Leptin deficiency-induced obesity affects the density of mast cells in abdominal fat depots and lymph nodes in mice

<p>Abstract</p> <p>Background</p> <p>Mast cells are implicated in the pathogenesis of obesity and insulin resistance. Here, we explored the effects of leptin deficiency-induced obesity on the density of mast cells in metabolic (abdominal fat depots, skeletal muscle, and liver) and lymphatic (abdominal lymph nodes, spleen, and thymus) organs. Fourteen-week-old male leptin-deficient <it>ob/ob </it>mice and their controls fed a standard chow were studied. Tissue sections were stained with toluidine blue to determine the density of mast cells. CD117/c-kit protein expression analysis was also carried out. Furthermore, mast cells containing immunoreactive tumor necrosis factor-α (TNF-α), a proinflammatory cytokine involved in obesity-linked insulin resistance, were identified by immunostaining.</p> <p>Results</p> <p><it>ob/ob </it>mice demonstrated adiposity and insulin resistance. In abdominal fat depots, mast cells were distributed differentially. While most prevalent in subcutaneous fat in controls, mast cells were most abundant in epididymal fat in <it>ob/ob </it>mice. Leptin deficiency-induced obesity was accompanied by a 20-fold increase in the density of mast cells in epididymal fat, but a 13-fold decrease in subcutaneous fat. This finding was confirmed by CD117/c-kit protein expression analysis. Furthermore, we found that a subset of mast cells in epididymal and subcutaneous fat were immunoreactive for TNF-α. The proportion of mast cells immunoreactive for TNF-α was higher in epididymal than in subcutaneous fat in both <it>ob/ob </it>and control mice. Mast cells were also distributed differentially in retroperitoneal, mesenteric, and inguinal lymph nodes. In both <it>ob/ob </it>mice and lean controls, mast cells were more prevalent in retroperitoneal than in mesenteric and inguinal lymph nodes. Leptin deficiency-induced obesity was accompanied by increased mast cell density in all lymph node stations examined. No significant difference in the density of mast cells in skeletal muscle, liver, spleen, and thymus was noted between <it>ob/ob </it>and control mice.</p> <p>Conclusions</p> <p>This study demonstrates that leptin deficiency-induced obesity is accompanied by alterations in the density of mast cells in abdominal fat depots. The divergent distribution of mast cells in subcutaneous versus visceral fat might partially account for their differential biological behavior. Mast cells might also play a role in adaptive immune response occurring in regional lymph nodes in obesity.</p

Apoptosis, mastocytosis, and diminished adipocytokine gene expression accompany reduced epididymal fat mass in long-standing diet-induced obese mice

<p>Abstract</p> <p>Background</p> <p>Obesity is characterized by increased cell death and inflammatory reactions in the adipose tissue. Here, we explored pathophysiological alterations taking place in the adipose tissue in long-standing obesity. In the epididymal fat of C57BL/6 mice fed a high-fat diet for 20 weeks, the prevalence and distribution of dead adipocytes (crown-like structures), mast cells (toluidine blue, mMCP6), macrophages (F4/80), and apoptotic cells (cleaved caspase-3) were measured. Moreover, gene and/or protein expression of several adipocytokines (leptin, adiponectin, TNF-α, IL-10, IL-6, MCP-1), F4/80, mMCP6, cleaved caspase-3 were determined.</p> <p>Results</p> <p>We observed that the epididymal fat mass was lower in obese than in lean mice. In obese mice, the epididymal fat mass correlated inversely with body weight and liver mass. Dead adipocytes, mast cells, macrophages, and apoptotic cells were abundant in the epididymal fat of obese mice, especially in the rostral vs. caudal zone. Accordingly, mMCP6, F4/80, and cleaved caspase-3 gene and/or protein expression was increased. Conversely, adiponectin, leptin, IL-6, and MCP-1 gene expression levels were lower in the epididymal fat of obese than lean mice. Although TNF-α and IL-10 gene expression was higher in the epididymal fat of obese mice, their expression relative to F4/80 and mMCP6 expression were lower in the heavily infiltrated rostral than caudal zone.</p> <p>Conclusions</p> <p>This study demonstrates that in mice with long-standing obesity diminished gene expression of several adipocytokines accompany apoptosis and reduced mass of the epididymal fat. Our findings suggest that this is due to both increased prevalence of dead adipocytes and altered immune cell activity. Differential distribution of metabolically challenged adipocytes is indicative of the presence of biologically diverse zones within the epididymal fat.</p

Establishment of Protein Delivery Systems Targeting Podocytes

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes

Podocytes [version 1; referees: 2 approved]

Podocytes are highly specialized cells of the kidney glomerulus that wrap around capillaries and that neighbor cells of the Bowman’s capsule. When it comes to glomerular filtration, podocytes play an active role in preventing plasma proteins from entering the urinary ultrafiltrate by providing a barrier comprising filtration slits between foot processes, which in aggregate represent a dynamic network of cellular extensions. Foot processes interdigitate with foot processes from adjacent podocytes and form a network of narrow and rather uniform gaps. The fenestrated endothelial cells retain blood cells but permit passage of small solutes and an overlying basement membrane less permeable to macromolecules, in particular to albumin. The cytoskeletal dynamics and structural plasticity of podocytes as well as the signaling between each of these distinct layers are essential for an efficient glomerular filtration and thus for proper renal function. The genetic or acquired impairment of podocytes may lead to foot process effacement (podocyte fusion or retraction), a morphological hallmark of proteinuric renal diseases. Here, we briefly discuss aspects of a contemporary view of podocytes in glomerular filtration, the patterns of structural changes in podocytes associated with common glomerular diseases, and the current state of basic and clinical research

Insight in Human Skin Microcirculation Using In Vivo Reflectance-Mode Confocal Laser Scanning Microscopy

Reflectance-mode confocal laser scanning microscopy allows in vivo imaging of the human skin. We hypothesized that this high-resolution technique enables observation of dynamic changes of the cutaneous microcirculation. Twenty-two volunteers were randomly divided in two groups. Group 1 was exposed to local heating and group 2 to local cold stress. Confocal microscopy was performed prior t0 (control), directly t1 and 5 min t2 after local temperature changes to evaluate quantitative blood cell flow, capillary loop diameter, and density of dermal capillaries. In group 1, blood flow increased at t1 (75.82 ± 2.86/min) and further at t2 (84.09 ± 3.39/min) compared to the control (61.09 ± 3.21/min). The control capillary size was 9.59 ± 0.25 μm, increased to 11.16 ± 0.21 μm (t1) and 11.57 ± 0.24 μm (t2). The dermal capillary density increased in t1 (7.26 ± 0.76/mm2) and t2 (8.16 ± 0.52/mm2), compared to the control (7.04 ± 0.62/mm2). In group 2, blood flow decreased at t1 (41.73 ± 2.61/min) and increased at t2 (83.27 ± 3.29/min) compared to the control (60.73 ± 2.90/min). The control capillary size was 9.55 ± 0.25 μm, decreased at t1 (7.78 ± 0.26 μm) and increased at t2 (11.38 ± 0.26 μm). Capillary density decreased at t1 (5.01 ± 0.49/mm2) and increased at t2 (7.28 ± 0.53/mm2) compared to the control (7.01 ± 0.52/mm2). Confocal microscopy is a sensitive and noninvasive imaging tool for characterizing and quantifying dynamic changes of cutaneous microcirculation on a histomorphological level

Quality of life and functional outcome following microsurgical fasciocutaneous vs. myocutaneous tissue transfer

Background: Coverage of soft tissue defects at the lower extremity may necessitate microsurgical tissue transfer, such as by fasciocutaneous anterolateral thigh (ALT) or myocutaneous musculus latissimus dorsi (MLD) flaps. Hitherto, these two flaps have not been compared systematically in terms of patient satisfaction and functional outcome. The purpose of this study was to compare patients’ satisfaction and functional outcome following ALT vs. MLD transfer. Methods: Thirty-six patients were divided into an ALT group (n=22, mean age: 42.0 years) and a MLD group (n=14, mean age: 55.5 years). Both groups were compared concerning isokinetic analysis (Biodex System III), circumference measurement, flap volume, scar size, complication rates, patients’ satisfaction, and functional outcome (SF-36 questionnaire, Foot and Ankle Outcome Score (FAOS)), hospital stay and duration of surgery.Results: Isokinetic force measurements showed a higher mean maximum force for dorsiflexion in the MLD group at an angular velocity of 60°/sec. (ALT 17.5±7.9 Nm, MLD 18.5±16.3 Nm; p<0.013) and higher strength endurance at 180°/sec (ALT 8.17±5.6 Nm, MLD 13.36±9.4 Nm; p<0.008), whereas no differences in all other measurements was found. The mean lower leg circumference was significantly larger in the MLD group (ALT 25.0±3.39 cm, MLD 30.36±2.14 cm; p<0.013). The mean difference in the circumference measurement between the covered and uninjured extremity within both groups differed significantly (ALT 2.55±1.71 cm, MLD 8.13±1.65 cm; p<0.001). The flap size in the ALT group was 148.77±6.58 cm² compared to 251.63±21.28 cm² in the MLD group (p<0.01). The scar size in the ALT group was 40.61± compared to 93.40±22.65 cm² in the MLD group (p<0.01). Superficial necrosis occurred in both groups (ALT n=3, MLD n=5). Donor area seroma was only seen in the MLD group (n=2). A complete flap failure was recorded only in the ALT group (n=2). SF-36 and FAOS indicated no significant differences between groups (p>0.05) in quality of life and functional outcome. Average hospital stay and duration of operation showed no differences between groups.Conclusion: In the present study, no significant differences in quality of life and functional outcome were found between ALT and MLD group. Only in isokinetic force measurement isolated significant differences with advantages of MLD group were detected. The current study provides additional information concerning ALT and MLD flaps and may support decision-making in selecting the appropriate free flap

Polyaniline grafted polyacylonitrile conductive composite fibers for reversible immobilization of enzymes: Stability and catalytic properties of invertase

WOS: 000267619700014Polyacylonitrile fibers (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The effect of aniline concentration on the grafting efficiency and on the electrical surface resistance of PAN/PANI composite fibers was investigated. The surface resistance of the conductive composite fibers in this work was found to be between 8.0 and 0.5 k Omega/cm. As the amount of grafted PANI increased on the PAN fibers the electrical resistance of composite fibers decreased. The PAN/PANI composite fibers were characterized by SEM and FTIR studies. Composite PAN/PANI fibers were used for reversible immobilization of invertase. The immobilization efficiency and the activity of the immobilized invertase (from 1.0 mg/mL invertase solution at pH 5.5) were increased with increasing PANI contents of the composite fibers. The maximum amount of immobilized enzyme onto composite fibers containing 2.0% PANI was about 76.6 mg/g. The optimum pH for the free enzyme was observed at 5.0. On the other hand, immobilized invertase yielded a broad optimum PH profile between pH 5.0 and 7.0. Immobilized invertase exhibited 83% of its original activity even after two months storage at 4 degrees C while the free enzyme showed only 7% of its initial activity. (C) 2009 Elsevier Ltd. All rights reserved