Phonopheresis Associated with Nanoparticle Gel from Phyllanthus amarus Relieves Pain by Reducing Oxidative Stress and Proinflammatory Markers in Adults with Knee Osteoarthritis

Objective: To determine the changes in serum levels of inflammatory biomarkers and antioxidant levels among the knee osteoarthritis(OA) patients after treatment with Phyllanthus amarus(PP) by nanoparticle gel phonophoresis. Methods: This study was a randomized, double-blind, placebo-control, parallel-group, clinical trial involving 30 subjects with mild-to-moderate degree of knee OA. The patients were allocated to two groups using a computer-generated random numbers, and received conventional ultrasound therapy(control group, 15 cases) and PP(treatment group, 15 cases) once daily for 10 sessions. The pain was evaluated by visual analogue scale(VAS). Serum levels of tumor necrosis factor-α(TNF-α) were determined by enzyme-linked immunosorbnent assay(ELISA). Nitric oxide(NO) was determined by modified Griess reagent. The antioxidant effects, including superoxide dismutase(SOD) and total antioxidant capacity(TAC), were also measured by ELISA assay. Results: The VAS score was significantly decreased in the treatment group compared with the control group after treatment(P0.01). The serum concentrations of TNF-α and NO were significantly reduced in the treatment group compared with the control group(P0.01) after treatment. However, the serum concentrations of SOD and TAC in the treatment group were significantly higher after treatment compared with the control group(P0.01). Conclusion: PP could alleviate knee pain and significantly reduce systemic antiinflammatory effects in knee OA patients.     

Can adiponectin predict gestational diabetes?

The aim of the present study was to evaluate whether adiponectin is a predictive factor for gestational diabetes mellitus (GDM) and is appropriate as a screening test for GDM. Three-hundred and fifty-nine women with singleton pregnancy and indications for GDM screening according to criteria of the American College of Obstetricians and Gynecologists were enrolled in the study between July 5, 2004 and March 11, 2005. After confirming gestational age (GA) and number of fetuses by ultrasound, all women underwent a 1-h glucose challenge test with 50 g glucose load (50-g GCT) between 21 and 27 weeks of GA. Blood samples for determination of adiponectin levels were also obtained on the same day. Subsequently, between 24 and 28 weeks of GA, the women underwent an oral glucose tolerance test with 100 g glucose load (100-g OGTT). The diagnosis of GDM was established when two or more of the following criteria were fulfilled: (1) fasting glucose >95 mg/dl; (2) 1-h glucose >180 mg/dl; (3) 2-h glucose >155 mg/dl; (4) 3-h glucose >140 mg/dl. Sixty women were diagnosed with GDM, a prevalence of 16.7%. There was no difference in age between the GDM and non-GDM groups. Pre-pregnancy and sampling-day body mass index (BMI), increase in weight and all blood glucose levels were greater in women with GDM than in those without (p < 0.05). Adiponectin concentrations were significantly negatively correlated with GA and plasma glucose levels of the GCT and each OGTT. Using logistic regression analyses, adiponectin, but not age, pre-pregnancy BMI and increase in weight, was demonstrated as an independent predictive factor for GDM. The area under the receiver-operator characteristic curve of adiponectin was significantly lower than that of the GCT [0.63 (95% confidence interval (CI) 0.53-0.67) vs. 0.73 (95% CI 0.71-0.80), p < 0.001]. At a cut-off value of 140 mg/dl of the 50-g GCT, the sensitivity and specificity of the test were 90% and 61%, respectively. The 50-g GCT could identify GDM in 54 (90%) out of 60 women. On the other hand, at an arbitrary cut-off value of 10 microg/ml for adiponectin, sensitivity of 91% and specificity of 31% were achieved. If this cut-off value was used for ruling in or out pregnant women for the GDM screening, 27% of all women could be eliminated from needing to perform an OGTT, with five women (8.3%) misclassified. In conclusion, this study demonstrated that adiponectin was an independent predictor for GDM. As for GDM screening, adiponectin was not as strong a predictor as GCT. However, with advantage of being less cumbersome, adiponectin could be used to rule out pregnant women at low risk of GDM.     

Association of Hb Q-Thailand with homozygous Hb E and heterozygous Hb Constant Spring in pregnancy

Hemoglobin (Hb) Q-Thailand [alpha74(EF3): Asp-->His] is an abnormal Hb found mainly in China and South-east Asian countries. Association of the alpha(Q-Thailand) allele with alpha-thalassemia has important implications in diagnosis. We report the hitherto undescribed conditions of this variant in two unrelated pregnant Thai women. Routine Hb analyses using high-performance liquid chromatography identified abnormal Hb migrating after Hb A(2) in addition to a homozygous Hb E in the proband 1 and to a heterozygous Hb Constant Spring (Hb CS) in the proband 2. Further alpha-globin gene analysis identified that the variant was caused by the GAC to CAC mutation at codon 74 of the alpha1-globin gene corresponding to the Hb Q-Thailand, detected in cis to the 4.2 kb deletional alpha-thalassemia 2 in both cases. Interaction of the alpha(Q-Thailand) with the beta(E) globin chains in the proband 1 leads to a Hb variant, namely the Hb QE. Family study of the proband 1 showed that her non-pregnant sister had the same genotype but her father was a double heterozygote for Hb E and Hb Q-Thailand in whom both Hb Q-Thailand and Hb QE were detected. Genotype-phenotype relationships observed in these families with complex hemoglobinopathies are presented and compared with those of simple homozygote for Hb E, heterozygote for Hb CS and heterozygote for Hb Q-Thailand found in other unrelated subjects. A simple DNA assay based on allele-specific polymerase chain reaction for simultaneous detection of the Hb Q-Thailand mutation and the 4.2 kb deletional alpha-thalassemia 2 determinant was developed and validated.     

Molecular characterization of thalassemia intermedia associated with HPFH-6/beta-thalassemia and HPFH-6/Hb E in Thai patients

We report the molecular and hematological characterizations of thalassemia caused by interactions of the hereditary persistence of fetal hemoglobin (HPFH)-6 with beta-thalassemia in 2 Thai patients and the HPFH-6 with Hb E in another Thai patient. Marked hypochromic microcytosis, characteristics of thalassemia intermedia, were obvious in the former 2 cases but the latter had much milder clinical phenotype with normal Hb and a slightly reduced mean corpuscular volume (MCV) value. Hb analysis revealed no Hb A but Hb A(2)F patterns in the compound HPFH-6/beta-thalassemia patients and the EF pattern in the HPFH-6/Hb E patient. The (G)gamma-globin chain predominated in all cases. Globin gene analyses demonstrated that all patients carried the 101-kb HPFH-6 deletion in trans to the beta-thalassemia genes with the IVS1#5 G-C mutation and the G insertion between codons 8/9 and the beta(E)-gene, respectively. Hematologic data of the patients were compared to those of the HPFH-6 heterozygotes found in their family members and different genotype-phenotype interactions of this HPFH determinant in these Thai patients are illustrated.     

Reversible short-term and delayed long-term cognitive impairment induced by chronic mild cerebral hypoperfusion in rats

Chronic cerebral hypoperfusion induced by aging in combination with vascular disorder potentially contributes to the development of vascular dementia. This study aimed to investigate the age-related changes in spatial performances in chronic mild cerebral hypoperfusion induced by permanent right common carotid artery occlusion (rCCAO) in rats. Four-month-old male Sprague–Dawley rats (n = 20) were randomly assigned into sham and rCCAO groups. Spatial performances of young adult rats (age 4–8 months) were evaluated repeatedly by the radial arm water maze at 6 days, and 1, 2 and 4 months after surgery. The spatial performance was re-assessed by the Morris water maze when the rats were 18 months old. The present results revealed that the rCCAO rats developed progressive deficit in spatial learning and memory, starting from day 6 and significant deficit was found at 2 months after rCCAO (p < 0.05). However, the spatial performance of the rCCAO rats was recovered at 4 months after surgery. Testing of the cognitive flexibility of the aged rCCAO rats (18 months old), indicated that the learning flexibility of the aged rCCAO rats was significantly impaired. This deficit was found in parallel with pronounced white matter damage in the corpus callosum and internal capsule and significant cell death in the dorsal hippocampus. Our results suggested that vascular risk insult in young adult rats resulted in spatial learning deficit which could be completely compensated later on. However, such previous vascular risk could be exacerbated by advancing age and subsequently lead to a deficit in cognitive flexibility with white matter damage and significant neuronal death in the dorsal hippocampus.     

Hb Paksé [(alpha2) codon 142 (TAA-->TAT or Term-->Tyr)J in Thai patients with EAbart's disease and Hb H Disease

Hb Paksé is caused by an alpha2-globin gene termination codon mutation, TAA-->TAT or Term-->Tyr, initially described in a Laotian family. We now report for the first time that the same mutation has been found in 14 Thai patients, seven with EABart's disease, four with Hb H disease, and three with alpha-thalassemia trait who were initially diagnosed as having Hb Constant Spring (Hb CS; alpha2-globin gene termination codon mutation TAA-->CAA or Term-->Gln). Co-inheritance of this mutation with alpha-thalassemia-1 (SEA type) leads to Hb H disease (hereafter designated as Hb H-Paksé disease) and to a complex thalassemia syndrome, namely EABart's-Paksé disease. Hematological data of these patients were compared with those of classical Hb H-CS and the EABart's patients. To facilitate epidemiological and diagnostic screening of Hb Paksé, a simple assay procedure based on allele specific polymerase chain reaction (PCR) amplifications was developed and validated. Using this allele specific PCR as a screening method, five additional individuals with Hb Paksé were found among 71 Thai subjects previously thought to have Hb CS.     

Multiplex allele-specific PCR assay for differential diagnosis of Hb S, Hb D-Punjab and Hb Tak

BACKGROUND: Apart from hemoglobin (Hb) E, Hb D-Punjab [beta121(GH4)Glu-Gln] and Hb Tak [beta147Term-Thr] are the two most common beta-chain variants among the Asian population. These two Hb variants have similar alkaline electrophoretic mobilities and HPLC profiles as those of the Hb S [beta6(A3)Glu-Val]. Differential diagnosis of these clinically relevant hemoglobinopathies is therefore problematic. Direct detection of the beta-globin gene mutations would be another diagnostic alternative. METHODS: A simultaneous DNA diagnosis of the three Hb variants was developed based on the multiplex allele-specific polymerase chain reaction (PCR) approach. The method was validated on 10 carriers of Hb D-Punjab, 5 carriers of Hb Tak, 2 carriers of Hb S and 50 normal individuals of Thai origin. RESULTS: The three abnormal Hbs could be correctly diagnosed with the simultaneous PCR approach, and a complete concordance with results using other established methods was obtained. CONCLUSIONS: The multiplex allele-specific PCR approach developed should prove useful in complementing routine Hb analysis for differential diagnosis of these three common Hb variants and should facilitate a program of hemoglobinopathy screening in the region.     

A simplified screening strategy for thalassaemia and haemoglobin E in rural communities in south-east Asia

OBJECTIVE: To evaluate a simple screening strategy for thalassaemia and haemoglobin (Hb) E in a prevention and control programme for thalassaemia in rural communities with limited resources. METHODS: Blood samples from 301 Thai-Khmer participants were screened for thalassaemia and Hb E using a combined modified one-tube osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) precipitation test. Results were evaluated with standard haematological analyses including erythrocyte indices, Hb typing and quantification and polymerase chain reaction (PCR) analysis of alpha-globin and beta-globin genes. FINDINGS: Participants were divided into four groups according to the results of the combined tests. Altogether, 104 of 301 participants (34.6%) had negative results on both tests; 48 (15.9%) were positive on the OF test but not the DCIP test; 40 (13.3%) were negative on the OF test but positive on DCIP test; and 109 (36.2%) were positive on both tests. No carrier of clinically significant forms of thalassaemia (alpha(o)-thalassaemia, beta-thalassaemia) or Hb E was found among the group that had negative results for both tests. All participants with Hb E had positive DCIP tests. Carriers of alpha+-thalassaemia or Hb Constant Spring could generate either positive or negative OF test results but they all had negative DCIP tests. Using both tests as a preliminary screening for the three important groups of carriers gave a sensitivity of 100% and a specificity of 69.8%. The positive predictive value of the combined test was 77.2%. The negative predictive value was 100%. Further evaluation of the screening system by local staff at three community hospitals found a sensitivity of 98.1-100% and a specificity of 65.4-88.4% with positive predictive values of 75.0-86.9% and negative predictive values of 98.1-100%. CONCLUSION: A combined test using OF and DCIP could be used as an effective preliminary screening alternative to an electronic blood cell count for identifying carriers with alpha(o)-thalassaemia, beta-thalassaemia and Hb E. The strategy should prove useful for population screening in prevention and control programmes in rural communities in south-east Asia where laboratory facilities and economic resources are limited.     

Prenatal detection of fetal hemoglobin E gene from maternal plasma

In order to provide a noninvasive prenatal diagnosis of the hemoglobin E (Hb E) related disorder, we have evaluated the possibility of identifying the fetal beta(E)-globin gene in maternal plasma. The analysis was performed during 8 to 18 weeks of gestation using DNA extracted from 200 micro L of plasma from pregnant women whose husbands carried Hb E. The beta(E)-globin mutation in maternal plasma was detected by a nested PCR amplification followed by the Mnl I restriction analysis. The result was compared with that of routine analysis of the CVS specimens. Among the five pregnant women examined, the fetal beta(E)-globin gene was identified in maternal plasma in three of them and the result was completely concordant with the conventional CVS analysis. This simple noninvasive prenatal detection of the fetal beta(E)-globin gene should prove useful in a prevention and control program of Hb E/beta-thalassemia in countries where the beta(E)-globin gene is prevalent.     

A simplified screening for alpha-thalassemia 1 (SEA type) using a combination of a modified osmotic fragility test and a direct PCR on whole blood cell lysates

In order to provide a rapid method for identifying alpha-thalassemia 1 in a region with massive population and limited resources, we have tested a rapid screening strategy. Preliminary screening was done using a modified one tube osmotic fragility test (OF test) followed by RBC indices; Hb analysis and detection of alpha-thalassemia 1 with the Southeast Asian deletion (SEA type) were performed by PCR. One hundred and seventy-five adult Thai subjects were studied. Fifty-one of the 175 subjects (29.1%) were positive for a modified OF test. They all had significantly lower MCV and MCH but higher RDW-CV values as compared to the OF negative group. A successful identification of alpha-thalassemia 1 deletion using a direct PCR on cell lysates was demonstrated. Among the 51 OF-test-positive subjects, 7 were found to be alpha-thalassemia 1 carriers, 3 of whom were also carriers of Hb E. No alpha-thalassemia 1 was detected in the OF-test-negative group. A combination of a modified OF test and a direct PCR analysis on whole blood cell lysates would therefore provide an effective screening for alpha-thalassemia 1 in the regions where a program of prevention and control of the disease remains underserved.