A Combination of Physical and Chemical Treatments Is More Effective in The Preparation of Acellular Uterine Scaffolds

Objective: Decellularized uterine scaffold, as a new achievement in tissue engineering, enables recellularization andregeneration of uterine tissues and supports pregnancy in a fashion comparable to the intact uterus. The acellularmethods are methods preferred in many respects due to their similarity to normal tissue, so it is necessary to try tointroduce an acellularization protocol with minimum disadvantages and maximum advantages. Therefore, this studyaimed to compare different protocols to achieve the optimal uterus decellularization method for future in vitro and invivo bioengineering experiments. Materials and Methods: In this experimental study, rat uteri were decellularized by four different protocols (P) usingsodium dodecyl sulfate (SDS), with different doses and time incubations (P1 and P2), SDS/Triton-X100 sequentially(P3), and a combination of physical (freeze/thaw) and chemical reagents (SDS/Triton X-100). The scaffolds wereexamined by histopathological staining, DNA quantification, MTT assay, blood compatibility assay, FESEM, andmechanical studies. Results: Histology assessment showed that only in P4, cell residues were completely removed. Masson’s trichromestaining demonstrated that in P3, collagen fibers were decreased; however, no damage was observed in the collagenbundles using other protocols. In indirect MTT assays, cell viabilities achieved by all used protocols were significantlyhigher than the native samples. The percentage of red blood cell (RBC) hemolysis in the presence of prepared scaffoldsfrom all 4 protocols was less than 2%. The mechanical properties of none of the obtained scaffolds were significantlydifferent from the native sample except for P3. Conclusion: Uteri decellularized with a combination of physical and chemical treatments (P4) was the most favorabletreatment in our study with the complete removal of cell residue, preservation of the three-dimensional structure,complete removal of detergents, and preservation of the mechanical property of the scaffolds

Neurosteroid-Mediated Regulation of Brain Innate Immunity in HIV/Aids: DHEA-S Suppresses Neurovirulence

Neurosteroids are cholesterol-derived molecules synthesized within the brain, which exert trophic and protective actions. Infection by human and feline immunodeficiency viruses (HIV and FIV, respectively) causes neuroinflammation and neurodegeneration, leading to neurological deficits. Secretion of neuroinflammatory host and viral factors by glia and infiltrating leukocytes mediates the principal neuropathogenic mechanisms during, although the effect of neurosteroids on these processes is unknown. We investigated the interactions between neurosteroid mediated effects and lentivirus infection outcomes. Analyses of HIV-infected uninfected human brains disclosed a reduction in neurosteroid synthesis enzyme expression. Human neurons exposed to supernatants from HIV macrophages exhibited suppressed enzyme expression without reduced cellular viability. HIV human macrophages treated with sulfated dehydroepiandrosterone (DHEA-S) showed suppression of inflammatory gene (IL-1, IL-6, TNF-) expression. IV-infected IV) animals treated daily with 15mg/kg body weight. DHEA-S treatment reduced inflammatory gene transcripts (IL-1, TNF-, CD3, GFAP) in brain compared to vehicle-(-cyclodextrin)-treated FIV animals similar to levels found in vehicle treated FIV animals. DHEA-S treatment also increased CD4T-cell levels and prevented neurobehavioral deficits and neuronal loss among FIV animals, compared to vehicle-treated FIV animals. Reduced neuronal neuro-steroid synthesis was evident in lentivirus infections, but treatment with DHEA-S limited neuroinflammation and prevented neurobehavioral deficits. Neurosteroid-derived therapies could be effective in the treatment of virus- or inflammation-mediated neurodegeneration

Effect of Hypoxia Preconditioned Adipose-Derived Mesenchymal Stem Cell Conditioned Medium on Cerulein-Induced Acute Pancreatitis in Mice

Purpose: Acute pancreatitis (AP) is an inflammatory disorder distinguished by tissue injury and inflammation of the pancreas. Using paracrine potential of mesenchymal stem cells (MSCs) provides a useful clinical approach in treating inflammatory diseases. We investigated the therapeutic effects of adipose-derived MSC conditioned medium (CM) and hypoxia preconditioned adipose-derived MSC conditioned medium (HCM) in cerulein-induced AP in mice. Methods: AP was induced in C57BL/6 mice by intraperitoneal injection of cerulein (75 μg/ kg/h × 7 times). One hour following the last injection of cerulein, mice were treated with intraperitoneal injection of CM and HCM (500 µL/mice/30 min × 3 times). Twelve hours following the treatment, serum levels of amylase and lipase were measured. In addition, pancreas pathological changes, immunohistochemical examinations for evaluation of IL-6 expression and pancreatic myeloperoxidase (MPO) enzyme activity were analyzed. Results: The in vitro results of the morphological, differentiation and immunophenotyping analyses confirmed that hypoxia preconditioned MSCs (HP-MSCs) conserve MSCs characteristics after preconditioning. However, HP-MSCs significantly expressed high mRNA level of hypoxia inducible factor 1-α and higher level of total protein. The in vivo findings of the current study showed that CM and HCM significantly reduced the amylase & lipase activity, the severity of pancreas tissue injury and the expression of IL-6 and MPO enzyme activity compared with the AP group. However, no significant difference between CM and HCM groups was demonstrated. Conclusion: Use of CM and HCM can attenuate cerulein-induced AP and decrease inflammation in the pancreas tissue in AP mice

Interactions between human immunodeficiency virus (HIV)-1 Vpr expression and innate immunity influence neurovirulence

<p>Abstract</p> <p>Background</p> <p>Viral diversity and abundance are defining properties of human immunodeficiency virus (HIV)-1's biology and pathogenicity. Despite the increasing availability of antiretroviral therapy, HIV-associated dementia (HAD) continues to be a devastating consequence of HIV-1 infection of the brain although the underlying disease mechanisms remain uncertain. Herein, molecular diversity within the HIV-1 non-structural gene, Vpr, was examined in RNA sequences derived from brain and blood of HIV/AIDS patients with or without HIV-associated dementia (HAD) together with the ensuing pathobiological effects.</p> <p>Results</p> <p>Cloned brain- and blood-derived full length <it>vpr </it>alleles revealed that amino acid residue 77 within the brain-derived alleles distinguished HAD (77Q) from non-demented (ND) HIV/AIDS patients (77R) (<it>p </it>< 0.05) although <it>vpr </it>transcripts were more frequently detected in HAD brains (<it>p </it>< 0.05). Full length HIV-1 clones encoding the 77R-ND residue induced higher <it>IFN-α</it>, <it>MX1 </it>and <it>BST-2 </it>transcript levels in human glia relative to the 77Q-HAD encoding virus (<it>p </it>< 0.05) but both viruses exhibited similar levels of gene expression and replication. Myeloid cells transfected with 77Q-(p<it>Vpr77Q-HAD</it>), 77R (p<it>Vpr77R-ND</it>) or Vpr null (p<it>Vpr</it>^<it>(-)</it>)-containing vectors showed that the p<it>Vpr77R-ND </it>vector induced higher levels of immune gene expression (<it>p </it>< 0.05) and increased neurotoxicity (<it>p </it>< 0.05). Vpr peptides (amino acids 70-96) containing the 77Q-HAD or 77R-ND motifs induced similar levels of cytosolic calcium activation when exposed to human neurons. Human glia exposed to the 77R-ND peptide activated higher transcript levels of <it>IFN-α</it>, <it>MX1</it>, <it>PRKRA </it>and <it>BST-2 </it>relative to 77Q-HAD peptide (<it>p </it>< 0.05). The Vpr 77R-ND peptide was also more neurotoxic in a concentration-dependent manner when exposed to human neurons (<it>p </it>< 0.05). Stereotaxic implantation of full length Vpr, 77Q-HAD or 77R-ND peptides into the basal ganglia of mice revealed that full length Vpr and the 77R-ND peptide caused greater neurobehavioral deficits and neuronal injury compared with 77Q-HAD peptide-implanted animals (<it>p </it>< 0.05).</p> <p>Conclusions</p> <p>These observations underscored the potent neuropathogenic properties of Vpr but also indicated viral diversity modulates innate neuroimmunity and neurodegeneration.</p

Proteinase-activated receptor 2 modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis

The proteinase-activated receptors (PARs) are widely recognized for their modulatory properties of inflammation and neurodegeneration. We investigated the role of PAR2 in the pathogenesis of multiple sclerosis (MS) in humans and experimental autoimmune encephalomyelitis (EAE) in mice. PAR2 expression was increased on astrocytes and infiltrating macrophages in human MS and murine EAE central nervous system (CNS) white matter (P < 0.05). Macrophages and astrocytes from PAR2 wild-type (WT) and knockout (KO) mice exhibited differential immune gene expression with PAR2 KO macrophages showing significantly higher interleukin 10 production after lipopolysaccharide stimulation (P < 0.001). PAR2 activation in macrophages resulted in the release of soluble oligodendrocyte cytotoxins (P < 0.01). Myelin oligodendrocyte glycoprotein–induced EAE caused more severe inflammatory gene expression in the CNS of PAR2 WT animals (P < 0.05), together with enhanced T cell proliferation and interferon γ production (P < 0.05), compared with KO littermates. Indeed, PAR2 WT animals showed markedly greater microglial activation and T lymphocyte infiltration accompanied by worsened demyelination and axonal injury in the CNS compared with their PAR2 KO littermates. Enhanced neuropathological changes were associated with a more severe progressive relapsing disease phenotype (P < 0.001) in WT animals. These findings reveal previously unreported pathogenic interactions between CNS PAR2 expression and neuroinflammation with ensuing demyelination and axonal injury

Allopregnanolone and neuroinflammation: a focus on multiple sclerosis

The progesterone derivative, allopregnanolone (ALLO), is one of the most widely studied compounds among neurosteroids. Through interactions with GABA-A receptors expressed by neurons and glial cells, ALLO has been shown to affect diverse aspects of neural cell physiology, including cell proliferation and survival, migration and gene expression. Recent data point to important roles for ALLO in different neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and multiple sclerosis (MS). Dysregulation in ALLO biosynthesis pathways has been reported in brain tissue from MS patients as well as in the central nervous system (CNS) tissue derived from MS animal models. Administration of ALLO has been shown to ameliorate neurobehavioral deficits together with neuropathology and inflammation in the CNS of animals with autoimmune demyelination. These findings are in line with previous reports indicating growth- and differentiation-promoting actions of ALLO on neurons and glial cells as well as its neuroprotective effects in the context of other CNS diseases. Nonetheless, these findings have also raised the possibility that ALLO might influence leukocyte biology and associated neuroinflammatory mechanisms independent of its neuroregenerative properties. Herein, we review the current knowledge regarding the role of ALLO in the pathogenesis of multiple sclerosis, and discuss the potential cell and molecular pathways that might be influenced by ALLO in the context of disease

Targeting proteinase-activated receptors: Therapeutic potential and challenges

Proteinase-activated receptors (PARs), a family of four seven-transmembrane G protein-coupled receptors, act as targets for signalling by various proteolytic enzymes. PARs are characterized by a unique activation mechanism involving the proteolytic unmasking of a tethered ligand that stimulates the receptor. Given the emerging roles of these receptors in cancer as well as in disorders of the cardiovascular, musculoskeletal, gastrointestinal, respiratory and central nervous system, PARs have become attractive targets for the development of novel therapeutics. In this Review we summarize the mechanisms by which PARs modulate cell function and the roles they can have in physiology and diseases. Furthermore, we provide an overview of possible strategies for developing PAR antagonists. © 2012 Macmillan Publishers Limited. All rights reserved

Investigating the Effect of rs3783605 Single-nucleotide Polymorphism on the Activity of VCAM-1 Promoter in Human Umbilical Vein Endohelial Cells

The interaction between immune cells and endothelial lining of blood vessels is vital in many processes such as inflammatory and immune responses as well as cancer cell metastasis. The expression level of VCAM-1 is regulated by many factors including the promoter activity that is possibly affected by the single nucleotide polymorphisms (SNPs) present in the promoter. There are previous reports suggesting an important role for rs3783605 at -420 position in the pathogenesis of VCAM1-associated diseases. This is possibly due to the effect of this SNP on promoter activity and gene expression. Therefore, present study was designed to investigate the effect of rs3783605 on the activity of VCAM-1 gene promoter in human umbilical vein endothelial cells (HUVEC). In this study, two appropriate expression vectors containing VCAM1 promoter with different alleles of rs3783605 were constructed to express the Green Fluorescent Protein (GFP). Expression vectors were transfected into HUVECs and their EGFP expression level was assessed by the fluorescent microscopy and real-time PCR. Bright green fluorescence was seen in the HUVECs transfected by expression vector containing CMV promoter. The expression level in the cells transfected by vector containing promoter with A allele of rs3783605 was 0.14888 folds and G allele was about 0.37851 folds of cells transfected by vector having CMV promoter (p<0.001). Moreover, HUVECs transfected by G allele of rs3783605 showed about 2-fold higher transcriptional activity compared with the A allele, (p=0.049). Our findings showed that rs3783605 polymorphism may play a role in VCAM-1 gene expression. Therefore, it is likely that it may have an important role in the pathogenesis of VCAM1-associated diseases and tumor metastases