Mutations in EXTL3 Cause Neuro-immuno-skeletal Dysplasia Syndrome.

EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal dysplasia syndrome, and to gain insight into the pathogenesis of the disorder, we analyzed the localization of EXTL3 in fibroblasts derived from affected individuals and determined glycosaminoglycan concentrations in these cells as well as in urine and blood. We observed abnormal glycosaminoglycan concentrations and increased concentrations of the non-sulfated chondroitin disaccharide D0a0 and the disaccharide D0a4 in serum and urine of all analyzed affected individuals. In summary, we show that biallelic mutations in EXTL3 disturb glycosaminoglycan synthesis and thus lead to a recognizable syndrome characterized by variable expression of skeletal, neurological, and immunological abnormalities.

Slc22a5 haploinsufficiency does not aggravate the phenotype of the long-chain acyl-CoA dehydrogenase KO mouse.

Secondary carnitine deficiency is commonly observed in inherited metabolic diseases characterised by the accumulation of acylcarnitines such as mitochondrial fatty acid oxidation (FAO) disorders. It is currently unclear if carnitine deficiency and/or acylcarnitine accumulation play a role in the pathophysiology of FAO disorders. The long-chain acyl-CoA dehydrogenase (LCAD) KO mouse is a model for long-chain FAO disorders and is characterised by decreased levels of tissue and plasma free carnitine. Tissue levels of carnitine are controlled by SLC22A5, the plasmalemmal carnitine transporter. Here, we have further decreased carnitine availability in the LCAD KO mouse through a genetic intervention by introducing one defective Slc22a5 allele (jvs). Slc22a5 haploinsufficiency decreased free carnitine levels in liver, kidney, and heart of LCAD KO animals. The resulting decrease in the tissue long-chain acylcarnitines levels had a similar magnitude as the decrease in free carnitine. Levels of cardiac deoxycarnitine, a carnitine biosynthesis intermediate, were elevated due to Slc22a5 haploinsufficiency in LCAD KO mice. A similar increase in heart and muscle deoxycarnitine was observed in an independent experiment using Slc22a5jvs/jvs mice. Cardiac hypertrophy, fasting-induced hypoglycemia and increased liver weight, the major phenotypes of the LCAD KO mouse, were not affected by Slc22a5 haploinsufficiency. This may suggest that secondary carnitine deficiency does not play a major role in the pathophysiology of these phenotypes. Similarly, our data do not support a major role for toxicity of long-chain acylcarnitines in the phenotype of the LCAD KO mouse.

Prediction of phenotypic severity in mucopolysaccharidosis type IIIA.

OBJECTIVE: Mucopolysaccharidosis IIIA or Sanfilippo disease type A is a progressive neurodegenerative disorder presenting in early childhood, caused by an inherited deficiency of the lysosomal hydrolase sulfamidase. New missense mutations, for which genotype-phenotype correlations are currently unknown, are frequently reported, hampering early prediction of phenotypic severity and efficacy assessment of new disease-modifying treatments. We aimed to design a method to determine phenotypic severity early in the disease course. METHODS: Fifty-three patients were included for whom skin fibroblasts and data on disease course and mutation analysis were available. Patients were phenotypically characterized on clinical data as rapidly progressing or slowly progressing. Sulfamidase activity was measured in fibroblasts cultured at 37 °C and at 30 °C. RESULTS: Sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a combination of known severe mutations remained below the limit of quantification under both culture conditions. In contrast, sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a known mild mutation increased above the limit of quantification when cultured at 30 °C. With division on the basis of the patients' phenotype, fibroblasts from slowly progressing patients could be separated from rapidly progressing patients by increase in sulfamidase activity when cultured at 30 °C (p < 0.001, sensitivity = 96%, specificity = 93%). INTERPRETATION: Phenotypic severity strongly correlates with the potential to increase sulfamidase activity in fibroblasts cultured at 30 °C, allowing reliable distinction between patients with rapidly progressing or slowly progressing phenotypes. This method may provide an essential tool for assessment of treatment effects and for health care and life planning decisions. Ann Neurol 2017;82:686-696.

Microvascular Permeability after an Acute and Chronic Salt Load in Healthy Subjects: A Randomized Open-label Crossover Intervention Study.

BACKGROUND: Sodium-induced microcirculatory changes, endothelial surface layer alterations in particular, may play an important role in sodium-mediated blood pressure elevation. However, effects of acute and chronic sodium loading on the endothelial surface layer and microcirculation in humans have not been established. The objective of this study was to assess sodium-induced changes in blood pressure and body weight as primary outcomes and also in microvascular permeability, sublingual microcirculatory dimensions, and urinary glycosaminoglycan excretion in healthy subjects. METHODS: Twelve normotensive males followed both a low-sodium diet (less than 50 mmol/day) and a high-sodium diet (more than 200 mmol/day) for eight days in randomized order, separated by a crossover period. After the low-sodium diet, hypertonic saline (5 mmol sodium/liter body water) was administered intravenously in 30 min. RESULTS: Both sodium interventions did not change blood pressure. Body weight increased with 2.5 (95% CI, 1.7 to 3.2) kg (P < 0.001) after dietary sodium loading. Acute intravenous sodium loading resulted in increased transcapillary escape rate of I-labeled albumin (2.7 [0.1 to 5.3] % cpm · g · h; P = 0.04), whereas chronic dietary sodium loading did not affect transcapillary escape rate of I-labeled albumin (-0.03 [-3.3 to 3.2] % cpm · g · h; P = 1.00), despite similar increases of plasma sodium and osmolality. Acute intravenous sodium loading coincided with significantly increased plasma volume, as assessed by the distribution volume of albumin, and significantly decreased urinary excretion of heparan sulfate and chondroitin sulfate. These changes were not observed after dietary sodium loading. CONCLUSIONS: Our results suggest that intravenous sodium loading has direct adverse effects on the endothelial surface layer, independent of blood pressure.

Newborn screening for mucopolysaccharidoses: a pilot study of measurement of glycosaminoglycans by tandem mass spectrometry.

BACKGROUND: Mucopolysaccharidoses (MPS) are a group of inborn errors of metabolism that are progressive and usually result in irreversible skeletal, visceral, and/or brain damage, highlighting a need for early diagnosis. METHODS: This pilot study analyzed 2862 dried blood spots (DBS) from newborns and 14 DBS from newborn patients with MPS (MPS I, n = 7; MPS II, n = 2; MPS III, n = 5). Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Heparan sulfate (0S, NS), dermatan sulfate (DS) and mono- and di-sulfated KS were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Median absolute deviation (MAD) was used to determine cutoffs to distinguish patients from controls. Cutoffs were defined as median + 7× MAD from general newborns. RESULTS: The cutoffs were as follows: HS-0S > 90 ng/mL; HS-NS > 23 ng/mL, DS > 88 ng/mL; mono-sulfated KS > 445 ng/mL; di-sulfated KS > 89 ng/mL and ratio di-KS in total KS > 32 %. All MPS I and II samples were above the cutoffs for HS-0S, HS-NS, and DS, and all MPS III samples were above cutoffs for HS-0S and HS-NS. The rate of false positives for MPS I and II was 0.03 % based on a combination of HS-0S, HS-NS, and DS, and for MPS III was 0.9 % based upon a combination of HS-0S and HS-NS. CONCLUSIONS: Combination of levels of two or more different GAGs improves separation of MPS patients from unaffected controls, indicating that GAG measurements are potentially valuable biomarkers for newborn screening for MPS.

Transorgan fluxes in a porcine model reveal a central role for liver in acylcarnitine metabolism.

Acylcarnitines are derived from mitochondrial acyl-CoA metabolism and have been associated with diet-induced insulin resistance. However, plasma acylcarnitine profiles have been shown to poorly reflect whole body acylcarnitine metabolism. We aimed to clarify the individual role of different organ compartments in whole body acylcarnitine metabolism in a fasted and postprandial state in a porcine transorgan arteriovenous model. Twelve cross-bred pigs underwent surgery where intravascular catheters were positioned before and after the liver, gut, hindquarter muscle compartment, and kidney. Before and after a mixed meal, we measured acylcarnitine profiles at several time points and calculated net transorgan acylcarnitine fluxes. Fasting plasma acylcarnitine concentrations correlated with net hepatic transorgan fluxes of free and C2- and C16-carnitine. Transorgan acylcarnitine fluxes were small, except for a pronounced net hepatic C2-carnitine production. The peak of the postprandial acylcarnitine fluxes was between 60 and 90 min. Acylcarnitine production or release was seen in the gut and liver and consisted mostly of C2-carnitine. Acylcarnitines were extracted by the kidney. No significant net muscle acylcarnitine flux was observed. We conclude that liver has a key role in acylcarnitine metabolism, with high net fluxes of C2-carnitine both in the fasted and fed state, whereas the contribution of skeletal muscle is minor. These results further clarify the role of different organ compartments in the metabolism of different acylcarnitine species.

Biomarker responses correlate with antibody status in mucopolysaccharidosis type I patients on long-term enzyme replacement therapy.

BACKGROUND: Antibody formation can interfere with effects of enzyme replacement therapy (ERT) in lysosomal storage diseases. Biomarkers are used as surrogate marker for disease burden in MPS I, but large systematic studies evaluating the response of biomarkers to ERT are lacking. We, for the first time, investigated the response of a large panel of biomarkers to long term ERT in MPS I patients and correlate these responses with antibody formation and antibody mediated cellular uptake inhibition. METHODS: A total of 428 blood and urine samples were collected during long-term ERT in 24 MPS I patients and an extensive set of biomarkers was analyzed, including heparan sulfate (HS) and dermatan sulfate (DS) derived disaccharides; total urinary GAGs (DMBu); urinary DS:CS ratio and serum heparin co-factor II thrombin levels (HCII-T). IgG antibody titers and the effect of antibodies on cellular uptake of the enzyme were determined for 23 patients. RESULTS: Median follow-up was 2.3 years. In blood, HS reached normal levels more frequently than DS (50% vs 12.5%, p=0.001), though normalization could take several years. DMBu normalized more rapidly than disaccharide levels in urine (p=0.02). Nineteen patients (83%) developed high antibody titers. Significant antibody-mediated inhibition of enzyme uptake was observed in 8 patients (35%), and this correlated strongly with a poorer biomarker response for HS and DS in blood and urine as well as for DMBu, DS:CS-ratio and HCII-T (all p<0.006). CONCLUSIONS: This study shows that, despite a response of all studied biomarkers to initiation of ERT, some biomarkers were less responsive than others, suggesting residual disease activity. In addition, the correlation of cellular uptake inhibitory antibodies with a decreased biomarker response demonstrates a functional role of these antibodies which may have important clinical consequences.

Supplementing pregnant mice with a specific mixture of nondigestible oligosaccharides reduces symptoms of allergic asthma in male offspring.

BACKGROUND: Previously, maternal supplementation with short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS; ratio 9:1) was shown to affect maternal and fetal immune status in mice. OBJECTIVE: This study was designed to test the long-term effects of supplementation of mice with scGOS/lcFOS before and during pregnancy on the immune response in the offspring, using an ovalbumin (OVA)-induced model for experimental allergic asthma. METHODS: Female Balb/c mice were fed a control diet or a diet supplemented with 3% scGOS/lcFOS and mated to C57BL/6 males. All dams were fed the control diet after delivery. At 6 wk, male offspring received an intraperitoneal injection of aluminum hydroxide and OVA (control and scGOS/lcFOS group) or saline (sham group). The acute allergic skin response (ASR) after intradermal challenge with OVA or saline was measured at 8 wk. After 3 airway challenges with nebulized OVA or saline, lung function was measured. RESULTS: The scGOS/lcFOS group had a significantly lower acute ASR (85 ± 9 µm) than the control group (124 ± 9 µm; P = 0.01). Lower lung resistance from a response to methacholine challenge was seen in the scGOS/lcFOS group. OVA-specific immunoglobulin (Ig)E concentrations in the control group [93 ± 45 arbitrary unit (AU)] and the scGOS/lcFOS group (67 ± 45 AU) were higher than in the sham group (11 ± 2 AU). OVA specific IgG2a concentrations in the scGOS/lcFOS (146 ± 24 AU) were higher than in the sham group (2 ± 0.3 AU) and control group (18 ± 3.5 AU; P < 0.05). Finally, the scGOS/lcFOS group had a higher percentage of regulatory T cells (1.11% ± 0.07%) than the sham group (0.14% ± 0.03%) and the control group (0.11% ± 0.02%; P < 0.05). CONCLUSION: Maternal supplementation of mice with scGOS/lcFOS during pregnancy leads to a significant decrease in allergic symptoms in the offspring.

Novel heparan sulfate assay by using automated high-throughput mass spectrometry: Application to monitoring and screening for mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are caused by deficiency of one of a group of specific lysosomal enzymes, resulting in excessive accumulation of glycosaminoglycans (GAGs). We previously developed GAG assay methods using liquid chromatography tandem mass spectrometry (LC-MS/MS); however, it takes 4-5 min per sample for analysis. For the large numbers of samples in a screening program, a more rapid process is desirable. The automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) integrates a solid phase extraction robot to concentrate and desalt samples prior to direction into the MS/MS without chromatographic separation; thereby allowing each sample to be processed within 10s (enabling screening of more than one million samples per year). The aim of this study was to develop a higher throughput system to assay heparan sulfate (HS) using HT-MS/MS, and to compare its reproducibility, sensitivity and specificity with conventional LC-MS/MS. HS levels were measured in the blood (plasma and serum) from control subjects and patients with MPS II, III, or IV and in dried blood spots (DBS) from newborn controls and patients with MPS I, II, or III. Results obtained from HT-MS/MS showed 1) that there was a strong correlation of levels of disaccharides derived from HS in the blood, between those calculated using conventional LC-MS/MS and HT-MS/MS, 2) that levels of HS in the blood were significantly elevated in patients with MPS II and III, but not in MPS IVA, 3) that the level of HS in patients with a severe form of MPS II was higher than that in an attenuated form, 4) that reduction of blood HS level was observed in MPS II patients treated with enzyme replacement therapy or hematopoietic stem cell transplantation, and 5) that levels of HS in newborn DBS were elevated in patients with MPS I, II or III, compared to those of control newborns. In conclusion, HT-MS/MS provides much higher throughput than LC-MS/MS-based methods with similar sensitivity and specificity in an HS assay, indicating that HT-MS/MS may be feasible for diagnosis, monitoring, and newborn screening of MPS.

Genistein increases glycosaminoglycan levels in mucopolysaccharidosis type I cell models.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder characterized by diminished degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which results in the accumulation of these GAGs and subsequent cellular dysfunction. Patients present with a variety of symptoms, including severe skeletal disease. Genistein has been shown previously to inhibit GAG synthesis in MPS fibroblasts, presumably through inhibition of tyrosine kinase activity of the epidermal growth factor receptor (EGFR). To determine the potentials of genistein for the treatment of skeletal disease, MPS I fibroblasts were induced into chondrocytes and osteoblasts and treated with genistein. Surprisingly, whereas tyrosine phosphorylation levels (as a measure for tyrosine kinase inhibition) were decreased in all treated cell lines, there was a 1.3 and 1.6 fold increase in GAG levels in MPS I chondrocytes and fibroblast, respectively (p < 0.05). Sulfate incorporation in treated MPS I fibroblasts was 2.6 fold increased (p < 0.05), indicating increased GAG synthesis despite tyrosine kinase inhibition. This suggests that GAG synthesis is not exclusively regulated through the tyrosine kinase activity of the EGFR. We hypothesize that the differences in outcomes between studies on the effect of genistein in MPS are caused by the different effects of genistein on different growth factor signaling pathways, which regulate GAG synthesis. More studies are needed to elucidate the precise signaling pathways which are affected by genistein and alter GAG metabolism in order to evaluate the therapeutic potential of genistein for MPS patients.

Effect of dietary lipid structure in early postnatal life on mouse adipose tissue development and function in adulthood.

Obese individuals have more (hyperplastic) and larger (hypertrophic) adipocytes in their white adipose tissue (WAT) than normal-weight individuals. The difference in cell number emerges early in childhood, suggesting that this is a critical period for being susceptible to obesity. Breast-feeding has been shown to be protective against obesity, and we have previously shown in mice that the physical structure of lipids in human milk may contribute to this protective effect. In the present study, we investigated how differences in the physical structure of lipids in the early diet may modulate adipose tissue development. Male mice were fed a diet containing control infant milk formula (Control IMF; Danone Research) or Nuturis® (Concept IMF with large phospholipid-coated lipid droplets; Danone Research) from postnatal day (PN)16 to 42. Subsequently, mice were challenged with a moderate Western-style diet (WSD) until PN98, and body composition was monitored by dual-energy X-ray absorptiometry. Epididymal WAT was analysed for adipocyte size, number and gene expression of metabolic transcription factors. Early Concept IMF exposure reduced fat accumulation during the WSD challenge by 30 % compared with the Control IMF. It reduced adipocyte size without affecting adipocyte number in adult mice. The Concept IMF decreased the expression of PPARγ, CCAAT/enhancer-binding protein and retinoid X receptor α in WAT in adulthood, key regulators of metabolic activity. In conclusion, Concept IMF exposure in early life reduced susceptibility to obesity in adult life, by preventing adipocyte hypertrophia upon adult dietary challenge without affecting adipogenesis. These data emphasise the importance of the physical properties of dietary lipids in early life in obesity risk later in life.

Plasma and urinary levels of dermatan sulfate and heparan sulfate derived disaccharides after long-term enzyme replacement therapy (ERT) in MPS I: correlation with the timing of ERT and with total urinary excretion of glycosaminoglycans.

INTRODUCTION: Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels. METHODS: Plasma and urine samples were collected at different time points relative to the weekly ERT for three non-consecutive weeks in seven MPS I patients who had been treated with ERT for at least 2.5 years. Heparan and dermatan sulfate in plasma and urine were enzymatically digested into disaccharides, and HS and DS levels were determined by HPLC-MS/MS analysis. uGAGs were measured by the DMB test. RESULTS: The levels of HS and DS were markedly decreased compared with the levels before the initiation of ERT. However, the concentrations of DS in plasma and of both HS and DS in urine remained significantly elevated in all studied patients, while in six patients the level of total uGAGs had normalized. The concentrations of plasma and urinary HS during the weekly ERT followed a U-shaped curve. However, the effect size is small. The concentrations of plasma and urinary DS and uGAGs appeared to be in a steady state. CONCLUSIONS: HS and DS are sensitive biomarkers for monitoring the biochemical treatment efficacy of ERT and remain elevated despite long-term treatment. This finding may be related to the labeled dose or antibody status of the patient. The timing of the sample collection is not relevant, at least at the current dose of 100 IU/kg/weekly.

In vitro alternative for reactogenicity assessment of outer membrane vesicle based vaccines.

Intrinsic or added immune activating molecules are key for most vaccines to provide desired immunity profiles but may increase systemic reactogenicity. Regulatory agencies require rabbit pyrogen testing (RPT) for demonstration of vaccine reactogenicity. Recently, the monocyte activation test (MAT) gained popularity as in vitro alternative, yet this assay was primarily designed to test pyrogen-free products. The aim was to adjust the MAT to enable testing of pyrogen containing vaccines in an early stage of development where no reference batch is yet available. The MAT and RPT were compared for assessing unknown safety profiles of pertussis outer membrane vesicle (OMV) vaccine candidates to those of Bexsero as surrogate reference vaccine. Pertussis OMVs with wild-type LPS predominantly activated TLR2 and TLR4 and were more reactogenic than Bexsero. However, this reactogenicity profile for pertussis OMVs could be equalized or drastically reduced compared to Bexsero or a whole-cell pertussis vaccine, respectively by dose changing, modifying the LPS, intranasal administration, or a combination of these. Importantly, except for LPS modified products, reactogenicity profiles obtained with the RPT and MAT were comparable. Overall, we demonstrated that this pertussis OMV vaccine candidate has an acceptable safety profile. Furthermore, the MAT proved its applicability to assess reactogenicity levels of pyrogen containing vaccines at multiple stages of vaccine development and could eventually replace rabbit pyrogen testing.

Heparan sulfate and dermatan sulfate derived disaccharides are sensitive markers for newborn screening for mucopolysaccharidoses types I, II and III.

INTRODUCTION: Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs) caused by a defect in the degradation of glycosaminoglycans (GAGs). The accumulation of GAGs in MPS patients results in extensive, severe and progressive disease. Disease modifying therapy is available for three of the MPSs and is being developed for the other types. Early initiation of treatment, before the onset of irreversible tissue damage, clearly provides a favorable disease outcome. However, early diagnosis is difficult due to the rarity of these disorders in combination with the wide variety of clinical symptoms. Newborn screening (NBS) is probably the optimal approach, and several screening techniques for different MPSs have been studied. Here we describe a relatively simple and sensitive method to measure levels of dermatan and heparan sulfate derived disaccharides in dried blood spots (DBS) with HPLC-MS/MS, and show that this reliably separates MPS I, II and MPS III newborns from controls and heterozygotes. METHODS: Newborn DBS of 11 MPS I, 1 MPS II, and 6 MPS III patients, with phenotypes ranging from severe to relatively attenuated, were collected and levels of dermatan and heparan sulfate derived disaccharides in these DBS were compared with levels in DBS of newborn MPS I and MPS III heterozygotes and controls. RESULTS: The levels of dermatan and heparan sulfate derived disaccharides were clearly elevated in all newborn DBS of MPS I, II and III patients when compared to controls. In contrast, DBS of MPS I and III heterozygotes showed similar disaccharide levels when compared to control DBS. CONCLUSIONS: Our study demonstrates that measurement of heparan and dermatan sulfate derived disaccharides in DBS may be suitable for NBS for MPS I, II and MPS III. We hypothesize that this same approach will also detect MPS VI, and VII patients, as heparan sulfate and/or dermatan sulfate is also the primary storage products in these disorders.

Dietary fatty acids affect the immune system in male mice sensitized to ovalbumin or vaccinated with influenza.

PUFA are precursor molecules for eicosanoids such as leukotrienes and prostaglandins and may influence immune function through other mechanisms involving membranes, cell signaling, and gene expression. Immune-modulating properties of diets containing different oils [sunflower oil, rich in linoleic acid; linseed oil, rich in α-linolenic acid; salmon oil, rich in marine (n-3) PUFA; and beef tallow, rich in SFA] were investigated in an influenza-vaccination model, in which the delayed-type hypersensitivity (DTH) response was studied in C57BL/6 mice, and an ovalbumin (OVA)-sensitization model for experimental allergy in BALB/c mice. Six-week-old mice were fed the different diets for 7 wk. The first vaccination or OVA sensitization was given 2 wk after the start of the dietary intervention. In the mice vaccinated with influenza, the DTH response to the vaccine was significantly higher in mice fed the marine (n-3) PUFA diet compared to all other groups, indicating that these PUFA promote a T helper-1 response. In the OVA-sensitized mice, those fed the marine (n-3) PUFA diet had a less severe acute allergic skin response (ASR), suggesting that (n-3) PUFA lessen the T helper-2 response. Mice fed the SFA-rich diet had the most severe ASR, indicating that a diet with high levels of SFA may contribute to increased severity of allergic symptoms. Whereas significant differences in in vivo immune responses were measured, in vitro responses did not differ among the dietary groups. In conclusion, using 2 different models of immune responses demonstrates potential benefits from marine (n-3) PUFA.

Perturbed body fluid distribution and osmoregulation in response to high salt intake in patients with hereditary multiple exostoses.

BACKGROUND: Hereditary Multiple Exostoses (HME) is a rare autosomal disorder characterized by the presence of multiple exostoses (osteochondromas) caused by a heterozygous loss of function mutation in EXT1 or EXT2; genes involved in heparan sulfate (HS) chain elongation. Considering that HS and other glycosaminoglycans play an important role in sodium and water homeostasis, we hypothesized that HME patients have perturbed whole body volume regulation and osmolality in response to high sodium conditions. METHODS: We performed a randomized cross-over study in 7 male HME patients and 12 healthy controls, matched for age, BMI, blood pressure and renal function. All subjects followed both an 8-day low sodium diet (LSD, <50 mmol/d) and high sodium diet (HSD, >200 mmol/d) in randomized order. After each diet, blood and urine samples were collected. Body fluid compartment measurements were performed by using the distribution curve of iohexol and 125I-albumin. RESULTS: In HME patients, HSD resulted in significant increase of intracellular fluid volume (ICFV) (1.2 L, p = 0.01). In this group, solute-mediated water clearance was significantly lower after HSD, and no changes in interstitial fluid volume (IFV), plasma sodium, and effective osmolality were observed. In healthy controls, HSD did not influence ICFV, but expanded IFV (1.8 L, p = 0.058) and increased plasma sodium and effective osmolality. CONCLUSION: HME patients show altered body fluid distribution and osmoregulation after HSD compared to controls. Our results might indicate reduced interstitial sodium accumulation capacity in HME, leading to ICFV increase. Therefore, this study provides additional support that HS is crucial for maintaining constancy of the internal environment.

Identification and characterization of a complete carnitine biosynthesis pathway in Candida albicans.

Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl units. Here we show that the yeast Candida albicans can synthesize carnitine de novo, and we identify the 4 genes of the pathway. Null mutants of orf19.4316 (trimethyllysine dioxygenase), orf19.6306 (trimethylaminobutyraldehyde dehydrogenase), and orf19.7131 (butyrobetaine dioxygenase) lacked their respective enzymatic activities and were unable to utilize fatty acids, acetate, or ethanol as a sole carbon source, in accordance with the strict requirement for carnitine-mediated transport under these growth conditions. The second enzyme of carnitine biosynthesis, hydroxytrimethyllysine aldolase, is encoded by orf19.6305, a member of the threonine aldolase (TA) family in C. albicans. A strain lacking orf19.6305 showed strongly reduced growth on fatty acids and was unable to utilize either acetate or ethanol, but TA activity was unaffected. Growth of the null mutants on nonfermentable carbon sources is restored only by carnitine biosynthesis intermediates after the predicted enzymatic block in the pathway, which provides independent evidence for a specific defect in carnitine biosynthesis for each of the mutants. In conclusion, we have genetically characterized a complete carnitine biosynthesis pathway in C. albicans and show that a TA family member is mainly involved in the aldolytic cleavage of hydroxytrimethyllysine in vivo.

The Role of Virulence Proteins in Protection Conferred by Bordetella pertussis Outer Membrane Vesicle Vaccines.

The limited protective immunity induced by acellular pertussis vaccines demands development of novel vaccines that induce broader and longer-lived immunity. In this study, we investigated the protective capacity of outer membrane vesicle pertussis vaccines (omvPV) with different antigenic composition in mice to gain insight into which antigens contribute to protection. We showed that total depletion of virulence factors (bvg(-) mode) in omvPV led to diminished protection despite the presence of high antibody levels. Antibody profiling revealed overlap in humoral responses induced by vaccines in bvg(-) and bvg(+) mode, but the potentially protective responses in the bvg(+) vaccine were mainly directed against virulence-associated outer membrane proteins (virOMPs) such as BrkA and Vag8. However, deletion of either BrkA or Vag8 in our outer membrane vesicle vaccines did not affect the level of protection. In addition, the vaccine-induced immunity profile, which encompasses broad antibody and mixed T-helper 1, 2 and 17 responses, was not changed. We conclude that the presence of multiple virOMPs in omvPV is crucial for protection against Bordetella pertussis. This protective immunity does not depend on individual proteins, as their absence or low abundance can be compensated for by other virOMPs.

Abnormal sodium and water homeostasis in mice with defective heparan sulfate polymerization.

Glycosaminoglycans in the skin interstitium and endothelial surface layer have been shown to be involved in local sodium accumulation without commensurate water retention. Dysfunction of heparan sulfate glycosaminoglycans may therefore disrupt sodium and water homeostasis. In this study, we investigated the effects of combined heterozygous loss of heparan sulfate polymerization genes (exostosin glycosyltransferase 1 and 2; Ext1+/-Ext2+/-) on sodium and water homeostasis. Sodium storage capacity was decreased in Ext1+/-Ext2+/- mice as reflected by a 77% reduction in endothelial surface layer thickness and a lower skin sodium-to-glycosaminoglycan ratio. Also, these mice were characterized by a higher heart rate, increased fluid intake, increased plasma osmolality and a decreased skin water and sodium content, suggesting volume depletion. Upon chronic high sodium intake, the initial volume depletion was restored but no blood pressure increase was observed. Acute hypertonic saline infusion resulted in a distinct blood pressure response: we observed a significant 15% decrease in control mice whereas blood pressure did not change in Ext1+/-Ext2+/- mice. This differential blood pressure response may be explained by the reduced capacity for sodium storage and/or the impaired vasodilation response, as measured by wire myography, which was observed in Ext1+/-Ext2+/- mice. Together, these data demonstrate that defective heparan sulfate glycosaminoglycan synthesis leads to abnormal sodium and water homeostasis and an abnormal response to sodium loading, most likely caused by inadequate capacity for local sodium storage.

PPAR alpha-activation results in enhanced carnitine biosynthesis and OCTN2-mediated hepatic carnitine accumulation.

In fasted rodents hepatic carnitine concentration increases considerably which is not observed in PPAR alpha-/- mice, indicating that PPAR alpha is involved in carnitine homeostasis. To investigate the mechanisms underlying the PPAR alpha-dependent hepatic carnitine accumulation we measured carnitine biosynthesis enzyme activities, levels of carnitine biosynthesis intermediates, acyl-carnitines and OCTN2 mRNA levels in tissues of untreated, fasted or Wy-14643-treated wild type and PPAR alpha-/- mice. Here we show that both enhancement of carnitine biosynthesis (due to increased gamma-butyrobetaine dioxygenase activity), extra-hepatic gamma-butyrobetaine synthesis and increased hepatic carnitine import (OCTN2 expression) contributes to the increased hepatic carnitine levels after fasting and that these processes are PPAR alpha-dependent.