Glial cell type-specific changes in spinal dipeptidyl peptidase 4 expression and effects of its inhibitors in inflammatory and neuropatic pain

Altered pain sensations such as hyperalgesia and allodynia are characteristic features of various pain states, and remain difficult to treat. We have shown previously that spinal application of dipeptidyl peptidase 4 (DPP4) inhibitors induces strong antihyperalgesic effect during inflammatory pain. In this study we observed low level of DPP4 mRNA in the rat spinal dorsal horn in physiological conditions, which did not change significantly either in carrageenan-induced inflammatory or partial nerve ligation-generated neuropathic states. In naive animals, microglia and astrocytes expressed DPP4 protein with one and two orders of magnitude higher than neurons, respectively. DPP4 significantly increased in astrocytes during inflammation and in microglia in neuropathy. Intrathecal application of two DPP4 inhibitors tripeptide isoleucin-prolin-isoleucin (IPI) and the antidiabetic drug vildagliptin resulted in robust opioid-dependent antihyperalgesic effect during inflammation, and milder but significant opioid-independent antihyperalgesic action in the neuropathic model. The opioid-mediated antihyperalgesic effect of IPI was exclusively related to mu-opioid receptors, while vildagliptin affected mainly delta-receptor activity, although mu- and kappa-receptors were also involved. None of the inhibitors influenced allodynia. Our results suggest pathology and glia-type specific changes of DPP4 activity in the spinal cord, which contribute to the development and maintenance of hyperalgesia and interact with endogenous opioid systems

Nesfatin-1/NUCB2 as a Potential New Element of Sleep Regulation in Rats.

STUDY OBJECTIVES: Millions suffer from sleep disorders that often accompany severe illnesses such as major depression; a leading psychiatric disorder characterized by appetite and rapid eye movement sleep (REMS) abnormalities. Melanin-concentrating hormone (MCH) and nesfatin-1/NUCB2 (nesfatin) are strongly co - expressed in the hypothalamus and are involved both in food intake regulation and depression. Since MCH was recognized earlier as a hypnogenic factor, we analyzed the potential role of nesfatin on vigilance. DESIGN: We subjected rats to a 72 h-long REMS deprivation using the classic flower pot method, followed by a 3 h-long 'rebound sleep'. Nesfatin mRNA and protein expressions as well as neuronal activity (Fos) were measured by quantitative in situ hybridization technique, ELISA and immunohistochemistry, respectively, in 'deprived' and 'rebound' groups, relative to controls sacrificed at the same time. We also analyzed electroencephalogram of rats treated by intracerebroventricularly administered nesfatin-1, or saline. RESULTS: REMS deprivation downregulated the expression of nesfatin (mRNA and protein), however, enhanced REMS during 'rebound' reversed this to control levels. Additionally, increased transcriptional activity (Fos) was demonstrated in nesfatin neurons during 'rebound'. Centrally administered nesfatin-1 at light on reduced REMS and intermediate stage of sleep, while increased passive wake for several hours and also caused a short-term increase in light slow wave sleep. CONCLUSIONS: The data designate nesfatin as a potential new factor in sleep regulation, which fact can also be relevant in the better understanding of the role of nesfatin in the pathomechanism of depression

Recruitment and Activation of Pancreatic Stellate Cells from the Bone Marrow in Pancreatic Cancer: A Model of Tumor-Host Interaction

BACKGROUND AND AIMS: Chronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC) contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas. METHODS: Whole bone marrow was harvested from male β-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA). Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation. RESULTS: GFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC) population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3. CONCLUSIONS: This study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that contribute to the activated PaSC population in chronic pancreatitis and pancreatic cancer have different phenotypes, and may play important roles in these diseases. Further, bone marrow transplantation may provide a useful model for the study of tumor-host interactions in cancer and pancreatitis

2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls

Abstract Background Chronic obstructive pulmonary disease (COPD) is associated with several extra-pulmonary effects of which skeletal muscle wasting is one of the most common and contributes to reduced quality of life, increased morbidity and mortality. The molecular mechanisms leading to muscle wasting are not fully understood. Proteomic analysis of human skeletal muscle is a useful approach for gaining insight into the molecular basis for normal and pathophysiological conditions. Methods To identify proteins involved in the process of muscle wasting in COPD, we searched differentially expressed proteins in the vastus lateralis of COPD patients with low fat free mass index (FFMI), as a surrogate of muscle mass (COPDL, n = 10) (FEV1 33 ± 4.3% predicted, FFMI 15 ± 0.2 Kg.m−2), in comparison to patients with COPD and normal FFMI (COPDN, n = 8) and a group of age, smoking history, and sex matched healthy controls (C, n = 9) using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, combined with mass spectrometry (MS). The effect of silencing DOT1L protein expression on markers of cell arrest was analyzed in skeletal muscle satellite cells (HSkMSCs) in vitro and assessed by qPCR and Western blotting. Results A subset of 7 proteins was differentially expressed in COPDL compared to both COPDN and C. We found an increased expression of proteins associated with muscle homeostasis and protection against oxidative stress, and a decreased expression of structural muscle proteins and proteins involved in myofibrillogenesis, cell proliferation, cell cycle arrest and energy production. Among these was a decreased expression of the histone methyltransferase DOT1L. In addition, silencing of the DOT1L gene in human skeletal muscle satellite cells in vitro was significantly related to up regulation of p21 WAF1/Cip1/CDKN1A, a marker of cell arrest and ageing. Conclusions 2D-DIGE coupled with MS identified differences in the expression of several proteins in the wasted vastus lateralis that are relevant to the disease process. Down regulation of DOT1L in the vastus lateralis of COPDL patients may mediate the muscle wasting process through up regulation of markers of cell arrest and senescence

Targets of wnt/ß-catenin transcription in penile carcinoma

Penile squamous cell carcinoma (PeCa) is a rare malignancy and little is known regarding the molecular mechanisms involved in carcinogenesis of PeCa. The Wnt signaling pathway, with the transcription activator ß-catenin as a major transducer, is a key cellular pathway during development and in disease, particularly cancer. We have used PeCa tissue arrays and multi-fluorophore labelled, quantitative, immunohistochemistry to interrogate the expression of WNT4, a Wnt ligand, and three targets of Wnt-ß-catenin transcription activation, namely, MMP7, cyclinD1 (CD1) and c-MYC in 141 penile tissue cores from 101 unique samples. The expression of all Wnt signaling proteins tested was increased by 1.6 to 3 fold in PeCa samples compared to control tissue (normal or cancer adjacent) samples (p<0.01). Expression of all proteins, except CD1, showed a significant decrease in grade II compared to grade I tumors. High magnification, deconvolved confocal images were used to measure differences in co-localization between the four proteins. Significant (p<0.04-0.0001) differences were observed for various permutations of the combinations of proteins and state of the tissue (control, tumor grades I and II). Wnt signaling may play an important role in PeCa and proteins of the Wnt signaling network could be useful targets for diagnosis and prognostic stratification of disease

Genome-wide mRNA expression profiling in vastus lateralis of COPD patients with low and normal fat free mass index and healthy controls

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) has significant systemic effects beyond the lungs amongst which muscle wasting is a prominent contributor to exercise limitation and an independent predictor of morbidity and mortality. The molecular mechanisms leading to skeletal muscle dysfunction/wasting are not fully understood and are likely to be multi-factorial. The need to develop therapeutic strategies aimed at improving skeletal muscle dysfunction/wasting requires a better understanding of the molecular mechanisms responsible for these abnormalities. Microarrays are powerful tools that allow the investigation of the expression of thousands of genes, virtually the whole genome, simultaneously. We aim at identifying genes and molecular pathways involved in skeletal muscle wasting in COPD. METHODS: We assessed and compared the vastus lateralis transcriptome of COPD patients with low fat free mass index (FFMI) as a surrogate of muscle mass (COPD(L)) (FEV(1) 30 ± 3.6%pred, FFMI 15 ± 0.2 Kg.m(−2)) with patients with COPD and normal FFMI (COPD(N)) (FEV(1) 44 ± 5.8%pred, FFMI 19 ± 0.5 Kg.m(−2)) and a group of age and sex matched healthy controls (C) (FEV(1) 95 ± 3.9%pred, FFMI 20 ± 0.8 Kg.m(−2)) using Agilent Human Whole Genome 4x44K microarrays. The altered expression of several of these genes was confirmed by real time TaqMan PCR. Protein levels of P21 were assessed by immunoblotting. RESULTS: A subset of 42 genes was differentially expressed in COPD(L) in comparison to both COPD(N) and C (PFP < 0.05; −1.5 ≥ FC ≥ 1.5). The altered expression of several of these genes was confirmed by real time TaqMan PCR and correlated with different functional and structural muscle parameters. Five of these genes (CDKN1A, GADD45A, PMP22, BEX2, CGREF1, CYR61), were associated with cell cycle arrest and growth regulation and had been previously identified in studies relating muscle wasting and ageing. Protein levels of CDKN1A, a recognized marker of premature ageing/cell cycle arrest, were also found to be increased in COPD(L). CONCLUSIONS: This study provides evidence of differentially expressed genes in peripheral muscle in COPD patients corresponding to relevant biological processes associated with skeletal muscle wasting and provides potential targets for future therapeutic interventions to prevent loss of muscle function and mass in COPD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12931-014-0139-5) contains supplementary material, which is available to authorized users

Multispectral Fluorescence Imaging Allows for Distinctive Topographic Assessment and Subclassification of Tumor-Infiltrating and Surrounding Immune Cells.

Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis.This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies