B Cell,Th17, and Neutrophil Related Cerebrospinal Fluid Cytokine/Chemokines Are Elevated in MOG Antibody Associated Demyelination

Background

Myelin oligodendrocyte glycoprotein antibody (MOG Ab) associated demyelination represents a subgroup of autoimmune demyelination that is separate from multiple sclerosis and aquaporin 4 IgG-positive NMO, and can have a relapsing course. Unlike NMO and MS, there is a paucity of literature on immunopathology and CSF cytokine/chemokines in MOG Ab associated demyelination.

Aim

To study the differences in immunopathogenesis based on cytokine/chemokine profile in MOG Ab-positive (POS) and -negative (NEG) groups.

Methods

We measured 34 cytokines/chemokines using multiplex immunoassay in CSF collected from paediatric patients with serum MOG Ab POS [acute disseminated encephalomyelitis (ADEM = 8), transverse myelitis (TM = 2) n = 10] and serum MOG Ab NEG (ADEM = 5, TM = 4, n = 9) demyelination. We generated normative data using CSF from 20 non-inflammatory neurological controls.

Results

The CSF cytokine and chemokine levels were higher in both MOG Ab POS and MOG Ab NEG demyelination groups compared to controls. The CSF in MOG Ab POS patients showed predominant elevation of B cell related cytokines/chemokines (CXCL13, APRIL, BAFF and CCL19) as well as some of Th17 related cytokines (IL-6 AND G-CSF) compared to MOG Ab NEG group (all p<0.01). In addition, patients with elevated CSF MOG antibodies had higher CSF CXCL13, CXCL12, CCL19, IL-17A and G-CSF than patients without CSF MOG antibodies.

Conclusion

Our findings suggest that MOG Ab POS patients have a more pronounced CNS inflammatory response with elevation of predominant humoral associated cytokines/chemokines, as well as some Th 17 and neutrophil related cytokines/chemokines suggesting a differential inflammatory pathogenesis associated with MOG antibody seropositivity. This cytokine/chemokine profiling provides new insight into disease pathogenesis, and improves our ability to monitor inflammation and response to treatment. In addition, some of these molecules may represent potential immunomodulatory targets.     

A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses

Introduction

Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III).

Methods

Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs.

Results

The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them.

Conclusion

The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay.     

Neuromyelitis Optica in Austria in 2011: To Bridge the Gap between Neuroepidemiological Research and Practice in a Study Population of 8.4 Million People

Background

In 2008 the Austrian Task Force for Neuromyelitis Optica (NMO) started a nation-wide network for information exchange and multi-centre collaboration. Their aim was to detect all patients with NMO or NMO spectrum disorders (NMO-SD) in Austria and to analyse their disease courses and response to treatment.

Methods

(1) As of March 2008, 1957 serum samples (of 1557 patients) have been tested with an established cell based immunofluorescence aquaporin-4 antibody (AQP4-ab) assay with a high sensitivity and specificity (both >95%). All tests were performed in a single reference laboratory (Clinical Dept. of Neurology of the Innsbruck Medical University). (2) A nation-wide survey with several calls for participation (via email newsletters, articles in the official journal of the Austrian Society of Neurology, and workshops) was initiated in 2008. All collected data will be presented in a way that allows that every individual patient can be traced back in order to ensure transparency and to avoid any data distortion in future meta-analyses. The careful and detailed presentation allows the visualization and comparison of the different disease courses in real time span. Failure and response to treatment are made visible at one glance. Database closure was 31 December 2011. All co-operators were offered co-authorship.

Results

All 71 NMO- or NMO-SD patients with AQP4-ab positivity (age range 12.3 to 79.6 years) were analysed in detail. Sex ratio (m:f = 1:7) and the proportion of patients without oligoclonal bands in cerebrospinal fluid (86.6%) were in line with previously published results. All identified patients were Caucasians.

Conclusions

A nationwide collaboration amongst Austrian neurologists with good network communications made it possible to establish a database of 71 AQP4-ab positive patients with NMO/NMO-SD. This database is presented in detail and provides the basis for further studies and international cooperation in order to investigate this rare disease.     

Connexin 43 Astrocytopathy Linked to Rapidly Progressive Multiple Sclerosis and Neuromyelitis Optica

Background

Multiple sclerosis (MS) and neuromyelitis optica (NMO) occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx) pathology and disease aggressiveness in Asian patients with MS and NMO.

Methods/Principal Findings

Samples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD), six with MS, and 20 with other neurological diseases (OND). Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59), chronic active (n=58) and chronic inactive (n=23). Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, P=0.0090). Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, P=0.0296). Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients.

Conclusions

These findings suggest that autoantibody-independent astrocytic Cx43 loss may relate to disease aggressiveness and distal oligodendrogliopathy in both MS and NMO.     

Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

Introduction

Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method

We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results

Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions

Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.     

Search for Allergens from the Pollen Proteome of Sunflower (Helianthus annuus L.): A Major Sensitizer for Respiratory Allergy Patients

Background

Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools.

Methodology

Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensional electrophoretic separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoallergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry.

Results

Prevalence of sunflower pollen sensitization was observed among 21% of the pollen allergic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient sera detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two pectate lyases and a cysteine protease.

Conclusion

Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level, which substantiated the clinical evidence of sunflower allergy. Further purification and recombinant expression of these allergens will improve component-resolved diagnosis and therapy of pollen allergy.     

The Impact of Stannous,Fluoride Ions and Its Combination on Enamel Pellicle Proteome and Dental Erosion Prevention

Objectives

To compare the effects of stannous (Sn) and fluoride (F) ions and their combination on acquired enamel pellicle (AEP) protein composition (proteome experiment), and protection against dental erosion (functional experiment).

Methods

In the proteome experiment, bovine enamel specimens were incubated in whole saliva supernatant for 24h for AEP formation. They were randomly assigned to 4 groups (n=10), according to the rinse treatment: Sn (800ppm/6.7mM, SnCl₂), F (225ppm/13mM, NaF), Sn and F combination (Sn+F) and deionized water (DIW, negative control). The specimens were immersed 3× in the test rinses for 2min, 2h apart. Pellicles were collected, digested, and analyzed for protein content using liquid chromatography electrospray ionization tandem mass spectrometry. In the functional experiment, bovine enamel specimens (n=10) were similarly treated for pellicle formation. Then, they were subjected to a five-day erosion cycling model, consisting of 5min erosive challenges (15.6 mM citric acid, pH 2.6, 6×/d) and 2min treatment with the rinses containing Sn, F or Sn+F (3×/d). Between the treatments, all specimens were incubated in whole saliva supernatant. Surface loss was determined by profilometry.

Results

Our proteome approach on bovine enamel identified 72 proteins that were common to all groups. AEP of enamel treated with Sn+F demonstrated higher abundance for most of the identified proteins than the other groups. The functional experiment showed reduction of enamel surface loss for Sn+F (89%), Sn (67%) and F (42%) compared to DIW (all significantly different, p<0.05).

Conclusion

This study highlighted that anti-erosion rinses (e.g. Sn+F) can modify quantitatively and qualitatively the AEP formed on bovine enamel. Moreover, our study demonstrated a combinatory effect that amplified the anti-erosive protection on tooth surface.     

Fatigue in Multiple Sclerosis: Assessing Pontine Involvement Using Proton MR Spectroscopic Imaging

Background/Objective

The underlying mechanism of fatigue in multiple sclerosis (MS) remains poorly understood. Our study investigates the involvement of the ascending reticular activating system (ARAS), originating in the pontine brainstem, in MS patients with symptoms of fatigue.

Methods

Female relapsing-remitting MS patients (n = 17) and controls (n = 15) underwent a magnetic resonance spectroscopic imaging protocol at 1.5T. Fatigue was assessed in every subject using the Fatigue Severity Scale (FSS). Using an FSS cut-off of 36, patients were categorized into a low (n = 9, 22 ± 10) or high (n = 10, 52 ± 6) fatigue group. The brain metabolites N-acetylaspartate (NAA) and total creatine (tCr) were measured from sixteen 5x5x10 mm³ spectroscopic imaging voxels in the rostral pons.

Results

MS patients with high fatigue had lower NAA/tCr concentration in the tegmental pons compared to control subjects. By using NAA and Cr values in the cerebellum for comparison, these NAA/tCr changes in the pons were driven by higher tCr concentration, and that these changes were focused in the WM regions.

Discussion/Conclusion

Since there were no changes in NAA concentration, the increase in tCr may be suggestive of gliosis, or an imbalanced equilibrium of the creatine and phosphocreatine ratio in the pons of relapsing-remitting MS patients with fatigue.     

Ecological Validity of Walking Capacity Tests in Multiple Sclerosis

Background

Ecological validity implicates in how far clinical assessments refer to real life. Short clinical gait tests up to ten meters and 2- or 6-Minutes Walking Tests (2MWT/6MWT) are used as performance-based outcomes in Multiple Sclerosis (MS) studies and considered as moderately associated with real life mobility.

Objective

To investigate the ecological validity of 10 Meter Walking Test (10mWT), 2MWT and 6MWT.

Methods

Persons with MS performed 10mWT, 6MWT including 2MWT and 7 recorded days by accelerometry. Ecological validity was assumed if walking tests represented a typical walking sequence in real-life and correlations with accelerometry parameters were strong.

Results

In this cohort (n=28, medians: age=45, EDSS=3.2, disease duration=9 years), uninterrupted walking of 2 or 6 minutes occurred not frequent in real life (2.61 and 0.35 sequences/day). 10mWT correlated only with slow walking speed quantiles in real life. 2MWT and 6MWT correlated moderately with most real life walking parameters.

Conclusion

Clinical gait tests over a few meters have a poor ecological validity while validity is moderate for 2MWT and 6MWT. Mobile accelerometry offers the opportunity to control and improve the ecological validity of MS mobility outcomes.     

Grey Matter Atrophy in Multiple Sclerosis: Clinical Interpretation Depends on Choice of Analysis Method

Background

Studies disagree on the location of grey matter (GM) atrophy in the multiple sclerosis (MS) brain.

Aim

To examine the consistency between FSL, FreeSurfer, SPM for GM atrophy measurement (for volumes, patient/control discrimination, and correlations with cognition).

Materials and Methods

127 MS patients and 50 controls were included and cortical and deep grey matter (DGM) volumetrics were performed. Consistency of volumes was assessed with Intraclass Correlation Coefficient/ICC. Consistency of patients/controls discrimination was assessed with Cohen’s d, t-tests, MANOVA and a penalized double-loop logistic classifier. Consistency of association with cognition was assessed with Pearson correlation coefficient and ANOVA. Voxel-based morphometry (SPM-VBM and FSL-VBM) and vertex-wise FreeSurfer were used for group-level comparisons.

Results

The highest volumetry ICC were between SPM and FreeSurfer for cortical regions, and the lowest between SPM and FreeSurfer for DGM. The caudate nucleus and temporal lobes had high consistency between all software, while amygdala had lowest volumetric consistency. Consistency of patients/controls discrimination was largest in the DGM for all software, especially for thalamus and pallidum. The penalized double-loop logistic classifier most often selected the thalamus, pallidum and amygdala for all software. FSL yielded the largest number of significant correlations. DGM yielded stronger correlations with cognition than cortical volumes. Bilateral putamen and left insula volumes correlated with cognition using all methods.

Conclusion

GM volumes from FreeSurfer, FSL and SPM are different, especially for cortical regions. While group-level separation between MS and controls is comparable, correlations between regional GM volumes and clinical/cognitive variables in MS should be cautiously interpreted.     

Proteomic Analysis of the Human Skin Proteome after In Vivo Treatment with Sodium Dodecyl Sulphate

Background

Skin has a variety of functions that are incompletely understood at the molecular level. As the most accessible tissue in the body it often reveals the first signs of inflammation or infection and also represents a potentially valuable source of biomarkers for several diseases. In this study we surveyed the skin proteome qualitatively using gel electrophoresis, liquid chromatography tandem mass spectrometry (GeLC-MS/MS) and quantitatively using an isobaric tagging strategy (iTRAQ) to characterise the response of human skin following exposure to sodium dodecyl sulphate (SDS).

Results

A total of 653 skin proteins were assigned, 159 of which were identified using GeLC-MS/MS and 616 using iTRAQ, representing the most comprehensive proteomic study in human skin tissue. Statistical analysis of the available iTRAQ data did not reveal any significant differences in the measured skin proteome after 4 hours exposure to the model irritant SDS.

Conclusions

This study represents the first step in defining the critical response to an irritant at the level of the proteome and provides a valuable resource for further studies at the later stages of irritant exposure.     

Ghrelin Gene Variants Influence on Metabolic Syndrome Components in Aged Spanish Population

Background

The role of genetic variations within the ghrelin gene on cardiometabolic profile and nutritional status is still not clear in humans, particularly in elderly people.

Objectives

We investigated six SNPs of the ghrelin gene and their relationship with metabolic syndrome (MS) components.

Subjects and Methods

824 subjects (413 men/411 women, age 77.31±5.04) participating in the Mataró aging study (n = 310) and the Hortega study (n = 514) were analyzed. Anthropometric variables, ghrelin, lipids, glucose and blood pressure levels were measured, and distribution of SNPs -994CT (rs26312), -604GA (rs27647), -501AC (rs26802), R51Q (rs34911341), M72L (rs696217) and L90G (rs4684677) of the ghrelin gene evaluated. Genotypes were determined by multiplex PCR and SNaPshot minisequencing. MS (IDF criteria) was found in 54.9%.

Results

No association between any of the SNPs and levels of total fasting circulating ghrelin levels was found. C/A-A/A genotype of M72L was associated with increased risk of central obesity according to IDF criteria, while G/A-G/G genotypes of -604GA with reduced risk. A/A genotype of -501AC polymorphism was associated to decreased BMI. In relation to lipid profile, the same genotypes of -604GA were associated with increased total cholesterol and LDL-cholesterol and -501AC with reduced triglycerides. There were no associations with systolic or diastolic blood pressure levels or with hypertension, glucose levels or diabetes and ghrelin polymorphisms. However, G/G genotype of -604GA was associated with glucose >100 mg/dL. Haplotype analysis showed that only one haplotype is associated with increased risk of waist circumference and central obesity. The analysis of subjects by gender showed an important and different association of these polymorphisms regarding MS parameters.

Conclusion

Ghrelin gene variants -604GA, -501AC and M72L are associated with certain components of MS, in particular to BMI and lipid profile in elderly Spanish subjects.     

Subcutaneous Progesterone Is Effective and Safe for Luteal Phase Support in IVF: An Individual Patient Data Meta-Analysis of the Phase III Trials

Objective

To summarize efficacy and safety data on a new progesterone compound which is available for subcutaneous administration as compared to vaginally administered progesterone for luteal phase support in patients undergoing IVF treatment.

Design

Data from two randomized phase III trials (07EU/Prg06 and 07USA/Prg05) performed according to GCP standards with a total sample size of 1435 per-protocol patients were meta-analyzed on an individual patient data level.

Setting

University affiliated reproductive medicine unit.

Patients

Subcutaneous progesterone was administered to a total of 714 subjects and vaginal progesterone was administered to a total of 721 subjects who underwent fresh embryo transfer after ovarian stimulation followed by IVF or ICSI. The subjects were between 18 and 42 years old and had a BMI <30kg/m².

Interventions

Subcutaneous progesterone 25 mg daily vs. either progesterone vaginal gel 90 mg daily (07EU/Prg06) or 100 mg intravaginal twice a day (07USA/Prg05) for luteal phase support in IVF patients.

Main outcome measures

Ongoing pregnancy rate beyond 10 gestational weeks, live birth rate and OHSS risk.

Results

The administration of subcutaneous progesterone versus intra-vaginal progesterone had no impact on ongoing pregnancy likelihood (OR = 0.865, 95% CI 0.694 to 1.077; P = n.s.), live birth likelihood (OR = 0.889, 95% CI 0.714 to 1.106; P = n.s.) or OHSS risk (OR = 0.995, 95% CI 0.565 to 1.754; P = n.s.) in regression analyses accounting for clustering of patients within trials, while adjusting for important confounders. Only female age and number of oocytes retrieved were significant predictors of live birth likelihood and OHSS risk.

Conclusion

No statistical significant or clinical significant differences exist between subcutaneous and vaginal progesterone for luteal phase support.     

Urinary Sex Steroids and Anthropometric Markers of Puberty - A Novel Approach to Characterising Within-Person Changes of Puberty Hormones

Background/Aims

The longitudinal relationships of within-individual hormone and anthropometric changes during puberty have not ever been fully described. The objectives of this study were to demonstrate that 3 monthly urine collection was feasible in young adolescents and to utilise liquid chromatography-tandem mass spectrometry assay methods for serum and urine testosterone (T), estradiol (E₂) and luteinizing hormone (LH) in adolescents by relating temporal changes in urine and serum hormones over 12 months to standard measures of pubertal development.

Methods

A community sample of 104 adolescents (57 female) was studied over 12 months with annual anthropometric assessment, blood sampling and self-rated Tanner staging and urine collected every 3 months. Serum and urine sex steroids (T, E₂) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LH by immunoassay.

Results

A high proportion (92%) of scheduled samples were obtained with low attrition rate of 6.7% over the 12 months. Urine hormone measurements correlated cross-sectionally and longitudinally with age, anthropometry and Tanner stage.

Conclusion

We have developed a feasible and valid sampling methodology and measurements for puberty hormones in urine, which allows a sampling frequency by which individual pubertal progression in adolescents can be described in depth.     

Randomized Phase II Study of 5-Fluorouracil Hepatic Arterial Infusion with or without Antineoplastons as an Adjuvant Therapy after Hepatectomy for Liver Metastases from Colorectal Cancer

Background

Antineoplastons are naturally occurring peptides and amino acid derivatives found in human blood and urine. Antineoplaston A10 and AS2-1 reportedly control neoplastic growth and do not significantly inhibit normal cell growth. Antineoplastons contain 3-phenylacetylamino-2, 6-piperidinedione (A10), phenylacetylglutamine plus phenylacetylisoglutamine (A10-I), and phenylacetylglutamine plus phenylacetate (AS2-1). This open label, non- blinded randomized phase II study compared the efficacy of hepatic arterial infusion (HAI) with 5-fluorouracil,with or without antineoplastons as a postoperative therapy for colorectal metastasis to the liver.

Methods

Sixty-five patients with histologically confirmed metastatic colon adenocarcinoma in liver, who had undergone hepatectomy, and/or thermal ablation for liver metastases were enrolled between 1998- 2004 in Kurume University Hospital. Patients were randomly assigned to receive systemic antineoplastons (A10-I infusion followed by per-oral AS2-1) plus HAI (AN arm) or HAI alone (control arm) based on the number of metastases and presence/ absence of extra-hepatic metastasis at the time of surgery. Primary endpoint was cancer-specific survival (CSS); secondary endpoints were relapse-free survival (RFS), status and extent of recurrence, salvage surgery (rate) and toxicity.

Findings

Overall survival was not statistically improved (p=0.105) in the AN arm (n=32). RFS was not significant (p=0.343). Nevertheless, the CSS rate was significantly higher in the AN arm versus the control arm (n=33) with a median survival time 67 months (95%CI 43-not calculated) versus 39 months (95%CI 28-47) (p=0.037) and 5 year CSS rate 60% versus 32% respectively. Cancer recurred more often in a single organ than in multiple organs in the AN arm versus the control arm. The limited extent of recurrent tumours in the AN arm meant more patients remained eligible for salvage surgery. Major adverse effects of antineoplastons were fullness of the stomach and phlebitis. No serious toxicity, including bone marrow suppression, liver or renal dysfunction, were found in the AN arm.

Interpretation

Antineoplastons (A10 Injection and AS2-1) might be useful as adjunctive therapy in addition to HAI after hepatectomy in colorectal metastases to the liver.

Trial registration information

ClinicalTrials.gov UMIN000012099     

Microbiological Screening Is Necessary to Distinguish Carriers of Plasmid-Mediated AmpC Beta-Lactamase-Producing Enterobacteriaceae and Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae because of Clinical Similarity

Objectives

Plasmid-mediated AmpC beta-lactamase-producing (pAmpC) Enterobacteriaceae are increasing worldwide, difficult to identify and often confounded with extended-spectrum beta-lactamase (ESBL) producers. The low prevalence precludes routine universal admission screening. Therefore, we evaluated potential risk factors for carriage of pAmpC-producing Enterobacteriaceae that would allow targeted screening to improve yield and reduce cost.

Patients and methods

We performed a case control study at a tertiary care center from 1/2006 to 12/2010. Cases were adult patients in whom pAmpC-producing Enterobacteriaceae were isolated; controls were chosen among carriers of ESBL-producing Enterobacteriaceae. Both infected and colonized patients were included.

Results

Over five years, we identified 40 pAmpC producers in 39 patients among 16,247 screened consecutive isolates of Enterobacteriaceae. The pAmpC prevalence was low (0.25%), but more than 30% of pAmpC carriers received incorrect empirical antibiotic treatment. When compared with 39 ESBL controls, pAmpC carriage was associated with clinically confirmed infections in 74% (versus 51%) (p=0.035), mainly of the urinary tract, previous antibiotic exposure in 63% (versus 36%) (p=0.035) and carriage of a nasogastric tube in 23% (versus 0%) (p=0.002). In the multivariate regression analysis only clinically confirmed infections remained significantly associated with pAmpC carriage (OR 1.44 (95%CI 1.15-2.57)). No other clinical and blood test-associated risk factor allowed discrimination of pAmpC-carrying patients from ESBL controls. The type of acquisition – nosocomial versus community-acquired – was also non-informative for resistance type, as 46% of pAmpC- and 44% of ESBL-producing Enterobacteriaceae were community-acquired.

Conclusions

This study could not identify a clinical profile that would allow targeted screening for pAmpC-producing Enterobacteriaceae when compared to ESBL carriers. Because empiric antimicrobial therapy was inappropriate in more than 30%, rapid identification of pAmpC carriers is needed. New microbiological methods are therefore required to simplify rapid and reliable detection of pAmpC carriers.     

A Multicenter,Open-Label,Controlled Phase II Study to Evaluate Safety and Immunogenicity of MVA Smallpox Vaccine (IMVAMUNE) in 18–40 Year Old Subjects with Diagnosed Atopic Dermatitis

Background

Replicating smallpox vaccines can cause severe complications in individuals with atopic dermatitis (AD). Prior studies evaluating Modified Vaccinia Ankara virus (MVA), a non-replicating vaccine in humans, showed a favorable safety and immunogenicity profile in healthy volunteers.

Objective

This Phase II study compared the safety and immunogenicity of MVA enrolling groups of 350 subjects with AD (SCORAD ≤ 30) and 282 healthy subjects.

Methods

Subjects were vaccinated twice with MVA, each dose given subcutaneously 4 weeks apart. Adverse events, cardiac parameters, and the development of vaccinia virus humoral immune responses were monitored.

Results

The overall safety of the vaccine was similar in both groups. Adverse events affecting skin were experienced significantly more often in subjects with AD, but the majority of these events were mild to moderate in intensity. Seroconversion rates and geometric mean titers for total and neutralizing vaccinia-specific antibodies in the AD group were non-inferior compared to the healthy subjects.

Limitations

The size of the study population limited the detection of serious adverse events occurring at a frequency less than 1%.

Conclusion

MVA has a favorable safety profile and the ability to elicit vaccinia-specific immune responses in subjects with AD.

Trial Registration

ClinicalTrials.gov NCT00316602     

Syringe-push membrane absorption as a simple rapid method of urine preparation for clinical proteomics

Background

The analysis of urinary proteome might reveal biomarkers of clinical value. However, current methods of urine preparation for down-stream proteomic analysis are complicated, time-consuming, and/or expensive. This study aims to develop a robust, simple, inexpensive and readily accessible urine preparation method to facilitate clinical proteomic workflow.

Result

Syringe-push membrane absorption (SPMA) was successfully developed by a combination of 5-ml medical syringe and protein-absorbable membrane. Comparing three membranes i.e., nitrocellulose, polyvinylidene difluoride (PVDF) and Whatman no.1, nitrocellulose combined with SPMA (nitrocellulose-SPMA) provided the greatest quality of proteome profile as demonstrated by 2-DE. The quality of the proteome profile and the performance of nitrocellulose-SPMA were systematically compared with three current methods of urine preparation (i.e., ultrafiltration, dialysis/lyophilization and precipitation). While different methods of urine preparation provided comparable proteome quality, nitrocellulose-SPMA had better working performance due to acceptable recovery yield, less workload, short working time, high accessibility and low unit cost. In addition, protein absorbed on nitrocellulose harvested from the SPMA procedure could be stored as a dried membrane at room temperature for at least 1-month without protein degradation or modification.

Conclusions

SPMA is a simple rapid method of preparing urine for downstream proteomic analysis. Because of it is highly accessible and has long storage duration, this technique holds potential benefit for large-scale multi-center research and future development of clinical investigation based upon urinary proteomic analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9087-4) contains supplementary material, which is available to authorized users.     

Vitamin D Status Is an Independent Risk Factor for Global Cognitive Impairment in Peritoneal Dialysis Patients

Objective

Vitamin D (VD) deficiency is an independent risk factor for cognitive impairment (CI) in the general population, but VD status in peritoneal dialysis (PD) patients has not been investigated. In this study, we aimed to investigate the relationship between serum VD levels and global and specific cognitive functions in PD patients.

Design and Setting

Cross-sectional study, simultaneously conducted at two PD centers.

Patients

Clinically stable patients (n = 273) undergoing PD for at least 3 months were enrolled over a period of one year.

Main outcome Measures

Demographic and comorbidity data were recorded, and routine biochemical parameters and serum 25-hydroxyvitamin D (25(OH) D) levels of overnight fasted patients were determined. Global cognitive function was assessed by the Modified Mini-Mental State Examination (3MS) score; executive function, by the trail making tests (Trails A and B); and immediate memory, delayed memory, and language ability by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) sub-tests.

Results

In the univariate analysis, serum 25(OH) D levels significantly correlated with 3MS scores (r = -0.139; P = 0.02), and Trail A (r = -0.188; P = 0.002) and B (r = -0.154; P = 0.01) completion times. In the multivariate analysis, 25(OH) D was found to be independently associated with global CI, but not with executive dysfunction. Serum 25(OH) D could not predict scores of immediate/delayed memory and language ability.

Conclusions

VD deficiency is highly prevalent in PD patients and is an independent risk factor for global CI in this patient cohort.     

European Ancestry Predominates in Neuromyelitis Optica and Multiple Sclerosis Patients from Brazil

Background

Neuromyelitis optica (NMO) is considered relatively more common in non-Whites, whereas multiple sclerosis (MS) presents a high prevalence rate, particularly in Whites from Western countries populations. However, no study has used ancestry informative markers (AIMs) to estimate the genetic ancestry contribution to NMO patients.

Methods

Twelve AIMs were selected based on the large allele frequency differences among European, African, and Amerindian populations, in order to investigate the genetic contribution of each ancestral group in 236 patients with MS and NMO, diagnosed using the McDonald and Wingerchuck criteria, respectively. All 128 MS patients were recruited at the Faculty of Medicine of Ribeirão Preto (MS-RP), Southeastern Brazil, as well as 108 healthy bone marrow donors considered as healthy controls. A total of 108 NMO patients were recruited from five Neurology centers from different Brazilian regions, including Ribeirão Preto (NMO-RP).

Principal Findings

European ancestry contribution was higher in MS-RP than in NMO-RP (78.5% vs. 68.7%) patients. In contrast, African ancestry estimates were higher in NMO-RP than in MS-RP (20.5% vs. 12.5%) patients. Moreover, principal component analyses showed that groups of NMO patients from different Brazilian regions were clustered close to the European ancestral population.

Conclusions

Our findings demonstrate that European genetic contribution predominates in NMO and MS patients from Brazil.