Functions of Liver Natural Killer Cells Are Dependent on the Severity of Liver Inflammation and Fibrosis in Chronic Hepatitis C

During chronic hepatitis C virus (HCV) infection, the role of intra-hepatic (IH) natural killer (NK) cells is still controversial. To clarify their functions, we investigated anti-viral and cytotoxic activity of NK cells in human fresh liver biopsies. We compared the functions of IH-NK cells in HCV-infected and NASH patients in physiological conditions as well as after stimulation using flow cytometric and immunohistochemical analyses. Interestingly, few IH-NK cells produced anti-viral cytokine IFN-γ in HCV-infected patients similarly as in non-infected individuals. Spontaneous degranulation activity was extremely low in peripheral NK cells compared to IH-NK cells, and was significantly higher in IH-NK cells from HCV-infected patients compared to non-infected individuals. Immunohistochemical analysis revealed that perforin granules were polarized at the apical pole of IH-NK cells. The presence of CD107a and perforin in IH-NK cells demonstrated that NK cells exerted a cytolytic activity at the site of infection. Importantly, IH-NK cell functions from HCV-infected patients were inducible by specific exogenous stimulations. Upon ex vivo K562 target cell stimulations, the number of degranulating NK cells was significantly increased in the pool of IH-NK cells compared to circulating NK cells. Interestingly, after stimulation, the frequency of IFN-γ-producing IH-NK cells in HCV-infected patients was significantly higher at early stage of inflammation whereas the spontaneous IH-NK cell degranulation activity was significantly impaired in patients with highest inflammation and fibrosis Metavir scores. Our study highlights that some IH-NK cells in HCV-infected patients are able to produce INF-γ and degranulate and that those two activities depend on liver environment including the severity of liver injury. Thus, we conclude that critical roles of IH-NK cells have to be taken into account in the course of the liver pathogenesis associated to chronic HCV infection.     

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.     

On stiffness of scaffolds for bone tissue engineering—a numerical study

Tissue scaffolds are typically designed and fabricated to match native bone properties. However, it is unclear if this would lead to the best tissue ingrowth outcome within the scaffold as neo-tissue keeps changing the stiffness of entire construct. This paper presents a numerical method to address this issue for design optimization and assessment of tissue scaffolds. The elasticity tensors of two different types of bones are weighted by different multipliers before being used as the targets in scaffold design. A cost function regarding the difference between the effective elasticity tensor, calculated by the homogenization technique, and the target tensor, is minimized by using topology optimization procedure. It is found that different stiffnesses can lead to different remodeling results. The comparison confirms that bone remodeling is at its best when the scaffold elastic tensor matches or is slightly higher than the elastic properties of the host bone.     

In vitro and in vivo antimuscarinic effects of (-)cis 2,3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl-1,5 benzothiazepin-4-(5H)one HCl (BTM-1086) in guinea pig peripheral tissues

The potency and selectivity of (-)cis-2,3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl-1,5 benzothiazepin-4-(5H)one HCl (BTM-1086) for muscarinic receptor subtypes was compared in functional assay systems, in guinea pig peripheral tissues, to known reference drugs: atropine (nonselective), pirenzepine (M1), AF-DX 116 (M2) and HHSiD (M3). Like atropine, BTM-1086 was a potent, nonselective, competitive muscarinic antagonist with no detectable antispasmodic activity in urinary bladder or ileal muscle. In vivo, in the guinea pig cystometrogram, BTM-1086 depressed intravesical bladder pressure (PvesP) with the same efficacy and potency as oxybutynin, a drug used clinically for the treatment of urinary incontinence. The pharmacological profile of BTM-1086, however, suggests that it may not be suitable for development for bladder dysfunction disorders.     

Isolation of proteins assembled in lipid body membranes during fat mobilization in cucumber cotyledons

Lipid bodies from fat-mobilizing cotyledons of cucumber and other Cucurbitaceae were investigated. Proteins and glycoproteins were analyzed by electrophoresis and then used to characterize the lipid body membrane at different stages of cell development. Contaminations by other membranes or organelles were ruled out by comparing the main constituents from the endoplasmic reticulum, cytosol, glyoxysomes and protein bodies with the pattern of the lipid body membrane, considering both the prevalent peptides and the dominating glycoproteins. Among the proteins of lipid body membranes in ripening and germinating cotyledons, a 90-kDa peptide was found as unique marker of lipid bodies at the stage of fat mobilization. The 90-kDa protein was purified, and antibodies against it were raised in rabbits. By means of immunoprecipitation and electrophoretic analysis it was demonstrated that the synthesis of the 90-kDa form located in lipid bodies shows a transient increase and subsequent decline, with maximal values being observed at day 3 of germination. At this stage, the rate of de novo synthesis was compared considering lipid body proteins and other organellar proteins. The 90-kDa protein appeared as the lipid body constituent that is synthesized and assembled in the organelle by far at the highest rates.     

Oxiranylphenyl esters from Pimpinella diversifolia

The major components of the essential oil from roots of Pimpinella diversifolia, gathered in the Kumaun Region of India, have been identified as the (+)-Z-2-methyl-2-butenoate (angelate) and (+)-isobutyrate esters of 4-methoxy-2-(E-3-methyloxiranyl)phenol. Aromatic ¹³C NMR resonances of these compounds and their synthetic acetate analog, as well as those of 2-methoxy-4-(E-3-methyloxiranyl)phenyl acetate prepared from isoeugenol, were found to be in excellent agreement with calculated values. Comparison of the EIMS of the natural and synthetic products with those reported for compounds previously identified as 2-methoxy-4-(E-3-methyloxiranyl)phenyl esters indicates that they also have the 4-methoxy-2-(E-3-methyloxiranyl)phenyl structure.