bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in blood microbiota profiles associated with liver fibrosis in obese patients: A pilot analysis
Authors: Lelouvier Benjamin , Servant Florence , Païssé Sandrine , Brunet Anne‐Claire , Benyahya Salah , Serino Matteo , Valle Carine , Ortiz Maria Rosa , Puig Josep , Courtney Michael , Federici Massimo , Fernández‐Real José‐Manuel , Burcelin Rémy , Amar Jacques
Published: September 2016

The early detection of liver fibrosis among patients with nonalcoholic fatty liver disease (NAFLD) is an important clinical need. In view of the suggested role played by bacterial translocation in liver disease and obesity, we sought to investigate the relationship between blood microbiota and liver fibrosis (LF) in European cohorts of patients with severe obesity. We carried out a cross‐sectional study of obese patients, well characterized with respect to the severity of the NAFLD, in the cohort FLORINASH. This cohort has been divided into a discovery cohort comprising 50 Spanish patients and then in a validation cohort of 71 Italian patients. Blood bacterial DNA was analyzed both quantitatively by 16S ribosomal DNA (rDNA) quantitative polymerase chain reaction and qualitatively by 16S rDNA targeted metagenomic sequencing and functional metagenome prediction. Spanish plasma bile acid contents were analyzed by liquid chromatography/mass spectrometry. The 16S rDNA concentration was significantly higher in patients of the discovery cohort with LF. By 16S sequencing, we found specific differences in the proportion of several bacterial taxa in both blood and feces that correlate with the presence of LF, thus defining a specific signature of the liver disease. Several secondary/primary bile acid ratios were also decreased with LF in the discovery cohort. We confirmed, in the validation cohort, the correlation between blood 16S rDNA concentration and LF, whereas we did not confirm the specific bacterial taxa signature, despite a similar trend in patients with more‐severe fibrosis. Conclusion: Changes in blood microbiota are associated with LF in obese patients. Blood microbiota analysis provides potential biomarkers for the detection of LF in this population. (Hepatology 2016;64:2015‐2027).
Forkhead box A2 regulates biliary heterogeneity and senescence during cholestatic liver injury in mice‡
Authors: McDaniel Kelly , Meng Fanyin , Wu Nan , Sato Keisaku , Venter Julie , Bernuzzi Francesca , Invernizzi Pietro , Zhou Tianhao , Kyritsi Konstantina , Wan Ying , Huang Qiaobing , Onori Paolo , Francis Heather , Gaudio Eugenio , Glaser Shannon , Alpini Gianfranco
Published: September 2016

Biliary‐committed progenitor cells (small mouse cholangiocytes; SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, forkhead box A2 (FoxA2), is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Messenger RNA expression in SMCCs and LGCCs was assessed by polymerase chain reaction (PCR) array analysis. Liver tissues and hepatic stellate cells (HSCs) from primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients were tested by real‐time PCR for methylation, senescence, and fibrosis markers. Bile duct ligation (BDL) and multidrug resistance protein 2 (MDR2) knockout mice (MDR2–/–) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes, DNA methyltransferase (DNMT) 1 and DNMT3B. Serum alanine aminotransferase and aspartate aminotransferase levels in nonobese diabetic/severe combined immunodeficiency mice engrafted with SMCCs post‐BDL showed significant changes compared to vehicle‐treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2–/– mouse liver as well as in isolated HSCs by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. Conclusion: The definitive endoderm marker and the positive regulator of biliary development, FoxA2, mediates the therapeutic effect of biliary‐committed progenitor cells during cholestatic liver injury. (Hepatology 2017;65:544‐559).
Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib through the laminin‐332/α3 integrin axis recovery of focal adhesion kinase ubiquitination
Authors: Azzariti Amalia , Mancarella Serena , Porcelli Letizia , Quatrale Anna Elisa , Caligiuri Alessandra , Lupo Luigi , Dituri Francesco , Giannelli Gianluigi
Published: September 2016

In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)‐332, produced by hepatic stellate cells (HSCs). Ln‐332 is the ligand of α3β1 and α6β4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln‐332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln‐332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC‐CM or Ln‐332 inhibited sorafenib's effectiveness in HCC cells expressing both α3β1 and α6β4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln‐332 and HSC‐CM. Hep3B cells expressing α6β4 but lacking the α3 integrin were insensitive to Ln‐332 and HSC‐CM protective effects. Hep3B α3‐positive, but not wild‐type and scramble transfected, cells acquired protection by sorafenib when plated on Ln‐332‐CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal‐regulated kinases 1/2, whereas Ln‐332 and HSC‐CM partially restored the pathways. Silencing FAK, but not extracellular signal‐regulated kinases 1/2, abrogated the protection induced by Ln‐332 and HSC‐CM, suggesting a specific role for FAK. Sorafenib down‐regulated total FAK, inducing its proteasomal degradation, while Ln‐332 and HSC‐CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. Conclusion: This study unveils a novel mechanism of sorafenib resistance depending on the α3β1/Ln‐332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103‐2117).
Extracellular vesicles released by hepatocytes from gastric infusion model of alcoholic liver disease contain a MicroRNA barcode that can be detected in blood
Authors: Eguchi Akiko , Lazaro Raul G. , Wang Jiaohong , Kim Jihoon , Povero Davide , Willliams Brandon , Ho Samuel B. , Stärkel Peter , Schnabl Bernd , Ohno‐Machado Lucila , Tsukamoto Hidekazu , Feldstein Ariel E.
Published: September 2016

Extracellular vesicles (EVs) released during cell stress, or demise, can contain a barcode of the cell origin, including specific microRNAs (miRNAs). Here, we tested the hypothesis that during early alcoholic steatohepatitis (ASH) development, hepatocytes (HCs) release EVs with an miRNA signature that can be measured in circulation. A time‐course experiment showed that after 2 weeks of intragastric infusion, a time point that results in isolated steatosis, there was no increase of blood EVs. After 4 weeks of infusion, mice developed features of early ASH accompanied by a marked increase in the level of EVs in blood (P < 0.05), as well as in culture media of isolated HCs (P < 0.001) and hepatic macrophages (P < 0.001), with HCs being the predominant source of EVs. The transcriptome analysis of HC‐EVs from ASH mice detected differentially expressed miRNAs, including nine significantly up‐regulated and four significantly down‐regulated miRNAs. Target prediction and pathway analyses of the up‐regulated miRNAs identified 121 potential target genes involved in inflammatory and cancer pathways, such as nuclear factor kappa B, EGF, Wnt, and B‐cell lymphoma 2. Three miRNAs, let7f, miR‐29a, and miR‐340, were increased in blood EVs from ASH mice (P < 0.05), but not in blood EVs from three other models of chronic liver injury, including bile duct ligation, nonalcoholic steatohepatitis, and obese mice, as well as EVs released from hepatocytes exposed to ethanol. Blood EV level (P < 0.01) and three miRNAs (P < 0.05) were significantly increased in patients with ambulatory mild ALD as compared to nonalcoholics. Conclusion: Damaged hepatocytes from ASH mice are a key EV source with a specific miRNA cargo, which are specific for ASH‐related liver injury. These findings uncover EVs as a potentially novel diagnostic for ASH. (Hepatology 2017;65:475‐490).
Sofosbuvir and ribavirin for 6 weeks is not effective among people with recent hepatitis C virus infection: The DARE‐C II study
Authors: Martinello Marianne , Gane Edward , Hellard Margaret , Sasadeusz Joe , Shaw David , Petoumenos Kathy , Applegate Tanya , Grebely Jason , Maire Laurence , Marks Philippa , Dore Gregory J. , Matthews Gail V.
Published: September 2016

While interferon‐based therapy has excellent efficacy in acute and recent hepatitis C virus (HCV) infection, the side effect profile limits implementation. Sofosbuvir and ribavirin for 12‐24 weeks is safe and well tolerated in chronic HCV, with efficacy dependent on genotype and disease stage. The aim of this study was to assess the efficacy of sofosbuvir and ribavirin for 6 weeks in individuals with recent HCV infection. In this open‐label study conducted in Australia and New Zealand, adults with recent HCV (duration of infection <12 months) received sofosbuvir 400 mg daily and weight‐based ribavirin (<75 kg, 1,000 mg/day; ≥75 kg, 1,200 mg/day) for 6 weeks. The primary efficacy endpoint was sustained virological response at posttreatment week 12 (SVR12). Nineteen participants commenced sofosbuvir and ribavirin (89% male, 74% with human immunodeficiency virus, 68% genotype 1a). Four (21%) reported a symptomatic HCV seroconversion illness, including 2 with jaundice. At baseline, median HCV RNA was 5.4 log10 IU/mL (interquartile range 4.4‐6.8) and median estimated duration of infection was 37 weeks (interquartile range 27‐41). At the end of treatment, HCV RNA was nonquantifiable in 89% (n = 17). SVR4 and SVR12 were 42% (n = 8) and 32% (n = 6), respectively. Treatment failure was due to nonresponse (n = 2), posttreatment relapse (n = 9), reinfection (n = 1), and loss to follow‐up (n = 1). The regimen was well tolerated with minimal hematological toxicity. SVR12 was related to baseline HCV RNA (≤6 log10 IU/mL, P = 0.018) and early on‐treatment viral kinetics (HCV RNA below the level of quantitation at week 1, P = 0.003). Conclusion: Six weeks of sofosbuvir and ribavirin was safe and well tolerated, but efficacy was suboptimal; further research is needed to determine whether more potent interferon‐free direct‐acting antiviral regimens will allow treatment duration to be shortened in recent, predominantly asymptomatic HCV infection. (Hepatology 2016;64:1911‐1921).
Spacial and Temporal Patterns of Gene Expression After Cardiac MEK1 Gene Transfer Improve Post‐Infarction Remodeling Without Inducing Global Hypertrophy
Authors: Fan Yanying , Yang Yi‐Lin , Yeh Che‐Chung , Mann Michael J.
Published: September 2016

Alteration of mitogen activated protein (MAP) kinase signaling in transgenic mice can ameliorate post‐myocardial infarction (MI) remodeling. However, pre‐existing changes in transgenic hearts and clinically unrealistic transgene expression likely affect the response to injury; it is unknown whether clinically relevant induction of transgene expression in an otherwise normal heart can yield similar benefits. Constitutively active MEK1 (aMEK1) or LacZ adeno‐associated virus 9 (AAV9) vectors were injected into the left ventricular (LV) chambers of mice either just before or after coronary ligation. Hearts were evaluated via Western blot, quantitative polymerase chain reaction, histology, and echocardiography. AAV9‐mediated aMEK1 delivery altered ERK1/2 expression/activation as in transgenic mice. Transgene expression was not immediately detectable but plateaued at 17 days, and therefore did not likely impact acute ischemia as it would in transgenics. With AAV9‐aMEK1 injection just prior to MI, robust expression in the infarct border zone during post‐MI remodeling increased border zone wall thickness and reduced infarct size versus controls at 4 weeks, but did not induce global hypertrophy. Significant improvements in local and global LV function were observed, as were trends toward a preservation of LV volume. Delivery after ligation significantly lowered transgene expression in the infarct border zone and did not yield structural or functional benefits. The primary benefits observed in transgenic mice, ameliorated remodeling, and reduced chronic infarct size, were achievable via clinically relevant gene transfer of aMEK1, supporting ongoing translational efforts. Important differences, however, were observed, and consideration must be given to the timing and distribution of transgene delivery and expression. J. Cell. Biochem. 118: 775–784, 2017. © 2016 Wiley Periodicals, Inc.
Identification of Domains for Efficient Notch Signaling Activity in Immobilized Notch Ligand Proteins
Authors: Liu Ledi , Wada Hiroe , Matsubara Natsuki , Hozumi Katsuto , Itoh Motoyuki
Published: September 2016

Notch is a critical signaling pathway that controls cell fate and tissue homeostasis, but the functional characterization of Notch ligand domains that activate Notch receptors remains incomplete. Here, we established a method for immobilizing Notch ligand proteins onto beads to measure time‐dependent Notch activity after the addition of Notch ligand‐coated beads. A comparison between activities by the Notch ligand found on the cell surface to that of the ligand immobilized on beads showed that immobilized Notch ligand protein produces comparable signal activity during the first 10 h. Follow‐up truncation studies showed that the N‐terminal epidermal growth factor (EGF) repeat three region of delta like canonical Notch ligand 4 (DLL4) or jagged 1 (JAG1) is the minimum region for activating Notch signaling, and the DLL4 EGF repeat three domain may have a role in activation through a mechanism other than by increasing binding affinity. In addition, we found that reconstruction of the DLL4 delta and OSM‐11 (DOS) motif (N257P) resulted in an increase in both binding affinity and signaling activity, which suggests that the role of the DOS motif is conserved among Notch ligands. Furthermore, active DLL4 protein on beads promoted T cell differentiation or inhibited B cell differentiation in vitro, whereas JAG1 proteins on beads did not have any effect. Taken together, our findings provide unambiguous evidence for the role of different Notch ligands and their domains in Notch signal activation, and may be potential tools for controlling Notch signaling activation. J. Cell. Biochem. 118: 785–796, 2017. © 2016 Wiley Periodicals, Inc.
Salivary Amylase as a Marker of Salivary Gland Function in Patients Undergoing Radiotherapy for Oral Cancer
Authors: Vedam V. K. Vaishnavi , Boaz Karen , Natarajan Srikant , Ganapathy Sivadas
Published: September 2016

Background

The aim of this study was to evaluate salivary amylase in patients with primary oral cancer undergoing radiotherapy as the main modality of treatment.

Materials/methods

The study was conducted on ten histologically proven cases of oral cancer undergoing radiotherapy. Stimulated whole saliva was collected at three stages of radiotherapy—0, 3, and 6 weeks. Salivary amylase was estimated using Henry‐Chiamori method and comparison was made with appropriate age‐ and gender‐matched controls.

Results

Salivary amylase levels showed significant decrease in healthy subjects when compared to oral cancer patients (P < 0.001). The latter group also showed changing trend with initial decrease from 0 to 3 weeks followed by increase from 3 to 6 weeks following radiotherapy (P < 0.0528).

Conclusions

The trend in changes in the levels of salivary amylase could be used as a surrogate marker of salivary gland function in patients with oral cancer undergoing radiotherapy as primary treatment.
Evaluation of the LABGEO PT10 Point‐Of‐Care Testing Device: Comparison of Analyte Measurements in Capillary Whole Blood and Lithium Heparin Whole Blood Samples With Those in Central Laboratory
Authors: Lim Jinsook , Kim Hyunjin , Koo Sun Hoe , Kwon Gye Cheol
Published: September 2016

Background

Point‐of‐care (POC) testing device has been widely used because of its rapid availability of results making diagnosis and management as early as possible. Capillary blood can reduce the difficulty of obtaining samples compared to venous blood and allows the prompt testing results. In this study, we evaluated the usefulness of capillary blood in Samsung LABGEO PT10.

Methods

Fifty‐one patients and 18 healthy adults aged between 20 and 65 were enrolled and their capillary and venous blood samples were collected. Venous blood samples were split into lithium heparin (LiHep) tube and serum‐separating tube. Measurements using capillary blood and LiHep whole blood were performed in LABGEO PT10. Serum was used for measurement by Toshiba 2000FR NEO in central laboratory.

Results

In comparison between measurements in LABGEO PT10 using capillary and LiHep whole blood, the slope ranged between 0.9289 and 1.0471, correlation coefficients (R2) were over 0.95 except albumin, high‐density lipoprotein, and total protein. Comparison of measurements in capillary and LiHep whole blood using LABGEO PT10 with those in the central laboratory revealed that the slope ranged between 0.6433 and 1.1364 for capillary whole blood and 0.6255 and 1.1602 for LiHep whole blood except alkaline phosphatase. For most of analytes, R2 were over 0.95.

Conclusion

Measurements in LABGEO PT10 using capillary blood was well correlated with those in LABGEO PT10 using LiHep whole blood and also with in the central laboratory. In conclusion, capillary blood provides reliable measurements and can be trustfully used in LABGEO PT10.
The Reference Intervals for Serum C‐Terminal Agrin Fragment in Healthy Individuals and as a Biomarker for Renal Function in Kidney Transplant Recipients
Authors: Yu Dan , Li Hai‐Xia , Liu Yi , Ying Ze‐Wei , Guo Jing‐Jing , Cao Chen‐Ying , Wang Jia , Li Yuan‐Fang , Yang Hui‐Rong
Published: September 2016

Background

C‐terminal agrin fragment (CAF) has been shown to be a promising new biomarker for kidney function. The aim of this study was to verify the reference intervals for CAF in Chinese healthy adults and to assess the efficiency of CAF for monitoring renal function after transplantation.

Methods

Serum samples were collected from 200 healthy adult subjects and 60 living donor kidney recipients before and on day 1, day 2 and at 6 months after transplantation. We measured serum CAF, creatinine, cystatin C and NGAL concentrations at each time. Estimated glomerular filtration rate (eGFR) was evaluated by Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) equation. Reference intervals for CAF were determined at 2.5th and 97.5th percentiles.

Results

Serum CAF concentrations were observed to be higher in females of old age groups while no significant differences were discovered in males between age groups. There were significant gender‐related differences in CAF in old age groups (50–64 and ≥65 years). Serum CAF correlated positively with serum creatinine, cystatin C and negatively with eGFR on day 1, day 2 and at 6 months after kidney transplantation. CAF and NGAL fell rapidly into the normal range on the second postoperative day, prior to creatinine and cystatin C.

Conclusions

This study verified the reference intervals for serum CAF. CAF could be a potential new biomarker for kidney function monitoring.
Valproic Acid and Lithium Meditate Anti‐Inflammatory Effects by Differentially Modulating Dendritic Cell Differentiation and Function
Authors: Leu Sy‐Jye , Yang Yi‐Yuan , Liu Hsing‐Cheng , Cheng Chieh‐Yu , Wu Yu‐Chen , Huang Ming‐Chyi , Lee Yuen‐Lun , Chen Chi‐Ching , Shen Winston W. , Liu Ko‐Jiunn
Published: September 2016

Valproic acid (VPA), with inhibition activity mainly toward histone deacetylase (HDAC) and Glycogen Synthase Kinase (GSK)‐3, and lithium, with inhibition activity mainly toward GSK‐3, are both prescribed in clinical as mood‐stabilizers and anticonvulsants for the control of bipolar disorder. This study aims to compare the immuno‐modulation activities of VPA and lithium, especially on the differentiation and functions of dendritic cells (DC). Our data show that treatment with VPA or lithium effectively alleviated the severity of collagen‐induced arthritis triggered by LPS in mice. Both agents reduced the serum level of IL‐6 and IL‐10 after LPS challenge in mice. VPA and lithium both induce significant down‐regulation of group I CD1 expression and secretion of IL‐6 during differentiation of human monocyte‐derived immature DC, while they differ in the induction of CD83 and CD86 expression, secretion of IL‐8, IL‐10, and TNF‐α. Upon stimulation of immature DC with LPS, VPA, and lithium both reduced the secretion of IL‐6 and TNF‐α. However, only lithium significantly increased the production of IL‐10, while VPA increased the production of IL‐8 but substantially reduce the secretion of IL‐10 and IL‐23. Treatment with VPA resulted in a reduced capacity of LPS‐stimulated DC to promote the differentiation of T helper 17 cells that are critical in the promotion of inflammatory responses. Taken together, our results suggest that VPA and lithium may differentially modulate inflammation through regulating the capacity of DC to mediate distinct T cell responses, and they may provide a complementary immunomodulatory effects for the treatment of inflammation‐related diseases. J. Cell. Physiol. 232: 1176–1186, 2017. © 2016 Wiley Periodicals, Inc.
Pulsed Electromagnetic Field Exposure Reduces Hypoxia and Inflammation Damage in Neuron‐Like and Microglial Cells
Authors: Vincenzi Fabrizio , Ravani Annalisa , Pasquini Silvia , Merighi Stefania , Gessi Stefania , Setti Stefania , Cadossi Ruggero , Borea Pier Andrea , Varani Katia
Published: September 2016

In the present study, the effect of low‐frequency, low‐energy pulsed electromagnetic fields (PEMFs) has been investigated by using different cell lines derived from neuron‐like cells and microglial cells. In particular, the primary aim was to evaluate the effect of PEMF exposure in inflammation‐ and hypoxia‐induced injury in two different neuronal cell models, the human neuroblastoma‐derived SH‐SY5Y cells and rat pheochromocytoma PC12 cells and in N9 microglial cells. In neuron‐like cells, live/dead and apoptosis assays were performed in hypoxia conditions from 2 to 48 h. Interestingly, PEMF exposure counteracted hypoxia damage significantly reducing cell death and apoptosis. In the same cell lines, PEMFs inhibited the activation of the hypoxia‐inducible factor 1α (HIF‐1α), the master transcriptional regulator of cellular response to hypoxia. The effect of PEMF exposure on reactive oxygen species (ROS) production in both neuron‐like and microglial cells was investigated considering their key role in ischemic injury. PEMFs significantly decreased hypoxia‐induced ROS generation in PC12, SH‐SY5Y, and N9 cells after 24 or 48 h of incubation. Moreover, PEMFs were able to reduce some of the most well‐known pro‐inflammatory cytokines such as tumor necrosis factor–α (TNF‐α), interleukin (IL)‐1β, IL‐6, and IL‐8 release in N9 microglial cells stimulated with different concentrations of LPS for 24 or 48 h of incubation time. These results show a protective effect of PEMFs on hypoxia damage in neuron‐like cells and an anti‐inflammatory effect in microglial cells suggesting that PEMFs could represent a potential therapeutic approach in cerebral ischemic conditions. J. Cell. Physiol. 232: 1200–1208, 2017. © 2016 Wiley Periodicals, Inc.
Krabbe disease: One Hundred years from the bedside to the bench to the bedside
Authors: Wenger David A. , Rafi Mohammad A. , Luzi Paola
Published: September 2016

This Review summarizes the progress in understanding the pathogenesis and treatment of Krabbe disease from the description of five patients in by Knud Krabbe until 2016. To determine the cause of this genetic disease, pathological and chemical analyses of tissues from the nervous systems of patients were performed. It was determined that these patients had a pathological feature known as globoid cell in the brain and that this consisted partially of galactosylceramide, a major sphingolipid component of myelin. The finding that these patients had a deficiency of galactocerebrosidase (GALC) activity opened the way to relatively simple diagnostic testing with easily obtainable tissue samples, studies leading to the purification of GALC, and cloning of the GALC cDNA and gene. The availability of the gene sequence led to the identification of mutations in patients and to the current studies involving the use of viral vectors containing the GALC cDNA to treat experimentally naturally occurring animal models, such as twitcher mice. Currently, treatment of presymptomatic human patients is limited to hematopoietic stem cell transplantation (HSCT). With recent studies showing successful treatment of animal models with a combination of HSCT and viral gene therapy, it is hoped that more effective treatments will soon be available for human patients. For this Review, it is not possible to reference all of the articles contributing to our current state of knowledge about this disease; however, we have chosen those that have influenced our studies by suggesting research paths to pursue. © 2016 Wiley Periodicals, Inc.
Chaperones as potential therapeutics for Krabbe disease
Authors: Graziano Adriana Carol Eleonora , Pannuzzo Giovanna , Avola Rosanna , Cardile Venera
Published: September 2016

Krabbe's disease (KD) is an autosomal recessive, neurodegenerative disorder. It is classified among the lysosomal storage diseases (LSDs). It was first described in , but the genetic defect for the galactocerebrosidase (GALC) gene was not discovered until the beginning of the 1970s, 20 years before the GALC cloning. Recently, in 2011, the crystal structures of the GALC enzyme and the GALC‐product complex were obtained. For this, compared with other LSDs, the research on possible therapeutic interventions is much more recent. Thus, it is not surprising that some treatment options are still under preclinical investigation, whereas their relevance for other pathologies of the same group has already been tested in clinical studies. This is specifically the case for pharmacological chaperone therapy (PCT), a promising strategy for selectively correcting defective protein folding and trafficking and for enhancing enzyme activity by small molecules. These compounds bind directly to a partially folded biosynthetic intermediate, stabilize the protein, and allow completion of the folding process to yield a functional protein. Here, we review the chaperones that have demonstrated potential therapeutics during preclinical studies for KD, underscoring the requirement to invigorate research for KD‐addressed PCT that will benefit from recent insights into the molecular understanding of GALC structure, drug design, and development in cellular models. © 2016 Wiley Periodicals, Inc.
New therapeutic approaches for Krabbe disease: The potential of pharmacological chaperones
Authors: Spratley Samantha J. , Deane Janet E.
Published: September 2016

Missense mutations in the lysosomal hydrolase β‐galactocerebrosidase (GALC) account for at least 40% of known cases of Krabbe disease (KD). Most of these missense mutations are predicted to disrupt the fold of the enzyme, preventing GALC in sufficient amounts from reaching its site of action in the lysosome. The predominant central nervous system (CNS) pathology and the absence of accumulated primary substrate within the lysosome mean that strategies used to treat other lysosomal storage disorders (LSDs) are insufficient in KD, highlighting the still unmet clinical requirement for successful KD therapeutics. Pharmacological chaperone therapy (PCT) is one strategy being explored to overcome defects in GALC caused by missense mutations. In recent studies, several small‐molecule inhibitors have been identified as promising chaperone candidates for GALC. This Review discusses new insights gained from these studies and highlights the importance of characterizing both the chaperone interaction and the underlying mutation to define properly a responsive population and to improve the translation of existing lead molecules into successful KD therapeutics. We also highlight the importance of using multiple complementary methods to monitor PCT effectiveness. Finally, we explore the exciting potential of using combination therapy to ameliorate disease through the use of PCT with existing therapies or with more generalized therapeutics, such as proteasomal inhibition, that have been shown to have synergistic effects in other LSDs. This, alongside advances in CNS delivery of recombinant enzyme and targeted rational drug design, provides a promising outlook for the development of KD therapeutics. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.
Diffusion tensor imaging: A biomarker of outcome in Krabbe's disease
Authors: Poretti Andrea , Meoded Avner , Fatemi Ali
Published: September 2016

Krabbe's disease is a rare autosomal recessive lysosomal disorder resulting from deficiency of β‐galactocerebrosidase that affects primarily cerebral white matter and peripheral nerves. Conventional magnetic resonance imaging (MRI) is sensitive to changes in white matter myelination, but its assessment is based purely on qualitative, visual inspection, and it is subject to interobserver variability and open to reader bias. Diffusion tensor imaging (DTI) is an advanced MRI technique that provides quantitative information about the microscopic structural organization of the white matter and changes in cell density and myelination, and it is a suitable MRI tool for studying Krabbe's disease. This Review discusses the available studies on the application of quantitative DTI analysis to assess white matter changes in patients with Krabbe's disease. Quantitative analysis of DTI scalars, especially radial diffusivity and fractional anisotropy, has been shown to be a sensitive in vivo biomarker of white matter microstructural damage in Krabbe's disease, to detect early white matter injury in asymptomatic neonates with Krabbe's disease, to predict motor and cognitive functions after hematopoietic stem cell transplantation (HSCT), and to serve as a measurement for monitoring effects of HSCT on white matter development in Krabbe's disease. © 2016 Wiley Periodicals, Inc.
Cellular transplant therapies for globoid cell leukodystrophy: Preclinical and clinical observations
Authors: Maher Keri R. , Yeager Andrew M.
Published: September 2016

Globoid cell leukodystrophy (GLD) is a progressive neurodegenerative disorder caused by the deficiency of galactocerebrosidase (GALC), resulting in accumulation of toxic metabolites in neural tissues. Clinically variable based on age of onset, infantile GLD is generally a rapidly fatal syndrome of progressive neurologic and cognitive decline, whereas later‐onset GLD has a more indolent, protracted clinical course. Animal models, particularly the twitcher mouse, have allowed investigation of both the pathophysiology of and the potential treatment modalities for GLD. Cellular therapy for GLD, notably hematopoietic cell transplantation (HCT; transplantation of bone marrow, peripheral blood stem cells, or umbilical cord blood cells) from a normal related or unrelated allogeneic donor provides a self‐renewing source of GALC in donor‐derived cells. The only currently available treatment option in human GLD, allogeneic HCT, can slow the progression of the disease and improve survival, especially when performed in presymptomatic infants. Because persistent neurologic dysfunction still occurs after HCT in GLD, preclinical studies are evaluating combinations of HCT with other treatment modalities. © 2016 Wiley Periodicals, Inc.
Evidence for improved survival in postsymptomatic stem cell‐transplanted patients with Krabbe's disease
Authors: Langan Thomas J. , Barcykowski Amy L. , Dare Jonathan , Pannullo Erin C. , Muscarella Leah , Carter Randy L.
Published: September 2016

Krabbe's disease (KD) is a severe neurodegenerative disorder affecting white matter in the brain and peripheral nerves. Transplantation of hematopoietic stem cells (HSCT), although not curative, has been shown to extend survival and alleviate neurodevelopmental symptoms when treatment precedes the onset of symptoms. Existing evidence, although not tested statistically, seems clearly to show that postsymptomatic transplantation does not improve neurodevelopmental outcomes. The impact of postsymptomatic HSCT treatment on survival, however, is an open question. This study uses a KD registry to examine the effect of HSCT on survival of symptomatic KD patients. Sixteen transplanted patients were matched by age of onset to 68 nontransplanted patients. The potential confounding effect of age of onset was, therefore, avoided. To quantify the effect of HSCT over time, we used Cox regression analysis, and we observed a sustained and nearly 2.2‐fold risk of death from KD in patients who were not transplanted relative to those who were transplanted (one‐tailed P = 0.0365; 95% lower bound = 1.07). The improvement of survival resulting from HSCT did not appear to depend on the age of symptom onset. Thus, these results establish a long‐term, quantitative benefit of HSCT even in patients who are already experiencing symptoms. They also provide a benchmark for improved survival that can be used for potential new treatments for KD. © 2016 Wiley Periodicals, Inc.
Suppression of galactocerebrosidase premature termination codon and rescue of galactocerebrosidase activity in twitcher cells
Authors: Luddi Alice , Crifasi Laura , Capaldo Angela , Piomboni Paola , Costantino‐Ceccarini Elvira
Published: September 2016

Krabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of β‐galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in β‐galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of β‐galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well‐characterized compound known to induce ribosomal read‐through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense‐mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose‐dependent manner. The low but sustained expression of β‐galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read‐through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc.
Substrate reduction therapy for Krabbe's disease
Authors: Sands Scott A. , LeVine Steven M.
Published: September 2016

Krabbe's disease (KD) is a lysosomal storage disorder in which galactosylceramide, a major glycosphingolipid of myelin, and psychosine (galactose‐sphingosine) cannot be adequately metabolized because of a deficiency in galactosylceramidase. Substrate reduction therapy (SRT) has been tested in preclinical studies. The premise of SRT is to reduce the synthesis of substrates that are not adequately digested so that the substrate burden is lowered, resulting in less accumulation of unmetabolized material. SRT is used for Gaucher's disease, in which inhibitors of the terminal biosynthetic step are used. Unfortunately, an inhibitor for the final step of galactosylceramide biosynthesis, i.e., UDP glycosyltransferase 8 (a.k.a. UDP‐galactose ceramide galactosyltransferase), has not been found. Approaches that inhibit an earlier biosynthetic step or that lessen the substrate burden by other means, such as genetic manipulations, have been tested in the twitcher mouse model of KD. Either as a stand‐alone therapy or in combination with other approaches, SRT slowed the disease course, indicating that this approach has potential therapeutic value. For instance, in individuals with adult‐onset disease, SRT theoretically could lessen the production of substrates so that residual enzymatic activity could adequately manage the lower substrate burden. In more severe forms of disease, SRT theoretically could be part of a combination therapy. However, SRT has the potential to impair normal function by reducing the synthesis of galactosylceramide to levels that impede myelin function, or SRT could have other deleterious effects. Thus, multiple issues need to be resolved before this approach is ready for testing in humans. © 2016 Wiley Periodicals, Inc.
Can psychosine and galactocerebrosidase activity predict early‐infantile Krabbe's disease presymptomatically?
Authors: Carter Randy L. , Wrabetz Lawrence , Jalal Kabir , Orsini Joseph J. , Barczykowski Amy L. , Matern Dietrich , Langan Thomas J.
Published: September 2016

Krabbe's disease (KD) is a fatal neurodegenerative disorder, with the early‐infantile form (EIKD) defined by onset of symptoms before age 6 months. Early and highly accurate identification of EIKD is required to maximize benefits of hematopoietic stem cell transplantation treatment. This study investigates the potential for accurate prediction of EIKD based on a novel newborn screening (NBS) tool developed from two biomarkers, galactocerebrosidase (GALC) enzyme activity and galactosylsphingosine concentration (psychosine [PSY]). Normative information about PSY and GALC, derived from distinct samples of normal newborns, was used to develop the novel diagnostic tool. Bivariate normal limits (BVNL) were constructed, assuming a multivariate normal distribution of natural logarithms of GALC and PSY of normal newborns. The (lnGALC, lnPSY) points for newborns in various “abnormal groups,” including one group of infants who subsequently suffered EIKD, were plotted on a graph of BVNL. The points for all EIKD patients fell outside of BVNL (100% sensitivity). In a simulation study to compare the false‐positive rate of existing univariate methods of diagnosis with our new BVNL‐based method, we generated 100 million normal newborn data points. All fell within BVNL (i.e., zero false positives), whereas 5,682 false positives were observed when applying a two‐tiered univariate method of the type suggested in the literature. These results suggest that (lnGALC, lnPSY) BVNLs will allow highly accurate prediction of EIKD, whereas two‐tiered univariate approaches will not. Redevelopment of the BVNL based on GALCs and PSYs measured on a common large sample of normal newborns is required for NBS use. © 2016 Wiley Periodicals, Inc.
Quantum dots and potential therapy for Krabbe's disease
Authors: Dawson Glyn
Published: September 2016

Enzyme replacement therapy and substrate reduction therapy have proved useful in reversing many pathological consequences of many nonneural lysosomal storage diseases but have not yet reversed pathology or influenced disease outcome in Krabbe's disease (KD). This Review discusses the relative merits of stem cell therapy, molecular chaperone therapy, gene therapy, substrate reduction therapy, enzyme replacement therapy, and combination therapy. Given the limitations of these approaches, this Review introduces the idea of using tiny, 6‐nm, intensely fluorescent quantum dots (QDs) to deliver a cell‐penetrating peptide and 6 histidine residue‐tagged β‐D‐galactocerebrosidase across the blood–brain barrier. We can therefore follow the fate of injected material and ensure that all targets are reached and that accumulated material is degraded. Uptake of lysosomal hydrolases is a complex process, and the cell‐penetrating peptide JB577 is uniquely able to promote endosomal egress of the QD cargo. This Review further shows that uptake may depend on the charge of the coating of the QD, specifically, that negative charge directs the cargo to neurons. Because KD involves primarily glia, specifically oligodendroglia, we experiment with many coatings and discover a coating (polyethylene glycol 600 amino) that has a positive charge and targets oligodendrocytes. A similar effect is achieved by treating with chondroitinase ABC to degrade the extracellular matrix, indicating that enzyme replacement has several hurdles to overcome before it can become a routine CNS therapy. © 2016 Wiley Periodicals, Inc.
Clinical, electrophysiological, and biochemical markers of peripheral and central nervous system disease in canine globoid cell leukodystrophy (Krabbe's disease)
Authors: Bradbury Allison M. , Bagel Jessica H. , Jiang Xuntian , Swain Gary P. , Prociuk Maria L. , Fitzgerald Caitlin A. , O'Donnell Patricia A. , Braund Kyle G. , Ory Daniel S. , Vite Charles H.
Published: September 2016

Globoid cell leukodystrophy (GLD), or Krabbe's disease, is a debilitating and always fatal pediatric neurodegenerative disease caused by a mutation in the gene encoding the hydrolytic enzyme galactosylceramidase (GALC). In the absence of GALC, progressive loss of myelin and accumulation of a neurotoxic substrate lead to incapacitating loss of motor and cognitive function and death, typically by 2 years of age. Currently, there is no cure. Recent convincing evidence of the therapeutic potential of combining gene and cell therapies in the murine model of GLD has accelerated the requirement for validated markers of disease to evaluate therapeutic efficacy. Here we demonstrate clinically relevant and quantifiable measures of central (CNS) and peripheral (PNS) nervous system disease progression in the naturally occurring canine model of GLD. As measured by brainstem auditory‐evoked response testing, GLD dogs demonstrated a significant increase in I‐V interpeak latency and hearing threshold at all time points. Motor nerve conduction velocities (NCVs) in GLD dogs were significantly lower than normal by 12–16 weeks of age, and sensory NCV was significantly lower than normal by 8–12 weeks of age, serving as a sensitive indicator of peripheral nerve dysfunction. Post‐mortem histological evaluations confirmed neuroimaging and electrodiagnostic assessments and detailed loss of myelin and accumulation of storage product in the CNS and the PNS. Additionally, cerebrospinal fluid psychosine concentrations were significantly elevated in GLD dogs, demonstrating potential as a biochemical marker of disease. These data demonstrate that CNS and PNS disease progression can be quantified over time in the canine model of GLD with tools identical to those used to assess human patients. © 2016 Wiley Periodicals, Inc.
Krabbe's leukodystrophy: Approaches and models in vitro
Authors: Avola Rosanna , Graziano Adriana Carol Eleonora , Pannuzzo Giovanna , Alvares Elisa , Cardile Venera
Published: September 2016

This Review describes some in vitro approaches used to investigate the mechanisms involved in Krabbe's disease, with particular regard to the cellular systems employed to study processes of inflammation, apoptosis, and angiogenesis. The aim was to update the knowledge on the results obtained from in vitro models of this neurodegenerative disorder and provide stimuli for future research. For a long time, the nonavailability of established neural cells has limited the understanding of neuropathogenic mechanisms in Krabbe's leukodystrophy. More recently, the development of new Krabbe's disease cell models has allowed the identification of neurologically relevant pathogenic cascades, including the major role of elevated psychosine levels. Thus, direct and/or indirect roles of psychosine in the release of cytokines, reactive oxygen species, and nitric oxide and in the activation of kinases, caspases, and angiogenic factors results should be clearer. In parallel, it is now understood that the presence of globoid cells precedes oligodendrocyte apoptosis and demyelination. The information described here will help to continue the research on Krabbe's leukodystrophy and on potential new therapeutic approaches for this disease that even today, despite numerous attempts, is without cure. © 2016 Wiley Periodicals, Inc.
Immunological considerations for treating globoid cell leukodystrophy
Authors: Karumuthil‐Melethil Subha , Gray Steven J.
Published: September 2016

Globoid cell leukodystrophy (GLD, or Krabbe's disease) is a severe inherited neurodegenerative disease caused by the lack of a lysosomal enzyme, GALC. The disease has been characterized in humans as well as three naturally occurring animal models, murine, canine, and nonhuman primate. Multiple treatment strategies have been explored for GLD, including enzyme replacement therapy, small‐molecule pharmacological approaches, gene therapy, and bone marrow transplant. No single therapeutic approach has proved to be entirely effective, and the reason for this is not well understood. It is unclear whether initiation of a neuroinflammatory cascade in GLD precedes demyelination, a hallmark of the disease, but it does precede overt symptoms. This Review explores what is known about the role of inflammation and the immune response in the progression of GLD as well as how various treatment strategies might interplay with innate and adaptive immune responses involved in GLD. The focus of this Review is on GLD, but these concepts may have relevance for other, related diseases. © 2016 Wiley Periodicals, Inc.