Levels Of Free Sialic Acid Research Articles

Changes in the levels of free sialic acid during ex vivo lung perfusion do not correlate with pulmonary function. Experimental model

BackgroundEx vivo lung perfusion (EVLP) constitutes a tool with great research potential due to its advantages over in vivo and in vitro models. Despite its important contribution to lung reconditioning, this technique has the disadvantage of incurring high costs and can induce pulmonary endothelial injury through perfusion and ventilation. The pulmonary endothelium is made up of endothelial glycocalyx (EG), a coating of proteoglycans (PG) on the luminal surface. PGs are glycoproteins linked to terminal sialic acids (Sia) that can affect homeostasis with responses leading to edema formation. This study evaluated the effect of two ex vivo perfusion solutions on lung function and endothelial injury.MethodsWe divided ten landrace swine into two groups and subjected them to EVLP for 120 min: Group I (n = 5) was perfused with Steen® solution, and Group II (n = 5) was perfused with low-potassium dextran-albumin solution. Ventilatory mechanics, histology, gravimetry, and sialic acid concentrations were evaluated.ResultsBoth groups showed changes in pulmonary vascular resistance and ventilatory mechanics (p < 0.05, Student’s t-test). In addition, the lung injury severity score was better in Group I than in Group II (p < 0.05, Mann–Whitney U); and both groups exhibited a significant increase in Sia concentrations in the perfusate (p < 0.05 t-Student) and Sia immunohistochemical expression.ConclusionsSia, as a product of EG disruption during EVLP, was found in all samples obtained in the system; however, the changes in its concentration showed no apparent correlation with lung function.

N-acetylneuraminate pyruvate lyase controls sialylation of muscle glycoproteins essential for muscle regeneration and function.

Deleterious variants in N-acetylneuraminate pyruvate lyase (NPL) cause skeletal myopathy and cardiac edema in humans and zebrafish, but its physiological role remains unknown. We report generation of mouse models of the disease: NplR63C, carrying the human p.Arg63Cys variant, and Npldel116 with a 116-bp exonic deletion. In both strains, NPL deficiency causes drastic increase in free sialic acid levels, reduction of skeletal muscle force and endurance, slower healing and smaller size of newly formed myofibers after cardiotoxin-induced muscle injury, increased glycolysis, partially impaired mitochondrial function, and aberrant sialylation of dystroglycan and mitochondrial LRP130 protein. NPL-catalyzed degradation of sialic acid in the muscle increases after fasting and injury and in human patient and mouse models with genetic muscle dystrophy, demonstrating that NPL is essential for muscle function and regeneration and serves as a general marker of muscle damage. Oral administration of N-acetylmannosamine rescues skeletal myopathy, as well as mitochondrial and structural abnormalities in NplR63C mice, suggesting a potential treatment for human patients.

A40 GUT MICROBIOTA PROMOTES NUTRIENT AVAILABLITY AND PATHOGENESIS OF AN ATTACHING/ EFFACING BACTERIAL PATHOGEN

Abstract Background Our gut microbiota plays an important role in protecting the gastrointestinal (GI) tract from invading enteric pathogens. Much of this colonization resistance is mediated by limiting nutrient availability, however, enteric pathogens have evolved strategies to subvert this competition, utilizing commensal metabolites to facilitate their infection. Access to nutrients is not only crucial for a pathogen’s metabolic fitness, but can also drive the expression of virulence factors, a process high in energy demands. In addition, enteric pathogens, such as the attaching and effacing (A/E) bacterium Citrobacter rodentium, must cross the colonic mucus layer that normally prevents their direct access to the underlying epithelium. Intestinal mucus is comprised of highly glycosylated mucins, with the sugar sialic acid frequently occupying the terminal position of their O-glycan side chains. We hypothesize that C. rodentium utilizes commensal-liberated mucin sugars, such as sialic acid, as nutrients and signals to promote its virulence. Purpose This study investigates the mechanisms by which A/E pathogens reach the colonic mucosal surface, and the role played by commensal microbes in facilitating the infection. Method Expression of virulence factors secreted by C. rodentium in the presence or absence of sialic acid was analyzed by SDS-PAGE and mass spectrometry. Next, we infected specific-pathogen free (SPF), germfree (GF), and previously GF C57Bl/6 mice mono-colonized with Bacteroides thetaotaomicron, a mucus-degrading commensal, to examine their susceptibility to C. rodentium and to measure the levels of free sialic acid in their feces. Result(s) Sensing of sialic acid by C. rodentium, was found to induce the secretion of several key virulence proteins, enhancing the pathogen’s migration across the colonic mucus layer and adhesion to the underlying epithelium. Access to sialic acid within the gut environment was enhanced in the presence of microbiota, as the levels of free sialic acid were low in GF mice. Interestingly, despite GF mice carrying very high C. rodentium burdens, passage across the mucus layer and infection of their colonic epithelium was impaired as compared to SPF mice. Notably, B. thetaotaomicron was found to degrade whole mucus in vitro, facilitating its consumption by C. rodentium for growth, while B. thetaotaomicron mono-colonized GF mice showed increased susceptibility to colonic infection by C. rodentium. Conclusion(s) We demonstrate that although commensal microbes promote colonization resistance, as an A/E pathogen infection establishes, specific commensal bacteria accelerate infection in the GI tract by releasing an important nutrient, ie. sialic acid, from mucus. Access to sialic acid promotes C. rodentium virulence by inducing the key virulence factors that facilitate its translocation across the mucus layer as well as adhesion to the epithelium, thereby expediting disease progression. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR, Other Please indicate your source of funding; CH.I.L.D. Foundation Disclosure of Interest None Declared

Increased Sphingomyelin and Free Sialic Acid in Cerebrospinal Fluid of Kearns-Sayre Syndrome: New Findings Using Untargeted Metabolomics

BackgroundKearns-Sayre syndrome (KSS) is caused by duplications and/or deletions of mitochondrial DNA (mtDNA) and is typically diagnosed based on a classic triad of symptoms with chronic progressive external ophthalmoplegia (CPEO), retinitis pigmentosa, and onset before age 20 years. The present study aimed to diagnose two patients, on suspicion of KSS. MethodsOne of the patients went through a diagnostic odyssey, with normal results from several mtDNA analyses, both in blood and muscle, before the diagnosis was confirmed genetically. ResultsTwo patients presented increased tau protein and low 5-methyltetrahydrofolate (5-MTHF) levels in the cerebrospinal fluid (CSF). Untargeted metabolomics on CSF samples also showed an increase in the levels of free sialic acid and sphingomyelin C16:0 (d18:1/C16:0), compared with four control groups (patients with mitochondrial disorders, nonmitochondrial disorders, low 5-MTHF, or increased tau proteins). ConclusionsIt is the first time that elevated sphingomyelin C16:0 (d18:1/C16:0) and tau protein in KSS are reported. Using an untargeted metabolomics approach and standard laboratory methods, the study could shed new light on metabolism in KSS to better understand its complexity. In addition, the findings may suggest the combination of elevated free sialic acid, sphingomyelin C16:0 (d18:1/C16:0), and tau protein as well as low 5-MTHF as new biomarkers in the diagnostics of KSS.

Association between Serum Sialic Acid Levels and Disease Severity in Psoriasis: A Case-control Study

Introduction: Psoriasis is a chronic inflammatory disease distributed worldwide with varying prevalence among different geographical areas and ethnic groups. It has been recently found that oxidative stress is one of the important factors in the pathogenesis of psoriasis. The varied effects of oxidative stress include changes in cellular uptake, altered enzymatic activity of proteins, increased predisposition to aggregation and proteolysis, which subsequently alter their immunogenicity. Sialic Acid (SA) is an acetylated derivative of neuramic acid. It is a marker for acute phase inflammatory response, with increased levels observed in inflammatory diseases. Aim: To estimate the levels of SA in psoriasis patients and to correlate with the severity of the disease. Materials and Methods: This case-control study was conducted among 50 patients, diagnosed with psoriasis, and 50 age and sex-matched subjects without psoriasis. General, systemic and dermatological examination was carried out. The severity of psoriasis was assessed according to Psoriasis Area Severity Index (PASI). The serum was treated with ethanol and centrifugation to precipitate proteins. The SA of both the precipitate and supernatant was estimated based on the reaction of SA with the ninhydrin reagent. Calculation of sialic acid was done by the formula: OD of test/ OD of standard x Concentration of the standard. To establish a correlation student’s t-test was used. Results: There were 39 males and 11 females in each case and control group. The mean±SD age of cases and controls was 44.04±10.9 years and 44.10±9.996, respectively. The mean PASI value was 23.1666 ± 18.47. Mean SA in cases and controls were 35.792±2.124 and 28.556±3.854 (p-values &lt;0.001). A positive correlation was observed between the free SA and psoriasis severity (p-value &lt;0.001, r = 0.460). Conclusion: Higher levels of free serum SA were significantly associated with more severe forms of psoriasis.

Cerebellum and temporal lobes of Alzheimer's disease patients present with an altered glycobiological state.

A major challenge facing Alzheimer's Disease (AD) research is the slowing or prevention of synaptic pruning, a pathology associated with driving the progression of AD's clinical symptoms. Currently, the mechanism by which pruning occurs is not well understood and whilst the complement cascade has been implicated (Hong etal., 2016), there are still questions regarding cellular targets and regulation. Therefore, exploration of additional regulatory factors is likely to offer novel targets for future therapeutic strategies. One key regulatory factor we propose is glycosylation, specifically the role of sialic acid 'caps'. Here, we show that glycobiological status of individual brain regions appears to be tied to AD pathology, suggesting an exciting avenue for novel therapeutic approaches. Temporal lobe and cerebellar tissue samples from AD (n=20) and age matched control (n=20) brains were homogenised in RIPA lysis buffer supplemented with phosphatase and protease inhibitors and assessed for Neuraminidase 2-4 and sialic acid levels. Neuraminidase levels were assessed using western blot whilst total, bound and free sialic acid levels were assessed using a fluorometric assay. Significant increases in free sialic acid were observed in the temporal lobes of AD patients compared to controls, with no significant differences detected in total and bound sialic acid. Conversely, both total and bound sialic acid were significantly increased in the cerebellum, while levels of free sialic acid were unchanged. Moreover, reductions in Neu 2 and Neu 4 were observed in the temporal region during AD, with no significant changes in the cerebellum. These findings show that mild increases in free sialic acid are associated with AD pathology in vulnerable brain regions such as the temporal lobe. Crucially, they show significant increases in total and bound sialic acid in the cerebellum, a region minimally impacted by AD pathology (Larner, 1997). This suggests there are key differences between cerebellar and temporal lobe sialylation in AD, that may in turn underpin the differential vulnerability of these brain regions to disease linked degenerative processes. Understanding more about the mechanisms that drive this sialylation may prove valuable in the quest for novel therapeutic targets for AD.

Elevated plasma free sialic acid levels in individuals with reduced glomerular filtration rates.

N-acetylneuraminic acid (Neu5Ac, sialic acid) is a negatively charged monosaccharide, and the predominant form of sialic acid in human cells (1). Sialic acid is typically found as the terminal monosaccharide on glycoconjugates, where it plays a role in various physiologic and pathologic interactions (1). Sialylated glycoconjugates are critical contributors to the polyanionic component of the glomerular glycocalyx, contributing to size and charge selectivity for plasma macromolecules (1,2). Podocyte foot process morphology is maintained by the anionic charged sialic acid residues on glycoconjugates in podocyte membranes (2). Free sialic acid is filtered but not reabsorbed by the human kidney, in a fashion similar to that for creatinine (3), but in contrast to the handling of other monosaccharides such as glucose, mannose, galactose, and fructose that are reabsorbed by tubular cells (4). Circulating sialic acid levels, both unbound and bound to glycoconjugates, have only been sporadically studied in different conditions, including some renal disorders (3,5−9); a possible causative link to reduced eGFRs has been seldom discussed or investigated. We designed this study to establish a correlation between eGFR and plasma free sialic acid across a range of subjects with glomerular disorders. This would not only emphasize this often-overlooked aspect of renal filtering of free sialic acid, but also inform our developmental program for glomerular diseases using the sialic acid precursor, N-acetylmannosamine (ManNAc) (10), which was expected to significantly increase plasma free sialic acid levels in subjects with reduced eGFR. Peripheral blood was obtained from eight subjects enrolled in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) natural history study 94-DK-0127 (ClinicalTrials.gov identifier, NT1392), “Pathogenesis of FSGS,” and from eight subjects enrolled in NIDDK study 16-DK-0036 (ClinicalTrials.gov identifier, NCT02639260), “A Phase 1 Multiple Ascending Dose Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of …

Poly(3,4-ethylenedioxythiophene) Bearing Pyridylboronic Acid Group for Specific Recognition of Sialic Acid.

Conducting polymers tethered with molecular recognition elements are good candidates for biosensing applications such as detecting a target molecule with selectivity. We develop a new monomer, namely, 3,4-ethylenedioxythiophene bearing a pyridylboronic acid moiety (EDOT-PyBA), for label-free detection of sialic acid as a cancer biomarker. PyBA, which is known to show specific binding to sialic acid in acid conditions is used as a synthetic ligand instead of lectins. PyBA confirms the enhanced binding affinity for sialic acid at pH 5.0-6.0 compared with traditional phenylboronic acid. Poly(EDOT-PyBA) is electrodeposited on a planar glassy carbon electrode and the obtained film is successfully characterized by X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, water contact angle measurements, and electrochemical impedance spectroscopy. The specific interaction of PyBA with sialic acid at the solution/electrode interface is detected by differential pulse voltammetry in a dynamic range 0.1-3.0 mM with a detection limit of 0.1 mM for a detection time of 3 min. The sensitivity covers the total level of free sialic acid in human serum and the assay time is the shorter than that of other methods. The poly(EDOT-PyBA) electrode successfully detects spiked sialic acid in human serum samples. Owing to its processability, mass productivity, and robustness, polythiophene conjugated with "boronolectin" is a candidate material for developing point-of-care and wearable biosensors.

A New Patient With Intermediate Severe Salla Disease With Hypomyelination: A Literature Review for Salla Disease

BackgroundLikely pathogenic variants in SLC17A5 results in allelic disorders of free sialic acid metabolism including (1) infantile free sialic acid storage disease with severe global developmental delay, coarse facial features, hepatosplenomegaly, and cardiomegaly; (2) intermediate severe Salla disease with moderate to severe global developmental delay, hypotonia, and hypomyelination with or without coarse facial features, and (3) Salla disease with normal appearance, mild cognitive dysfunction, and spasticity. Patient DescriptionThis five-year-old girl presented with infantile-onset severe global developmental delay, truncal hypotonia, and generalized dystonia following normal development during her first six months of life. Brain magnetic resonance imaging showed marked hypomyelination and a thin corpus callosum at age 19 months, both unchanged on follow-up at age 28 months. Urine free sialic acid was moderately elevated. Cerebrospinal fluid free sialic acid was marginally elevated. Sequencing of SLC17A5 revealed compound heterozygous likely pathogenic variants, namely, a known missense (c.291G>A) variant and a novel truncating (c.819+1G>A) variant, confirming the diagnosis of Salla disease at age 3.5 years. ConclusionWe report a new patient with intermediate severe Salla disease. Normal or marginally elevated urine or cerebrospinal fluid free sialic acid levels cannot exclude Salla disease. In patients with progressive global developmental delay and hypomyelination on brain magnetic resonance imaging, Salla disease should be included into the differential diagnosis.

Rapid regulation of sialidase activity in response to neural activity and sialic acid removal during memory processing in rat hippocampus

Sialidase cleaves sialic acids on the extracellular cell surface as well as inside the cell and is necessary for normal long-term potentiation (LTP) at mossy fiber-CA3 pyramidal cell synapses and for hippocampus-dependent spatial memory. Here, we investigated in detail the role of sialidase in memory processing. Sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac) or 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB was increased by high-K+-induced membrane depolarization. Sialidase activity was also increased by chemical LTP induction with forskolin and activation of BDNF signaling, non-NMDA receptors, or NMDA receptors. The increase in sialidase activity with neural excitation appears to be caused not by secreted sialidase or by an increase in sialidase expression but by a change in the subcellular localization of sialidase. Astrocytes as well as neurons are also involved in the neural activity-dependent increase in sialidase activity. Sialidase activity visualized with a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe for sialidase activity, at the CA3 stratum lucidum of rat acute hippocampal slices was immediately increased in response to LTP-inducible high-frequency stimulation on a time scale of seconds. To obtain direct evidence for sialic acid removal on the extracellular cell surface during neural excitation, the extracellular free sialic acid level in the hippocampus was monitored using in vivo microdialysis. The free sialic acid level was increased by high-K+-induced membrane depolarization. Desialylation also occurred during hippocampus-dependent memory formation in a contextual fear-conditioning paradigm. Our results show that neural activity-dependent desialylation by sialidase may be involved in hippocampal memory processing.

Substantial deficiency of free sialic acid in muscles of patients with GNE myopathy and in a mouse model.

GNE myopathy (GNEM), also known as hereditary inclusion body myopathy (HIBM), is a late- onset, progressive myopathy caused by mutations in the GNE gene encoding the enzyme responsible for the first regulated step in the biosynthesis of sialic acid (SA). The disease is characterized by distal muscle weakness in both the lower and upper extremities, with the quadriceps muscle relatively spared until the late stages of disease. To explore the role of SA synthesis in the disease, we conducted a comprehensive and systematic analysis of both free and total SA levels in a large cohort of GNEM patients and a mouse model. A sensitive LC/MS/MS assay was developed to quantify SA in serum and muscle homogenates. Mean serum free SA level was 0.166 μg/mL in patients and 18% lower (p<0.001) than that of age-matched control samples (0.203 μg/mL). In biopsies obtained from patients, mean free SA levels of different muscles ranged from 0.046–0.075 μg/μmol Cr and were markedly lower by 72–85% (p<0.001) than free SA from normal controls. Free SA was shown to constitute a small fraction (3–7%) of the total SA pool in muscle tissue. Differences in mean total SA levels in muscle from patients compared with normal controls were less distinct and more variable between different muscles, suggesting a small subset of sialylation targets could be responsible for the pathogenesis of GNEM. Normal quadriceps had significantly lower levels of free SA (reduced by 39%) and total SA (reduced by 53%) compared to normal gastrocnemius. A lower SA requirement for quadriceps may be linked to the reported quadriceps sparing in GNEM. Analysis of SA levels in GneM743T/M743T mutant mice corroborated the human study results. These results show that serum and muscle free SA is severely reduced in GNEM, which is consistent with the biochemical defect in SA synthesis associated with GNE mutations. These results therefore support the approach of reversing SA depletion as a potential treatment for GNEM patients.

Identification of a large intronic transposal insertion in SLC17A5 causing sialic acid storage disease

BackgroundSialic acid storage diseases are neurodegenerative disorders characterized by accumulation of sialic acid in the lysosome. These disorders are caused by mutations in SLC17A5, the gene encoding sialin, a sialic acid transporter located in the lysosomal membrane. The most common form of sialic acid storage disease is the slowly progressive Salla disease, presenting with hypotonia, ataxia, epilepsy, nystagmus and findings of cerebral and cerebellar atrophy. Hypomyelination and corpus callosum hypoplasia are typical as well. We report a 16 year-old boy with an atypically mild clinical phenotype of sialic acid storage disease characterized by psychomotor retardation and a mixture of spasticity and rigidity but no ataxia, and only weak features of hypomyelination and thinning of corpus callosum on MRI of the brain.ResultsThe thiobarbituric acid method showed elevated levels of free sialic acid in urine and fibroblasts, indicating sialic acid storage disease. Initial Sanger sequencing of SLC17A5 coding regions did not show any pathogenic variants, although exon 9 could not be sequenced. Whole exome sequencing followed by RNA and genomic DNA analysis identified a homozygous 6040 bp insertion in intron 9 of SLC17A5 corresponding to a long interspersed element-1 retrotransposon (KF425758.1). This insertion adds two splice sites, both resulting in a frameshift which in turn creates a premature stop codon 4 bp into intron 9.ConclusionsThis study describes a novel pathogenic variant in SLC17A5, namely an intronic transposal insertion, in a patient with mild biochemical and clinical phenotypes. The presence of a small fraction of normal transcript may explain the mild phenotype. This case illustrates the importance of including lysosomal sialic acid storage disease in the differential diagnosis of developmental delay with postnatal onset and hypomyelination, as well as intronic regions in the genetic investigation of inborn errors of metabolism.

Association between salivary sialic acid and periodontal health status among smokers.

BackgroundSmoking is an environmental risk factor causing poor dental health. Sialic acid is a salivary marker of oxidative stress for research of periodontal diseases.AimsTo identify diagnostic sialic acid fraction and its scavenger effect for periodontal diseases among smokers and periodental health status.Subject and methodThis study carried out in the Khanzad specialized dental center – Erbil city. The study population is composed of 62 convenient samples. A structured interview questionnaire form was used to collect data about socio-demographic properties and smoking history. Clinical measurements were carried out to measure periodontal health status. Un-stimulated whole saliva samples were collected for measuring sialic acid fractions. Statistical package for social science (SPSS, version 18), was used for analysis and odds ratio.ResultsRisk of smoking increased significantly in young to mid ages, which included most of the current smokers, with periodontal diseases, and high total free sialic acid. Risk of periodontitis and teeth missing increased significantly by long duration of smoking, bad tooth brushing, and poor eating habits. Risk of teeth mobility and loss decreased significantly by early smoking cessation and low income. High levels of free sialic acid correlated significantly in current smokers with medium and deep pocket depth.ConclusionSalivary free sialic acid may be used as a diagnostic oxidative stress biomarker for periodontal diseases among young current smokers. Cumulative destructive effect of long duration of smoking on the periodontum can be controlled by smoking cessation, good oral hygiene and diet habit in early old ages.

Galactooligosaccharide and Sialyllactose Content in Commercial Lactose Powders from Goat and Cow Milk.

The most commonly used infant formulas contain lactose originating from cow milk. Goat milk has recently been claimed to be nutritionally more effective for infants than other milks. In baby foods, much emphasis is placed on the concentrations of intestinal microflora-promoting oligosaccharides, which are generally transferred into lactose from milk during crystallization process. Here we show that higher level of free sialic acid is present in goat lactose powder compared to cow lactose powder. Without proteinase K treatment, the amount of 3-sialyllactose and 6-sialyllactose were similar in goat and cow lactose powders. However, after proteolysis, 6-sialyllactose was present at higher levels in goat than in cow lactose powder. Galactooligosaccharides, a group of prebiotics, are present in milk in the form of glycoproteins. Galactooligosaccharide content was also higher in goat lactose powder than in cow lactose powder.

Free and bound forms of sialic acid in blood plasma of patients with chronic lymphocytic leukemia

The level of free and bound forms of sialic acids (SА) was investigated in chronic lymphocytic leukemia (CLL) and at different stages of receiving chemotherapy. Determination of sialic acid in blood plasma of patients with CLL before treatment were carried out on the first day and 2 months after taking chemotherapy drugs for combination schemes. Hematologically healthy donors represented the control group. Determination of SA was conducted by thiobarbituric method using trichloroacetic acid for distribution of total sialic acids onto free, protein bound sialic acid, and oligo bound sialic acid. Levelof free sialic acid in serum in chronic lymphocytic leukemia was equal to 43.2% compared to the total number of plasma. On the first day of chemotherapy on background of the general level of oligo bound sialic acids we observed increase in free sialic acid by 24.2% compared to patients not receiving treatment. The level of sialic acids increased 2.6 times compared to norm and featured no significant changes at different stages of treatment. The concentration of sialic acids bound to proteins at 73.4% was lower compared to the control group. After receiving chemotherapy on the first day there was a decline of this indicator to 56.2% compared to groups of patients before treatment. Two months after the treatment the level of all parameters under study returned back to the values obtained at the start of treatment. This data can serve as an additional efficiency criterion of the chemotherapy.

The early detection of Salla disease through second-tier tests in newborn screening: How to face incidental findings

We describe here a 34 months child, practically asymptomatic which presented with high levels of free sialic acid in urine by biochemical detection in second-tier tests newborn screening and with two disease causing mutations in SLC17A5 gene. SLC17A5 mutation analysis showed p.Tyr306* previously decribed and the novel mutation p.Leu167Pro. This early onset diagnosis allowed us to perform a fast and accurate genetic counseling to the family, helped to better understanding the natural history of this rare disease and probably it could promote cost reduction in future diagnostic tests in the hypothetic case of starting symptoms without diagnosis established. Moreover, an early diagnosis could save family from a long period of time until achieving a definitive diagnostic and to develop an early symptomatic and supportive management of patient to attenuate, as much as possible, disease complications. But, above all, this case illustrates the huge ethical dilemma which arises from any secondary finding (second tier) in newborn screening.

Sialic acid level reflects the disturbances of glycosylation and acute-phase reaction in rheumatic diseases

In the rheumatic diseases, the changes in the carbohydrate part of serum glycoproteins occur and these abnormalities can be monitored by serum level of total and free sialic acid. The aim of this study was to evaluate the total and free sialic acid level as a marker of inflammation activity (TSA) and the changes in glycosylation of blood glycoproteins (FSA) in rheumatoid arthritis (RA), systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). Studies were carried out in 50 patients with RA, 24 with SLE and 32 with SSc. TSA concentration was measured with an enzymatic, colorimetric method and FSA with a thiobarbituric method. The serum levels of TSA in RA and SLE patients were significantly increased compared to controls and in RA patients were higher than that in SSc patients. The mean serum level of FSA in RA patients was significantly higher, but in SSc patients significantly lower than that in the controls, and in RA patients was significantly higher than in SLE and in SSc patients. All acute-phase proteins were changed: Positive acute-phase proteins were elevated, and the negative protein was decreased. The positive acute-phase proteins positively correlated with the levels of TSA and FSA in RA and SSc patients. In SLE patients, TSA positively correlated with haptoglobin and α1-antitrypsin. In RA patients, there was the positive correlation of TSA and FSA with DAS 28. The changes in the serum levels of TSA and FSA in the course of rheumatic diseases could reflect the abnormalities in glycosylation/sialylation patterns of glycoproteins induced by acute-phase response.

Diminished complement-activating capacity through the classical pathway in sera from type 2 diabetes mellitus

Complement-activating capacity through the classical pathway in type 2 diabetes mellitus (T2DM) was examined in the context of free sialic acid as a potential modulator of complement activation. Complement-activating capacity was investigated in an incubation study of heat-aggregated IgG (HAG) and sera from 42 T2DM patients. The study demonstrated diminished in-vitro complement-activating capacity through the classical pathway in T2DM. Various doses of N-acetyl neuraminic acid (NANA) were incubated with normal serum and HAG. Complement activation product levels decreased in a NANA dose-dependent manner. Isoelectrofocusing analysis in a mixture of NANA and purified C3 indicated that C3 changed pI dose-dependently, resulting in the downregulation of complement activation. The serum levels of free sialic acid were determined by fluorometric assay in the 42 T2DM sera samples, and were significantly increased in patients with diminished complement activation. These data indicate that increased serum sialic acid may become a candidate for decreasing complement-activating capacity in T2DM.

Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens

The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens1–4. How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella typhimurium and Clostridium difficile, employ a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo. Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo. Colonization of gnotobiotic mice with a sialidase-deficient mutant of the model gut symbiont Bacteroides thetaiotaomicron (Bt) reduces free sialic acid levels resulting in a downregulation of C. difficile’s sialic acid catabolic pathway and impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new possibilities for therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

P.3.7 A prodrug strategy to increase oral absorption and bioavailability of Sialic acid in an HIBM mouse model

Sialic acid (SA) is intended for use as a substrate replacement therapy for the treatment of hereditary inclusion body myopathy (HIBM or also DMRV, now known as GNE Myopathy), a severe, neuromuscular disease caused by a defect in the sialic acid biosynthetic pathway. Oral SA provided in the drinking water to an HIBM mouse model is effective in restoring biochemical sialylation and reducing pathology while also preventing the progressive decline in muscle function. However, the pharmaceutical properties of SA in terms of absorption and delivery are not optimal, and the potential exists to make a therapeutically more potent compound for substrate replacement therapy. A series of prodrugs of SA were synthesized and tested in order to achieve a higher degree of absorption, increase delivery to muscle and potentially improve the therapeutic efficacy in humans. Three prodrugs candidates were developed and selected based on their ability to increase uptake in vitro and in vivo. Uptake into induced C2C12 mouse myotube cultures indicated that the prodrugs achieved many fold higher SA levels in the myotubes compared to SA treatment at the same concentration. In HIBM mice after 14 days of treatment with oral prodrugs or SA (BID, 200 mg/kg/day), results showed that the prodrugs consistently had higher levels of free SA in the serum compared to SA and the prodrugs had a substantially prolonged half-life over SA. The high free SA levels achieved suggest that the prodrugs are accumulating in tissues, including muscle, and act as a reservoir to release SA into the serum. Additionally, levels of uncleaved prodrugs were relatively low in the serum suggesting that the prodrug candidates are being metabolized appropriately in the tissues. A 60 day study in HIBM mice is currently underway to confirm the previous results and to evaluate the efficacy and potential toxicity of the three prodrug candidates as compared to SA.