BackgroundWhite adipose tissue (WAT) is a complex, diffuse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defense, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT by breaking up the matrix of collagen fibres; however, it is unclear to what extent adipocyte number in primary cultures correlates with their number in intact WAT, since recovery and viability are often unknown.Experimental DesignEpididymal WAT of four young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of glucose uptake and lactate, glycerol and NEFA excretion rates up to 48 h. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were measured. The presence of non-nucleated cells (erythrocytes) was also estimated.ResultsCell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70–75%. Cells showed even higher metabolic activity in the second than in the first day of incubation. Adipocytes were 7%, erythrocytes 66% and other stromal (nucleated cells) 27% of total WAT cells. However, their overall volumes were 90%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 1.3% of WAT.ConclusionsThe methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have also found that the “live cell mass” of adipose tissue is very small: about 13 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood (the rats were killed by exsanguination). These data translate (with respect to the actual “live cytoplasm” size) into an extremely high metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

Background. White adipose tissue (WAT) is a complex, disperse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defence, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT breaking up the matrix collagen fibres, but primary cultures of these cells could not be easily correlated to intact WAT, since often recovery and viability are unknown. Experimental design. Epididymal WAT of 4-6 young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of lactate production. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were also measured. The presence of non-nucleated cells (erythrocytes) was also estimated. Results. Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70-75%. Adipocytes were 7%, erythrocytes 68% and other stromal (nucleated cells) 24% of total WAT cells. However, their overall volumes were, 91%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 2.5% of WAT. Conclusions. The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have found, also, that the "live cell mass" of adipose tissue is very small (about 25 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood). This fact, translates into an extremely high (with respect to the actual "live cytoplasm" size) metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

Colonization by NTHi is high in this Mediterranean coast region with remarkable ampicillin resistant. Younger age and the winter period were facilitating factors.

Background Fatty acid metabolism in the hypothalamus has an important role in food intake, but its specific role in AgRP neurons is poorly understood. Here, we examined whether carnitinea palmitoyltransferase 1A (CPT1A), a key enzyme in mitochondrial fatty acid oxidation, affects energy balance. Methods To obtain Cpt1aKO mice and their control littermates, Cpt1a(flox/flox) mice were crossed with tamoxifen-inducible AgRPCreERT2 mice. Food intake and body weight were analyzed weekly in both males and females. At 12 weeks of age, metabolic flexibility was determined by ghrelin-induced food intake and fasting–refeeding satiety tests. Energy expenditure was analyzed by calorimetric system and thermogenic activity of brown adipose tissue. To study fluid balance the analysis of urine and water intake volumes; osmolality of urine and plasma; as well as serum levels of angiotensin and components of RAAS (renin–angiotensin–aldosterone system) were measured. At the central level, changes in AgRP neurons were determined by: (1) analyzing specific AgRP gene expression in RiboTag–Cpt1aKO mice obtained by crossing Cpt1aKO mice with RiboTag mice; (2) measuring presynaptic terminal formation in the AgRP neurons with the injection of the AAV1-EF1a-DIO-synaptophysin-GFP in the arcuate nucleus of the hypothalamus; (3) analyzing AgRP neuronal viability and spine formations by the injection AAV9-EF1a-DIO-mCherry in the arcuate nucleus of the hypothalamus; (4) analyzing in situ the specific AgRP mitochondria in the ZsGreen-Cpt1aKO obtained by breeding ZsGreen mice with Cpt1aKO mice. Two-way ANOVA analyses were performed to determine the contributions of the effect of lack of CPT1A in AgRP neurons in the sex. Results Changes in food intake were just seen in male Cpt1aKO mice while only female Cpt1aKO mice increased energy expenditure. The lack of Cpt1a in the AgRP neurons enhanced brown adipose tissue activity, mainly in females, and induced a substantial reduction in fat deposits and body weight. Strikingly, both male and female Cpt1aKO mice showed polydipsia and polyuria, with more reduced serum vasopressin levels in females and without osmolality alterations, indicating a direct involvement of Cpt1a in AgRP neurons in fluid balance. AgRP neurons from Cpt1aKO mice showed a sex-dependent gene expression pattern, reduced mitochondria and decreased presynaptic innervation to the paraventricular nucleus, without neuronal viability alterations. Conclusions Our results highlight that fatty acid metabolism and CPT1A in AgRP neurons show marked sex differences and play a relevant role in the neuronal processes necessary for the maintenance of whole-body fluid and energy balance.

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Background: An epidemiological study of Streptococcus pneumoniae nasopharyngeal carriage in healthy children was carried out five years after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13). Objectives: Study the impact of pediatric vaccination with PCV13, and other associated epidemiological factors on the status of nasopharyngeal carriage, the circulating pneumococcal serotypes, and the antibiotic susceptibility to more frequently used antibiotics. Methods: A multi-center study was carried out in Primary Health Care, which included 1821 healthy children aged 1 to 4 years old. All isolates were sent to the Spanish Pneumococcal Reference Laboratory for serotyping and antimicrobial susceptibility testing. Results: At least one dose of PCV13 had been received by 71.9% of children and carriage pneumococcal prevalence was 19.7%. The proportion of PCV13 serotypes was low (14.4%), with an observed predominance of non-vaccine serotypes, 23B, 11A, 10A, 35B/F, and 23A were the five most frequent. A high rate of resistance to penicillin, erythromycin, and trimethoprim sulfamethoxazole was found. Conclusions: A low proportion of PCV13 serotypes were detected, confirming the impact of pediatric vaccination for reducing the serotypes vaccine carriage. High resistance rates to clinically important antibiotics were observed.

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