Molecular, hematological and clinical aspects of thalassemia major and thalassemia intermedia associated with Hb E-beta-thalassemia in Northeast Thailand

Hb E-beta-thalassemia is the most common form of beta-thalassemia found in Thailand. The disease exhibits a varied clinical expression ranging from severe transfusion dependence to relatively mild thalassemia intermedia. We evaluated the effects of primary and secondary genetic factors in modulating the hematological and clinical presentation of 148 northeast Thai patients including 103 severe thalassemia major (TM) and 45 thalassemia intermedia (TI). Among 148 cases examined, eleven different mutations including two novel ones; (beta(33/34 (-G)) and beta(IVS2#815 C-T)) were identified in trans to the beta(E) gene in two TM cases. The other 9 known mutations included beta(41/42), beta(17), beta(IVS2#654), beta(-28), beta(71/72), beta(35), beta(IVS1#5), beta(IVS1#1) and beta(41). Except for the beta(-28) mutation which was found only in the TI group, others mutations were identified in both TM and TI. Co-inheritance of alpha-thalassemia as a phenotype modulating factor was not evident in this study, nor was the presence of the -158 (G)gamma-globin Xmn I polymorphism. Further analysis of the polymorphic (TG)n(CG)m repeats within the IVS2 of the two gamma-globin genes revealed no different proportions of the polymorphic patterns among TM and TI groups of patients either. Our data reveals that in the majority of these Hb E-beta-thalassemia patients, it is very hard to predict the clinical phenotype of the patients from the beta-globin mutations and these secondary genetic modifiers.     

The diverse molecular basis and hematological features of Hb H and AEBart's diseases in Northeast Thailand

We defined the molecular basis and correlated the hematological phenotypes with the globin genotypes in 52 patients with Hb H disease and 29 patients with AEBart's disease of northeast Thailand. Among the former group, the most prevalent molecular defect was found to be the interaction of alpha-thalassemia 1 (SEA type) with the Hb Constant Spring (Hb CS; 35 of 52 patients), followed by the deletion of three alpha-globin genes with the SEA type alpha-thalassemia 1 and the 3.7- or 4.2-kb deletion of alpha-thalassemia 2 (14 of 52 patients) and the interaction of the SEA alpha-thalassemia 1 with the Hb Paksé which was found in the remaining 3 patients. Among the 29 patients of the latter group, in 18 disease was caused by interactions of Hb E heterozygotes with the SEA alpha-thalassemia 1 and Hb CS. Interaction of Hb E heterozygotes with a deletional form of Hb H disease was detected in 7 patients and the Hb Paksé AEBart's disease was found in another 3 patients. A remaining patient with an unusually severe form of AEBart's disease with a lower Hb E level and observable Hb H was associated with a hitherto undescribed condition, the interaction of Hb E heterozygote with alpha-thalassemia 1 and an alpha2 codon 30 (GAG) deletion. Hematological characterization of the patients demonstrated that although disease in most of them was associated with thalassemia intermedia phenotypes, it was apparent that association with the nondeletional form of alpha-thalassemia 2 produced a more severe phenotype than that of the deletional one. Therefore, alpha-globin gene analysis of Hb H and AEBart's disease patients would be useful for predicting the clinical outcome and improving genetic counseling.     

Different profiles of hepatic alkoxyresorufin O‐dealkylase activities in small rodents

The aims of the present study were to determine cytochrome P450 enzyme activity in six strains of experimental rodents (n = 5/sex/species): ICR, C57BL/6 and DBA/2 mice; Sprague Dawley and Wistar rats; and Dunkin Hartley guinea pigs. After animals were treated with the typical inducers β‐naphthoflavone (BNF), dexamethasone (DEX) and phenobarbital (PB), the levels of O‐dealkylation of ethoxyresorufin (EROD), methoxyresorufin (MROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) activity were determined using responsive catalytic reactions to study CYP1A1, CYP1A2 and CYP2B, respectively. A maximal induction of EROD and MROD was found in BNF‐treated animals from all strains (2.4‐ to 15.1‐fold) except DBA/2 (0.9‐ to 1.8‐fold). C57BL/6 mice had the strongest BNF‐induced EROD (15.1‐fold) and MROD (8.3‐fold) activities. No differences in BNF‐induced EROD and MROD activities were observed between males and females. However, the EROD activity of Wistar rats and the MROD activity of Sprague Dawley rats were higher in males than females. DEX induced PROD activity only in mice (1.3‐ to 7.1‐fold), but not in rats and guinea pigs (0.2‐ to 1.1‐fold). However, induction of BROD activity was found in DEX‐treated mice and rats (1.5 to 12.5‐fold), but not in guinea pigs (0.3 to 0.4‐fold). PB caused a significant elevation of PROD (1.7‐ to 10.4‐fold) and BROD (31‐ to 13.2‐fold) activities in all the animals. PB‐induced BROD activity was higher in females than males in Sprague Dawley rats. These observations strongly suggest that the choice of experimental animal strain, species and inducer is of critical importance for studies of drug metabolism and interaction. Copyright © 2012 John Wiley & Sons, Ltd.     

Parenteral Nutrition Decreases Paneth Cell Function and Intestinal Bactericidal Activity While Increasing Susceptibility to Bacterial Enteroinvasion

Introduction: Parenteral nutrition (PN) increases the risk of infection in patients with contraindication to enteral feeding. Paneth cells produce and secrete antimicrobial products that protect the mucosa from pathogens. Their loss is associated with increased host‐pathogen interactions, mucosal inflammation, and altered microbiome composition. Hypothesis: We hypothesized that PN reduces Paneth cell product expression, and these changes would reduce bactericidal properties of tissue secretions following cholinergic stimulation, increase mucosal enteroinvasion, and shift the intestinal microbiome. Method: Experiment 1: Male ICR mice were randomized to Chow (n = 8) or PN (n = 8). Ileum tissue was collected for Paneth cell antimicrobial expression using RT‐PCR, stimulated with a cholinergic agonist degranulate Paneth cells bactericidal activity, or used to assess bacterial enteroinvasion in EVISC. Experiment 2: Mice were randomized to Chow (n = 11) or PN (n = 8) and ileum washing was collected for 16s pyrosequencing analysis. Results: Compared to Chow, PN decreased tissue expression of REGIII‐g (p < 0.002), lysozyme (p < 0.002), and cryptdin‐4 (p < 0.03). At the phylum level, PN decreased total Firmicutes but increased total Bacteroidetes, and Proteobacteria. Functionally, secretions from PN tissue was less bactericidal (p < 0.03) and demonstrated increased susceptibility to enteroinvasion by E coli (p < 0.02). Conclusion: PN without enteral nutrition impairs innate mucosal immune function. Tissue expression of Paneth cell antimicrobial proteins decreases associated with compositional shifts to the microbiome, decreased bactericidal activity of mucosal secretions and greater susceptibility of to enteroinvasion by E coli. These changes may explain in‐part the increased risk of infection in parenterally fed patients.     

Triple heterozygosity of a hemoglobin variant: hemoglobin Pyrgos with other hemoglobinopathies

This article is the first report of hemoglobin (Hb) Pyrgos along with other Hbs forming triple-heterozvgous patterns. Of 2 cases, the first occurred in a Thai girl with thalassemic facies, marked anemia, and hepatosplenomegaly, who had Hb Pyrgos in association with Hb H disease with Hb Constant Spring (CS). This case represents a triple heterozygosity comprising Hb Pyrgos, alpha-thalassemia 1, and Hb CS. Hb electrophoresis revealed an abnormal Hb in addition to Hbs CS, A2, A, Bart's, and H. This abnormal Hb moved slightly faster than Hb A but more slowly than Hb Bart's. Polymerase chain reaction revealed that the abnormal Hb was caused by a missense mutation within codon 83 of the beta-globin gene (GGC to GAC) resulting in a glycine-to-aspartic acid substitution, which corresponds to Hb Pyrgos. The patient required blood transfusions by the age of 3 years. A splenectomy was performed when she was 5 years old, after which her hematocrit level remained above 32%. The second case was the patient's older sister who was also triple heterozygous (Hb Pyrgos, E, and CS) but was healthy.     

Epileptogenicity of focal malformations due to abnormal cortical development: direct electrocorticographic-histopathologic correlations

PURPOSE: Malformations due to abnormal cortical development (MCDs) are common pathologic substrates of medically intractable epilepsy. The in situ epileptogenicity of these lesions as well as its relation to histopathologic changes remains unknown. The purpose of this study was to correlate the cellular patterns of MCDs with the expression of focal cortical epileptogenicity as assessed by direct extraoperative electrocorticographic (ECoG) recordings by using subdural grids. METHODS: Fifteen patients with drug-resistant focal epilepsy due to pathologically confirmed MCD who underwent subdural electrode placement for extraoperative seizure localization and cortical mapping between 1997 and 2000 were included in the study. Areas of interictal spiking and ictal-onset patterns were identified and separated during surgery for further pathologic characterization (cellular and architectural). Three pathologic groups were identified: type I; architectural disorganization with/without giant neurons, type IIA; architectural disorganization with dysmorphic neurons, and type IIB; architectural disorganization, dysmorphic neurons, and balloon cells (BCs). The focal histopathologic subtypes of MCDs in cortical tissue resected were then retrospectively correlated with in situ extraoperative ECoG patterns. RESULTS: Cortical areas with histopathologic subtype IIA showed significantly higher numbers of slow repetitive spike pattern in comparison with histopathologic type I (p = 0.007) and normal pathology (p = 0.002). The ictal onset came mainly from cortical areas with histopathologic type IIA (nine of 15 patients). None of the seizures originated from neocortical areas that showed BC-containing MCD (type IIB). CONCLUSIONS: This study shows that areas containing BCs are less epileptogenic than are closely located dysplastic regions. These results suggest a possible protective effect of BCs or a severe disruption in the neuronal networks in BCs containing dysplastic lesions. Further studies are needed to elucidate the nature and the potential role(s) of balloon cells in MCD-induced epileptogenicity.     

Cardiac autonomic modulation and blood pressure responses to isometric handgrip and submaximal cycling exercise in individuals with down syndrome

Purpose

Individuals with Down syndrome (DS) exhibit autonomic dysfunction, manifested as attenuated heart rate (HR) and blood pressure (BP) responses to sympathoexcitation. Whether a subgroup of individuals with DS with a normal HR response would have normal autonomic responses to sympathoexcitation remains unclear.

Methods

We compared autonomic modulation using HR variability (HRV) and BP responses in individuals with and without DS (controls) matched for the HR change to isometric handgrip (HG) (10 DS, 8 controls) and submaximal cycling exercise (CE) (9 DS, 9 controls). HG was performed for 2 min at 30 % of maximal voluntary contraction. CE included two 6-min stages at 0 W and at 50 % of body weight. Beat-to-beat HR and BP were recorded. HRV variables were natural log transformation (Ln) of low frequency (LF), high frequency (HF), LF/HF ratio, total power (TP), and the root mean square of successive differences (RMSSD).

Results

In the HG study, although individuals with DS exhibited an overall lower systolic BP, LF/HF ratio, and LnLF/LnHF, their BP and HRV responses to HG were similar to those of the controls. In the CE study, individuals with DS exhibited lower resting LnLF and an overall lower systolic BP and mean arterial pressure compared with controls. During the CE, individuals with DS exhibited an increased diastolic BP and a smaller reduction in LnTP than controls. These differences disappeared after controlling for confounders.

Conclusions

Our results suggest that despite normal HR responses to sympathoexcitatory tasks, HRV was largely similar to controls, with some evidence of autonomic dysfunction in individuals with DS.

     

Impact of intramuscular administration of lipid-soluble and water-soluble vehicles into regenerating muscle at the distinct phases of skeletal muscle regeneration

Interpretation on the effectiveness of potential substances to enhance skeletal muscle regeneration is difficult if an inappropriate vehicle is administered, since vehicle administration can directly enhance or suppress regenerative capacity. In the current study, intramuscular administration of lipid-soluble and water-soluble vehicles into regenerating muscle at the distinct phases of skeletal muscle regeneration (regenerative vs. remodeling) were investigated. Tested vehicles included lipid-soluble [olive oil, (0.1, 1, 5, and 40%) dimethyl sulfoxide (DMSO), and 40% propylene glycol (PG)] and water-soluble [0.9% NaCl, PBS, 0.1% ethanol, and distilled water]. Skeletal muscle regeneration was induced by 1.2% BaCl₂ injection to the tibialis anterior muscle of 10-week-old C57BL/6 male mice. Histological features, skeletal muscle stem cell activity, regenerating muscle fiber formation, angiogenesis, extracellular matrix remodeling, and macrophage infiltration were examined. The results revealed repeated administration of 40% DMSO and 40% PG causes significant recurrent muscle injury, which is pronounced during the remodeling phase compared to the regenerative phase. These findings were supported by (1) massive infiltration of F4/80⁺ macrophages; (2) significant increase of skeletal muscle stem cell re-activation and nascent regenerating muscle fiber formation; (3) excess fibrous formation; and (4) decreased regenerating muscle fiber cross-sectional area. These deleterious effects were comparable to 2% trypsin (degenerative substance) administration and less pronounced with a single administration. Nevertheless, recurrent muscle injury was still presented with 5% DMSO administration but it can be alleviated when 0.1% DMSO was administered during the remodeling phase. In contrast, none of the tested vehicles enhanced regenerative capacity compared with IGF-1 administration. Altogether, intramuscular administration of vehicle containing high concentration of DMSO or PG could impair skeletal muscle regenerative capacity and potentially affect validation of the investigational substance.     

Effects of Metformin on Hepatic Steatosis in Adults with Nonalcoholic Fatty Liver Disease and Diabetes: Insights from the Cellular to Patient Levels

Nonalcoholic fatty liver disease (NAFLD) patients with diabetes constitute a subgroup of patients with a high rate of liver-related complications. Currently, there are no specific drug recommendations for these patients. Metformin, a conventional insulin sensitizer agent, has been widely prescribed in patients with diabetes. Metformin treatment has been shown to be effective at alleviating hepatic lipogenesis in animal models of NAFLD, with a variety of mechanisms being deemed responsible. To date, most studies have enrolled diabetic patients who are treated with metformin, with the drug being taken continuously throughout the study. Although evidence exists regarding the benefits of metformin for NAFLD in preclinical studies, reports on the efficacy of metformin in adult NAFLD patients have had some discrepancies regarding changes in liver biochemistry and hepatic fat content. Evidence has also suggested possible effects of metformin as regards the prevention of hepatocellular carcinoma tumorigenesis. This review was performed to comprehensively summarize the available in vitro, in vivo and clinical studies regarding the effects of metformin on liver steatosis for the treatment of adult NAFLD patients with diabetes. Consistent reports as well as controversial findings are included in this review, and the mechanistic insights are also provided. In addition, this review focuses on the efficacy of metformin as a monotherapy and as a combined therapy with other antidiabetic medications.     

Vobasinyl-iboga bisindole alkaloids, potent acetylcholinesterase inhibitors from Tabernaemontana divaricata root

The roots of the Thai medicinal plant, Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schult., were investigated for their content of acetylcholinesterase inhibitors. Bioassay-guided fractionation using the Ellman colorimetric method led to the isolation of two bisindole alkaloids, 19,20-dihydrotabernamine and 19,20-dihydroervahanine A. The compounds showed higher inhibitory activity on acetylcholinesterase in comparison with galanthamine, a well-known acetylcholinesterase inhibitor. The inhibitory activity of 19,20-dihydroervahanine A was proved to be specific, reversible and competitive. During the separation process, two inactive bisindole alkaloids, conodurine and tabernaelegantine A, were also isolated. The data suggest that the substitutions at the carbons 11', 12' and 16' might affect the acetylcholinesterase inhibitory activity.