Relative Units Research Articles

Salivary Desmoglein Enzyme-Linked Immunosorbent Assay for Diagnosis of Pemphigus Vulgaris: A Noninvasive Alternative Test to Serum Assessment

Background. Serum desmoglein enzyme-linked immunosorbent assay (ELISA) is used for the diagnosis and monitoring of pemphigus diseases. Objectives. To compare the diagnostic accuracy of salivary antidesmoglein (Dsg) 1 and 3 ELISA in the diagnosis of pemphigus vulgaris (PV) patients with that of serum desmogleins ELISA. Methods. Eighty-six untreated PV patients and 180 age- and sex-matched PV-free controls were recruited in this case-control study. PV was diagnosed based on clinical, histopathological, and direct immunofluorescence findings. After processing, serum and salivary anti-Dsg 1 and 3 were measured by the ELISA method using Euroimmun kit (Lübeck, Germany). Results. Using the cut-off point of 20 relative units (RU)/mL, the serum anti-Dsg 1 and 3 ELISA were positive in 62 (72.1%) and 83 (96.5%) patients, respectively, and the salivary anti-Dsg 1 and 3 ELISA were positive in 31 (36.1%) and 63 (73.3%) patients, respectively. The specificity of salivary anti-Dsg 1 and anti-Dsg 3 were both 98.9%. Optimal cut-off values of 7.7 and 13.4 RU/mL were determined for the salivary anti-Dsg 1 and anti-Dsg 3 ELISA, respectively. Conclusion. Salivary anti-Dsg 1 and 3 ELISA with high specificities (98.9%) could be suggested as safe and noninvasive methods for the diagnosis of PV when obtaining a blood sample is difficult.

EXPRESSION OF TYPE 2A PROTEIN PHOSPHATASES IN CARDIAC HEALTH AND DISEASE

Cardiac physiology and hypertrophy is regulated by the phosphorylation status of many proteins, which is regulated (in part) by activity of the type 2A serine/threonine protein phosphatase family. Phosphatase activity of this family is conferred by the homologous PP2ACα/β, PP4C and PP6C catalytic subunits. Using quantitative PCR, gene expression of type 2A phosphatases showed that PP6C mRNA (5.07) expressed as relative units (RU) was higher than PP2ACα (3.52), PP2ACβ (3.24) and PP4C (4.54) in H9c2 cardiomyocytes. However, in adult rat ventricular myocytes (ARVM), PP4C mRNA (6.42) was higher than PP2ACα (2.52), PP2ACβ (3.93) and PP6C (4.96). Using Western immunoblotting, PP2ACα/β protein expression was similar in both H9c2 cardiomyocytes and ARVM. PP6C protein expression in ARVM was significantly higher (P<0.05) when compared to H9c2 cardiomyocytes, while PP4C protein expression in ARVM was undetectable. Additionally, we showed that 28 days of transverse aortic constriction in the mouse induced a ∼61% increase in the left ventricular (LV) mass index and a significant increase (p<0.01) in the expression of PP2ACα/β protein when compared to sham-operated LV myocardium. PP4C protein expression was not detectable  in sham or hypertrophied LV myocardium, whereas PP6C protein expression was similar in both groups. This study illustrates the differences in the expression of the type 2A protein phosphatases in both H9c2 cardiomyocytes, ARVM and also highlights the importance of PP2A in cardiac pathological hypertrophy. Future work will aim to identify substrates of PP2ACα, PP2ACβ, PP4C and PP6C related to calcium regulation and hypertrophy in cardiomyocytes using small interfering RNA.

Adiponectin predicts MMP-2 activity independently of obesity.

Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, have been identified in atherosclerotic plaques and have been directly associated with plaque remodelling and vulnerability. Cardiovascular disease (CVD) is related to insulin resistance (IR) and obesity, characterized by changes in plasma levels of inflammatory markers, such as adiponectin and C-reactive protein (CRP). Our aim was to evaluate the impact of both proteins on MMP-2 and MMP-9 behaviour in individuals with IR. Plasma MMP-2 and MMP-9 activity, adiponectin and hs-CRP concentration and lipoprotein profile were determined in 52 patients with metabolic syndrome (MS) and 27 controls. Patients with MS presented significantly higher MMP-2 activity than controls: 0·95 ± 0·12 vs. 0·77 ± 0·15 relative units (RU) (P < 0·001), while MMP-9 activity was not detectable. MMP-2 activity decreased across quartiles of adiponectin, being significantly reduced in individuals with the highest levels of adiponectin in compared with the lowest levels (0·75 ± 0·17 vs. 0·93 ± 0·09 RU, P < 0·005). This difference persisted significant after adjusting by obesity markers. MMP-2 activity was significantly increased in individuals with the highest levels (G3) compared with those with the lowest levels (G1) of hs-CRP (0·94 ± 0·12 vs. 0·86 ± 0·12, P = 0·041) CONCLUSION: In this study, we observed that adiponectin levels predicted MMP-2 plasma activity independently of obesity. This finding suggests that the inflammatory process, associated with the highest CVD risk, would be involved in MMPs vascular production.

SMT19969 as a treatment for Clostridium difficile infection: an assessment of antimicrobial activity using conventional susceptibility testing and an in vitro gut model.

ObjectivesWe investigated the efficacy of the novel antimicrobial agent SMT19969 in treating simulated Clostridium difficile infection using an in vitro human gut model.MethodsConcentrations of the predominant cultivable members of the indigenous gut microfloras and C. difficile (total and spore counts) were determined by viable counting. Cytotoxin titres were determined using cell cytotoxicity and expressed as log10 relative units (RU). Clindamycin was used to induce simulated C. difficile PCR ribotype 027 infection. Once high-level cytotoxin titres (≥4 RU) were observed, SMT19969 was instilled for 7 days. Two SMT19969 dosing regimens (31.25 and 62.5 mg/L four times daily) were evaluated simultaneously in separate experiments. MICs of SMT19969 were determined against 30 genotypically distinct C. difficile ribotypes.ResultsSMT19969 was 7- and 17-fold more active against C. difficile than metronidazole and vancomycin, respectively, against a panel of genotypically distinct isolates (P < 0.05). Both SMT19969 dosing regimens demonstrated little antimicrobial activity against indigenous gut microflora groups except clostridia. SMT19969 inhibited C. difficile growth and repressed C. difficile cytotoxin titres in the gut model.ConclusionsThese data suggest that SMT19969 is a narrow-spectrum and potent antimicrobial agent against C. difficile. Additional studies evaluating SMT19969 in other models of C. difficile infection are warranted, with human studies to place these gut model observations in context.

Plasma FGF23 and the risk of stroke

To examine fibroblast growth factor 23 (FGF23) as a risk factor for incident stroke in a racially/ethnically diverse population-based urban cohort. Stroke-free Northern Manhattan Study participants with FGF23 measurements (n = 2,525) were followed for a mean of 12 (±5) years to detect incident strokes. We used Cox proportional hazards models to estimate the association of baseline FGF23 with incident total, ischemic, and hemorrhagic stroke. Median FGF23 was 57 relative units (RU)/mL (interquartile range = 44-81 RU/mL). Each unit increase of natural log-transformed FGF23 conferred a 40% greater overall stroke risk after adjusting for estimated glomerular filtration rate and sociodemographic and vascular risk factors (hazard ratio = 1.4, 95% confidence interval 1.1-1.6, p = 0.004). Penalized spline analysis revealed a linear association with overall stroke risk at ≥90 RU/mL FGF23, compared with <90 RU/mL (hazard ratio = 1.5, 95% confidence interval = 1.2-2.1, p = 0.004). Greater FGF23 conferred a doubling of intracerebral hemorrhage (ICH) risk but no significant increased risk of ischemic stroke. The associations of elevated FGF23 levels with greater risks of overall stroke and ICH events were independent of phosphate and parathyroid hormone levels and were similar among participants without chronic kidney disease. Elevated FGF23 was a risk factor for overall stroke and ICH events, in particular in a racially and ethnically diverse urban community, independent of chronic kidney disease.

High Transcript Levels of Vitamin D Receptor Are Correlated with Higher mRNA Expression of Human Beta Defensins and IL-10 in Mucosa of HIV-1-Exposed Seronegative Individuals

Vitamin D (VitD) is an endogenous immunomodulator that could protect from HIV-1 infection reducing immune activation and inducing the expression of anti-HIV-1 peptides. To establish a correlation between VitD and natural resistance to HIV-1 infection, a case-control study using blood and mucosa samples of 58 HIV-1-exposed but seronegative (HESN) individuals, 43 HIV-1 seropositives (SPs) and 59 non-exposed healthy controls (HCs) was carried out. The VitD concentration in plasma was determined by ELISA, and mRNA relative units (RU) of VDR, IL-10, TGF-β, TNF-α and IL-1β in peripheral blood mononuclear cells (PBMCs), oral and genital mucosa was quantified by qRT-PCR. mRNA levels of human beta-defensin (HBD) -2 and -3 were previously reported and used for correlations. Significantly higher levels of VitD were found in plasma as well as higher mRNA RU of VDR in PBMCs, and in genital mucosa from HESN compared to HCs. In addition, higher mRNA RU of TNF-α, IL-1β and IL-10, and lower mRNA RU of TGF-β were found in PBMC from HESNs compared to HCs. We also observed higher IL-10 mRNA RU in genital mucosa of HESNs compared to HCs, and the mRNA levels of TNF-α in oral and genital mucosa of SPs were higher compared to HESNs. Furthermore, positive correlations between VDR and IL-10 mRNA RU in PBMCs and genital mucosa of HESNs were found. Finally, HBD-2 and HBD-3 mRNA RU were positively correlated with VDR mRNA expression in oral mucosa from HESNs. These results suggest that high levels of VitD and its receptor are associated with natural resistance to HIV-1 infection. Up-regulation of the anti-inflammatory IL-10, and the induction of anti-HIV-1 defensins in mucosa might be part of the mechanisms involved in this association. However, further studies are required to define causal associations.

A validated model for predicting outcome after liver transplantation: implications on transplanting the extremely sick

Given the organ shortage, there is a need to optimize outcome after liver transplantation (LT). We defined posttransplant hospital length of stay > 60 days (LOS > 60) as a surrogate of suboptimal outcome. In the first phase of the study, a 'Study cohort' (SC) of 643 patients was used to identify risk factors and construct a mathematical model to identify recipients with anticipated inferior results. In the second phase, a cohort of 417 patients was used for validation of the model ['Validation Cohort' (VC)]. In the SC, 65 patients (10.1%) had LOS > 60 days. One- and 3-year patient/graft survival rates were 81.9%/76.1% and 73.4%/67.4%, respectively. Patient and graft survival rates of those with LOS > 60 days were inferior (P < 0.0001), while transplant cost was greater [3.42 relative units (RU) vs. 1 RU, P < 0.0001]. In a multivariable analysis, pretransplant dialysis (P < 0.001), mechanical ventilation (P < 0.015), MELD (P < 0.003), and age (P < 0.009) were predictors of LOS > 60 days [ROC curve - 0.75 (95% CI 0.70, 0.81)]. In the VC, 53 patients (12.7%) were expected to have adverse outcome by the model. These patients had longer LOS (P < 0.0001), higher cost (<0.0001), and inferior patient and graft survival (P < 0.007).

Antibodies against gonadotropin-releasing hormone (GnRH) in patients with diabetes mellitus is associated with lower body weight and autonomic neuropathy

BackgroundEsophageal dysmotility and gastroparesis are common secondary complications in patients with diabetes mellitus. Patients with dysmotility express antibodies against gonadotropin-releasing hormone (GnRH) in serum. The aim of the present study was to scrutinize patients with diabetes mellitus with regard to the presence of GnRH antibodies, and to examine associations between antibodies and clinical findings.ResultsThirty-nine consecutive patients with diabetes mellitus were included in the study after clinical examination and examination by esophageal manometry and gastric emptying scintigraphy. Serum was analyzed for the presence of antibodies against GnRH using an ELISA, and values are expressed as relative units (RU). Two age- and gender-matched healthy subjects per each patient served as controls. The prevalence of IgM GnRH antibodies in patients was 33% compared to 14% in controls (p = 0.027), with a higher antibody titer; 1.2 (0.6-5.0) and 0.2 (0.1-0.3) RU, respectively (p = 0.000). The expression of IgG antibodies was 15% in patients and none in controls (p = 0.000). Lower body mass index was associated with the presence of IgM antibodies (OR = 0.835, 95% CI = 0.699–0.998), and autonomic neuropathy with the presence IgG antibodies (OR = 9.000, 95% CI = 1.327–61.025). Esophageal dysmotility (69%) or gastroparesis (18%) were not associated with the presence of IgM antibodies (OR = 0.589, 95% CI = 0.143–2.424 and OR = 3.407, 95% CI = 0.633–18.350, respectively). Neither was esophageal dysmotility associated with IgG antibodies (OR = 2.500, 95% CI = 0.259–24.096).ConclusionsAntibodies against GnRH are more common in patients with diabetes mellitus compared with healthy controls. IgM antibodies are associated with lower body mass index and IgG antibodies are associated with autonomic neuropathy.

Antibodies against Gonadotropin-Releasing Hormone in Patients with Posterior Laryngitis

Patients with functional gastrointestinal disorders express antibodies against gonadotropin-releasing hormone (GnRH) in serum. One common cause of posterior laryngitis (PL) is extra-esophageal reflux, but a functional etiology has also been suggested. The aim of this study was to scrutinize patients with PL with regard to the presence of GnRH antibodies and to examine the association between antibodies and symptoms and reflux. Consecutive PL patients were included after examination. Serum was analyzed for the presence of antibodies using an enzyme-linked immunosorbent assay (ELISA) method and expressed as relative units (RU). Two age- and gender-matched healthy subjects per case served as controls. The prevalence of IgM GnRH antibodies in patients was 35% compared with 28% in controls (P = 0.06), with higher levels in patients (0.8 (0.3–2.2) RU) than in controls (0.2 (0.1–0.6) RU) (P = 0.007). The corresponding IgG antibody prevalences were 43% and 4%, respectively (P = 0.001), with no difference in levels (P = 0.70). There was no association between antibodies and clinical findings.

FGF-23 and the Progression of Coronary Arterial Calcification in Patients New to Dialysis

Fibroblast growth factor 23 (FGF-23), a regulator of phosphorus metabolism, is a risk marker in CKD. FGF-23 has been associated with coronary arterial calcification (CAC), but it is not known whether FGF-23 predicts CAC progression in CKD. The aim of this study was to evaluate the association of FGF-23 with CAC progression in advanced CKD. FGF-23 levels and CAC were measured by electrocardiography-triggered multislice computed tomography in 99 individuals initiating dialysis. Patients were enrolled in the study from April 2008 to July 2010. CAC was calculated using Agatston and calcium volume score. Sixty-seven study participants had repeat CAC measures at 1 year. Linear regression was used to assess the association of FGF-23 with CAC. The mean age of study participants was 50 years; 33% were women, and 64% were black. The median FGF-23 level was 1238 relative units (RU)/ml (interquartile range, 515-2218 RU/ml). According to Agatston score, FGF-23 was not associated with baseline CAC (P=0.14) but was significantly associated with CAC progression. There was a 192.3-Agatston unit change in CAC score per 1-SD change in FGF-23 (P=0.008) in models adjusting for known risk factors for CAC and serum phosphate. This association persisted after adjustment for high-sensitivity C-reactive protein, 25-OH vitamin D levels, and the use of phosphorus binders. Results were similar when change in calcium volume score was used. In individuals with advanced CKD, serum FGF-23 is strongly associated with CAC progression. FGF-23 may be a marker of cardiovascular risk in CKD.

Does Increased Blood Pressure Rather Than Aging Influence Retinal Pulse Wave Velocity?

It was demonstrated previously that retinal pulse wave velocity (rPWV) as a measure of retinal arterial stiffness is increased in aged anamnestically healthy volunteers compared with young healthy subjects. Using novel methodology of rPWV assessment this finding was confirmed and investigated whether it might relate to the increased blood pressure usually accompanying the aging process, rather than to the aging itself. A total of 12 young 25.5-year-old (24.0-28.8) [median(1st quartile-3rd quartile)] and 12 senior 68.5-year-old (63.8-71.8) anamnestically healthy volunteers; and 12 senior 63.0-year-old (60.8-65.0) validated healthy volunteers and 12 young 33.0-year-old (29.5-35.0) hypertensive patients were examined. Time-dependent alterations of vessel diameter were assessed by the Dynamic Vessel Analyzer in a retinal artery of each subject. The data were filtered and processed using mathematical signal analysis and rPWVs were calculated. rPWV amounted to 1200 (990-1470) RU (relative units)/s in the hypertensive group and to 1040 (700-2230) RU/s in anamnestically healthy seniors. These differed significantly from rPWVs in young healthy group (410 [280-500] RU/s) and in validated healthy seniors (400 [320-510] RU/s). rPWV associated with age and mean arterial pressure (MAP) in the pooled cohort excluded validated healthy seniors. In a regression model these associations remain when alternately adjusted for MAP and age. When including validated healthy seniors in the pooled cohort only association with MAP remains. Both aging (with not excluded cardiovascular risk factors) and mild hypertension are associated with elevated rPWV. rPWV increases to a similar extent both in young mildly hypertensive subjects and in aged anamnestically healthy persons. Healthy aging is not associated with increased rPWV.

Anticitrullinated Protein Antibodies and Radiological Progression in Juvenile Idiopathic Arthritis

The aim of the study was to investigate whether determination of anticitrullinated protein antibodies (ACPA) provides predictive information on severity of disease course and joint destruction in children with juvenile idiopathic arthritis (JIA). Sera from 74 children with JIA were examined for ACPA using the ELISA test. To assess joint destruction, plain radiographs of both hands were scored twice according to the Steinbrocker scale: at the beginning of observation and after 8.9 to 15.2 months (median 11.5 months) of the followup. Correlations between ACPA serum levels and the disease characteristics (type of JIA onset, disease activity, disease duration, radiological status) were investigated. Twenty-six out of 74 examined children with JIA (35.0%) were ACPA-positive [> 5 relative units (RU)/ml]. ACPA were present in all types of JIA onset, including 36.6% of children with early stage JIA (disease duration < 6 months). All of the IgM-rheumatoid factor (RF)-positive children with polyarticular type of JIA onset were simultaneously positive for ACPA. ACPA levels correlated positively with disease activity at the beginning of the study (rho = 0.7196; p < 0.0001) and after followup (rho = 0.2485; p = 0.0486). Disease duration did not significantly affect ACPA serum levels. ACPA levels correlated positively with radiological joint destruction in children with JIA, both at the beginning of the study (rho = 0.4599; p = 0.0004) and after the followup period (rho = 0.5523; p < 0.0001). ACPA were superior to IgM-RF in diagnosing JIA and provided predictive information on severity of disease course and radiological outcome.

Identification of An Abnormal JAK2 WT CD34+ Cell Population Characterized by High Bcl-Xl Expression Levels Both in JAK2 Positive and Negative PV, ET and PMF Patients,

Abstract 3472 Background:The JAK2V617F gene mutation is a common feature of Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF); however the disease can occur without the JAK2 mutation suggesting that ET, PV and PMF share a common molecular mechanisms which is still unknown. In addition, in patients with the JAK2V617F mutation a substantial proportion of progenitor cells do not bear the JAK2V617F mutation and it is still unclear whether this population is normal or not. Methods: 64 patients affected by MPNs and 12 healthy subjects were included in the study. Twenty three patients were affected by ET, 25 by PV and 22 by PMF. We analyzed the expression, of the anti-apoptotic gene Bcl-xL, both at mRNA and protein level by qRT-PCR and immunofluorescence assay with specific antibodies. Erythroblasts, CD34+ and CD34+CD38-cell populations were selected and analyzed. Moreover, we designed specific PNA (Peptide Nucleic Acid) for JAK2V617F and for Bcl-xL isoform to identify their presence/expression at single CD34+ cell level. Results: Bcl-xL protein and mRNA were found progressively over-expressed in erythroblasts, in CD34+ and in CD34+ CD38- cells from ET, PV and PMF patients without any differences between JAK2V617F and wild type (WT) patients. The median protein level in erythroblasts increased from 22 Relative Units (RU) in ET, to 68,5 in PV and to 85 in PMF. Similarly, in CD34+ Bcl-xL increased from 30 RU in ET, to 89 RU in PV, and to 135 RU in PMF. Finally, CD34+CD38-increased from 17.4 RU in ET, to 45.5 RU in PV, to 101.7 RU in PMF. In addition, analysis by PNA identified distinct progenitor cells population. In patients bearing the JAK2 mutation, three CD34+ cell sub-populations were found: (a) one JAK2 positive and with high levels of Bcl-xL; (b) one JAK2 negative and with high levels of Bcl-xL. Thus, the cell population showing high levels of Bcl-xL is always greater than the JAK2 positive one; (c) one double negative population. In patients without JAK2 mutation, two CD34+ cell populations are identified: (a) one characterized by high levels of Bcl-xL and (b) one double negative. The distribution of these cell subpopulations is different in the distinct MPN. Conclusions: We have identified a previously undisclosed CD34+ cell population which is characterized by high level of Bcl-xL expression and represents the majority of CD34+ cells both in wild type and mutated samples. Interestingly, in mutated samples, this population encompasses the JAK2 mutated one. These findings suggest that Bcl-xL plays an important role in MPN, although it remains undisclosed whether this activation is dependent from an upstream oncogenetic signal. Finally, with this technique we can now identify a putative residual normal CD34+ cells in MPN which may be useful for monitoring patients undergoing targeted therapy with inhibitors. Altogether, these findings disclose unfolded aspects of pathophysiology of MPN and may contribute to a better understanding of the pathogenesis of MPN. Disclosures:Barosi:Novartis: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Dissecting the Hematopoietic Niche to Improve Transplant: Hedgehog and Wnt Pathways Activation in Fetal Mesenchymal Cells (MSC) Are Associated with Better Support to Hematopoietic Progenitors and Higher Proliferative Capacity Compared to Adult MSC

Abstract 4809 Background.Mesenchymal Stem Cells (MSC) are among major players of the hematopoietic niche. In addition, they have shown to be useful in clinical studies both as immune-modulators as well as in regenerative medicine. However, a major obstacle for proving their usefulness is to obtain a clinically effective cell dose, since often less than 10^6 /kg of adult MSC have been used. Thus, their potential efficacy can be either disregarded or not formally proved. In addition, forcing in vitro the proliferative potential of adult BM derived MSC (BM-MSC) implies the risk of generating chromosomal abnormalities.The aim of the study is to investigate the potential use of fetal derived MSC (FT-MSC) which could provide means to overcome the above limits. In addition, we attempted to identify some molecular mechanisms regulating the proliferative potential of FT-MSC and their supportive capacity of hematopoietic progenitors. Methods:Specimens from 5 fetal samples were collected after mechanically induced abortion (median gestational age 11 weeks). FT-MSC were obtained from kidneys and femurs and adult MSC from normal BM. After 15 days of culture adherent cells were sub-cultured until 80% confluence. Immunophenotype and karyotype were evaluated. The telomere length was evaluated using the Telomere PNA system. FT-MSC were injected into NOD/SCID mice to rule out any tumorigenic potential. The immunosuppressive activity of FT-MSC and adult BM-MSC was evaluated by standard assays. To quantify the supportive capacity of FT-MSC we weekly measured the colony output using normal BM MNC layered over confluent FT-MSC compared to stromal cell line MS-5 and to BM-MSC. Protein expression of the key regulators of Hedgehog and Wnt pathways were investigated in FT-MSC and BM-MSC by immunofluorescence using monoclonal antibodies against Indian Hedghog (IHh), Sonic Hedgehog (SHh), Desert Hedgehog (DHh) and b-catenin. Fluorescent signal was detected by confocal microscope. Results.MSC successfully grew from 100% of fetal samples. FT-MSC maintained the typical MSC markers (CD73, CD105, CD44, CD106, CD166, HLA class I, negative for HLA class II) and possessed the same capacity to inhibit T cell proliferation as BM-MSC. FT-MSC compared to adult BM-MSC showed a higher proliferative potential: they reached the fifteenth generation and had a median cumulative population doubling of 26 (range 4–47) while BM-MSC grew until the sixth passage showing a median cumulative population doubling of 4 (range 2–5) (p<0.05 at the fourth, fifth and sixth passage). This might be ascribed to both Hedghog and Wnt pathways which were highly activated in FT-MSC compared to BM-MSC. The mean value of SHh protein expression was 11,5 RU (relative units) compared to 5,5 RU in BM-MSC and b-catenin was 17 RU in FT-MSC vs 3.7 RU in adult. Differently from BM-MSC, FT-MSC maintained a stable karyotype without any sign of tumorigenic potential in the mouse model and stable telomere length during culture. By contrast, 5 out of 32 adult MSC developed genetic abnormalities during in vitro expansion. Importantly, FT-MSC display a better support of hematopoiesis compared to BM-MSC or MS-5 cell line, in fact, during five weeks of culture normal bone marrow samples produced an overall colony number higher when FT-MSC were used as feeder layer compared to BM-MSC or MS-5 (p= 0.07 for both). IHh was significantly higher in FT-MSC compared to BM-MSC (121 RU vs 9 RU). This is in keeping with what already reported by Kobune et al. (Blood 2006) regarding the role of IHh in supporting hematopoiesis and provides the molecular basis for the high supportive capacity of FT-MSC.In conclusion, FT-MSC seem to be candidate for being used to support hematopoiesis in transplant settings. FT-MSC overcome the limitation imposed by adult MSC providing a remarkable yield in culture and they show a reassuring safety profile. FT-MSC show a peculiar profile of Hedgehog and Wnt activation possibly responsible for their biological properties described in this study. Disclosures:Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Relation of Sex and Estrogen Therapy to Serum Fibroblast Growth Factor 23, Serum Phosphorus, and Urine Phosphorus: The Heart and Soul Study

Menopause is associated with urine phosphorus retention, which is mitigated by estrogen therapy. Fibroblast growth factor 23 (FGF-23) is a hormone originating from bone that regulates urine phosphorus excretion. Whether sex or estrogen therapy is associated with different FGF-23 levels is unknown. Cross-sectional study of ambulatory individuals with prevalent cardiovascular disease. Sex and, in women, use or nonuse of estrogen. Serum phosphorus, tubular maximum reabsorption of phosphorus indexed to glomerular filtration rate (TMP/GFR), and plasma FGF-23 concentrations. For 987 participants, mean age was 67 ± 11 years, 182 (18%) were women, and 46 (25%) were using estrogen. Mean estimated GFR was 71 ± 23 (SD) mL/min/1.73 m(2). Compared with women who were not using estrogen, both women on estrogen therapy and men had significantly lower serum phosphorus concentrations, lower TMP/GFR values (indicating higher urine phosphorus excretion), and lower FGF-23 concentrations with adjustment for age, demographics, and kidney function (P < 0.001 for each). Mean FGF-23 levels were 68.7 (95% CI, 59.7-79.0) relative units (RU)/mL in non-estrogen-using women, 43.8 (95% CI, 41.2-46.5) RU/mL in men, and 45.1 (95% CI, 35.2-57.4) RU/mL in women using estrogen in adjusted analysis (P < 0.001). Most participants were men. Estrogen therapy was not randomly assigned. Older women who are not using estrogen have higher FGF-23 levels than either men or women using estrogen. In the context of prior literature, these data suggest that postmenopausal phosphorus retention may stimulate higher FGF-23 concentrations after menopause.

Value of Golgi protein 73 monoclonal antibody in diagnosis of hepatocellular carcinoma.

To explore the sensitivity and specificity of Golgi protein 73 (GP73) monoclonal antibody in the diagnosis of hepatocellular carcinoma (HCC). Self-prepared GP73 monoclonal antibody was used as the primary antibody for detecting the serum GP73 levels in healthy controls(n=31)and HCC patients (n=59). The baseline level of the healthy controls was determined by semiquantitative analysis. The results were compared with those from GP73 polyclonal antibody and alpha-fetoprotein (AFP). The GP73 level of healthy controls was 1.2 (0.9-1.7) relative unit (RU), which was significantly lower than that of HCC patients [5.7 (2.5-7.8) RU] (P<0.001) with monoclonal antibody. Using polyclonal antibody, the GP73 level of HCC patients was also significantly higher than healthy controls [7.8 (3.0-12.4) RU vs. 1.1 (1.0-2.0) RU, P<0.001]. The sensitivity and specificity of GP73 monoclonal antibody in diagnosis of HCC were 84.7% and 93.5%; on the contrary, those of GP73 polyclonal antibody were 78.0% and 93.5%, respectively. The sensitivity and specificity of AFP (67.8% and 74.2%, respectively) in the HCC patients were markedly lower than those of GP73. Logistic regression analysis showed that the odds ratio (OR) of GP73 monoclonal antibody was 7.18 and that of GP73 polyclonal antibody was 1.51. Our self-prepared monoclonal antibody can effectively detect GP73 serum level in HCC patients, and has higher sensitivity and specificity than AFP. It may be superior to the currently used GP73 polyclonal antibody. The results lay the foundation for the further development of ELISA methods by using this monoclonal antibody.

Clinical significance of glutamic acid decarboxylase antibodies in patients with epilepsy

Glutamic acid decarboxylase antibodies (GADAs) have been detected in patients with epilepsy, but the clinical determinants of epilepsy associated with GADA have not been defined. We analyzed GADA with a radioimmunoassay in sera of 253 well-characterized patients with epilepsy and 200 control subjects. The positive samples were confirmed by immunohistochemistry and western blotting (WB). Sera were screened for other autoantibodies. GADA were detected in 15 patients (5.9%) and in three control subjects (1.5%) (p = 0.026). Seven patients (2.8%) had high GADA titers [>or=1,000 relative units (RUs)/ml], six of whom had temporal lobe epilepsy (TLE). All three GADA-positive control subjects had low titers. Two of the five patients with high GADA titers and available cerebrospinal fluid (CSF) samples had intrathecal synthesis (IS) of GADA; one patient had CSF oligoclonal bands. The prevalence of increased levels of GADA tended to be higher in patients with TLE than in patients with extra-TLE [odds ratio (OR) 1.32, 95% confidence interval (CI) 0.39-4.42; p = 0.657]. The patients with high GADA titers had significantly higher number of other autoantibodies compared to the patients with low GADA titers (p = 0.001) and the patients with normal GADA (p < 0.001). High GADA titers were present in a subgroup of patients; close to 90% had TLE. The immunologic profile of these patients suggests that the most probable origin of their epilepsy is autoimmune. A positive IS of GADA may be a marker of an ongoing immune response that could identify those patients in whom a trial with immunosuppressive therapy might be warranted.

A Step-wise Approach for Transfer of Immunogenicity Assays during Clinical Drug Development

We designed a three-step statistical approach to transfer bioanalytical assays (ELISA and Biacore) which evaluates the (1) average equivalence between the two labs (2) concordance in individual sample results between the two labs, and (3) long-term stability of assay performance. Each experimental design evaluated the contribution of four critical variables to the overall variability. Two lots of each variable were examined in a controlled experiment. The variables tested for ELISA were analyst, plate washer, biotinylated-therapeutic protein, and streptavidin-horseradish peroxidase; and for Biacore were analyst, instrument, chip lot, and conjugation chemistry reagent lots. Equivalence in the mean signal to noise (S/N) or mean relative units (RU) between the two labs was established through statistical evaluation of the assay performance characteristics across multiple assay variables. Concordance between the two labs in the individual sample results was subsequently verified both quantitatively and qualitatively. The long-term maintenance of assay stability was monitored by performance testing of a predefined set of samples which were prepared in sufficient quantities to last several years. The process of method validation for biomarker testing in clinical trials is to analyze the variability of the assay performance. However, different factors contribute to this variability and need to be evaluated when the method is transferred to another site/lab. Lack of understanding the critical variables can potentially result in unexpected problems and delays. The three-step statistical approach of assay transfer provides a robust process for transferring complex biological assays.

Glycoengineered Acid α-Glucosidase With Improved Efficacy at Correcting the Metabolic Aberrations and Motor Function Deficits in a Mouse Model of Pompe Disease

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.

In vivo, high-field, 3-Tesla 1H MR spectroscopic assessment of liver fibrosis in HCV-correlated chronic liver disease

Histology is the gold standard by which to diagnose and score hepatic fibrosis. Recently, it has been proposed that hepatic magnetic resonance spectroscopy (MRS) could provide an accurate representation of the disease process. The aim of this study was to correlate the in vivo high-field (3-Tesla) (1)H MRS features of noncirrhotic chronic hepatitis C patients stratified according to the histopathological stages of fibrosis. Six healthy controls and 23 patients with biopsy-proven precirrhotic hepatitic C virus (HCV)-related liver disease were included. The subdivision of patients into the histopathological stages of fibrosis was based on the Ishak fibrosis (F) scoring system: mild hepatitis (0< or =F< or =1), moderate (2< or =F< or =3) and severe hepatitis (4< or =F< or =5). For correlation analysis, the Spearman nonparametric test was used. Differences between groups were calculated with the nonparametric Mann-Whitney U test. A p value <0.05 was considered significant. The particular metabolite content was evaluated in relative units (RU), according to the pattern metabolite/H(2)O=area of the metabolite x1,000/area of nonsuppressed water. A significant statistical difference was observed between control vs. mild and moderate vs. severe disease severity in choline-containing compounds (CCC)/H(2)O ratios (p=0.0379 and p=0.0003) and in glutamine/glutamate (Glx)/H(2)O ratios (p=0.004 and p<0.0001), whereas a statistically significant difference in the lipid/H(2)O ratios was achieved only between control vs. moderate and between moderate vs. severe stages of disease (p=0.011 and p=0.0030). High-field (1)H MRS successfully differentiates between mild/moderate vs. severe stages of chronic hepatitis and can be considered a complement to most standard imaging protocols in the liver.