HTRA1 in Placental Cell Models: A Possible Role in Preeclampsia.

The HtrA serine peptidase 1 (HTRA1) is a multidomain secretory protein with serine-protease activity involved in the regulation of many cellular processes in both physiological and pathological conditions. HTRA1 is normally expressed in the human placenta, and its expression is higher in the first trimester compared to the third trimester, suggesting an important role of this serine protease in the early phases of human placenta development. The aim of this study was to evaluate the functional role of HTRA1 in in vitro models of human placenta in order to define the role of this serine protease in preeclampsia (PE). BeWo and HTR8/SVneo cells expressing HTRA1 were used as syncytiotrophoblast and cytotrophoblast models, respectively. Oxidative stress was induced by treating BeWo and HTR8/SVneo cells with H2O2 to mimic PE conditions in order to evaluate its effect on HTRA1 expression. In addition, HTRA1 overexpression and silencing experiments were performed to evaluate the effects on syncytialization, cell mobility, and invasion processes. Our main data showed that oxidative stress significantly increased HTRA1 expression in both BeWo and HTR8/SVneo cells. In addition, we demonstrated that HTRA1 has a pivotal role in cell motility and invasion processes. In particular, HTRA1 overexpression increased while HTRA1 silencing decreased cell motility and invasion in HTR8/SVneo cell model. In conclusion, our results suggest an important role of HTRA1 in regulating extravillous cytotrophoblast invasion and motility during the early stage of placentation in the first trimester of gestation, suggesting a key role of this serine protease in PE onset.

ZO-1 expression in normal human macula densa: Immunohistochemical and immunofluorescence investigations.

The macula densa (MD) is an anatomical structure having a plaque shape, placed in the distal end of thick ascending limb of each nephron and belonging to juxtaglomerular apparatus (JGA). The aim of the present investigation is to investigate the presence of ZO-1, a specific marker of tight juncions (TJs), in MD cells. Six samples of normal human renal tissue were embedded in paraffin for ZO-1 expression analysis by immunohistochemical and immunofluorescence techniques. We detected ZO-1 expression in the apical part of cell membrane in MD cells by immunohistochemistry. In addition, ZO-1 and nNOS expressions (a specific marker of MD) were colocalized in MD cells providing clear evidence of TJs presence in normal human MD. Since ZO-1 is responsible for diffusion barrier formation, its presence in the MD supports the existence of a tubulomesangial barrier that ensures a regulated exchange between MD and JGA effectors in renal and glomerular haemodynamic homeostasis.

Butyrylcholinesterase distribution in the mouse gastrointestinal tract: An immunohistochemical study.

Butyrylcholinesterase (BChE) is a hydrolytic enzyme that together with acetylcholinesterase (AChE) belongs to the cholinesterase family. Whereas AChE has a well-established role in regulating cholinergic neurotransmission in central and peripheral synapses, the physiological role of BChE remains elusive. In this morphological immunohistochemical and double-label confocal microscopy study we investigated the distribution of BChE in the mouse gastrointestinal tract. BChE-positive cells were detected in the liver (both in hepatocytes and cholangiocytes), in the keratinised layers of the squamous epithelium of the oesophagus and forestomach, in the oxyntic mucosa of the stomach, in the mucus-secreting cells of duodenal Brunner glands and the small and large intestinal mucosa. Interestingly, BChE-positive cells were often detected close to gastrointestinal proliferative niches. In the oxyntic mucosa, the close proximity of ghrelin-producing and BChE-positive parietal cells suggests that BChE may be involved in ghrelin hydrolysation through paracrine action. To our knowledge, this is the first comprehensive morphological study performed to gain insight into the physiological role of BChE in the gastrointestinal tract.

Palmitoylethanolamide dampens neuroinflammation and anxiety-like behavior in obese mice.

High-fat diet (HFD) consumption leads to obesity and a chronic state of low-grade inflammation, named metainflammation. Notably, metainflammation contributes to neuroinflammation due to the increased levels of circulating free fatty acids and cytokines. It indicates a strict interplay between peripheral and central counterparts in the pathogenic mechanisms of obesity-related mood disorders. In this context, the impairment of internal hypothalamic circuitry runs in tandem with the alteration of other brain areas associated with emotional processing (i.e., hippocampus and amygdala). Palmitoylethanolamide (PEA), an endogenous lipid mediator belonging to the N-acylethanolamines family, has been extensively studied for its pleiotropic effects both at central and peripheral level. Our study aimed to elucidate PEA capability in limiting obesity-induced anxiety-like behavior and neuroinflammation-related features in an experimental model of HFD-fed obese mice. PEA treatment promoted an improvement in anxiety-like behavior of obese mice and the systemic inflammation, reducing serum pro-inflammatory mediators (i.e., TNF-α, IL-1ß, MCP-1, LPS). In the amygdala, PEA increased dopamine turnover, as well as GABA levels. PEA also counteracted the overactivation of HPA axis, reducing the expression of hypothalamic corticotropin-releasing hormone and its type 1 receptor. Moreover, PEA attenuated the immunoreactivity of Iba-1 and GFAP and reduced pro-inflammatory pathways and cytokine production in both the hypothalamus and hippocampus. This finding, together with the reduced transcription of mast cell markers (chymase 1 and tryptase ß2) in the hippocampus, indicated the weakening of immune cell activation underlying the neuroprotective effect of PEA. Obesity-driven neuroinflammation was also associated with the disruption of blood-brain barrier (BBB) in the hippocampus. PEA limited the albumin extravasation and restored tight junction transcription modified by HFD. To gain mechanistic insight, we designed an in vitro model of metabolic injury using human neuroblastoma SH-SY5Y cells insulted by a mix of glucosamine and glucose. Here, PEA directly counteracted inflammation and mitochondrial dysfunction in a PPAR-α-dependent manner since the pharmacological blockade of the receptor reverted its effects. Our results strengthen the therapeutic potential of PEA in obesity-related neuropsychiatric comorbidities, controlling neuroinflammation, BBB disruption, and neurotransmitter imbalance involved in behavioral dysfunctions.

High-Fat Diet Impairs Mouse Median Eminence: A Study by Transmission and Scanning Electron Microscopy Coupled with Raman Spectroscopy.

Hypothalamic dysfunction is an initial event following diet-induced obesity, primarily involving areas regulating energy balance such as arcuate nucleus (Arc) and median eminence (ME). To gain insights into the early hypothalamic diet-induced alterations, adult CD1 mice fed a high-fat diet (HFD) for 6 weeks were studied and compared with normo-fed controls. Transmission and scanning electron microscopy and histological staining were employed for morphological studies of the ME, while Raman spectroscopy was applied for the biochemical analysis of the Arc-ME complex. In HFD mice, ME ß2-tanycytes, glial cells dedicated to blood-liquor crosstalk, exhibited remarkable ultrastructural anomalies, including altered alignment, reduced junctions, degenerating organelles, and higher content of lipid droplets, lysosomes, and autophagosomes. Degenerating tanycytes also displayed an electron transparent cytoplasm filled with numerous vesicles, and they were surrounded by dilated extracellular spaces extending up to the subependymal layer. Consistently, Raman spectroscopy analysis of the Arc-ME complex revealed higher glycogen, collagen, and lipid bands in HFD mice compared with controls, and there was also a higher band corresponding to the cyanide group in the former compared to the last. Collectively, these data show that ME ß2-tanycytes exhibit early structural and chemical alterations due to HFD and reveal for the first-time hypothalamic cyanide presence following high dietary lipids consumption, which is a novel aspect with potential implications in the field of obesity.

Activation of a non-neuronal cholinergic system in visceral white adipose tissue of obese mice and humans.

BACKGROUND AND OBJECTIVES: Since white adipose tissue (WAT) lacks parasympathetic cholinergic innervation, the source of the acetylcholine (ACh) acting on white adipocyte cholinergic receptors is unknown. This study was designed to identify ACh-producing cells in mouse and human visceral WAT and to determine whether a non-neuronal cholinergic system becomes activated in obese inflamed WAT. METHODS: Mouse epididymal WAT (eWAT) and human omental fat were studied in normal and obese subjects. The expression of the key molecules involved in cholinergic signaling was evaluated by qRT-PCR and western blotting whereas their tissue distribution and cellular localization were investigated by immunohistochemistry, confocal microscopy and in situ hybridization. ACh levels were measured by liquid chromatography/tandem mass spectrometry. The cellular effects of ACh were assessed in cultured human multipotent adipose-derived stem cell (hMADS) adipocytes. RESULTS: In mouse eWAT, diet-induced obesity modulated the expression of key cholinergic molecular components and, especially, raised the expression of choline acetyltransferase (ChAT), the ACh-synthesizing enzyme, which was chiefly detected in interstitial macrophages, in macrophages forming crown-like structures (CLSs), and in multinucleated giant cells (MGCs). The stromal vascular fraction of obese mouse eWAT contained significantly higher ACh and choline levels than that of control mice. ChAT was undetectable in omental fat from healthy subjects, whereas it was expressed in a number of interstitial macrophages, CLSs, and MGCs from some obese individuals. In hMADS adipocytes stressed with tumor necrosis factor α, ACh, alone or combined with rivastigmine, significantly blunted monocyte chemoattractant protein 1 and interleukin 6 expression, it partially but significantly, restored adiponectin and GLUT4 expression, and promoted glucose uptake. CONCLUSIONS: In mouse and human visceral WAT, obesity induces activation of a macrophage-dependent non-neuronal cholinergic system that is capable of exerting anti-inflammatory and insulin-sensitizing effects on white adipocytes.

Adeno-Associated Virus Type 5 Infection via PDGFRα Is Associated With Interstitial Lung Disease in Systemic Sclerosis and Generates Composite Peptides and Epitopes Recognized by the Agonistic Immunoglobulins Present in Patients With Systemic Sclerosis.

OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.

Neuronal nitric oxyde synthase positive neurons in human indusium griseum.

The study was designed to analyze the nNOS positive neurons present in the indusium griseum by describing their distribution and morphology. To this purpose, sagittal serial sections from paraffin or frozen autopsy specimens of corpus callosum including the overlying indusium griseum were processed by immunohistochemistry and immunofluorescence, using an antibody against the neuronal form of the enzyme nitric oxyde synthase. To test the specificity of the antibody used, Western Blot was performed in the indusium griseum of the same specimens. The stainings revealed the presence of many neuronal nitric oxyde synthase-immunopositive neurons in human indusium griseum, located along both rostral-caudal and medio-lateral directions. In particular, they were more numerous 1 mm apart from the midline, and their number peaked over the body of the corpus callosum. They showed different morphologies; in some cases, they were located at the boundary between indusium griseum and corpus callosum, more densely packed in proximity to the pial arteries penetrating into the corpus callosum. The significant presence and distribution of neuronal nitric oxyde synthase-immunopositive neurons suggests that indusium griseum likely plays a functional role in the neurovascular regulation within the corpus callosum. Schematic representation of human adult IG and the neurovascular unit originating from sopracallosal artery (Sca) that branches into smaller arterioles (Br) (created in PowerPoint). The arterioles cross the three layers of IG (layers I, II and III) and penetrate into the CC separated from IG by the Virchow-Robin space (VRs). As the arterioles go deeper, this space disappears and the vascular basement membrane comes into direct contact with the astrocytic end-feets (intracallosal arterioles and capillaries). nNOS-immunopositive neurons (nNOSIP N) surround the arterioles and control the vasomotore tone secreting nitric oxyde (NO). Two morphological types of nNOSIP N can be appreciated by the use of different colors: fusiform (blue) and ovoidal (pink). Also NeuN-immunopositive neurons (N) and many astrocytes (As) are present, more numerous in IG than in CC.

Brown Fat Anatomy in Humans and Rodents.

The Brown Adipose Tissue (BAT) is composed by mitochondrial rich, multilocular adipocytes, in strict topographical and functional relation with vasculature and noradrenergic nerves. Brown adipocytes are able to dissipate energy to produce heat, in a process known as non-shivering thermogenesis. Due to its contribution to energy expenditure, BAT is intensely studied for its potential to counteract metabolic diseases such as obesity, type 2 diabetes, dyslipidemia and cardiovascular diseases. BAT displays specific morphological characteristics that allow to assess its functional state. In this chapter we describe methodologies to properly dissect BAT depots, evaluate their gross anatomy, and assess its activation by light microscopy using peroxidase immunostaining and by laser scanning confocal microscopy using immunofluorescence. We also describe methodologies to study BAT ultrastructure by transmission and scanning electron microscopy, to visualize peroxidase immunostaining reactions at an ultrastructural level and to perform immunofluorescence reactions on paraffin-embedded samples, more often available in the clinical setting (due to the possibility to store them long-term) as opposed to fresh samples. The described techniques can be employed to study BAT morphology and activation in response to various stimuli (e.g., cold exposure; specific dietary composition) and in different pathological conditions (e.g., obesity; type 2 diabetes).

Ciliary Neurotrophic Factor Modulates Multiple Downstream Signaling Pathways in Prostate Cancer Inhibiting Cell Invasiveness.

BACKGROUND: Prostate cancer (PCa) remains the most common diagnosed tumor and is the second-leading cause of cancer-related death in men. If the cancer is organ-confined it can be treated by various ablative therapies such as RP (radical prostatectomy), RT (radiation therapy), brachytherapy, cryosurgery or HIFU (High-Intensity Focused Ultrasound). However, advanced or metastatic PCa treatment requires systemic therapy involving androgen deprivation, but such patients typically progress to refractory disease designated as castration-resistant prostate cancer (CRPC). Interleukin-6 (IL-6) has been established as a driver of prostate carcinogenesis and tumor progression while less is known about the role of ciliary neurotrophic factor (CNTF), a member of the IL-6 cytokine family in prostate cancer. Moreover, MAPK/ERK, AKT/PI3K and Jak/STAT pathways that regulate proliferative, invasive and glucose-uptake processes in cancer progression are triggered by CNTF. METHODS: We investigate CNTF and its receptor CNTFRα expressions in human androgen-responsive and castration-resistant prostate cancer (CRPC) by immunohistochemistry. Moreover, we investigated the role of CNTF in proliferative, invasive processes as well as glucose uptake using two cell models mimicking the PCa (LNCaP cell line) and CRPC (22Rv1 cell line). CONCLUSIONS: Our results showed that CNTF and CNTFRa were expressed in PCa and CRPC tissues and that CNTF has a pivotal role in prostate cancer environment remodeling and as a negative modulator of invasion processes of CRPC cell models.

Senescent macrophages in the human adipose tissue as a source of inflammaging.

Obesity is a major risk factor for type 2 diabetes and a trigger of chronic and systemic inflammation. Recent evidence suggests that an increased burden of senescent cells (SCs) in the adipose tissue of obese/diabetic animal models might underlie such pro-inflammatory phenotype. However, the role of macrophages as candidate SCs, their phenotype, the distribution of SCs among fat depots, and clinical relevance are debated. The senescence marker ß-galactosidase and the macrophage marker CD68 were scored in visceral (vWAT) and subcutaneous (scWAT) adipose tissue from obese patients (n=17) undergoing bariatric surgery and control patients (n=4) subjected to cholecystectomy. A correlation was made between the number of SCs and BMI, serum insulin, and the insulin resistance (IR) index HOMA. The monocyte cell line (THP-1) was cultured in vitro in high glucose milieu (60 mM D-glucose) and subsequently co-cultured with human adipocytes (hMADS) to investigate the reciprocal inflammatory activation. In obese patients, a significantly higher number of SCs was observed in vWAT compared to scWAT; about 70% of these cells expressed the macrophage marker CD68; and the number of SCs in vWAT, but not in scWAT, positively correlated with BMI, HOMA-IR, and insulin. THP-1 cultured in vitro in high glucose milieu acquired a senescent-like phenotype (HgSMs), characterized by a polarization toward a mixed M1/M2-like secretory phenotype. Co-culturing HgSMs with hMADS elicited pro-inflammatory cytokine expression in both cell types, and defective insulin signaling in hMADS. In morbid obesity, expansion of visceral adipose depots involves an increased burden of macrophages with senescent-like phenotype that may promote a pro-inflammatory profile and impair insulin signaling in adipocytes, supporting a framework where senescent macrophages fuel obesity-induced systemic inflammation and possibly contribute to the development of IR.

Palmitoylethanolamide Promotes White-to-Beige Conversion and Metabolic Reprogramming of Adipocytes: Contribution of PPAR-α.

The potential role of brown and beige adipose tissue against obesity has been recognized. Browning, or beiging of white adipose tissue (WAT) is associated with the remodeling of adipocytes and the improvement of their metabolic and secretory functions. Here, palmitoylethanolamide (PEA) restore the plasticity of brown and white adipocytes impaired in mice on a high-fat diet (HFD). Young male C57Bl/6J mice were fed with control (STD) diet or HFD for 12 weeks. Ultramicronized PEA (30 mg/kg/die p.o.) was administered for an additional 7 weeks, together with HFD. PEA recovered interscapular brown fat morphology and function, increasing UCP1 positivity, noradrenergic innervation, and inducing the mRNA transcription of several specialized thermogenic genes. PEA promotes the beige-conversion of the subcutaneous WAT, increasing thermogenic markers and restoring leptin signaling and tissue hormone sensitivity. The pivotal role of lipid-sensing peroxisome proliferator-activated receptor (PPAR)-α in PEA effects was determined in mature 3T3-L1. Moreover, PEA improved mitochondrial bioenergetics in mature adipocytes measured by a Seahorse analyzer and induced metabolic machinery via AMPK phosphorylation. All these outcomes were dampened by the receptor antagonist GW6471. Finally, PEA induced adipogenic differentiation and increased AMPK phosphorylation in human adipose-derived stromal cells (ASCs) obtained from subcutaneous WAT of normal-weight patients and patients with obesity. We identify PEA and PPAR-α activation as the main mechanism by which PEA can rewire energy-storing white into energy-consuming brown-like adipocytes via multiple and converging effects that restore WAT homeostasis and metabolic flexibility.

Hypoxia-induced expression of neuronal nitric oxide synthase in astrocytes of human corpus callosum.

Nitric oxide (NO) is a gaseous neurotransmitter largely diffused in the brain; among other functions, it regulates the cerebral blood flow in response to hypoxia. NO can be synthetized by three different isoforms of the enzyme NO synthase: neuronal (nNOS), typical of neurons, endothelial and inducible. The aim of this study was to assess nNOS expression in human corpus callosum (CC) astrocytes, and its relationship with the hypoxia duration. Autoptic samples of CC from adult human subjects have been processed with immunohistochemistry and immunofluorescence using antibodies anti-nNOS and anti-glial fibrillary acidic protein (GFAP), the astrocyte marker. Results demonstrated for the first time the presence of nNOS-immunopositive astrocytes in the human CC. In particular, nNOS-positive astrocytes were absent in subjects deceased after a short hypoxia; their number and labeling intensity, however, increased with hypoxia prolongation. Neuronal NOS immunopositivity of CC astrocytes seems thus related to the hypoxia duration and the consequent brain damage.

Palmitoylethanolamide counteracts brain fog improving depressive-like behaviour in obese mice: Possible role of synaptic plasticity and neurogenesis.

BACKGROUND AND PURPOSE: High-fat diet (HFD)-induced obesity is accompanied by metabolic and neurochemical changes that have been associated with depression. Recent studies indicate that palmitoylethanolamide (PEA) exerts metabolic effects and holds neuroprotective potential. However, studies on HFD exposure in mice which investigate the effects of PEA on monoamine system and synaptic plasticity are limited. EXPERIMENTAL APPROACH: In C57Bl/6J male mice, obesity was established by HFD feeding for 12 weeks. Then, mice were treated with ultra-micronized PEA (30 mg·kg-1 daily p.o.) or vehicle for 7 weeks along with HFD. Mice receiving chow diet and vehicle served as controls. Thereafter, depressive-, anhedonic-like behaviour and cognitive performance were measured. Monoamine analyses were performed on brain areas (nucleus accumbens, Nac; prefrontal cortex, PFC; hippocampus), and markers of synaptic plasticity and neurogenesis were evaluated in hippocampus. KEY RESULTS: PEA limited depressive- and anhedonic-like behaviour, and cognitive deficits induced by HFD. PEA induced an increase in 5-HT levels in PFC, and a reduction of dopamine and 5-HT turnover in Nac and PFC, respectively. Moreover, PEA increased dopamine levels in the hippocampus and PFC. At a molecular level, PEA restored brain-derived neurotrophic factor signalling pathway in hippocampus and PFC, indicating an improvement of synaptic plasticity. In particular, PEA counteracted the reduction of glutamatergic synaptic density induced by HFD in the stratum radiatum of the CA1 of the hippocampus, where it also exhibited neurogenesis-promoting abilities. CONCLUSION AND IMPLICATIONS: PEA may represent an adjuvant therapy to limit depressive-like behaviours and memory deficit, affecting monoamine homeostasis, synaptic plasticity and neurogenesis. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.4/issuetoc.

Ciliary neurotrophic factor (CNTF) and its receptor (CNTFRα) signal through MAPK/ERK pathway in human prostate tissues: a morphological and biomolecular study.

Ciliary neurotrophic factor (CNTF) is a member of interleukin-6 type cytokine family. The CNTF receptor complex is a heterodimer including gp130 and CNTF receptor α (CNTFRα) proteins triggering the activation of multiple intracellular signaling pathways including AKT/PI3K, MAPK/ERK and Jak/STAT pathways. At present no data are available on the localization of CNTF and CNTFRα in prostate as well as on the role of CNTF in this organ. In this study we have analyzed the localization of CNTF and CNTFRα by immunohistochemistry and we have used PWR-1E cell line as a model for normal glandular cell to investigate the role of this cytokine. Our results show that CNTF and CNTFRa are expressed in the staminal compart of the prostate and that CNTF selectively inhibits ERK pathway. In conclusion, we suggest that CNTF could be considered as key molecule to maintenance epithelium homeostasis via pERK downregulation by an autocrine mechanism. Further CNTF studies in prostate cancer could be useful to verify the potential role of this cytokine in carcinogenesis.

A large proportion of mediastinal and perirenal visceral fat of Siberian adult people is formed by UCP1 immunoreactive multilocular and paucilocular adipocytes.

Many deleterious consequences for health of excessive fat accumulation are due to visceral fat. Browning of visceral fat is mainly cold dependent and has been proposed as a possible tool for future therapies of obesity and related disorders. In this paper, we studied the composition of mediastinal and perirenal visceral fat, collected at necropsy, of human adults that lived in Siberia, one of the coldest regions of the earth. Data showed that a consistent part of the mediastinal and perirenal fat (up to about 40%) had the morphology typical of brown adipocytes and that a relevant percentage of them (up to about 30%) also expressed the functional marker uncoupling protein 1 (UCP1). Patients living mainly outdoor had higher percentage of brown-like adipocytes with more intensely UCP1 immunoreactive cells. The presence of numerous UCP1 immunoreactive paucilocular cells, a transitional stage of transdifferentiating adipocytes, supports the idea that visceral fat can be converted to brown adipose tissue in adult humans in physiological conditions. Tyrosine hydroxylase immunoreactive noradrenergic parenchymal nerve fibers were positively correlated to the number of multilocular adipocytes in mediastinal fat, and a similar trend was also observed in the perirenal fat.

Melatonin Supplementation Decreases Hypertrophic Obesity and Inflammation Induced by High-Fat Diet in Mice.

Obesity results from critical periods of positive energy balance characterized by caloric intake greater than energy expenditure. This disbalance promotes adipose tissue dysfunction which is related to other comorbidities. Melatonin is a low-cost therapeutic agent and studies indicate that its use may improve obesity-related disorders. To evaluate if the melatonin is efficient in delaying or even blocking the damages caused by excessive ingestion of a high-fat diet (HFD) in mice, as well as improving the inflammatory profile triggered by obesity herein, male C57BL/6 mice of 8 weeks were induced to obesity by a HFD and treated for 10 weeks with melatonin. The results demonstrate that melatonin supplementation attenuated serum triglyceride levels and total and LDL cholesterol and prevented body mass gain through a decreased lipogenesis rate and increased lipolytic capacity in white adipocytes, with a concomitant increment in oxygen consumption and Pgc1a and Prdm16 expression. Altogether, these effects prevented adipocyte hypertrophy caused by HFD and reflected in decreased adiposity. Finally, melatonin supplementation reduced the crown-like-structure (CLS) formation, characteristic of the inflammatory process by macrophage infiltration into white adipose tissue of obese subjects, as well as decreased the gene expression of inflammation-related factors, such as leptin and MCP1. Thus, the melatonin can be considered a potential therapeutic agent to attenuate the metabolic and inflammatory disorders triggered by obesity.

Adipocyte-secreted BMP8b mediates adrenergic-induced remodeling of the neuro-vascular network in adipose tissue.

Activation of brown adipose tissue-mediated thermogenesis is a strategy for tackling obesity and promoting metabolic health. BMP8b is secreted by brown/beige adipocytes and enhances energy dissipation. Here we show that adipocyte-secreted BMP8b contributes to adrenergic-induced remodeling of the neuro-vascular network in adipose tissue (AT). Overexpression of bmp8b in AT enhances browning of the subcutaneous depot and maximal thermogenic capacity. Moreover, BMP8b-induced browning, increased sympathetic innervation and vascularization of AT were maintained at 28 °C, a condition of low adrenergic output. This reinforces the local trophic effect of BMP8b. Innervation and vascular remodeling effects required BMP8b signaling through the adipocytes to 1) secrete neuregulin-4 (NRG4), which promotes sympathetic axon growth and branching in vitro, and 2) induce a pro-angiogenic transcriptional and secretory profile that promotes vascular sprouting. Thus, BMP8b and NRG4 can be considered as interconnected regulators of neuro-vascular remodeling in AT and are potential therapeutic targets in obesity.

Design of a new tracking device for on-line beam range monitor in carbon therapy.

Charged particle therapy is a technique for cancer treatment that exploits hadron beams, mostly protons and carbon ions. A critical issue is the monitoring of the beam range so to check the correct dose deposition to the tumor and surrounding tissues. The design of a new tracking device for beam range real-time monitoring in pencil beam carbon ion therapy is presented. The proposed device tracks secondary charged particles produced by beam interactions in the patient tissue and exploits the correlation of the charged particle emission profile with the spatial dose deposition and the Bragg peak position. The detector, currently under construction, uses the information provided by 12 layers of scintillating fibers followed by a plastic scintillator and a pixelated Lutetium Fine Silicate (LFS) crystal calorimeter. An algorithm to account and correct for emission profile distortion due to charged secondaries absorption inside the patient tissue is also proposed. Finally detector reconstruction efficiency for charged particle emission profile is evaluated using a Monte Carlo simulation considering a quasi-realistic case of a non-homogenous phantom.

Action of Administered Ciliary Neurotrophic Factor on the Mouse Dorsal Vagal Complex.

Ciliary neurotrophic factor (CNTF) induces weight loss in obese rodents and humans through activation of the hypothalamic Jak-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway. Here, we tested the hypothesis that CNTF also affects the brainstem centers involved in feeding and energy balance regulation. To this end, wild-type and leptin-deficient (ob/ob and db/db) obese mice were acutely treated with intraperitoneal recombinant CNTF. Coronal brainstem sections were processed for immunohistochemical detection of STAT3, STAT1, STAT5 phosphorylation and c-Fos. In wild-type mice, CNTF treatment for 45 min induced STAT3, STAT1, and STAT5 phosphorylation in neurons as well as glial cells of the area postrema; here, the majority of CNTF-responsive cells activated multiple STAT isoforms, and a significant proportion of CNTF-responsive glial cells bore the immaturity and plasticity markers nestin and vimentin. After 120 min CNTF treatment, c-Fos expression was intense in glial cells and weak in neurons of the area postrema, it was intense in several neurons of the rostral and caudal solitary tract nucleus (NTS), and weak in some cholinergic neurons of the dorsal motor nucleus of the vagus. In the ob/ob and db/db mice, Jak-STAT activation and c-Fos expression were similar to those induced in wild-type mouse brainstem. Treatment with CNTF (120 min, to induce c-Fos expression) and leptin (25 min, to induce STAT3 phosphorylation) demonstrated the co-localization of the two transcription factors in a small neuron population in the caudal NTS portion. Finally, weak immunohistochemical CNTF staining, detected in funiculus separans, and meningeal glial cells, matched the modest amount of CNTF found by RT-qPCR in micropunched area postrema tissue, which in contrast exhibited a very high amount of CNTF receptor. Collectively, the present findings show that the area postrema and the NTS exhibit high, distinctive responsiveness to circulating exogenous and, probably, endogenous CNTF.