Interleukin 21 Receptor Affects Adipogenesis of Human Adipose-Derived Stem/Stromal Cells

Dysfunctions in adipose tissue cells are responsible for several obesity-related metabolic diseases. Understanding the process of adipocyte formation is thus fundamental for understanding these diseases. The adipocyte differentiation of adipose-derived stem/stromal cells (ADSCs) showed a reduction in the mRNA level of the interleukin 21 receptor (IL21R) during this process. Although the receptor has been associated with metabolic diseases, few studies have examined its function in stem cells. In this study, we used confocal immunofluorescence assays to determine that IL21R colocalizes with mitochondrial protein ATP5B, ALDH4A1, and the nucleus of human ADSCs. We demonstrated that silencing and overexpression of IL21R did not affect the cell proliferation and mitochondrial activity of ADSCs. However, IL21R silencing did reduce ADSC adipogenic capacity. Further studies are needed to understand the mechanism involved between IL21R and the adipogenic differentiation process.

Imaging Flow Cytometry and Confocal Immunofluorescence Microscopy of Virus-Host Cell Interactions

Viruses are diverse pathogens that use host factors to enter cells and cause disease. Imaging the entry and replication phases of viruses and their interactions with host factors is key to fully understanding viral infections. This review will discuss how confocal microscopy and imaging flow cytometry are used to investigate virus entry and replication mechanisms in fixed and live cells. Quantification of viral images and the use of cryo-electron microscopy to gather structural information of viruses is also explored. Using imaging to understand how viruses replicate and interact with host factors, we gain insight into cellular processes and identify novel targets to develop antiviral therapeutics and vaccines.

Preliminary Observations on Skeletal Muscle Adaptation and Plasticity in Homer 2-/- Mice

Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR, confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2-/- mice detected the exclusive presence of the adult ε-nAChR isoform excluding denervation. As for NMJ morphology, data were not conclusive, and further work is needed to ascertain whether the null Homer 2 phenotype induces any endplate remodelling. Within the context of adaptation and plasticity, the present data show that Homer 2 is a co-regulator of the normotrophic status in a muscle specific fashion.

Epilepsy-causing Reelin mutations result in impaired secretion and intracellular degradation of mutant proteins

Autosomal dominant lateral temporal epilepsy (ADLTE) is a genetically heterogeneous neurologic disorder clinically characterized by focal seizures with auditory symptoms and/or aphasia. About 20% of ADLTE families segregate disease-causing heterozygous mutations in RELN, a brain-expressed gene encoding the secreted protein Reelin. Using a cell-based secretion assay, we show that pathogenic RELN mutations abolish or significantly reduce secretion of mutant proteins, and that this secretion defect results from impaired trafficking of mutant Reelin along the secretory pathway. Confocal immunofluorescence analysis of transiently transfected cells shows that Reelin mutant proteins are degraded by the autophagy system, as revealed by increased formation of autophagosomes immunoreacting with the autophagy markers p62 and LC3. In addition, LC3 immunoblotting shows a significant increase of autophagy flux due to mutant overexpression. Finally, we show that the secretion defect of mutant proteins can be partially rescued by small-molecule correctors. Altogether, these results suggest that Reelin mutant proteins are not properly secreted and that they are degraded through the autophagy pathway.

HIV Increases the Inhibitory Impact of Morphine and Antiretrovirals on Autophagy in Primary Human Macrophages: Contributions to Neuropathogenesis

HIV enters the CNS early after peripheral infection, establishing reservoirs in perivascular macrophages that contribute to development of HIV-associated neurocognitive disorders (HAND) in 15–40% of people with HIV (PWH) despite effective antiretroviral therapy (ART). Opioid use may contribute to dysregulated macrophage functions resulting in more severe neurocognitive symptoms in PWH taking opioids. Macroautophagy helps maintain quality control in long-lived cell types, such as macrophages, and has been shown to regulate, in part, some macrophage functions in the CNS that contribute to HAND. Using Western blotting and confocal immunofluorescence in primary human macrophages, we demonstrated that morphine and a commonly prescribed ART regimen induce bulk autophagy. Morphine and ART also inhibited completion of autophagy. HIV infection increased these inhibitory effects. We also examined two types of selective autophagy that degrade aggregated proteins (aggrephagy) and dysfunctional mitochondria (mitophagy). Morphine and ART inhibited selective autophagy mediated by p62 regardless of HIV infection, and morphine inhibited mitophagic flux in HIV-infected cells demonstrating potential mitotoxicity. These results indicate that inhibition of autophagy, both in bulk and selective, in CNS macrophages may mediate neurocognitive dysfunction in PWH using opioids. Increasing autophagic activity in the context of HIV may represent a novel therapeutic strategy for reducing HAND in these individuals.

Efficient antibacterial polyphosphazene material with potential to prominent wound healing

Reducing the toxicity of silver-based antibacterial agents used in gels and improving their biocompatibility are of great significance in the development of wound dressings. Thus, a highly crosslinked organic–inorganic hybrid porous polyphosphazene nanospheres (PRV-HMSs) were prepared via precipitation polymerization. The structures and properties were investigated. The results indicated that PRV-HMSs have uniform particle size about 500 nm, excellent thermal stability, high silver loading (26.5 wt.%) and remarkable sustained-release properties after loading AgNO3 (up to 2 weeks). The antibacterial effects were investigated with zone of inhibition testing, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), Live/Dead bacterial staining assay and bacterial growth curve assays. The silver-loaded nanospheres have excellent bactericidal effects for E. coli or S. aureus and an average bactericidal rate of 94.02% and 94.67% in the 120 hour long-acting antibacterial test. The cytotoxicity and laser confocal immunofluorescence staining experiments indicate that the simple combination of nanospheres and agar not only give low cytotoxicity, but also significantly promote the activation of macrophages, which showing potential application in the wound dressings based on hydrogel.

Misrouting of Glucagon and Stathmin-2 Towards Lysosomal System of α-Cells in Glucagon Hypersecretion of Diabetes

Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as a protein that resides in α-cell secretory granules, and showed that it regulates glucagon secretion by directing glucagon towards the endolysosomal system in αTC1-6 cells. Here, we hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ) to induce diabetes, Arg-stimulated secretion of glucagon and Stmn2 was augmented. However, cell glucagon content was significantly increased (p<0.001), but Stmn2 levels were reduced (p<0.01) in STZ-treated mice, as measured by both ELISA and immunofluorescence intensity. Expression of Gcg mRNA increased ~4.5 times, while Stmn2 mRNA levels did not change. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A+ lysosomes was dramatically reduced (p<0.001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p<0.01) increased. Further studies were conducted in αTC1-6 cells cultured in media containing high glucose (16.7 mM) for two weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of αTC1-6 cells with the lysosomal inhibitor bafilomycin A1 reduced K+-induced glucagon secretion, suggesting that high glucose may induce glucagon secretion from another lysosomal compartment. Both glucagon and Stmn2 co-localized with Lamp1, which marks secretory lysosomes, in cells cultured in high glucose. We propose that, in addition to enhanced trafficking and secretion through the regulated secretory pathway, the hyperglucagonemia of diabetes may also be due to re-routing of glucagon from the degradative Lamp2A+ lysosome towards the secretory Lamp1+ lysosome.

Liver involvement in patients with coeliac disease: proof of causality using IgA/anti-TG2 colocalisation techniques

AimsDespite clinical evidence of liver involvement in patients with coeliac disease (CeD), there is a lack of a method to prove this association.MethodsOf 146 treatment-naive patients with CeD, 26 had liver dysfunction. Liver biopsies and corresponding small intestinal biopsies were obtained from these 26 patients. Multicolour immunohistochemical and immunofluorescence confocal microscopic studies were performed on paraffin-embedded tissue to detect the IgA/anti-TG2 deposits. Follow-up liver biopsies were taken after a gluten-free diet.ResultsTwenty-six out of the 146 patients (17.8%) with suspected coeliac-associated liver disease on histological examination revealed irregular sinusoidal dilatation in 15 (57.6%), steatohepatitis in 4 (15.3%), non-specific chronic hepatitis in 3 (11.5%), autoimmune hepatitis in 2 (7.6%) biopsies, including cirrhosis in one of them, irregular perisinusoidal fibrosis and changes of non-cirrhotic portal fibrosis in one biopsy each (3.8%). IgA/anti-tTG deposits were observed in 22 (84.6%) liver biopsies by dual immunohistochemistry technique, and in 24 (92.3%) by confocal immunofluorescence technique and in all corresponding duodenal biopsies (100%). Overall, IgA/anti-tTG deposits showed 100% sensitivity, 77% specificity and 85% positive predictive value for establishing an association of extraintestinal pathology and CeD using archived tissues. Follow-up liver biopsies could be obtained in five patients; four of them showed not only resolution of the histological lesions but disappearance of IgA/anti-tTG co-localisation.ConclusionsData of the present study adds to the body of evidence that liver lesions in patients with CeD are disease related and may have been caused by a similar pathogenic mechanism that causes intestinal changes.

A Novel Inflammatory Dendritic Cell That Is Abundant and Contiguous to T Cells in the Kidneys of Patients With Lupus Nephritis

The mechanisms that promote local inflammatory injury during lupus nephritis (LN) flare are largely unknown. Understanding the key immune cells that drive intrarenal inflammation will advance our knowledge of disease pathogenesis and inform the development of new therapeutics for LN management. In this study, we analyzed kidney biopsies from patients with proliferative LN and identified a novel inflammatory dendritic cell (infDC) population that is highly expressed in the LN kidney, but minimally present in healthy human kidneys. During an agnostic evaluation of immune transcript expression in the kidneys of patients with proliferative LN, the most abundantly overexpressed transcript from isolated glomeruli was FCER1G, which encodes the Fc receptor gamma chain (FcRγ). To identify the cell types expressing FcRγ that infiltrate the kidney in LN, studies were done on kidney biopsies from patients with active LN using confocal immunofluorescence (IF) microscopy. This showed that FcRγ is abundantly present in the periglomerular (PG) region of the kidney and to a lesser extent in the tubulointerstitium (TI). Further investigation of the surface markers of these cells showed that they were FcRγ+, MHC II+, CD11c+, CD163+, CD5−, DC-SIGN+, CD64+, CD14+, CD16+, SIRPα+, CD206−, CD68−, CD123−, CD3−, and CD11b−, suggesting the cells were infDCs. Quantification of the infDCs showed an average 10-fold higher level of infDCs in the LN kidney compared to the healthy kidneys. Importantly, IF identified CD3+ T cells to be adjacent to these infDCs in the PG space of the LN kidney, whereas both cell types are minimally present in the healthy kidney. Thus, we have identified a previously undescribed DC in lupus kidneys that may interact with intrarenal T cells and play a role in the pathogenesis of kidney injury during LN flare.

FKBP10 promotes proliferation of glioma cells via activating AKT-CREB-PCNA axis

Background Although the availability of therapeutic options including temozolomide, radiotherapy and some target agents following neurosurgery, the prognosis of glioma patients remains poor. Thus, there is an urgent need to explore possible targets for clinical treatment of this disease. Methods Tissue microarrays and immunohistochemistry were used to detect FKBP10, Hsp47, p-AKT (Ser473), p-CREB (Ser133) and PCNA expression in glioma tissues and xenografts. CCK-8 tests, colony formation assays and xenograft model were performed to test proliferation ability of FKBP10 in glioma cells in vitro and in vivo. Quantitative reverse transcriptase-PCR, western-blotting, GST-pull down, co-immunoprecipitation and confocal-immunofluorescence staining assay were used to explore the molecular mechanism underlying the functions of overexpressed FKBP10 in glioma cells. Results FKBP10 was highly expressed in glioma tissues and its expression was positively correlates with grade, poor prognosis. FKBP10-knockdown suppressed glioma cell proliferation in vitro and subcutaneous/orthotopic xenograft tumor growth in vivo. Silencing of FKBP10 reduced p-AKT (Ser473), p-CREB (Ser133), PCNA mRNA and PCNA protein expression in glioma cells. FKBP10 interacting with Hsp47 enhanced the proliferation ability of glioma cells via AKT-CREB-PCNA cascade. In addition, correlation between these molecules were also found in xenograft tumor and glioma tissues. Conclusions We showed for the first time that FKBP10 is overexpressed in glioma and involved in proliferation of glioma cells by interacting with Hsp47 and activating AKT-CREB-PCNA signaling pathways. Our findings suggest that inhibition of FKBP10 related signaling might offer a potential therapeutic option for glioma patients.