A novel DNAJB6 mutation causes dominantly inherited distal-onset myopathy and compromises DNAJB6 function.

BACKGROUND: Mutations in the DNAJB6 gene have been identified as a rare cause of dominantly inherited limb-girdle muscular dystrophy or distal-onset myopathy. MATERIALS AND METHODS: Exome sequencing was performed to investigate a Taiwanese family with a dominantly inherited distal-onset myopathy. Functional effects of the causal mutation were investigated in vitro. RESULTS: Exome sequencing of the two affected individuals in this family identified a heterozygous mutation, c.287C>T (p.Pro96Leu) in the DNAJB6 gene, which co-segregated with the myopathy within all 12 family members. Notably, this mutation is novel and localizes within the glycine and phenylalanine-rich (G/F) domain and alters an amino acid residue previously reported with a different mutation. Furthermore, immunofluorescence analyses and filter trap assay demonstrated that the c.287C>T (p.Pro96Leu) mutation possessed a dominant negative effect on the anti-aggregation function of DNAJB6 protein. CONCLUSION: This study expands the molecular spectrum of DNAJB6 mutations and also emphasizes the pathogenic role of DNAJB6 dysfunction in distal-onset myopathy.

Phosphorylation of glycogen synthase kinase-3ß in metabolically abnormal obesity affects immune stimulation-induced cytokine production.

BACKGROUND: Obesity is a severe health problem worldwide, which leads to multiple comorbidities including type 2 diabetes mellitus and cardiovascular diseases. Inflammation has been found to be an important characteristic of adipose tissue in obese subjects. However, obesity is also associated with compromised immune responses to infections and the impact of obesity on immune function has not been fully understood. SUBJECTS/METHODS: To clarify the role of obesity in the immune responses, we investigated the Toll-like receptor (TLR)-induced cytokine secretion by leukocytes from obese and lean subjects. We also investigated the relationship between insulin-induced intracellular signaling and cytokine production using peripheral blood mononuclear cells (PBMCs) and a monocytic cell line THP-1. RESULTS: We found decreased TLR-induced interferon-γ, interleukin-6 (IL-6) and tumor necrosis factor-α secretions and elevated IL-10 secretion by leukocytes from obese subjects when compared with lean controls. PBMCs from obese subjects showed enhanced basal Akt and glycogen synthase kinase-3ß (GSK-3ß) phosphorylation, which did not further increase with insulin and lipopolysaccharide (LPS) stimulation. We also found that LPS-induced IκBα degradation was inhibited in PBMCs from obese subjects. By using THP-1 cells with GSK-3ß knockdown or cells treated with hyperinsulinemic and high-fatty acid conditions, we found that LPS-induced nuclear factor κB (NF-κB) activation was inhibited and cyclic adenosine monophosphate response element-binding protein (CREB) activation was enhanced. CONCLUSIONS: These findings indicate that GSK-3ß is important in the regulation of NF-κB and CREB activation in leukocytes under the metabolic condition of obesity. Our study revealed a key mechanism through which metabolic abnormalities compromise leukocyte functions in people with obesity.

The metabolome profiling and pathway analysis in metabolic healthy and abnormal obesity.

OBJECTIVES: Mechanisms of the development of abnormal metabolic phenotypes among obese population are not yet clear. In this study, we aimed to screen metabolomes of both healthy and subjects with abnormal obesity to identify potential metabolic pathways that may regulate the different metabolic characteristics of obesity. METHODS: We recruited subjects with body mass index (BMI) over 25 from the weight-loss clinic of a central hospital in Taiwan. Metabolic healthy obesity (MHO) is defined as without having any form of hyperglycemia, hypertension and dyslipidemia, while metabolic abnormal obesity (MAO) is defined as having one or more abnormal metabolic indexes. Serum-based metabolomic profiling using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of 34 MHO and MAO individuals with matching age, sex and BMI was performed. Conditional logistic regression and partial least squares discriminant analysis were applied to identify significant metabolites between the two groups. Pathway enrichment and topology analyses were conducted to evaluate the regulated pathways. RESULTS: A differential metabolite panel was identified to be significantly differed in MHO and MAO groups, including L-kynurenine, glycerophosphocholine (GPC), glycerol 1-phosphate, glycolic acid, tagatose, methyl palmitate and uric acid. Moreover, several metabolic pathways were relevant in distinguishing MHO from MAO groups, including fatty acid biosynthesis, phenylalanine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation. CONCLUSION: Different metabolomic profiles and metabolic pathways are important for distinguishing between MHO and MAO groups. We have identified and discussed the key metabolites and pathways that may prove important in the regulation of metabolic traits among the obese, which could provide useful clues to study the underlying mechanisms of the development of abnormal metabolic phenotypes.

Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification.

Thrips, the sole vector of plant Tospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

Development of Species-Specific Primers for Agronomical Thrips and Multiplex Assay for Quarantine Identification of Western Flower Thrips.

While morphological identification of thrips species has been difficult because of their minute size and a lack of easily recognizable characteristics, molecular identification based on the development of specific molecular markers can be easily and reliably carried out. Among the known molecular markers, the nuclear internal transcribed spacer (ITS) exhibits distinguishable variations among thrips species. In this study, sequences of ITS2 region of 10 agriculturally important thrips were established to design species-specific primers for polymerase chain reaction (PCR). ITS2 sequence variations within these species were far less than those among species, indicating the suitability of this marker for species-specific primers design. These primers, though with one or two sporadically variable positions, showed a good efficacy within species. The specificity of these primers, examined on thrips species belonging to 15 genera, proved satisfactory. Furthermore, a multiplex PCR was used successfully for identifying Frankliniella occidentalis (Pergande), an insect pest monitored for quarantine purpose, and three additional thrips also commonly found in imported agricultural products and field samples, i.e., Thrips tabaci Lindeman, Thrips hawaiiensis (Morgan), and Frankliniella intonsa (Trybom). This study has demonstrated that specific primers and multiplex PCR based on ITS2 are reliable, convenient, and diagnostic tool to discriminate thrips species of quarantine and agricultural importance.

Toll-like receptor 2 deficiency improves insulin sensitivity and hepatic insulin signalling in the mouse.

AIMS/HYPOTHESIS: Substantial evidence suggests a link between elevated inflammation and development of insulin resistance. Toll-like receptor 2 (TLR2) recognises a large number of lipid-containing molecules and transduces inflammatory signalling in a variety of cell types, including insulin-responsive cells. Considering the contribution of the fatty acid composition in TLR2-depedent signalling, we hypothesised that the inflammatory signals transduced by TLR2 contribute to insulin resistance. METHODS: Mice deficient in TLR2 were used to investigate the in vivo roles of TLR2 in initiating and maintaining inflammation-associated insulin resistance and energy homeostasis. RESULTS: We first recapitulated the observation with elevated expression of TLR2 and inflammatory cytokines in white adipose tissue and liver of ob/ob mice. Aged or high-fat-fed TLR2-deficient mice were protected from obesity and adipocyte hypertrophy compared with wild-type mice. Moreover, mice lacking TLR2 exhibited improved glucose tolerance and insulin sensitivity regardless of feeding them regular chow or a high-fat diet. This is accompanied by reductions in expression of inflammatory cytokines and activation of extracellular signal-regulated kinase (ERK) in a liver-specific manner. The attenuated hepatic inflammatory cytokine expression and related signalling are correlated with increased insulin action specifically in the liver in TLR2-deficient mice, reflected by increased insulin-stimulated protein kinase B (Akt) phosphorylation and IRS1 tyrosine phosphorylation and increased insulin-suppressed hepatocyte glucose production. CONCLUSIONS/INTERPRETATION: The absence of TLR2 attenuates local inflammatory cytokine expression and related signalling and increases insulin action specifically in the liver. Thus, our work has identified TLR2 as a key mediator of hepatic inflammation-related signalling and insulin resistance.

Lower ataxia telangiectasia mutated (ATM) mRNA expression is correlated with poor outcome of laryngeal and pharyngeal cancer patients.

BACKGROUND: Ataxia telangiectasia mutated (ATM) kinase is a critical regulator in initiating DNA damage response and activating DNA repair. However, the correlation between ATM expression and the outcome of laryngopharyngeal cancer patients is unknown. We hypothesize that ATM expression is correlated with a worse outcome in laryngopharyngeal cancer patients. PATIENTS AND METHODS: The ATM messenger RNA (mRNA) expression of 80 tumors of laryngeal and pharyngeal cancer was examined by real-time quantitative RT-PCR. Overall survival rates were measured using Kaplan-Meier estimates and the log-rank tests. The adjusted hazard rate ratios (HRRs) were computed by multivariate Cox regressions. RESULTS: Reduced ATM mRNA was found in 65 of 80 studied cases. Lower ATM expression [tumor/normal <0.3, HRR = 2.49; 95% confidence interval (CI) 1.27-4.88], younger age (<55 years, HRR = 2.71; 95% CI 1.16-6.32), and larger tumor (T(3)/T(4), HRR = 2.21; 95% CI 1.10-4.44) were independent risk factors for survival. Patients with lower ATM and younger age (HRR = 6.51; 95% CI 2.05-20.66) or with lower ATM and T(3)/T(4) tumor (HRR = 5.23; 95% CI 2.04-13.40) exhibited the poorest outcome. CONCLUSION: The expression of ATM mRNA, which is frequently downregulated in laryngeal and pharyngeal cancers, could be a valuable prognostic marker.

Comparative study of percutaneous nephrostomy using catheters with and without locking strings.

BACKGROUND: Although it is generally felt that a catheter with a locking string can achieve better fixation and thus prevent catheter displacement, no formal study has ever substantiated this. METHODS: We retrospectively reviewed the charts from 80 patients (mean age of 64.6 ± 14.76 y) who underwent percutaneous nephrostomy (PCN) over a 1-year period. RESULTS: Most patients had catheters without locking strings and only 17 patients (21.3%) had catheters with locking strings. The median duration of catheter placement was 29 days (interquartile range 14 - 57 d). There were no significant differences in patients' characteristics or catheter outcomes between catheters with and catheters without locking strings (p > 0.05). In addition, no significant difference in the catheter 90-day survival between catheter types was found (log rank test, p = 0.638). On univariate analysis, tumor as an indication for PCN (p = 0.018), obstruction (p = 0.021) and displacement (p = 0.007) were associated with reduced catheter survival. The multivariate analysis indicated that tumor as an indication for PCN (HR: 0.28, 95% CI: 0.13 - 0.63, p = 0.002), obstruction (HR: 0.25, 95% CI: 0.08 - 0.77, p = 0.015) and catheter displacement (HR: 0.09, 95% CI: 0.03 - 0.31, p < 0.001) were independent hazard factors for reduced catheter 90-day survival. CONCLUSION: No significant difference in either complication rate or 90-day survival was found between catheters with or without locking strings. These findings may prove helpful to the clinician in deciding the type of catheter to use during PCN.

Effects of Exercise Alone or in Combination with High-Protein Diet on Muscle Function, Aerobic Capacity, and Physical Function in Middle-Aged Obese Adults: A Randomized Controlled Trial.

BACKGROUND: Obesity accelerates and exacerbates the age-related changes on muscle function and exercise capacity. In addition, the middle-aged population is often overlooked when talking about the prevention of sarcopenia. This study investigated the effects of exercise alone or in combination with a high-protein diet on muscle function and physical fitness in middle-aged obese adults. MATERIALS AND METHODS: Sixty-nine middle-aged (50-64 years old) obese adults were randomly assigned to one of the following groups: control group (C; n=23), exercise group (E; n=23) or exercise plus high-protein group (EP; n=23). Individuals within the E and EP groups received 12 weeks of exercise training; whereas, the individuals in the EP group also received a high-protein diet intervention (1.6g/kg/day). Individuals within the C group were asked to maintain their lifestyle for 12 weeks. Participants were evaluated before and after the intervention. Outcome measures included maximal exercise capacity, muscle function and functional physical performance. Analysis of covariance was used to determine the effects of the intervention. RESULTS: After the intervention, the E and EP groups had greater maximal work rate, peak oxygen consumption, and muscle power during muscle contractions at 180°/sec than that in the C group (P<0.05). The EP group, but not the E group, showed significant improvement in the sit-to-stand test and climbing stairs test than the C group after the intervention (P<0.05). Within group comparisons showed that the anaerobic threshold only increased in the EP group (+12% from pre-test). CONCLUSIONS: For middle-aged obese adults, exercise with a high-protein diet not only improved muscle power and exercise capacity but also enhanced their functional physical performance.

Prevalence of antibiotics resistance and OXA carbapenemases genes in multidrug-resistant Acinetobacter baumannii isolates in central Taiwan.

This study analyzed the prevalence of antibiotics resistance and the distribution of genes responsible for carbapenems resistance in Acinetobacter baumannii isolates. Clinical A. baumannii isolates were cultured, identified, and collected during the period from May 2007 to February 2009. Antibiotics resistance rates of the clinical isolates were analyzed by antimicrobial susceptibility testing. The distribution of carbapenemase alleles were investigated in the multidrug-resistant (MDR) A. baumannii isolates by multiplex polymerase chain reaction (PCR) techniques. A total of 1,265 independent A. baumannii isolates were identified. Approximately 70% of the clinical isolates were resistant to ampicillin/sulbactam, followed by imipenem, meropenem, cefepime, piperacillin/tazobactam, ceftazidime, and cefoperazone. Overall, 15.18% (192/1,265) of the isolates were characterized as MDR strains. All of the MDR A. baumannii isolates carried the bla (OXA51-like) allele. The detection rate of the bla (OXA23-like) and bla (OXA24-like) alleles was 96.35% (185/192) and 0.52% (1/192), respectively. Most of the isolates (185/192, 96.35%) carried genes which encode more than one carbapenemase. This report demonstrated that approximately 15% of A. baumannii clinical isolates in central Taiwan are MDR strains, with most of them harboring multiple carbapenemases. This study provides updated data regarding the prevalence of beta-lactam resistance and genotyping information of carbapenems resistance of A. baumannii in central Taiwan.

Macrophage activation syndrome with lung involvement complicating adult-onset Still's disease.

Adult-onset Still's disease (AOSD) can progress into macrophage activation syndrome (MAS), which may be fatal. We report on a 19-year-old Han Chinese female, who presented with MAS-related pulmonary parenchymal involvement complicating AOSD, and further associated with disseminated intravascular coagulation and generalized tonic-clonic seizure. She was managed by high-dose corticosteroids and pulse cyclophosphamide therapy with a complete recovery of disease activity.

T2 Relaxometry MRI Predicts Cerebral Palsy in Preterm Infants.

BACKGROUND AND PURPOSE: T2-relaxometry brain MR imaging enables objective measurement of brain maturation based on the water-macromolecule ratio in white matter, but the outcome correlation is not established in preterm infants. Our study aimed to predict neurodevelopment with T2-relaxation values of brain MR imaging among preterm infants. MATERIALS AND METHODS: From January 1, 2012, to May 31, 2015, preterm infants who underwent both T2-relaxometry brain MR imaging and neurodevelopmental follow-up were retrospectively reviewed. T2-relaxation values were measured over the periventricular white matter, including sections through the frontal horns, midbody of the lateral ventricles, and centrum semiovale. Periventricular T2 relaxometry in relation to corrected age was analyzed with restricted cubic spline regression. Prediction of cerebral palsy was examined with the receiver operating characteristic curve. RESULTS: Thirty-eight preterm infants were enrolled for analysis. Twenty patients (52.6%) had neurodevelopmental abnormalities, including 8 (21%) with developmental delay without cerebral palsy and 12 (31.6%) with cerebral palsy. The periventricular T2-relaxation values in relation to age were curvilinear in preterm infants with normal development, linear in those with developmental delay without cerebral palsy, and flat in those with cerebral palsy. When MR imaging was performed at >1 month corrected age, cerebral palsy could be predicted with T2 relaxometry of the periventricular white matter on sections through the midbody of the lateral ventricles (area under the receiver operating characteristic curve = 0.738; cutoff value of >217.4 with 63.6% sensitivity and 100.0% specificity). CONCLUSIONS: T2-relaxometry brain MR imaging could provide prognostic prediction of neurodevelopmental outcomes in premature infants. Age-dependent and area-selective interpretation in preterm brains should be emphasized.

Epigenetic silencing of the dual-role signal mediator, ANGPTL4 in tumor tissues and its overexpression in the urothelial carcinoma microenvironment.

Urothelial carcinoma (UC) carcinogenesis has been hypothesized to occur through epigenetic repression of tumor-suppressor genes (TSGs). By quantitative real-time polymerase chain reaction array, we found that one potential TSG, angiopoietin-like 4 (ANGPTL4), was expressed at very low levels in all bladder cancer cell lines we examined. Previous studies had demonstrated that ANGPTL4 is highly expressed in some cancers, but downregulated, by DNA methylation, in others. Consequently, owing to these seemingly conflicting functions in distinct cancers, the precise role of ANGPTL4 in the etiology of UC remains unclear. In this study, using methylation-specific PCR and bisulfite pyrosequencing, we show that ANGPTL4 is transcriptionally repressed by DNA methylation in UC cell lines and primary tumor samples, as compared with adjacent noncancerous bladder epithelium. Functional studies further demonstrated that ectopic expression of ANGPTL4 potently suppressed UC cell proliferation, monolayer colony formation in vitro, and invasion, migration, and xenograft formation in vivo. Surprisingly, circulating ANGPTL4 was significantly higher in plasma samples from UC patients than normal control, suggesting it might be secreted from other cell types. Interestingly, our data also indicated that exogenous cANGPTL4 could promote cell proliferation and cell migration via activation of signaling through the Erk/focal adhesion kinase axis. We further confirmed that mouse xenograft tumor growth could be promoted by administration of exogenous cANGPTL4. Finally, immunohistochemistry demonstrated that ANGPTL4 was downregulated in tumor cells but overexpressed in tumor adjacent stromal tissues of muscle-invasive UC tissue samples. In conclusion, our data support dual roles for ANGPTL4 in UC progression, either as a tumor suppressor or oncogene, in response to microenvironmental context.

Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells.

A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.

Infrared spectra of phospholipid membranes: interfacial dehydration by volatile anesthetics and phase transition.

Fourier-transform infrared attenuated total reflection (ATR) spectroscopy was used to study the effect of volatile anesthetics on fully hydrated dipalmitoylphosphatidylcholine (DPPC) vesicle membranes. The main phase transition was monitored by the change in the C-H2 asymmetric stretching frequencies of the lipid tails. The surface property was analyzed by the changes in the P = O stretching, (CH3)3-N+ stretching of the hydrophilic head, and C = O stretching of the glycerol skeleton. The partial pressures of those agents that decreased the transition temperature 1.0 C degree were halothane 0.75, enflurane 1.90 and CCl4 0.85 kPa. At a 2:1 lipid/anesthetic mole ratio, the polar anesthetics, halothane and enflurane, increased the ratio of (P = O stretching band area)/((CH3)3-N+ stretching band area) by 26.3% and 21.1%, respectively, whereas apolar CCl4 increased it 10.5%. The water molecules bound to the P = O moiety are apparently replaced by the anesthetic molecules. The deconvoluted C = O spectra showed two peaks: free sn-1 that is closer to the lipid core and hydrogen-bonded sn-2 that is closer to the polar head. Addition of halothane and enflurane, but not CCl4, increased the number of peaks to three. The third peak is free sn-2, formed by disrupting hydrogen-bonding to water. Because the temperature-induced spectral change was limited to C-H2 stretching at the main phase transition, the effects of anesthetics on the lipid membrane structure are not identical to temperature elevation. Among anesthetics, the effects of apolar and polar molecules on the interfacial properties are different.

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles.

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.

Supplementation with vitamins C and E enhances cytokine production by peripheral blood mononuclear cells in healthy adults.

The effect of supplementation with vitamins C and E on cytokine production of healthy adult volunteers was studied in a single-blind trial. Ten subjects in each group received daily vitamin C (1 g ascorbic acid), vitamin E (400 mg dl-alpha-tocopheryl acetate), or vitamins C and E for 28 d. Plasma concentrations of alpha-tocopherol, ascorbate, and lipid peroxides as well as the production of cytokines by peripheral blood mononuclear cells (PBMCs) were measured before, during, and at the end of the supplementation and 1 wk later. PBMCs were cultured in the presence of absence of lipopolysaccharide for 24 h. The interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) in the culture supernates were assayed by enzyme-linked immunosorbent assay methods. Production of IL-1 beta and TNF-alpha in the group supplemented with vitamins C and E was significantly higher (P < 0.05) than that of the groups given vitamin E or vitamin C alone. The enhancing effect of supplementation with a combination of vitamins E and C coincided with peak plasma alpha-tocopherol and ascorbate concentrations and the lowest plasma lipid peroxide concentrations (P < 0.05) on day 14. In addition, an in vitro experiment with PBMCs showed that vitamins E and C reduced lipopolysaccharide-induced prostaglandin E2 production and enhanced TNF-alpha production. These results indicate that combined supplementation with vitamins C and E is more immunopotentiating than supplementation with either vitamin alone in healthy adults.