster ovary (CHO) cells expressing the rat angiotensin II AT(1) receptor was investigated. The cells were transfected with a cDNA fragment containing the complete coding region of the angiotensin II AT(1A) receptor gene, as well as 56 bp of its 3'- and 52 bp of its 5'-untranslated regions. These cells (CHO-AT(1)) responded to angiotensin II by increases in intracellular Ca(2+) concentration and inositol phosphate turnover, which were inhibited upon repeated administrations, characterizing the tachyphylaxis phenomenon. In contrast to smooth muscle cells, which are rendered tachyphylactic to angiotensin II but not to [2-lysine]angiotensin II ([Lys(2)]angiotensin II), this analogue induced responses in CHO-AT(1) cells that were also inhibited upon repeated administrations. A smooth muscle cell line, which showed tachyphylaxis only to angiotensin II, became tachyphylactic also to [Lys(2)]angiotensin II after transfection with the angiotensin II AT(1) receptor gene. Our findings suggest that posttranscriptional control directed by the 3'- or the 5'-untranslated regions in the angiotensin II AT(1) receptor gene may play a role in modulating the signal transduction pathways involved in the mechanism of angiotensin II tachyphylaxis.