Molecular cloning and expression of leptin in gray and harbor seal blubber, bone marrow, and lung and its potential role in marine mammal respiratory physiology

Leptin is a multifunctional hormone, produced predominantly in adipocytes. It regulates energy balance through its impact on appetite and fat metabolism, and its concentration indicates the size of body fat reserves. Leptin also plays a vital role in stretch-induced surfactant production during alveolar development in the fetus. The structure, expression pattern, and role of leptin have not previously been explored in marine mammals. Phocid seals undergo cyclical changes in body composition as a result of prolonged fasting and intensive foraging bouts and experience rapid, dramatic, and repeated changes in lung volume during diving. Here, we report the tissue-specific expression pattern of leptin in these animals. This is the first demonstration of leptin expression in the lung tissue of a mature mammal, in addition to its expression in the blubber and bone marrow, in common with other animals. We propose a role for leptin in seal pulmonary surfactant production, in addition to its likely role in long-term energy balance. We identify substitutions in the phocine leptin sequence in regions normally highly conserved between widely distinct vertebrate groups, and, using a purified seal leptin antiserum, we confirm the presence of the leptin protein in gray seal lung and serum fractions. Finally, we report the substantial inadequacies of using heterologous antibodies to measure leptin in unextracted gray seal serum.

Multiple endocrine neoplasia, the old and the new: a mini review

Multiple endocrine neoplasia syndromes have since been classified as types 1 and 2, each with specific phenotypic patterns. MEN1 is usually associated with pituitary, parathyroid and paraneoplastic neuroendocrine tumours. The hallmark of MEN2 is a very high lifetime risk of developing medullary thyroid carcinoma (MTC) more than 95% in untreated patients. Three clinical subtypesdMEN2A, MEN2B, and familial MTC (FMTC) have been defined based on the risk of pheochromocytoma, hyperparathyroidism, and the presence or absence of characteristic physical features). MEN2 occurs as a result of germline activating missense mutations of the RET (REarranged during Transfection) proto-oncogene. MEN2-associated mutations are almost always located in exons 10, 11, or 13 through 16. Strong genotype-phenotype correlations exist with respect to clinical subtype, age at onset, and aggressiveness of MTC in MEN2. These are used to determine the age at which prophylactic thyroidectomy should occur and whether screening for pheochromocytoma or hyperparathyroidism is necessary. Specific RET mutations can also impact management in patients presenting with apparently sporadic MTC. Therefore, genetic testing should be performed before surgical intervention in all patients diagnosed with MTC. Recently, Pellegata et al. have reported that germline mutations in CDKN1B can predispose to the development of multiple endocrine tumours in both rats and humans and this new MEN syndrome is named MENX and MEN4, respectively. CDKN1B. A recent report showed that in sporadic MTC, CDKN1B V109G polymorphism correlates with a more favorable disease progression than the wild-type allele and might be considered a new promising prognostic marker. New insights on MEN syndrome pathogenesis and related inherited endocrine disorders are of particular interest for an adequate surgical and therapeutic approach.

Long-term outcomes following “presumed” total parathyroidectomy for secondary hyperparathyroidism of chronic kidney disease

Aim. The most efficacious surgical treatment for renal hyperparathyroidism is still subject of research. Considering its low incidence rate of long-term relapse, “presumed” total parathyroidectomy without autotrasplantation (TP) may be indicated for secondary hyperparathyroidism (2HPT) in patients with chronic kidney disease (CKD), not eligible for kidney transplantation. The aim of this study was to analyse the TP long-term results in 2HPT haemodialysis (HD) patients. Method. Between January 2004 and October 2009, 25 2HPT HD patients, not eligible for kidney transplantation, underwent TP of at least four parathyroid glands. Clinical status and intact parathyroid hormone (iPTH) serum levels were assessed intraoperatively and during a 36-month follow-up. Results. TP improved the typical clinical symptoms and a significant reduction of iPTH serum levels was achieved in each patient. Aparathyroidism was never observed; in case of severe postoperative hypocalcemia, hypocalcemic seizures were never reported and the long-term recurrence rate was 8%. Only one patient received a kidney transplantation. Postoperative cardiovascular events (hypertension, peripheral artery disease, arrhythmia, coronary or cerebrovascular disease) were observed in 32% of cases and mortality rate was 16%. Conclusions. Considering its low long-term relapse rate and the absence of postoperative aparathyroidism, TP may still be considered the treatment of choice in patients with aggressive forms of 2HPT or of advanced dialytic vintage, with no access to renal transplantation. In case of postoperative hypoparathyroidism, hypocalcaemia can be effectively managed by medical treatment.

Seizure and Profound Hypokalemia: Unusual Presentation of Primary Hyperparathyroidism

A 68-year-old man was admitted because of tonic–clonic convulsion. He had been receiving 200 mg itraconazole for 10 days. He had hypokalaemia (2.2 mEq/l), hypercalcaemia (Cacorr 11.0 mg/dl) and elevated serum parathyroid hormone (PTH, 95 pg/ml). Ultrasound examination of the neck revealed a low echoic tumour. Cessation of itraconazole and fluid supplementation eradicated clinical symptoms and profound hypokalaemia, but serum potassium remained low normal (3.4 mEq/l) and the mild hypercalcaemia and elevated PTH were unchanged. To conclude, a small amount of itraconazole (200 mg) precipitated profound hypokalaemia and seizure in a patient with mild hyperparathyroidism and low normal serum potassium.

Effect of vitamin D supplementation on bone status, glucose homeostasis and immune function in children with vitamin D deficiency

Background: Between 1961-1971 vitamin D deficiency was recognized as a public health issue in the UK, because of the lack of effective sunlight and the population mix [1, 2]. In recent years, health care professionals have cited evidence suggesting a re-emergence of the vitamin D deficiency linked to a number of health consequences as a concern [3-6]. Evidence from observational studies has linked low vitamin D status with impairment in glucose homeostasis and immune dysfunction [7-9]. However, interventional studies, particularly those focused on paediatric populations, have been limited and inconsistent. There is a need for detailed studies, to clarify the therapeutic benefits of vitamin D in these important clinical areas. Objective: The aims of this PhD thesis were two-fold. Firstly, to perform preliminary work assessing the association between vitamin D deficiency and bone status, glucose homeostasis and immune function, and to explore any changes in these parameters following short term vitamin D3 replacement therapy. Secondly, to assess the effectiveness of an electronic surveillance system (ScotPSU) as a tool to determine the current incidence of hospital-based presentation of childhood vitamin D deficiency in Scotland. Methods: Active surveillance was performed for a period of two years as a part of an electronic web-based surveillance programme performed by the Scottish Paediatric Surveillance Unit (ScotPSU). The validity of the system was assessed by identifying cases with profound vitamin D deficiency (in Glasgow and Edinburgh) from the regional laboratory. All clinical details were checked against those identified using the surveillance system. Thirty-seven children aged 3 months to 10 years, who had been diagnosed with vitamin D deficiency, were recruited for the bone, glucose and immunity studies over a period of 24 months. Twenty-five samples were analysed for the glucose and bone studies; of these, 18 samples were further analysed for immune study. Treatment consisted of six weeks taking 5000 IU units cholecalciferol orally once a day. At baseline and after completion of treatment, 25 hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), alkaline phosphatase (ALP), collagen type 1 cross-linked C-telopeptide (CTX), osteocalcin (OCN), calcium, phosphate, insulin, glucose, homeostasis model assessment index, estimated insulin resistance (HOMA IR), glycated hemoglobin (HbA1c), sex hormone binding globulin (SHBG), lipids profiles, T helper 1 (Th1) cytokines (interleukin-2 ( IL-2), tumor necrosis factors-alpha (TNF-α), interferon-gamma (INF-γ)), T helper 2 (Th2) cytokines (interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6)), T helper 17 (Th17) cytokine (interleukin-17 (IL-17)), Regulatory T (Treg) cytokine (interleukin-10 (IL-10)) and chemokines/cytokines, linked with Th1/Th2 subset balance and/or differentiation (interleukin-8 (IL-8), interleukin-12 (IL-12), eosinophil chemotactic protein ( EOTAXIN), macrophage inflammatory proteins-1beta (MIP-1β), interferon-gamma-induced protein-10 (IP-10), regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1(MCP-1)) were measured. Leukoocyte subset analysis was performed for T cells, B cells and T regulatory cells and a luminex assay was used to measure the cytokiens. Results: Between September 2009 and August 2011, 163 cases of vitamin D deficiency were brought to the attention of the ScotPSU, and the majority of cases (n = 82) were reported in Glasgow. The cross-validation checking in Glasgow and Edinburgh over a one-year period revealed only 3 (11%) cases of clearly symptomatic vitamin D deficiency, which had been missed by the ScotPSU survey in Glasgow. While 16 (67%) symptomatic cases had failed to be reported through the ScotPSU survey in Edinburgh. For the 23 children who are included in bone and glucose studies, 22 (96%) children had basal serum 25(OH)D in the deficiency range (< 50 nmol/l) and one (4%) child had serum 25(OH)D in the insufficiency range (51-75 nmol/l). Following vitamin D3 treatment, 2 (9%) children had final serum 25(OH)D lower than 50 nmol/l, 6 (26%) children had final serum 25(OH)D between >50-75 nmol/l, 12 (52%) children reached a final serum 25(OH)D >75-150 nmol/l and finally 3 (13%) exceeded the normal reference range with a final 25(OH)D >150 nmol/l. Markers for remodelling ALP and PTH had significantly decreased (p = 0.001 and <0.0001 for ALP and PTH respectively). In 17 patients for whom insulin and HOMA IR data were available and enrolled in glucose study, significant improvements in insulin resistance (p = 0.04) with a trend toward a reduction in serum insulin (p = 0.05) was observed. Of those 14 children who had their cytokines profile data analysed and enrolled in the immunity study, insulin and HOMA IR data were missed in one child. A significant increase in the main Th2 secreted cytokine IL-4 (p = 0.001) and a tendency for significant increases in other Th2 secreted cytokines IL-5 (p = 0.05) and IL-6 (p = 0.05) was observed following vitamin D3 supplementation. Conclusion: An electronic surveillance system can provide data for studying the epidemiology of vitamin D deficiency. However, it may underestimate the number of positive cases. Improving vitamin D status in vitamin D deficient otherwise healthy children significantly improved their vitamin D deficient status, and was associated with an improvement in bone profile, improvements in insulin resistance and an alteration in main Th2 secreting cytokines.

Treadmill walking exercise modulates bone mineral status and inflammatory cytokines in obese asthmatic patients with long term intake of corticosteroids.

Background: Obesity and asthma are an important public health problem in Saudi Arabia. An increasing body of data supports the hypothesis that obesity is a risk factor for asthma. Asthma appears to be associated with low bone mineral density (BMD) due to long-term use of corticosteroids. Studies recently showed that weight bearing exercise training can increase mineral bone density, reduce weight and improve metabolic control. Objective: The present study aimed to measure the effects of treadmill walking exercises on bone mineral status and inflammatory cytokines in obese asthmatic patients treated with long term intake of corticosteroids. Methods: Eighty obese asthmatic patients of both sexes, their age ranged from 41 to 53 years. Subjects were divided into two equal groups: training group (group A) received aerobic exercise training on treadmill for six months in addition to the medical treatment where, the control group (group B) received only the medical treatment. Results: The results of this study indicated a significant increase in BMD of the lumbar spine & the radius, serum calcium and high density lipoprotein cholesterol (HDL-c) & significant reduction in parathyroid hormone, leptin, tumor necrosis factor– alpha(TNF-α), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), low density lipoprotein cholesterol (LDL-c), triglycerides (TG) and body mass index (BMI) in group (A), while these changes were not significant in group (B).Also; there was a significant difference between both groups at the end of the study. Conclusion: Treadmill walking exercise training is an effective treatment policy to improve bone mineral status and modulates inflammatory cytokines and blood lipids profile in obese asthmatic patients with long term intake of corticosteroids.

Alterações à fisiologia e infeção por Plasmodium berghei do mosquito Anopheles gambiae mediadas pela ativação de recetores GPCR

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Characterization of bone pathologies in the ins2+/akita mouse, a new model for diabetes insulindependent: contribution for a better understanding of the disease in humans

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Association of vitamin D receptor gene variants with polycystic ovary syndrome: A case control study

Background: Vitamin D and insulin play an important role in susceptibility to polycystic ovary syndrome (PCOS), and therefore vitamin D receptor (VDR), parathyroid hormone (PTH), and insulin receptor (INSR) gene variants might be involved in the pathogenesis of PCOS. Objective: The present study was designed to investigate the possible associations between polymorphisms in VDR, PTH, and INSR genes and the risk of PCOS. Materials and Methods: VDR, PTH, and INSR gene variants were genotyped in 35 women with PCOS and 35 controls using Polymerase chain reaction – Restriction fragment length polymorphism method. Furthermore, serum levels of glucose and insulin were measured in all participants. Results: No significant differences were observed for the VDR FokI, VDR Tru9I, VDR TaqI,, PTH DraII, INSR NsiI, and INSR PmlI gene polymorphisms between the women with PCOS and controls. However, after adjustment for confounding factors, the VDR BsmI “Bb” genotype and the VDR ApaI "Aa" genotype were significantly under transmitted to the patients (p= 0.016; OR= 0.250; 95% CI= 0.081-0.769, and p= 0.017; OR= 0.260; 95% CI= 0.086-0.788, respectively). Furthermore, in the women with PCOS, insulin levels were lower in the participants with the INSR NsiI "NN" genotype compared with those with the "Nn + nn" genotypes (P= 0.045). Conclusion: The results showed an association between the VDR gene BsmI and ApaI polymorphisms and PCOS risk. These data also indicated that the INSR "NN" genotype was a marker of decreased insulin in women with PCOS. Our findings, however, do not lend support to the hypothesis that PTH gene DraII variant plays a role in susceptibility to PCOS.