Phenotype diversity in type 1 Gaucher disease: discovering the genetic basis of Gaucher disease/hematologic malignancy phenotype by individual genome analysis

Gaucher disease (GD), an inherited macrophage glycosphingolipidosis, manifests with an extraordinary variety of phenotypes that show imperfect correlation with mutations in the GBA gene. In addition to the classic manifestations, patients suffer from increased susceptibility to hematologic and nonhematologic malignancies. The mechanism(s) underlying malignancy in GD is not known, but is postulated to be secondary to macrophage dysfunction and immune dysregulation arising from lysosomal accumulation of glucocerebroside. However, there is weak correlation between GD/cancer phenotype and the systemic burden of glucocerebroside-laden macrophages. Therefore, we hypothesized that genetic modifier(s) may underlie the GD/cancer phenotype. In the present study, the genetic basis of GD/T-cell acute lymphoblastic lymphoma in 2 affected siblings was deciphered through genomic analysis. GBA gene sequencing revealed homozygosity for a novel mutation, D137N. Whole-exome capture and massively parallel sequencing combined with homozygosity mapping identified a homozygous novel mutation in the MSH6 gene that leads to constitutional mismatch repair deficiency syndrome and increased cancer risk. Enzyme studies demonstrated that the D137N mutation in GBA is a pathogenic mutation, and immunohistochemistry confirmed the absence of the MSH6 protein. Therefore, precise phenotype annotation followed by individual genome analysis has the potential to identify genetic modifiers of GD, facilitate personalized management, and provide novel insights into disease pathophysiology.     

Neutrophil cytochemistry in bacterial infection.

A new cytochemical technique, sensitive to altered lysosomal membrane permeability of blood neutrophils, has been evaluated as a screening test for bacterial infection. This technique, for the lysosomal enzymes acid phosphatase and chloroacetate esterase, was compared with the neutrophil alkaline phosphatase and nitroblue tetrazolium tests. The mean score for each method was significantly higher in infected patients than in normal controls. There was, however, considerable overlap of individual scores between infected patients and ill, but uninfected, patients. This overlap limits the diagnostic value of existing cytochemical screening methods.     

Angiotensin II-mediated oxidative DNA damage accelerates cellular senescence in cultured human vascular smooth muscle cells via telomere-dependent and independent pathways

Angiotensin II (Ang II) induces reactive oxygen species (ROS) production by human vascular smooth muscle cells (hVSMCs). ROS have been implicated in the development of both acute stress-induced premature senescence (SIPS) and chronic replicative senescence. Global oxidative DNA damage triggers SIPS and telomere DNA damage accelerates replicative senescence, both mediated via p53. This study tests the hypothesis that DNA is an important target for Ang II-induced ROS leading to senescence via telomere-dependent and independent pathways. DNA damage was quantified using the Comet assay, telomere DNA length by Southern blotting and hVSMC senescence by senescence-associated beta-galactosidase staining. Exposure to Ang II increased DNA damage in hVSMCs within 4 hours. Inhibition by an AT1 receptor antagonist (losartan metabolite: E3174) or catalase, confirmed that Ang II-induced DNA damage was AT1 receptor-mediated, via the induction of ROS. Acute exposure to Ang II resulted in SIPS within 24 hours that was prevented by coincubation with E3174 or catalase. SIPS was associated with increased p53 expression but was not dependent on telomere attrition because overexpression of human telomerase did not prevent Ang II-induced SIPS. Exposure to Ang II over several population doublings accelerated the rate of telomere attrition (by >2-fold) and induced premature replicative senescence of hVSMCs--an effect that was also attenuated by E3174 or catalase. These data demonstrate that Ang II-induced ROS-mediated DNA damage results in accelerated biological aging of hVSMCs via 2 mechanisms: (1) Acute SIPS, which is telomere independent, and (2) accelerated replicative senescence which is associated with accelerated telomere attrition.     

Treatment of achalasia: the short-term response to botulinum toxin injection seems to be independent of any kind of pretreatment

Background  

It has been suggested that intrasphincteric injection of botulinum toxin (BTX) may represent an alternative therapy to balloon dilatation in achalasia. The aim of the present study was to test the effectiveness of botulinum toxin injections in achalasia patients, as assessed using lower oesophageal sphincter pressure (LOSP) and symptom scores, and to compare the response in patients with different types of pretreatment (no previous treatment, balloon dilatation, myotomy, BTX injection).     

Update on the management of neuromuscular block: focus on sugammadex

Steroidal neuromuscular blocking agents (NMBAs), such as rocuronium, are widely used in clinical anesthesia and emergency medicine to facilitate endotracheal intubation and artificial ventilation and to allow surgical access to body cavities. Reversal of neuromuscular blockade is important for the acceleration of patient recovery and prevention of postoperative residual neuromuscular blockade and reduces the incidence of severe morbidity and mortality associated with anesthesia management. Sugammadex is the first selective relaxant binding agent (SRBA) and has been designed to reverse the steroidal neuromuscular blocking drug rocuronium. Encapsulation of the rocuronium molecule by sugammadex results in a rapid decrease in free rocuronium in the plasma and subsequently at the nicotinic receptor at the motor endplate. After encapsulation, rocuronium is not available to bind to the nicotinic receptor in the neuromuscular junction. This promotes the liberation of acetylcholine receptors, and muscle activity reappears. This new concept of reversal of neuromuscular block induced by rocuronium (or vecuronium) led to impressive results in animal and phase 1 and 2 studies. Sugammadex is currently in phase 3 clinical studies and may be commercially available by 2008.     

Large‐scale brain network abnormalities in Huntington's disease revealed by structural covariance

Huntington's disease (HD) is a progressive neurodegenerative disorder that can be diagnosed with certainty decades before symptom onset. Studies using structural MRI have identified grey matter (GM) loss predominantly in the striatum, but also involving various cortical areas. So far, voxel‐based morphometric studies have examined each brain region in isolation and are thus unable to assess the changes in the interrelation of brain regions. Here, we examined the structural covariance in GM volumes in pre‐specified motor, working memory, cognitive flexibility, and social‐affective networks in 99 patients with manifest HD (mHD), 106 presymptomatic gene mutation carriers (pre‐HD), and 108 healthy controls (HC). After correction for global differences in brain volume, we found that increased GM volume in one region was associated with increased GM volume in another. When statistically comparing the groups, no differences between HC and pre‐HD were observed, but increased positive correlations were evident for mHD, relative to pre‐HD and HC. These findings could be explained by a HD‐related neuronal loss heterogeneously affecting the examined network at the pre‐HD stage, which starts to dominate structural covariance globally at the manifest stage. Follow‐up analyses identified structural connections between frontoparietal motor regions to be linearly modified by disease burden score (DBS). Moderator effects of disease load burden became significant at a DBS level typically associated with the onset of unequivocal HD motor signs. Together with existing findings from functional connectivity analyses, our data indicates a critical role of these frontoparietal regions for the onset of HD motor signs. Hum Brain Mapp 37:67–80, 2016. © 2015 Wiley Periodicals, Inc.     

ASRI2005-88 Comparable levels of CD4+CD25+ CTLA4+ Treg cells in peripheral blood of pre-eclampsia and normal pregnant patients

The acceptance of the semiallogeneic fetus within the maternal environment requires tolerance mechanisms not fully characterized yet. Normal pregnancy is known to be associated with a Th2 profile. Furthermore, T-regulatory cells were proposed to regulate the Th2/Th1 balance at early stages of pregnancy. Treg may avoid the shift to a Th1 profile preventing miscarriage. Accordingly, spontaneous abortion is characterized by a Th1 dominance and diminished levels of Tregulatory cells (Treg). The major aim of the present work was to investigate if pre-eclampsia, a late immunological complication of pregnancy, is characterized by similar hallmarks. Therefore, we measured the surface antigens CD4, CD25, CD8, CTLA4 (as well as the secretion of IL-10) in peripheral blood from patients suffering from pre-eclampsia (n = 8) and age-matched patients undergoing normal pregnancies (n = 9) by 4-colour flow-cytometry. We were not able to find any significant differences in the levels of CD4⁺, CD25⁺, CD8⁺, CTLA4, CD4⁺/CD25⁺, CD4⁺/CD25^bright, CD4⁺/CTLA4, CD25⁺/CTLA4, CD4⁺/CD25⁺/CTLA4, CD8⁺/CD25⁺, CD8⁺/CTLA4 or CD8⁺/CD25⁺/CTLA4 cell subsets. Our data suggest that Treg may not participate in the onset of pre-eclampsia and suggest other regulatory mechanisms during late pregnancy.     

Degradation and biocompatibility of a poly(propylene fumarate)-based/alumoxane nanocomposite for bone tissue engineering

In this work, we evaluated the in vitro cytotoxicity and in vivo biocompatibility of a novel poly(propylene fumarate) (PPF)-based/alumoxane nanocomposite for bone tissue engineering applications. The incorporation of functionalized alumoxane nanoparticles into the PPF-based polymer was previously shown to significantly increase the material's flexural mechanical properties. In the current study, samples underwent accelerated in vitro degradation to allow the study of biological responses to these materials over the course of their degradation on a shortened timescale. The polymer, a macrocomposite composed of the polymer and micron-sized particles, and the nanocomposite were evaluated at three stages of degradation for in vitro cytotoxicity with a fibroblast cell line and in vivo soft-tissue response after 3 and 12 weeks of implantation in adult goats. All three material groups experienced mass loss during degradation, but the nanocomposite group eroded significantly faster than the other groups. Nondegraded materials demonstrated minimal cytotoxicity and a minor inflammatory response in soft tissue. On the contrary, predegraded samples elicited more pronounced responses, though these were due to the increase in surface area, surface roughness, and fragmentation associated with the degradation process. The presence of alumoxane nanoparticles in the PPF-based nanocomposite did not significantly affect its cytotoxicity or biocompatibility.     

Induced sputum and bronchial mucosal expression of interleukin-13 is not increased in chronic obstructive pulmonary disease

Background:  The Th2 cytokine interleukin-13 (IL-13) has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). We sought to examine IL-13 expression in COPD subjects in induced sputum and bronchus specimens. We hypothesized that inflammatory cells expressing IL-13 localize to the airway smooth muscle bundle and bronchial glands.
Methods:  Interleukin-13 was measured in sputum samples from subjects with COPD ( n  = 34) across a range of severity (Global initiative for chronic Obstructive Lung Disease 2–4) and controls ( n  = 14) using ELISA. IL-13+ cells and inflammatory cells were enumerated within surgically resected proximal airway using immunohistochemical techniques from subjects with COPD ( n  = 10), smoking ( n  = 10) and nonsmoking controls ( n  = 8).
Results:  Sputum IL-13 was measurable in only 6/34 subjects with COPD and was not found in the smoking or nonsmoking control subjects. In subjects with COPD and controls there was a paucity of IL-13+ cells. The distribution of inflammatory cells within different airway compartments was similar in COPD and controls except for an increase in CD3⁺ lymphocytes within bronchial glands in COPD ( P  = 0.04).
Conclusions:  Our findings do not support a role for IL-13 in COPD. However, the tissue localization of inflammatory cells to airway compartments, particularly the increase of T cells in glands in COPD may be important in disease.