Repositioning the Early Pathology of Type 1 Diabetes to the Extraislet Vasculature.

Type 1 diabetes (T1D) is a prototypic T cell-mediated autoimmune disease. Because the islets of Langerhans are insulated from blood vessels by a double basement membrane and lack detectable lymphatic drainage, interactions between endocrine and circulating T cells are not permitted. Thus, we hypothesized that initiation and progression of anti-islet immunity required islet neolymphangiogenesis to allow T cell access to the islet. Combining microscopy and single cell approaches, the timing of this phenomenon in mice was situated between 5 and 8 wk of age when activated anti-insulin CD4 T cells became detectable in peripheral blood while peri-islet pathology developed. This "peri-insulitis," dominated by CD4 T cells, respected the islet basement membrane and was limited on the outside by lymphatic endothelial cells that gave it the attributes of a tertiary lymphoid structure. As in most tissues, lymphangiogenesis seemed to be secondary to local segmental endothelial inflammation at the collecting postcapillary venule. In addition to classic markers of inflammation such as CD29, V-CAM, and NOS, MHC class II molecules were expressed by nonhematopoietic cells in the same location both in mouse and human islets. This CD45- MHC class II+ cell population was capable of spontaneously presenting islet Ags to CD4 T cells. Altogether, these observations favor an alternative model for the initiation of T1D, outside of the islet, in which a vascular-associated cell appears to be an important MHC class II-expressing and -presenting cell.

Measuring anti-islet autoimmunity in mouse and human by profiling peripheral blood antigen-specific CD4 T cells.

The endocrine pancreas is one of the most inaccessible organs of the human body. Its autoimmune attack leads to type 1 diabetes (T1D) in a genetically susceptible population and a lifelong need for exogenous insulin replacement. Monitoring disease progression by sampling peripheral blood would provide key insights into T1D immune-mediated mechanisms and potentially change preclinical diagnosis and the evaluation of therapeutic interventions. This effort has been limited to the measurement of circulating anti-islet antibodies, which despite a recognized diagnostic value, remain poorly predictive at the individual level for a fundamentally CD4 T cell-dependent disease. Here, peptide-major histocompatibility complex tetramers were used to profile blood anti-insulin CD4 T cells in mice and humans. While percentages of these were not directly informative, the state of activation of anti-insulin T cells measured by RNA and protein profiling was able to distinguish the absence of autoimmunity versus disease progression. Activated anti-insulin CD4 T cell were detected not only at time of diagnosis but also in patients with established disease and in some at-risk individuals. These results support the concept that antigen-specific CD4 T cells might be used to monitor autoimmunity in real time. This advance can inform our approach to T1D diagnosis and therapeutic interventions in the preclinical phase of anti-islet autoimmunity.

Brain transplantation of genetically corrected Sanfilippo type B neural stem cells induces partial cross-correction of the disease.

Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB) is a recessive genetic disorder that severely affects the brain due to a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intra-lysosomal accumulation of partially degraded heparan sulfate. There are no effective treatments for this disorder. In this project, we carried out an ex vivo correction of neural stem cells derived from Naglu -/- mice (iNSCs) induced pluripotent stem cells (iPSC) using a modified enzyme in which human NAGLU is fused to an insulin-like growth factor II receptor binding peptide in order to improve enzyme uptake. After brain transplantation of corrected iNSCs into Naglu -/- mice and long-term evaluation of their impact, we successfully detected NAGLU-IGFII activity in all transplanted animals. We found decreased lysosomal accumulation and reduced astrocytosis and microglial activation throughout transplanted brains. We also identified a novel neuropathological phenotype in untreated Naglu -/- brains with decreased levels of the neuronal marker Map2 and accumulation of synaptophysin-positive aggregates. Upon transplantation, we restored levels of Map2 expression and significantly reduced formation of synaptophysin-positive aggregates. Our findings suggest that genetically engineered iNSCs can be used to effectively deliver the missing enzyme to the brain and treat Sanfilippo type B-associated neuropathology.

Genetically Corrected iPSC-Derived Neural Stem Cell Grafts Deliver Enzyme Replacement to Affect CNS Disease in Sanfilippo B Mice.

Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB [MPS IIIB]) is a lysosomal storage disorder primarily affecting the brain that is caused by a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intralysosomal accumulation of heparan sulfate. There are currently no treatments for this disorder. Here we report that, ex vivo, lentiviral correction of Naglu-/- neural stem cells derived from Naglu-/- mice (iNSCs) corrected their lysosomal pathology and allowed them to secrete a functional NAGLU enzyme that could be taken up by deficient cells. Following long-term transplantation of these corrected iNSCs into Naglu-/- mice, we detected NAGLU activity in the majority of engrafted animals. Successfully transplanted Naglu-/- mice showed a significant decrease in storage material, a reduction in astrocyte activation, and complete prevention of microglial activation within the area of engrafted cells and neighboring regions, with beneficial effects extending partway along the rostrocaudal axis of the brain. Our results demonstrate long-term engraftment of iNSCs in the brain that are capable of cross-correcting pathology in Naglu-/- mice. Our findings suggest that genetically engineered iNSCs could potentially be used to deliver enzymes and treat MPS IIIB.

A Humoral Immune Response Alters the Distribution of Enzyme Replacement Therapy in Murine Mucopolysaccharidosis Type I.

Antibodies against recombinant proteins can significantly reduce their effectiveness in unanticipated ways. We evaluated the humoral response of mice with the lysosomal storage disease mucopolysaccharidosis type I treated with weekly intravenous recombinant human alpha-l-iduronidase (rhIDU). Unlike patients, the majority of whom develop antibodies to recombinant human alpha-l-iduronidase, only approximately half of the treated mice developed antibodies against recombinant human alpha-l-iduronidase and levels were low. Serum from antibody-positive mice inhibited uptake of recombinant human alpha-l-iduronidase into human fibroblasts by partial inhibition compared to control serum. Tissue and cellular distributions of rhIDU were altered in antibody-positive mice compared to either antibody-negative or naive mice, with significantly less recombinant human alpha-l-iduronidase activity in the heart and kidney in antibody-positive mice. In the liver, recombinant human alpha-l-iduronidase was preferentially found in sinusoidal cells rather than in hepatocytes in antibody-positive mice. Antibodies against recombinant human alpha-l-iduronidase enhanced uptake of recombinant human alpha-l-iduronidase into macrophages obtained from MPS I mice. Collectively, these results imply that a humoral immune response against a therapeutic protein can shift its distribution preferentially into macrophage-lineage cells, causing decreased availability of the protein to the cells that are its therapeutic targets.

Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation.

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.

Development of a food compositional database for the estimation of dietary intake of phyto-oestrogens in a group of postmenopausal women previously treated for breast cancer and validation with urinary excretion.

The scientific literature contains evidence suggesting that women who have been treated for breast cancer may, as a result of their diagnosis, increase their phyto-oestrogen (PE) intake. In the present paper, we describe the creation of a dietary analysis database (based on Dietplan6) for the determination of dietary intakes of specific PE (daidzein, genistein, glycitein, formononetin, biochanin A, coumestrol, matairesinol and secoisolariciresinol), in a group of women previously diagnosed and treated for postmenopausal breast cancer. The design of the database, data evaluation criteria, literature data entry for 551 foods and primary analysis by LC­MS/MS of an additional thirty-four foods for which there were no published data are described. The dietary intake of 316 women previously treated for postmenopausal breast cancer informed the identification of potential food and beverage sources of PE and the bespoke dietary analysis database was created to, ultimately, quantify their PE intake. In order that PE exposure could be comprehensively described, fifty-four of the 316 subjects completed a 24 h urine collection, and their urinary excretion results allowed for the description of exposure to include those identified as 'equol producers'.

Reliability of impact forces, hip angles and velocities during simulated forward falls using a novel Propelled Upper Limb fall ARrest Impact System (PULARIS).

Previous forward fall simulation methods have provided good kinematic and kinetic data, but are limited in that they have started the falls from a stationary position and have primarily simulated uni-directional motion. Therefore, a novel Propelled Upper Limb fall ARest Impact System (PULARIS) was designed to address these issues during assessments of a variety of fall scenarios. The purpose of this study was to present PULARIS and evaluate its ability to impact the upper extremities of participants with repeatable velocities, hand forces and hip angles in postures and with vertical and horizontal motion consistent with forward fall arrest. PULARIS consists of four steel tubing crossbars in a scissor-like arrangement that ride on metal trolleys within c-channel tracks in the ceiling. Participants are suspended beneath PULARIS by the legs and torso in a prone position and propelled horizontally via a motor and chain drive until they are quick released, and then impact floor-mounted force platforms with both hands. PULARIS velocity, hip angles and velocities and impact hand forces of ten participants (five male, five female) were collected during three fall types (straight-arm, self-selected and bent-arm) and two fall heights (0.05 m and 0.10 m) to assess the reliability of the impact conditions provided by the system. PULARIS and participant hip velocities were found to be quite repeatable (mean ICC = 0.81) with small between trial errors (mean = 0.03 m/s). The ratio of horizontal to vertical hip velocity components (~0.75) agreed well with previously reported data (0.70-0.80). Peak vertical hand impact forces were also found to be relatively consistent between trials with a mean ICC of 0.73 and mean between trial error of 13.4 N. Up to 83% of the horizontal hand impact forces displayed good to excellent reliability (ICC > 0.6) with small between trial differences. Finally, the ICCs for between trial hip angles were all classified as good to excellent. Overall, PULARIS is a reliable method and is appropriate for studying the response of the distal upper extremity to impact loading during non-stationary, multi-directional movements indicative of a forward fall. This system performed well at different fall heights, and allows for a variety of upper and lower extremity, and hip postures to be tested successfully in different landing scenarios consistent with elderly and sport-related falls.

Progress towards the characterisation of faecal compounds.

Intestinal nitrosation produces ATNCs (Apparent Total N-nitroso Compounds) and these have been linked with an increased risk of colon cancer from eating red meat. Modern LC-MS instrumentation makes direct detection of ATNC components in faecal water a possibility. The difficulty is in determining which of the many compounds present are N-nitrosamines before embarking on efforts to characterise them. We have assumed that any in vivo nitrosation of alimentary tract contents will be non-specific and depend on the amount and basicity of amine present, with concentration of nitrosating agent being the limiting factor. By further nitrosating faecal waters (and ileostomy fluids) we can increase the amount of ATNC and readily access these compounds. The amount and the number of nitrosamines generated depend on the concentration of individual amines present. By derivatisation separately using both 14N and 15N labelled nitrite, we demonstrated that inspecting chromatograms in parallel for a unit mass difference provides a novel and practicable means for identifying unknown ATNC in faecal and ileostomy samples. MS procedures were linked with the traditional approaches of thermal energy analyser (TEA), preparative HPLC, visualisation of nitrosamines with Griess reagent and degradation of N-nitroso compounds by UV irradiation. We have demonstrated that this approach is repeatable and have used it to identify 30 putative N-nitroso compounds (as protonated parent masses [M + H]+ at: 242, 258, 312, 313, 333, 348, 365, 377, 382, 386, 392, 412, 414, 421, 434, 442, 466, 467, 483, 493, 572, 582, 625, 636, 637, 656, 662, 752, 808, and 870.

Mass spectral studies towards more reliable measurement of perfluorooctanesulfonic acid and other perfluorinated chemicals (PFCs) in food matrices using liquid chromatography/tandem mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) experiments are described, leading to a reliable method for the measurement of perfluorooctanesulfonic acid (PFOS) and other perfluorinated chemicals (PFCs) in foods. Separations were performed on new fluorinated stationary phases, RP Octyl (-C(8)F(17)) or propyl-perfluorobenzene (-C(3)H(6)-C(6)F(5)), to ensure resolution of PFOS and interfering taurohydroxycholate isomers. Aqueous ammonium formate (5 mM) and methanol were used as the mobile phases. The mass spectrometer was operated in negative electrospray ionisation mode, recording two transitions for each analyte and one for each internal standard. The purities of the analytical standards for the eleven target perfluoro analytes (C(7) to C(12) carboxylic acids, C(4), C(6) and C(8) sulfonic acids, and octanesulfonamide (PFOSA)) were found to be in close agreement with the supplied values; the lowest purity was 91%. Five candidate internal standards were investigated, (13)C(4)-PFOS, (13)C(4)-perfluorooctanoic acid, (13)C(2)-perfluorodecanoic acid, D(9)-n-ethylperfluorooctanesulfonamidoethanol (D(9)-n-Et-FOSE) and D(3)-n-methylperfluorooctanesulfonamide (D(3)-n-Me-FOSA); the purities were all >98%. The use of tetrahydro-PFOS generated backgrounds (>1 microg/kg) for perfluoroheptanoic acid and perfluorobutanesulfonic acid. Similarly D(9)-n-Et-FOSE was unacceptable and D(3)-n-Me-FOSA was volatile, leaving no clear candidate for normalisation of the measurement of PFOSA. Severe matrix-induced suppression and enhancement effects influenced ionisation, making external calibration and quantification problematic. This was addressed by a parallel standard addition and matrix-matching approach, comparing ionisation in methanol, in procedural blanks and in food-based extracts. The limits of detection (LODs) of 0.001-0.01 microg/kg in solvent and 0.01-1 microg/kg in foods demonstrate that this method is suitable for the determination of PFCs in all food to the required 1 microg/kg reporting level.

Reliéva, a Mahonia aquifolium extract for the treatment of adult patients with atopic dermatitis.

This clinical study was conducted to determine the efficacy and safety of Reliéva cream in adult patients with atopic dermatitis (eczema). This was an open-label trial in 42 patients with atopic dermatitis treated for 12 weeks with Reliéva cream (a homeopathic product containing Psorberine, a proprietary Mahonia aquifolium extract). Efficacy and safety was assessed using Eczema Area and Severity Index scores and a Subject Reported Evaluation of Treatment. The results showed significant (P < 0.05) improvements with respect to Eczema Area and Severity Index scores by comparison to subjects' baseline scores. In addition, subjects responding to a posttreatment evaluation questionnaire indicated a substantial benefit when rating effectiveness, itching, and appearance as a result of using the study preparation. Reliéva cream appears to be a safe and effective treatment for adult patients with atopic dermatitis (eczema).

Influence of 10 wk of soy consumption on plasma concentrations and excretion of isoflavonoids and on gut microflora metabolism in healthy adults.

BACKGROUND: Little information is currently available on the role of the gut microflora in modulating isoflavone bioavailability or on sex differences in isoflavone metabolism and bioavailability. OBJECTIVE: We sought to determine whether chronic soy consumption influences isoflavone bioavailability as judged by plasma isoflavone concentrations and modified gut microflora activities [beta-glucoside hydrolysis and equol and O-desmethylangolensin (O-DMA) production]. We also examined whether sex differences in isoflavone metabolism exist. DESIGN: A randomized, parallel, controlled study design was used to compare a high-soy diet (104 +/- 24 mg total isoflavones/d) with a low-soy diet (0.54 +/- 0.58 mg total isoflavones/d) in 76 healthy young adults for 10 wk. RESULTS: Concentrations of isoflavones and their gut microflora metabolites in the plasma, urine, and feces were significantly higher in the subjects who consumed the high-soy diet than in those who consumed the low-soy diet. Concentrations of O-DMA in plasma and urine were higher in the men than in the women. Fecal bacteria from subjects consuming both diets could convert daidzein to equol ex vivo. Fecal beta-glucosidase activity was significantly higher in the subjects who consumed the high-soy diet than in those who consumed the low-soy diet. CONCLUSIONS: Although interindividual variation in isoflavone metabolism was high, intraindividual variation was low. Only concentrations of O-DMA in plasma and urine appeared to be influenced by sex. Chronic soy consumption does not appear to induce many significant changes to the gut metabolism of isoflavones other than higher beta-glucosidase activity.

Influence of soya-based infant formula consumption on isoflavone and gut microflora metabolite concentrations in urine and on faecal microflora composition and metabolic activity in infants and children.

The urinary excretion of soya isoflavones and gut microflora metabolites was investigated in infants and children who had been fed soya-based infant formulas in early infancy. These infants and children were compared with cows'-milk formula-fed controls, to determine at what age gut microflora metabolism of daidzein to equol and/or O-desmethylangolensin (O-DMA) was established, and whether exposure to isoflavones in early infancy influences their metabolism at a later stage of development. Sixty infants and children (aged 4 months-7 years) participated in the study; thirty in each of the soya and control groups. There were four age groups. These were: 4-6 months (seven in the soya group and seven in the control group); 7-12 months (seven in the soya group and nine in the control group); 1-3 years (six in the soya group and eight in the control group); 3-7 years (ten in the soya group and six in the control group). Urine samples were collected to measure isoflavonoids by MS, and faecal samples were collected to measure gut-health-related bacterial composition, by fluorescent in situ hybridisation with oligonucleotide probes, and metabolic activity. A soya challenge (typically a soya yoghurt alternative product containing 4.8 g soya protein and on average 22 mg total isoflavones) was given to control-group infants (>6 months) and children, and also to soya-group children that were no longer consuming soya, to determine their ability to produce equol and/or O-DMA. Urinary genistein, daidzein and glycitein were detected in all infants (4-6 months) fed soya-based infant formula; O-DMA was detected in 75 % of infants but equol was detected in only 25 %. In the controls (4-6 months), urinary isoflavonoids were very low or not detected. In the older age groups (7 months-7 years), O-DMA was found in the urine samples of 75 % of the soya group and 50 % of the controls, after the soya challenge. Equol excretion was detected in 19 % of the soya-group infants and children, and in only 5 % of the controls. However, in the oldest (3-7 years) children, the proportion excreting O-DMA and equol was similar in both groups. Faecal bacterial numbers for bifidobacteria (P<0.001), bacteroides and clostridia (P<0.05) were significantly lower for the soya group compared with the control group. There appears to be no lasting effect of early-life isoflavone exposure on isoflavone metabolism.

Assessment of performance of laboratories in determining acrylamide in crispbread.

A proficiency testing round was undertaken to assess the performance of laboratories to measure acrylamide in a sample of crispbread. Retail samples of crispbread were ground to a fine powder and after thorough mixing were packed in 40 g units for distribution. Ten samples were selected at random and analyzed in duplicate for acrylamide by liquid chromatography/mass spectrometry (LC/MS). Standard statistical tests showed that the material was homogeneous for the purposes of proficiency testing. Test samples were distributed to 55 laboratories in 16 countries in Europe, North America, Australia, and the Middle East. The results were analyzed by standard proficiency testing statistical procedures, and laboratories were awarded z-scores on the basis of their reported results. Based on a target standard deviation (sigmap value) taken from the Horwitz equation, for a robust mean value of 1.2 mg/kg acrylamide, satisfactory results (z-score within +/- 2 for those between 0.8 and 1.6 mg/kg) were obtained by 86% of the 37 laboratories that returned results. Only 1 laboratory was unsatisfactory and 4 had questionable results. About equal numbers of laboratories used gas chromatography (GC)/MS and LC/MS procedures with about 25% using MS/MS and one using GC with electron capture detection. There was no evident trend in performance or bias in results. GC/MS and LC/MS data were evenly distributed across the population of laboratories reporting results.

Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with [13C(3)]isoflavone internal standards.

A method has been developed for the analysis of phytoestrogens and their conjugates in human urine using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotopically labeled [13C(3)]daidzein and [13C(3)]genistein were synthesized and used as internal standards for isotope dilution mass spectrometry. Free aglycons and intact glucuronide, sulfate, diglucuronide, disulfate, and mixed sulfoglucuronide conjugates of isoflavones and lignans were observed in naturally incurred urine samples. Sample pretreatment was not necessary, other than addition of internal standards and pH adjustment. Urine was injected directly onto the analytical column. The limits of detection were generally <50ng/ml, precision was generally <10% CV for conjugates. Total hydrolyzed daidzein and genistein were measured against quality assurance urine sample and were accurate to within 12%. The accuracy of conjugate measurement can not be ascertained, as no reference samples are available. The mean sum of daidzein and its conjugates was within 20% of the hydrolyzed value. Concentrations of the free aglycons of up to 22% of genistein and 18% of daidzein were observed. The average pattern was ca. 54% 7-glucuronide, 25% 4(')-glucuronide, 13% monosulfates, 7% free daidzein, 0.9% sulfoglucuronides, 0.4% diglucuronide, and <0.1% disulfate. Selective enzymatic deconjugation with glucuronidase and mixed glucuronidase/sulfatase were used to validate the accuracy of the quantitation of the intact daidzein conjugates. There were no apparent sex differences, or conditioning effects on the conjugation profile of isoflavones after chronic dosing.

Isoflavone aglycon and glucoconjugate content of high- and low-soy U.K. foods used in nutritional studies.

The isoflavone aglycon and glucoconjugate content of commercially prepared and "home-prepared" high- and low-soy foods selected for use in an on-going nutritional study were measured by LC-MS. The daidzin, daidzein, 6"-O-malonyldaidzin, 6"-O-acetyldaidzin, genistein, genistin, 6"-O-malonylgenistin, 6"-O-acetylgenistin, glycitin, glycitein, 6"-O-malonylglycitin, and 6"-O-acetylglycitin content are expressed in terms of individual isoflavones, total isoflavone equivalents, and milligrams of isoflavones per portion served. Soybeans (774 mg x kg(-1) total isoflavones) and soybean-containing foods had the highest isoflavone content of the foods examined. The low-soy foods all contained very low concentrations (<8 mg x kg(-1) total isoflavones) of the isoflavone aglycons and glucoconjugates. High- and low-soy 11 day rotating menus were constructed from the analyzed foods to deliver 100.0 and 0.5 mg of isoflavones per day, respectively.