Variations in bovine milk oligosaccharides after calving using p-aminobenzoic ethyl ester closed-ring labeling and negative ion electrospray LC/MS/MS.

A strategy was proposed to analyze bovine milk oligosaccharides using p-aminobenzoic ethyl ester (ABEE) closed-ring labeling and C18 capillary liquid chromatography negative ion electrospray tandem mass spectrometry. Linkage specific fragment ions were used to identify oligosaccharide isomers. By constructing the mass chromatograms using linkage specific fragment ions, isomers were differentiated based on m/z values as well as temporal separation provided by liquid chromatography. In addition to disialyllactose and the single isomer lacto-N-neohexaose, four pairs of linkage isomers including 3'/6'-sialyllactose (3'/6'-SL), 3'/6'-sialyllactosamine (3'/6'-SLN), 3'/6'-sialylgalactosyl-lactose (3'/6'-SGL), and lacto-N-tetraose/lacto-N-neotetraose (LNT/LNnT) in bovine milk were investigated. Variations of bovine milk oligosaccharides in a lactation period of 72 h after calving were studied. Sialylated oligosaccharide was found to be distinctively more abundant in milk of the first 24 h, decreasing in successive milkings. For the first time, the variation of lacto-N-tetraose in bovine milk was reported.

Safe needling depths of upper back acupoints in children: a retrospective study.

BACKGROUND: Acupuncture is applied for treating numerous conditions in children, but few studies have examined the safe needling depth of acupoints in the pediatric population. In this study, we investigated the depths to which acupuncture needles can be inserted safely in the upper back acupoints of children and the variations in safe depth according to sex, age, weight, and body mass index (BMI). METHODS: We retrospectively studied computed tomography (CT) images of patients aged 4 to 18 years who underwent chest CT at China Medical University Hospital between December 2004 and May 2013. The safe depths of 23 upper back acupoints in the Governor Vessel (GV), Bladder Meridian (BL), Small Intestine Meridian (SI), Gallbladder Meridian (GB) and Spleen Meridian (SP) were measured directly from the CT images. The relationships between the safe depths of these acupoints and sex, age, body weight, and BMI were analyzed. RESULTS: The results indicated significant differences in safe needling depth between boys and girls in most upper back acupoints, except at BL42, BL44, BL45, BL46, GB21 and SP21. Safe depths differed significantly depending on age (p < 0.001), weight (p ≤ 0.01), and BMI (p < 0.05). Multiple regression analysis revealed that weight was the most crucial factor in determining the safe depth. CONCLUSIONS: Sex, age, weight, and BMI are relevant factors in determining the safe needling depths of upper back acupoints in children. Physicians should pay attention to wide variations in needle depth when performing acupuncture.

Traditional Chinese medicine therapy improves the survival of systemic lupus erythematosus patients.

OBJECTIVE: No previous studies have evaluated the effect of traditional Chinese medicine (TCM) treatment on the survival of patients with systemic lupus erythematosus (SLE). Hence, in this study, we determined whether TCM treatment affects the survival of SLE patients. METHODS: This nationwide population-based retrospective cohort study assessed 23,084 patients newly diagnosed with SLE between 1999 and 2009, using the database of the Taiwan National Health Insurance program. RESULTS: Among these patients, 9267 (40.15%) used TCM for SLE treatment and exhibited a significantly decreased risk of death [hazard ratio (HR) = 0.73; 95% confidence interval (CI): 0.68-0.78], with multivariate adjustment, compared with those without TCM use. A similar significant protective effect of TCM use was found across various subgroups of comorbidities. TCM use 1 year before diagnosis also reduced the risk of death. Our study findings indicated that Zhi Bo Di Huang Wan (HR = 0.54; 95% CI: 0.32-0.91), Jia Wei Xiao Yao San (HR = 0.35; 95% CI: 0.16-0.73), Liu Wei Di Huang Wan (HR = 0.51; 95% CI: 0.28-0.93), Gan Lu Yin (HR = 0.40; 95% CI: 0.17-0.96), and Yin Qiao San (HR = 0.22; 95% CI: 0.05-0.86) were the most effective TCM agents that improved survival. CONCLUSIONS: This nationwide retrospective cohort study provided information that combined therapy with TCM may improve the survival in SLE patients. This study also suggests that TCM may be used as an integral element of effective therapy for SLE.

Time-trend analysis of prevalence, incidence and traditional Chinese medicine use among children with asthma: a population-based study.

BACKGROUND: This study determined annual prevalence and incidence trends of asthma among children in Taiwan from 2002 to 2008. Risk factors and traditional Chinese medicine (TCM) use were examined. METHODS: A random sample was selected for a population-based study with a selection probability of 0.5 from all 3-18 years insurants. The annual prevalence and incidence of asthma were estimated according to age, sex, insurance premium and degree of urbanization. RESULTS: The prevalence of asthma increased from 12.99% in 2002 to 16.86% in 2008. The increase was greatest in 2008, among boys, 11-15 years, ≥medium insurance premium, and high- and medium-density urban area. TCM use in asthma-prevalent children decreased from 1.16% in 2002 to 0.59% in 2008. The incidence fluctuated, ranging from 1.01% in 2002 to 1.49% in 2005. The highest was in 2005, among boys, 3-5 years, ≥medium insurance premium and high-density urban area. TCM use in asthma-incident children decreased from 3.59% in 2002 to 1.69% in 2008. CONCLUSION: This study demonstrated a substantial increase in annual prevalence of asthma among children in Taiwan from 2002 to 2008. The incidence fluctuated. The TCM use showed a decreasing linear trend and was higher in incident than in prevalent cases.

The Depths from Skin to the Major Organs at Chest Acupoints of Pediatric Patients.

Background. Acupuncture is applied to treat numerous diseases in pediatric patients. Few reports have been published on the depth to which it is safe to insert needle acupoints in pediatric patients. We evaluated the depths to which acupuncture needles can be inserted safely in chest acupoints in pediatric patients and the variations in safe depth according to sex, age, body weight, and body mass index (BMI). Methods. We retrospectively studied computed tomography (CT) images of pediatric patients aged 4 to 18 years who had undergone chest CT at China Medical University Hospital from December 2004 to May 2013. The safe depth of chest acupoints was directly measured from the CT images. The relationships between the safe depth of these acupoints and sex, age, body weight, and BMI were analyzed. Results. The results demonstrated significant differences in depth among boys and girls at KI25 (kidney meridian), ST16 (stomach meridian), ST18, SP17 (spleen meridian), SP19, SP20, PC1 (pericardium meridian), LU2 (lung meridian), and GB22 (gallbladder meridian). Safe depth significantly differed among the age groups (P < 0.001), weight groups (P < 0.05), and BMI groups (P < 0.05). Conclusion. Physicians should focus on large variations in needle depth during acupuncture for achieving optimal therapeutic effect and preventing complications.

Time Trend Analysis of the Prevalence and Incidence of Diagnosed Asthma and Traditional Chinese Medicine Use among Adults in Taiwan from 2000 to 2011: A Population-Based Study.

BACKGROUND: The aim of this study was to determine the annual trends of traditional Chinese medicine (TCM) use for prevalent and incident asthmatic adults in Taiwan from 2000 to 2011. The annual prevalence and incidence of asthma in adults among subgroups of sociodemographic factors were also investigated. METHODS: A population-based study was conducted using a random sample with one million beneficiaries of all residents aged ≥18 years enrolled in the National Health Insurance program. Adults diagnosed with asthma were identified from the National Health Insurance Research Database. The annual prevalence and incidence of asthma in the adult population were estimated by using International Classification of Diseases, Ninth Revision, Clinical Modification diagnostic codes to identify relevant cases from 2000 to 2011. RESULTS: The number of annual prevalent cases of diagnosed asthma increased from 56,885 in 2000 to 101,535 in 2011. The prevalence increased significantly on annual basis, whereas the incidence rate fluctuated over time. The prevalence of TCM use by adults with asthma decreased significantly (p<0.05), from 38.58% in 2000 to 29.26% in 2011. The number of annual incident cases of diagnosed asthma decreased from 3,896 in 2000 to 2,684 in 2011. TCM use rates in asthma incident adults decreased significantly (p<0.05), from 54.24% in 2000 to 38.19% in 2011. CONCLUSION: The prevalence of TCM utilization is high among adults with asthma in Taiwan. However, our study demonstrated a substantial decrease in the annual prevalence of TCM use by prevalent and incident asthmatic adults in Taiwan from 2000 to 2011. In addition, the prevalence of TCM use was higher among incident cases, compared with those with prevalent cases.

Comparative study of sialyl glycoprotein with multiple glycosylation sites using isotope labeling and capillary liquid chromatography/mass spectrometry.

RATIONALE: A comparative strategy has been demonstrated using RNase B, a single-site N-linked high-mannose glycoprotein. Glycoproteins are more common with multiple glycosylation sites and with complex glycans. A strategy capable of differentiating the changes caused by glycoprotein concentration, glycosylation site occupancy, and a glycoform profile of complex glycoproteins would be beneficial. METHODS: Tryptic-digested glycoproteins were labeled using 12 C,H-formaldehyde and 13 C, D-formaldehyde, purified, and then analyzed using capillary reversed-phase liquid chromatography/mass spectrometry (RPLC/MS). The relative intensity of non-glycosylated peptides provided information on glycoprotein concentration variation. A site-specific glycoform profile variation was obtained by comparing the glycoform profile of CH2 and 13 CD2 glycopeptides. Determining the protein concentration and glycoform profile variations allows the glycosylation site occupancy variation to be calculated. RESULTS: A strong correlation between the observed and prepared ratios for fetuin glycopeptides from 0.2 to 5 was obtained. Two fetuin samples with different glycoprotein concentrations (4-fold change), glycoform profiles (normal and modified), and glycosylation site occupancies (100% and 50%) were prepared, labeled, mixed, purified, and analyzed using RPLC/MS. The results of the comparative study had a strong correlation with the prepared values. CONCLUSIONS: In this report, we demonstrated a comparative analysis of fetuin, a glycoprotein with multiple glycosylation sites and complex sialyl glycans. Compared to our previous approach, we made several modifications including the use of RPLC, a larger mass difference isotope tag, and isotope overlapping correction. The modified approach is expected to be applicable to most glycoproteins. Copyright © 2013 John Wiley & Sons, Ltd.

Estimation of the measurement uncertainty in quantitative determination of ketamine and norketamine in urine using a one-point calibration method.

An approach was proposed for the estimation of measurement uncertainty for analytical methods based on one-point calibration. The proposed approach is similar to the popular multiple-point calibration approach. However, the standard deviation of calibration was estimated externally. The approach was applied to the estimation of measurement uncertainty for the quantitative determination of ketamine (K) and norketamine (NK) at a 100 ng/mL threshold concentration in urine. In addition to uncertainty due to calibration, sample analysis was the other major source of uncertainty. To include the variation due to matrix effect and temporal effect in sample analysis, different blank urines were spiked with K and NK and analyzed at equal time intervals within and between batches. The expanded uncertainties (k = 2) were estimated to be 10 and 8 ng/mL for K and NK, respectively.

A comparative study of glycoprotein concentration, glycoform profile and glycosylation site occupancy using isotope labeling and electrospray linear ion trap mass spectrometry.

A strategy is presented for comparative analysis of glycoproteins in which the variation of protein concentration, variation of glycosylation site occupancy and variation of glycoform profile can be determined. A comparative study was performed using stable isotope labeling of glycopeptides and peptides by formaldehyde-H(2) and formaldehyde-D(2) and analysis by ESI-MS analysis. The relative intensity of the nonglycosylated peptide provided information about protein concentration variation. Variation of the glycoform profile was obtained by comparing the glycoform profile of d(0)- and d(4)-dimethyl labeled glycopeptides. By knowing the variation of protein concentration and the variation of glycoform profile, the variation of glycosylation site occupancy could be calculated. The utility of the proposed strategy was demonstrated with ribonuclease B with different protein concentrations, different levels of glycosylation site occupancy and different glycoform profiles.

Prednisolone oral solution plus inhaled procaterol for acute asthma in children: a double-blind randomized controlled trial.

BACKGROUND: To evaluate the efficacy of prednisolone sodium phosphate oral solution plus inhaled procaterol in the treatment of acute asthma in children. METHODS: Forty-three patients aged 6 to 12 years with an acute exacerbation of asthma were double-blind randomized into one of two treatment groups in a 1:1 ratio:1) prednisolone oral solution +placebo tablets + procaterol MDI or 2) prednisolone tablets +placebo oral solution + procaterol MDI, all given three times daily for 7 days. Peak expiratory flow rate (PEFR), 24-hour reflective asthma symptom scores, spirometry and pulmonary index score (PIS) were recorded before and after treatment. Net changes in PEFR, symptom score, PIS, Forced Expiratory Volume in the first second (FEV1), FEV1/forced vital capacity (FVC), forced expiratory flow between 25 and 75 percent of the forced vital capacity (FEF(25-75%)) (before and after treatment) and global assessment by the investigator and the subjects or their parents were analyzed. RESULTS: The two groups were statistically similar at baseline values of these parameters. After a 7-day course of treatment, the net change of PEFR before and after treatment was significantly improved in both groups, but there was no significant difference in the net change of PEFR between the two groups (57.27+/-31.44 L/min vs. 54.29 +/-30.04 L/min, difference 2.99 +/-30.76 L/min, mean +/-SD, P=0.752). The net change in PIS and total symptom score did not differ between the two groups (P=0.091 and 0.827, respectively). Similarly, the FEV1, FEV1/FVC and FEF25-75% all improved with either treatment, and neither group was significantly superior to the other group (P=0.162, 0.48 and 0.081, respectively). Global assessment by the investigator and the subjects or their parents at the end of study indicated an essentially comparable result. CONCLUSIONS: Prednisolone sodium phosphate oral solution plus inhaled procaterol is as efficacious as prednisolone tablets plus inhaled procaterol in the management of acute asthma in children.

X-linked hyper-immunoglobulin M syndrome: molecular genetic study and long-time follow-up of three generations of a Chinese family.

BACKGROUND: X-linked hyper-immunoglobulin M (IgM) syndrome (XHIGM) is a rare immunodeficiency disease caused by mutations of the CD40 ligand gene. Patients are subject to recurrent infections and have normal or elevated levels of IgM but markedly decreased serum IgG. OBJECTIVE: We describe molecular genetic studies and clinical manifestations in three generations of one family, as well as results of long-term treatment of 2 young men with the disorder. METHODS: Of 37 living family members, mutational analysis of the CD40 ligand gene was performed in 36 members. Laboratory data for patients and carriers were reviewed. RESULTS: Four male family members had died of unexplained causes. The 3 patients with XHIGM syndrome and the 5 carriers all had a novel mutation located at Tyr 169 Asn (T526A) in exon 5, the tumor necrosis factor domain of the CD40 ligand gene. In the 3 patients, CD40 ligand expression in activated CD4+ T cells was below 1%. In the carriers, about half of activated CD4+ cells expressed CD40 ligand. One carrier had malignant lymphoma. Long-term (>20 years) intravenous immunoglobulin therapy in 2 patients improved IgG levels but did not fully suppress the high levels of IgM, nor did it prevent late complications (bronchiectasis and sclerosing cholangitis). CONCLUSIONS: Diagnosis of a genetic immunodeficiency, especially an X-linked disease such as XHIGM syndrome, should prompt a survey of the entire family.

Wiskott-Aldrich syndrome complicated by an atypical lymphoproliferative disorder: a case report.

Wiskott-Aldrich syndrome (WAS) is an X-linked syndrome consisting of eczema, recurrent pyogenic infection, and thrombocytopenia with decreased platelet volume. Immunologic studies reveal normal immunoglobulin G (IgG), decreased IgM, elevated IgA and IgE levels, and decreased T-cell function. Patients with WAS often have increased susceptibility to lymphoproliferative disorders (LPDs). We report a 3-year-old boy who had persistent thrombocytopenia with bleeding, recurrent infections, and chronic eczema with frequent skin infections since birth. A blood smear revealed small platelets (50% of normal size). Immunologic studies showed normal IgG (1880 mg/dL), decreased IgM (76 mg/dL) and increased IgA (228 mg/dL) and IgE (14,282 IU/mL) levels. The relative proportions of immune cells were CD2 52.2%, CD3 41.1%, CD4 23.4%, CD8 16.8%, CD19 8.0%, CD57 7.7% and active T cells 14.6%. T-cell dysfunction was detected on the multitest for cell-mediated immunity. The WAS diagnosis was confirmed by mutation analysis which demonstrated a 4-base pair deletion in WAS protein gene exon 1. His thrombocytopenia was uncontrolled despite intravenous immunoglobulin infusions, so splenectomy was performed. The platelet count then rose to about 60,000 to 80,000/microL. However, about 2 weeks after splenectomy, he developed generalized lymphadenopathy and lymphoma was misdiagnosed based on lymph node biopsy at another hospital where he was admitted for urgent care. However, our analysis of his lymph node pathology led to the diagnosis of atypical LPD (ALPD). The lymphadenopathy regressed spontaneously 1 month later without chemotherapy. Early and correct diagnosis of WAS complicated with ALPD is important to avoid unnecessary chemotherapy.

Hyper-IgM1 syndrome with interstitial pneumonia and diarrhea caused by coxsackievirus B4 in a 3-month-old infant.

BACKGROUND: Hyper-IgM1 syndrome is a rare genetic primary immunodeficiency disease caused by mutations of the CD40 ligand gene. It is characterized by normal or elevated levels of IgM and markedly decreased serum IgG, IgA, and IgE levels. Patients with this syndrome often easily develop infections. During the past decade, it has become clear that enteroviral infections may also occur as a manifestation of hyper-IgM1 syndrome. OBJECTIVE: To report a case of hyper-IgM1 syndrome in a 3-month-old boy who had interstitial pneumonia and intractable diarrhea. METHODS: Chest radiography, bronchoscopy, immune studies, and open lung biopsy were performed. RESULTS: Chest radiography revealed diffuse bilateral infiltrates. Immune studies revealed the following proportions of lymphocyte markers: CD3, 5,976/microL; CD4, 5,015/microL; CD8, 866/microL; CD19, 1,325/microL; CD16 + 56, 935/microL; and active T cells, 225/microL. The IgG level was 190 mg/dL; IgA, 2 mg/dL; IgM, 34 mg/dL; IgE, 1 IU/dL; and CH50, 23.8/mL. CD40L expression was less than 1%, and a Tyr 169 Asn (t526a) mutation in the exon 5 tumor necrosis factor domain of the CD40L gene was found. The patient was treated with intravenous immunoglobulin and had a dramatic improvement in symptoms. Open lung biopsy failed to demonstrate pneumocystis, and there was no evidence of cryptosporidium in the stool. However, coxsackievirus B4 was isolated by viral throat culture. CONCLUSION: Interstitial pneumonia and diarrhea caused by coxsackievirus B4 may be a complication of hyper-IgM1 syndrome.

Recurrent acalculous cholecystitis and sclerosing cholangitis in a patient with X-linked hyper-immunoglobulin M syndrome.

X-linked hyper-immunoglobulin M (IgM) syndrome (XHIGM) is a rare genetic primary immunodeficiency disease caused by mutations of the CD40 ligand (CD40L) gene with normal or elevated levels of IgM and markedly decreased serum IgG, IgA, and IgE. Liver disease may occur as a clinical manifestation in XHIGM. This complication appears to increase with age. We report an 18-year-old male patient who had recurrent episodes of acalculous cholecystitis (AC) and sclerosing cholangitis (SC). The diagnosis of XHIGM was confirmed by the finding of CD40L expression < 1% of normal and a tyrosine 169 asparaginase (t526a) mutation in exon 5 (the tumor necrosis factor domain) of the CD40L gene. The patient had direct hyperbilirubinemia (direct bilirubin 5.5 mg/dL, total bilirubin 8.7 mg/dL), cholestasis (alkaline phosphatase 1133 U/L, gamma-glutamyl transferase 1019 U/L) and elevated transaminases (aspartate aminotransferase 70 U/L, alanine aminotransferase 101 U/L). Findings on abdominal ultrasound and abdominal computed tomography were compatible with AC. After the fourth episode of cholecystitis, cholecystectomy and liver biopsy were performed. Operative cholangiography revealed poor opacification of the hepatic duct and proximal common bile duct; the upstream intrahepatic bile ducts were not visualized. The biopsy specimen showed marked fibrosis of the portal areas. Enterococcus species was cultured from the bile. Children or adolescents with recurrent AC and SC should be evaluated for an underlying immunodeficiency syndrome such as XHIGM.

Focal seizures as an unusual presentation of neonatal lupus erythematosus.

Neonatal lupus erythematosus is an uncommon passive autoimmune disease in which there is a transplacental passage of anti-Ro/SSA and/or anti-La/SSB maternal autoantibodies. Common clinical manifestations include cardiac disease, notably congenital heart block, cutaneous lupus lesions, hematologic disorders, and hepatobiliary disease. During the past decade, however, it has become clear that central nervous disease may also be a manifestation of neonatal lupus. We report a male neonate with the disease who had focal seizures in addition to cutaneous lupus, anemia, and thrombocytopenia. Brain ultrasound revealed normal ventricular size without a midline shift or intracranial or intraventricular hemorrhage. A brain CT showed generalized low density involving the periventricular and deep white matter. A sleep EEG revealed rare spikes axial to the right parietal lobe. The neonate had a high titer of antinuclear antibodies (1:640) with a speckled pattern, anti-Ro/SSA and anti-La/SSB antibodies, but no anti-dsDNA antibodies. He was given anti-convulsant drugs with dramatic improvement of his symptoms. One month later, a sleep EEG was normal, and he had no further seizures.

De novo mutation causing X-linked hyper-IgM syndrome: a family study in Taiwan.

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency disorder caused by mutations of the gene encoding the CD40 ligand (CD40L). It is characterized by recurrent infections with markedly decreased serum IgG, IgA and IgE levels but normal or elevated IgM levels. We report the clinical manifestations and complete immune studies in the first family with molecularly proven XHIM in Taiwan. A 5-month-old boy presented with rapidly progressive pneumonia which responded poorly to antibiotics. High levels of IgM and very low levels of IgG, IgA, and IgE were noted in his plasma specimen: IgM, 128 mg/dl; IgG, 18 mg/dl; IgA, 4 mg/dl); IgE, 1 IU/ml. Whole blood flow cytometry when he was 21 months old showed that only a small percentage (0.48%) of his in vitro-activated CD4+ T cells expressed CD40L. When he was 3 years old, repeated flow cytometry showed essentially the same result (0.4%), compared with his father's CD40L expression of over 85%. The patient's mother had moderately decreased CD40L expression (74.4%). Hyper-IgM syndrome was confirmed by CD40L mutation analysis in the boy, which revealed a Lys 96 stop (nucleotide A307T) in exon 2 of CD40L, with a truncated protein resulting in the loss of the entire TNF domain. His mother was a carrier and apparently the individual in whom the mutation originated. Eleven other family members, including the patient's father, sister, and grandmother, and the mother's sisters and their children, all had normal results on CD40L mutation analysis. The patient has remained without significant bacterial infection on a regimen of monthly IVIG infusion and oral trimethoprim-sulfamethoxazole for Pneumocystis carinii pneumonia (PCP) prophylaxis, although he has had recurrent oral ulcers and neutropenia. Bone marrow transplantation is planned.

Hyper-IgM syndrome: report of one case.

The hyper-IgM syndrome (HIM) is a rare primary immunodeficiency disorder caused by defects in the CD40 ligand (CD40L)/CD40-signaling pathway. It is characterized by recurrent infections with markedly decreased IgG, IgA and IgE levels but normal or elevated serum IgM levels. A 5-month-old boy presented with rapidly progressive pneumonia which responded poorly to antibiotics. High levels of IgM and very low levels of IgG, IgE and IgA were noted in his plasma specimen (IgM, 128 mg/dl; IgG, 18 mg/dl; IgE, 1 IU/ml; IgA, 4 mg/dl). The relative proportions of immune cells were CD3 24.6%, CD4 10.3%, CD8 2.2%, CD19 30.2%, CD57 1.0% and active T cells 1.1%. After IVIG treatment, the pneumonia improved. Repeat assessment at the age of 15 months showed IgM decreased to the normal range (32 mg/dl). Whole blood flow cytometry assay for CD40L expression confirmed the diagnosis of hyper-lgM syndrome when he was 21 months old. Only a small percentage (0.48%) of the patient's in vitro activated CD4+ T cells expressed CD40L, compared with 33.54% from a healthy control. The patient's father, mother and sister all had a normal CD40L expression activation patterns (43.52%, 40.78%, 34.11%, respectively). On a regimen of monthly IVIG infusion and oral trimethoprim-sulfamethoxazole for Pneumocystis carinii pneumonia (PCP) prophylaxis, the patient has had no recurrent infections.