In vitro immunomodulatory activities of a newly concocted traditional Chinese medicine formula: VI-28.

Previous studies have suggested that Vigconic VI-28, an anti-aging traditional Chinese medicine (TCM) formula containing Radix Ginseng and Cornu Cervi Pantotrichum, possesses immunological efficacy. This in vitro study further investigated the immunomodulatory effects of the hot water extracts of VI-28. The study included (1) colorimetric 5-bromo-2'-deoxy-uridine proliferation ELISA for estimating mitogenicity in human peripheral blood mononuclear cells (PBMC), (2) immunofluorescence staining for measuring the expression of IL-2 receptor alpha (CD25) on lymphocytes, (3) cytometric bead array (CBA) for quantifying cytokine liberation from PBMC, and (4) intracellular immunophenotyping for macrophage phagocytosis and hydrogen peroxide (H(2)O(2)) production from monocytes. The results demonstrated that VI-28 (1) could dose-dependently inhibited the proliferation of unstimulated and lipopolysaccharide-activated PBMC but enhanced the proliferation of phytohemagglutinin-activated PBMC at concentrations of <1 mg/mL, (2) significantly augmented the expression of CD25 on lymphocytes at concentrations of 0.4 mg/mL or above (p < 0.05), (3) dose dependently (0.1-1.0 mg/mL) activated macrophage phagocytosis and monocyte synthesis of H(2)O(2) and (4) significantly increased the production of cytokines IL-8, IL-10, IL-12 and IL-1beta at various concentrations of VI-28 (p < 0.05). The results suggest that VI-28 is a potential immunomodulator which probably acts through the activation of lymphocytes and monocytes.

Immunomodulatory effects of a traditional Chinese medicine with potential antiviral activity: a self-control study.

Traditional Chinese medicine (TCM) has been used for prevention and treatment of severe acute respiratory syndrome (SARS) in Hong Kong during the outbreak in spring 2003. We investigated the immunomodulating effects of an innovative TCM regimen derived from two herbal formulas (Sang Ju Yin and Yu Ping Feng San) for treating febrile diseases. Thirty-seven healthy volunteers were given the oral TCM regimen daily for 14 days. Peripheral venous blood samples were taken on days 0, 15 and 29 for hematology, biochemistry and immunology tests, including the measurement of blood lymphocyte subsets and plasma T-helper lymphocyte types 1 and 2 cytokines and receptor. After 3 months, 23 of the volunteers participated in a control study without TCM treatment for the same time course of blood tests. Two volunteers withdrew on day 2, due to headache and dizziness. All others remained well without any side effects. No participants showed significant changes in their blood test results, except that the T-lymphocyte CD4/CD8 ratio increased significantly from 1.31 +/- 0.50 (mean +/- SD) on day 0 to 1.41 +/- 0.63 on day 15 (p < 0.02), and reduced to 1.32 +/- 0.47 on day 29 (p < 0.05). In the control study, there were no changes in the CD4/CD8 ratio. The transient increase in CD4/CD8 ratio was likely due to the TCM intake. We postulate that the administration of the innovative TCM may have beneficial immunomodulatory effects for preventing viral infections including SARS.

Detection of an intense resonance at 2.4 ppm in 1H MR spectra of patients with severe late-delayed, radiation-induced brain injuries.

Proton MRS and MRI were used to monitor the progression of severe cerebral radiation injuries in 10 patients over a period of 18 months. An unknown resonance (Px) in the 2.37-2.40 ppm region was consistently detected in the affected temporal lobes of four patients. The detection of Px was only confined to spectra with lactate (Lac) and in patients with the highest severity grading of radiation injury. The incidence of Px in Lac-positive spectra was 42.8% (15/35) and in lesions with highest injury grading was 46.8% (15/32). Lesions with Px had significantly higher Lac/creatine (Cr) ratios and more extensive mass effect changes when compared to lesions without Px. The probable identity of Px was examined in the context of anaerobic glycolysis producing pyruvate (2.37 ppm) and the model of metabolic changes in brain abscess formation implicating succinate (2.40 ppm).

Rapid analysis of CGG repeat length in the FMR1 gene.

The number of trinucleotide CGG repeats at the 5' untranslated region of the FMR1 gene is associated with the fragile X syndrome of mental retardation. We screened for the CGG repeat length in the FMR1 gene of the X-chromosomes from unrelated normal Chinese subjects recruited in Hong Kong and Dalian, a southern and a northern Chinese city respectively. These cities are about 3000 km apart and the residents have few historical interactions. Genomic DNA was analysed by PCR and detected by Southern hybridisation with a radiolabelled (CGG)5 probe for the CGG repeat number. A different distribution pattern of CGG allele size from the Caucasians is observed. It is a bimodal pattern with the most common CGG repeats allele at 29 against 30 in the Caucasians. Among the Hong Kong subjects, five alleles of more than 50 CGG repeats were detected, and four of those were in heterozygous females. There was no difference in the repeat patterns in subjects from the two cities, suggesting no genotypic variation in FMR1 between northern and southern Chinese.

MECP2 mutation in male patients with non-specific X-linked mental retardation.

In contrast to the preponderance of affected males in families with X-linked mental retardation, Rett syndrome (RTT) is a neurological disorder occurring almost exclusively in females. The near complete absence of affected males in RTT families has been explained by the lethal effect of an X-linked gene mutation in hemizygous affected males. We report here on a novel mutation (A140V) in the MECP2 gene detected in one female with mild mental retardation. In a family study, the A140V mutation was found to segregate in the affected daughter and in four adult sons with severe mental retardation. These results indicate that MECP2 mutations are not necessarily lethal in males and that they can be causative of non-specific X-linked mental retardation.

The apolipoprotein E epsilon4 allele is unlikely to be a major risk factor of age-related macular degeneration in Chinese.

Apolipoprotein E (ApoE) is a major transporter of lipids and cholesterol in the nervous system. Age-related macular degeneration (ARMD), characterized by drusen containing lipids, was reported to show a lower frequency of the ApoE epsilon4 allele than control subjects. We sought to examine the association of this polymorphism with ARMD in Hong Kong Chinese. Among 98 ARMD subjects, the frequency of epsilon4 carriers showed a trend toward a decrease compared to controls, but it was not significant (11.2 vs. 15.0%, p < 0.52). The association of epsilon4 with an apparent reduced risk of ARMD was reported previously in the exudative form of the disease, however among 39 exudative ARMD patients there was also no significant difference in epsilon4 frequency (12.8%, p < 0.93). The lack of a statistically significant effect of epsilon4 may be due to the lower frequency of epsilon4 in Chinese than Europeans. Thus we cannot exclude a possible effect of this allele on ARMD risk, but we can conclude that this allele is likely not a major factor influencing ARMD risk in the Chinese.

Trinucleotide CGG repeat in the FMR1 gene in Chinese mentally retarded patients.

The fragile X syndrome of mental retardation is related to the number of trinucleotide CGG repeats at the 5'-untranslated region of the FMR1 gene located on the X-chromosome. We have studied X-chromosomes from 649 unaffected Chinese subjects and 324 patients with mild mental retardation. All study subjects were unrelated. The CGG repeat number was analysed by electrophoresis of a polymerase chain reaction followed by gel transfer and hybridisation with a 32P-labeled (CCG)5 probe. The DNA samples having detectable CGG expansion were further analysed by Southern blot analysis with probe StB12.3 after restriction digestion by EcoR I and Eag I. For the unaffected Chinese subjects, a different distribution pattern of CGG allele size from Caucasians was observed. It was a bimodal pattern and the CGG repeat number ranged from 19 to 54. The most common CGG repeat allele was 29 compared with 30 in Caucasians. The second mode appeared at 36 repeats. There was mild statistical difference in the repeat patterns between the mentally retarded patients and unaffected subjects, although the essential features were similar. Among the mentally retarded patients, one male had an unmethylated full mutation and one female had a full mutation. The fragile X prevalence was 0.6%, which is lower than two previous studies in Chinese mentally retarded patients utilising cytogenetic analysis. Our results indicate that a large-scale screening program would be worthwhile to determine the prevalence of the fragile X syndrome in the Chinese population.

FRAXAC1 and DXS548 polymorphisms in the Chinese population.

The fragile X syndrome is the most common inherited form of mental retardation. Haplotype studies using FRAXAC1 and DXS548 polymorphic markers flanking the fragile site have demonstrated linkage disequilibrium at the FMR1 locus. We investigated the association of the FRAXAC1, DXS548 and CGG alleles between normal subjects and mentally retarded (MR) patients of unspecified cause who do have fragile X syndrome. We have evaluated the FRAXAC1 site in 390 normal subjects and 321 MR patients and the DXS548 site in 146 normal and 319 MR subjects. Both FRAXAC1 and DXS548 alleles were determined by application of the polymerase chain reaction. When compared with Caucasians, the normal Chinese population has a different FRAXAC1 allele distribution. There are more AC18 repeat alleles and fewer AC19 repeat alleles. The DXS548 allele distributions were similar between Chinese and Caucasians. The same distribution pattern of FRAXAC1 alleles was found in both normal subjects and MR patients, but there were significant differences in the distribution patterns of DXS548 alleles. The FMR1 CGG-DXS548 and FRAXAC1-DXS548 haplotype distribution between normal subjects and MR patients also differed significantly. Our results suggest a possible association between DXS548 alleles and non-FRAXA mental retardation.

Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma.

BACKGROUND: The ability to determine fetal RhD Status noninvasively is useful in the treatment of RhD-sensitized pregnant women whose partners are heterozygous for the RhD gene. The recent demonstration of fetal DNA in maternal plasma raises the possibility that fetal RhD genotyping may be possible with the use of maternal plasma. METHODS: We studied 57 RhD-negative pregnant women and their singleton fetuses. DNA extracted from maternal plasma was analyzed for the RhD gene with a fluorescence-based polymerase-chain-reaction (PCR) test sensitive enough to detect the RhD gene in a single cell. Fetal RhD status was determined directly by serologic analysis of cord blood or PCR analysis of amniotic fluid. RESULTS: Among the 57 RhD-negative women, 12 were in their first trimester of pregnancy, 30 were in their second trimester, and 15 were in their third trimester. Thirty-nine fetuses were RhD-positive, and 18 were RhD-negative. In the samples obtained from women in their second or third trimester of pregnancy, the results of RhD PCR analysis of maternal plasma DNA were completely concordant with the results of serologic analysis. Among the maternal plasma samples collected in the first trimester, 2 contained no RhD DNA, but the fetuses were RhD-positive; the results in the other 10 samples were concordant (7 were RhD-positive, and 3 RhD-negative). CONCLUSIONS: Noninvasive fetal RhD genotyping can be performed rapidly and reliably with the use of maternal plasma beginning in the second trimester of pregnancy.

CGG repeat interruptions in the FMR1 gene in patients with infantile autism.

We determined the CGG repeat length and AGG interruptions in the FMR1 gene in normal Chinese subjects and patients with infantile autism and mild mental retardation. Genomic DNA was investigated by PCR and Southern hybridisation for CGG repeat number and PCR with Mnl I restriction analysis for AGG interruption. Both the normal subjects and the patients with autism have 53 CGG repeats in FMR1, and the majority have two interspersed AGG. Our normal Chinese subjects have a similar number of interspersed AGG as other populations. When compared with the normal subjects, the autism patients have less AGG interruptions and a different pattern of AGG distribution. There was a significant difference in the CGG configurations between normal subjects and patients with autism. The latter had less interspersed AGG, as in fragile X patients, but they did not have fragile X. A study on mentally retarded patients with no infantile autism should also be carried out to ascertain whether mental retardation alone may have contributed to such AGG pattern.

Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.

We have developed a real-time quantitative PCR assay to measure the concentration of fetal DNA in maternal plasma and serum. Our results show that fetal DNA is present in high concentrations in maternal plasma, reaching a mean of 25.4 genome equivalents/ml (range 3.3-69. 4) in early pregnancy and 292.2 genome equivalents/ml (range 76. 9-769) in late pregnancy. These concentrations correspond to 3.4% (range 0.39%-11.9%) and 6.2% (range 2.33%-11.4%) of the total plasma DNA in early and late pregnancy, respectively. Sequential follow-up study of women who conceived by in vitro fertilization shows that fetal DNA can be detected in maternal serum as early as the 7th wk of gestation and that it then increases in concentration as pregnancy progresses. These data suggest that fetal DNA can be readily detected in maternal plasma and serum and may be a valuable source of material for noninvasive prenatal diagnosis.

Gas chromatographic-mass fragmentographic determination of serum 1 alpha,25 dihydroxyvitamin D3.

We extracted 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] from 10 mL serum using Sep-Pak C18 and Sep-Pak Silica mini-columns and normal-phase high performance liquid chromatography (HPLC) separation for analysis by gas chromatography-mass fragmentography (GC-MS). A GC-MS method was optimised using manual tuning for ion mass calibration and selective ion monitoring (SIM) for quantitation. Serum 1 alpha,25(OH)2D3 was identified by superimposition of the m/z 452 and 501 ion peaks and by overlapping the m/z 452 ion peak with that of its authentic standard. It was quantitated from the relative peak areas of its m/z 452 ion and the m/z 363 ion of vitamin D2, the internal standard. Twenty picograms of 1 alpha,25(OH)2D3 gave a peak with a signal-to-noise ratio of 26:1. Between-batch coefficient of variation (CV) for 1 alpha,25(OH)2D3 standard was < 13%. However, serum analysis was less precise, within-batch CV being 20%. The analytical recovery was about 70% and detection limit 0.5 pg/mL. When compared with a commercial radioreceptor assay we still found our method to be sensitive, specific, and adequate for confirmative and semiquantitative analysis of serum 1 alpha,25(OH)2D3.