Functional Characterisation of Alpha-Galactosidase A Mutations as a Basis for a New Classification System in Fabry Disease

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The study has been supported partially by an unrestricted scientific grant from Shire Human Genetic Therapies (Germany

Subsequent cultivation of chondrocytes and mesenchymal stem cells on the devitalised tissue

The regeneration of cartilage lesions still represents a major challenge. Cartilage has a tissue-specific architecture, complicating recreation by synthetic biomaterials. A novel approach for reconstruction is the use of devitalised cartilage. Treatment with high hydrostatic pressure (HHP) achieves devitalisation while biomechanical properties are remained. Therefore, in the present study, cartilage was devitalised using HHP treatment and the potential for revitalisation with chondrocytes and mesenchymal stem cells (MSCs) was investigated. The devitalisation of cartilage was performed by application of 480 MPa over 10 minutes. Effective cellular inactivation was demonstrated by the trypan blue exclusion test and DNA quantification. Histology and electron microscopy examinations showed undamaged cartilage structure after HHP treatment. For revitalisation chondrocytes and MSCs were cultured on devitalised cartilage without supplementation of chondrogenic growth factors. Both chondrocytes and MSCs significantly increased expression of cartilage- specific genes. ECM stainings showed neocartilage-like structure with positive AZAN staining as well as collagen type II and aggrecan deposition after three weeks of cultivation. Our results showed that HHP treatment caused devitalisation of cartilage tissue. ECM proteins were not influenced, thus, providing a scaffold for chondrogenic differentiation of MSCs and chondrocytes. Therefore, using HHP-treated tissue might be a promising approach for cartilage repair

The role of microbiota in primary sclerosing cholangitis and related biliary malignancies

Primary sclerosing cholangitis (PSC) is an immune-related cholangiopathy characterized by biliary inflammation, cholestasis, and multifocal bile duct strictures. It is associated with high rates of progression to end-stage liver disease as well as a significant risk of cholangiocarcinoma (CCA), gallbladder cancer, and colorectal carcinoma. Currently, no effective medical treatment with an impact on the overall survival is available, and liver transplantation is the only curative treatment option. Emerging evidence indicates that gut microbiota is associated with disease pathogenesis. Several studies analyzing fecal and mucosal samples demonstrate a distinct gut microbiome in individuals with PSC compared to healthy controls and individuals with inflammatory bowel disease (IBD) without PSC. Experimental mouse and observational human data suggest that a diverse set of microbial functions may be relevant, including microbial metabolites and bacterial processing of pharmacological agents, bile acids, or dietary compounds, altogether driving the intrahepatic inflammation. Despite critical progress in this field over the past years, further functional characterization of the role of the microbiota in PSC and related malignancies is needed. In this review, we discuss the available data on the role of the gut microbiome and elucidate important insights into underlying pathogenic mechanisms and possible microbe-altering interventions

NLRP3 inflammasome activation is required for fibrosis development in NAFLD

NLR inflammasomes, caspase 1 activation platforms critical for processing key pro-inflammatory cytokines, have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). As the direct role of the NLRP3 inflammasome remains unclear, we tested effects of persistent NLRP3 activation as a contributor to NAFLD development and, in particular, as a modulator of progression from benign hepatic steatosis to steatohepatitis during diet-induced NAFLD. Gain of function tamoxifen-inducible Nlrp3 knock-in mice allowing for in vivo temporal control of NLRP3 activation and loss of function Nlrp3 knockout mice were placed on short-term choline-deficient amino acid-defined (CDAA) diet, to induce isolated hepatic steatosis or long-term CDAA exposure, to induce severe steatohepatitis and fibrosis, respectively. Expression of NLRP3 associated proteins was assessed in liver biopsies of a well-characterized group of patients with the full spectrum of NAFLD. Nlrp3−/− mice were protected from long-term feeding CDAA-induced hepatomegaly, liver injury, and infiltration of activated macrophages. More importantly, Nlrp3−/− mice showed marked protection from CDAA-induced liver fibrosis. After 4 weeks on CDAA diet, wild-type (WT) animals showed isolated hepatic steatosis while Nlrp3 knock-in mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis. In the liver samples of patients with NAFLD, inflammasome components were significantly increased in those patients with nonalcoholic steatohepatitis (NASH) when compared to those with non-NASH NAFLD with mRNA levels of pro-IL1 beta correlated to levels of COL1A1. Our study uncovers a crucial role for the NLRP3 inflammasome in the development of NAFLD. These findings may lead to novel therapeutic strategies aimed at halting the progression of hepatic steatosis to the more severe forms of this disease.Fil: Wree, Alexander. University of California at San Diego; Estados Unidos. University Hospital Essen; AlemaniaFil: McGeough, Matthew D.. University of California at San Diego; Estados UnidosFil: Peña, Carla A.. University of California at San Diego; Estados UnidosFil: Schlattjan, Martin. University Hospital Essen; AlemaniaFil: Li, Hongying. University of California at San Diego; Estados UnidosFil: Inzaugarat, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Messer, Karen. University of California at San Diego; Estados UnidosFil: Canbay, Ali. University Hospital Essen; AlemaniaFil: Hoffman, Hal M.. University of California at San Diego; Estados Unidos. Ludwig Institute of Cancer Research; Estados UnidosFil: Feldstein, Ariel E.. University of California at San Diego; Estados Unido

Ultrahigh field MRI determination of water diffusion rates in ex vivo human lenses of different age

BACKGROUND: The development of presbyopia is correlated with increased lens stiffness. To reveal structural changes with age, ultrahigh field magnetic resonance imaging (UHF-MRI) was used to analyze water diffusion in differently aged human lenses ex vivo. METHODS: After enucleation lens extractions were performed. Lenses were photographed, weighed, and embedded in 0.5% agarose dissolved in culture medium. UHF-MRI was conducted to analyze anatomical characteristics of the lens using T2-weighted Turbo-RARE imaging and to obtain apparent diffusion coefficients (ADC) measurements. A Gaussian fit routine was used to examine the ADC histograms. RESULTS: An age-dependent increase in lens wet weight, lens thickness, and lens diameter was found (P<0.001). T2-weighted images revealed a hyperintense lens cortex and a gradually negative gradient in signal intensity towards the nucleus. ADC histograms of the lens showed bimodal distributions (lower ADC values mainly located in the nucleus and higher ADC values mainly located in the cortex), which did not change significantly with age [βPeak1=1.96E-7 (-20E-7, 10E-7), P=0.804 or βPeak2=15.4E-7 (-10E-7, 40E-7), P=0.276; respectively]. CONCLUSIONS: Clinically relevant age dependent lens hardening is probably not correlated with ADC changes within the nucleus, which could be confirmed by further measurements

MIPModDB: a central resource for the superfamily of major intrinsic proteins

The channel proteins belonging to the major intrinsic proteins (MIP) superfamily are diverse and are found in all forms of life. Water-transporting aquaporin and glycerol-specific aquaglyceroporin are the prototype members of the MIP superfamily. MIPs have also been shown to transport other neutral molecules and gases across the membrane. They have internal homology and possess conserved sequence motifs. By analyzing a large number of publicly available genome sequences, we have identified more than 1000 MIPs from diverse organisms. We have developed a database MIPModDB which will be a unified resource for all MIPs. For each MIP entry, this database contains information about the source, gene structure, sequence features, substitutions in the conserved NPA motifs, structural model, the residues forming the selectivity filter and channel radius profile. For selected set of MIPs, it is possible to derive structure-based sequence alignment and evolutionary relationship. Sequences and structures of selected MIPs can be downloaded from MIPModDB database which is freely available at http://bioinfo.iitk.ac.in/MIPModDB

Cytoarchitectonic and chemoarchitectonic characterization of the prefrontal cortical areas in the mouse

This study describes cytoarchitectonic criteria to define the prefrontal cortical areas in the mouse brain (C57BL/6 strain). Currently, well-illustrated mouse brain stereotaxic atlases are available, which, however, do not provide a description of the distinctive cytoarchitectonic characteristics of individual prefrontal areas. Such a description is of importance for stereological, neuronal tracing, and physiological, molecular and neuroimaging studies in which a precise parcellation of the prefrontal cortex (PFC) is required. The present study describes and illustrates: the medial prefrontal areas, i.e., the infralimbic, prelimbic, dorsal and ventral anterior cingulate and Fr2 area; areas of the lateral PFC, i.e., the dorsal agranular insular cortical areas and areas of the ventral PFC, i.e., the lateral, ventrolateral, ventral and medial orbital areas. Each cytoarchitectonically defined boundary is corroborated by one or more chemoarchitectonic stainings, i.e., acetylcholine esterase, SMI32, SMI311, dopamine, parvalbumin, calbindin and myelin staining

The neurobiology of Etruscan shrew active touch

The Etruscan shrew, Suncus etruscus, is not only the smallest terrestrial mammal, but also one of the fastest and most tactile hunters described to date. The shrew's skeletal muscle consists entirely of fast-twitch types and lacks slow fibres. Etruscan shrews detect, overwhelm, and kill insect prey in large numbers in darkness. The cricket prey is exquisitely mechanosensitive and fast-moving, and is as big as the shrew itself. Experiments with prey replica show that shape cues are both necessary and sufficient for evoking attacks. Shrew attacks are whisker guided by motion- and size-invariant Gestalt-like prey representations. Shrews often attack their prey prior to any signs of evasive manoeuvres. Shrews whisk at frequencies of approximately 14 Hz and can react with latencies as short as 25–30 ms to prey movement. The speed of attacks suggests that shrews identify and classify prey with a single touch. Large parts of the shrew's brain respond to vibrissal touch, which is represented in at least four cortical areas comprising collectively about a third of the cortical volume. Etruscan shrews can enter a torpid state and reduce their body temperature; we observed that cortical response latencies become two to three times longer when body temperature drops from 36°C to 24°C, suggesting that endothermy contributes to the animal's high-speed sensorimotor performance. We argue that small size, high-speed behaviour and extreme dependence on touch are not coincidental, but reflect an evolutionary strategy, in which the metabolic costs of small body size are outweighed by the advantages of being a short-range high-speed touch and kill predator

Are Metastases from Metastases Clinical Relevant? Computer Modelling of Cancer Spread in a Case of Hepatocellular Carcinoma

Background: Metastasis formation remains an enigmatic process and one of the main questions recently asked is whether metastases are able to generate further metastases. Different models have been proposed to answer this question; however, their clinical significance remains unclear. Therefore a computer model was developed that permits comparison of the different models quantitatively with clinical data and that additionally predicts the outcome of treatment interventions. Methods: The computer model is based on discrete events simulation approach. On the basis of a case from an untreated patient with hepatocellular carcinoma and its multiple metastases in the liver, it was evaluated whether metastases are able to metastasise and in particular if late disseminated tumour cells are still capable to form metastases. Additionally, the resection of the primary tumour was simulated. The simulation results were compared with clinical data. Results: The simulation results reveal that the number of metastases varies significantly between scenarios where metastases metastasise and scenarios where they do not. In contrast, the total tumour mass is nearly unaffected by the two different modes of metastasis formation. Furthermore, the results provide evidence that metastasis formation is an early event and that late disseminated tumour cells are still capable of forming metastases. Simulations also allow estimating how the resection of the primary tumour delays the patient’s death. Conclusion: The simulation results indicate that for this particular case of a hepatocellular carcinoma late metastases, i.e.

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

Background & Aims: NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods: We fed foz/foz and wild-type mice an atherogenic diet for 16 weeks, gavaged MCC950 or vehicle until 24 weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6 weeks and determined the effects on liver fibrosis. Results: In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24 weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion: MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH.Supported by NIH Project grants R2 AA023574 and U01 AA022489 (to AEF), Australian NHMRC project grants 1084136 and 1044288 (to GCF), and 1086786 (to MAC and AABR), and Deutsche Forschungsgemeinschaft WR 173/3-1 (to AW). Matthew Cooper is an NHMRC Principle Research Fellow (1059354