An independent comparison of the Novolytix salivary melatonin radioimmunoassay with the new Novolytix salivary melatonin enzyme-linked immunosorbent assay.

The dim light melatonin onset (DLMO) is the current gold standard biomarker of the timing of the central circadian clock in humans and is often assessed from saliva samples. To date, only one commercially available salivary melatonin assay is considered accurate at the low daytime levels required to accurately detect the DLMO (Novolytix RIA RK-DSM2). The aim of this study was to conduct the first independent evaluation of a newly improved enzyme-linked immunosorbent assay (ELISA; Novolytix MLTN-96) and compare it with the recommended radioimmunoassay (RIA)-both in terms of melatonin concentrations and derived DLMOs. Twenty participants (15 females, 18-59 years old) provided saliva samples every 30 min in dim light starting 6 h before their habitual bedtime, yielding a total of 260 saliva samples. Both the RIA and ELISA yielded daytime melatonin concentrations <2 pg/mL, indicating adequate accuracy to detect the DLMO. The melatonin concentrations from the two assays were highly correlated (r = .94, p < .001), although the RIA yielded lower levels of melatonin concentration than the ELISA, on average by 0.70 pg/mL (p = .006). Seventeen DLMOs were calculated from the melatonin profiles and the DLMOs from both assays were not statistically different (p = .36) and were highly correlated (r = .97, p < .001). Two DLMOs derived from the RIA occurred more than 30 min earlier than the DLMO derived from the ELISA. These results indicate that the new Novolytix ELISA is an appropriate assay to use if the Novolytix RIA is not feasible or available.

The effect of angiotensin receptor neprilysin inhibitor, sacubitril/valsartan, on central nervous system amyloid-ß concentrations and clearance in the cynomolgus monkey.

Sacubitril/valsartan (LCZ696) is the first angiotensin receptor neprilysin inhibitor approved to reduce cardiovascular mortality and hospitalization in patients with heart failure with reduced ejection fraction. As neprilysin (NEP) is one of several enzymes known to degrade amyloid-ß (Aß), there is a theoretical risk of Aß accumulation following long-term NEP inhibition. The primary objective of this study was to evaluate the potential effects of sacubitril/valsartan on central nervous system clearance of Aß isoforms in cynomolgus monkeys using the sensitive Stable Isotope Labeling Kinetics (SILK™)-Aß methodology. The in vitro selectivity of valsartan, sacubitril, and its active metabolite sacubitrilat was established; sacubitrilat did not inhibit other human Aß-degrading metalloproteases. In a 2-week study, sacubitril/valsartan (50mg/kg/day) or vehicle was orally administered to female cynomolgus monkeys in conjunction with SILK™-Aß. Despite low cerebrospinal fluid (CSF) and brain penetration, CSF exposure to sacubitril was sufficient to inhibit NEP and resulted in an increase in the elimination half-life of Aß1-42 (65.3%; p=0.026), Aß1-40 (35.2%; p=0.04) and Aßtotal (29.8%; p=0.04) acutely; this returned to normal as expected with repeated dosing for 15days. CSF concentrations of newly generated Aß (AUC(0-24h)) indicated elevations in the more aggregable form Aß1-42 on day 1 (20.4%; p=0.039) and day 15 (34.7%; p=0.0003) and in shorter forms Aß1-40 (23.4%; p=0.009), Aß1-38 (64.1%; p=0.0001) and Aßtotal (50.45%; p=0.00002) on day 15. However, there were no elevations in any Aß isoforms in the brains of these monkeys on day 16. In a second study cynomolgus monkeys were administered sacubitril/valsartan (300mg/kg) or vehicle control for 39weeks; no microscopic brain changes or Aß deposition, as assessed by immunohistochemical staining, were present. Further clinical studies are planned to address the relevance of these findings.

Differentiating Glomerular Inflammation from Fibrosis in a Bone Marrow Chimera for Rat Anti-Glomerular Basement Membrane Glomerulonephritis.

BACKGROUND: Many types of glomerulonephritis (GN) undergo tandem connected phases: inflammation and fibrosis. Fibrosis in human GNs leads to irreversible end-stage disease. This study investigated how these 2 phases were controlled. METHODS: Using a rat anti-glomerular basement membrane GN model, we established bone marrow (BM) chimeras between GN-resistant Lewis (LEW) and GN-susceptible Wistar Kyoto (WKY) rats. Glomerular inflammation and fibrosis were compared between chimeras. RESULTS: LEW's BM to WKY chimeras with or without co-transfer of host WKY's T cells were GN-resistant. On the other hand, WKY's BM to LEW (LEW(WKY)) chimeras developed glomerular inflammation and albuminuria upon immunization. Quantitative analysis showed that the number and composition of inflammatory cells in glomeruli of immunized LEW(WKY) chimeras were similar to those in immunized WKY rats at their inflammatory peak. Thus, glomerular inflammation was controlled by BM-derived non-T cell populations. However, unlike WKY rats, LEW(WKY) rats did not develop fibrosis until the end of experiments (84 days) in spite of persistent inflammation and albuminuria. CONCLUSION: Inflammation alone was not sufficient to trigger fibrosis, suggesting a critical role of glomerular cells in the fibrotic process. As LEW(WKY) chimera allows us to separate glomerular inflammation from fibrosis, this model provides a useful tool to study how fibrosis is initiated following inflammation.

Do Patients Accurately Recall Their Pain Levels Following Epidural Steroid Injection? A Cohort Study of Recall Bias in Patient-Reported Outcomes.

BACKGROUND: Although patient-reported outcomes (PROs) have become important in the evaluation of spine surgery patients, the accuracy of patient recall of pre- or post-intervention  symptoms following epidural steroid injection remains unknown. OBJECTIVES: The purpose of this study was to: 1) characterize the accuracy of patient recollection of back/leg pain following epidural steroid injection; 2) characterize the direction and magnitude of recall bias; and 3) characterize factors that impact patient recollection. STUDY DESIGN: A prospective cohort study. SETTING: Level 1 Academic Medical Center. METHODS: Using standardized questionnaires, we recorded numeric pain scores for patients undergoing lumbar epidural steroid injections at our institution. Baseline pain scores were obtained prior to injection, 4-hours and 24-hours postinjection. At a minimum of 2 weeks following the injection, patients were asked to recall their symptoms preinjection and at 4 hours and 24-hours postinjection. Actual and recalled scores, at each time point, were compared using paired t tests. Multivariable linear regression was used to identify factors that impacted recollection. RESULTS: Sixty-one patients with a mean age of 61.4 years (56% women) were included. Compared to their preinjection pain score, patients showed considerable improvement at both 4 hours (Mean Difference [MD] = 2.18, 95% Confidence Interval [CI] 1.42 to 2.94) and 24 hours (MD = 2.64, 95% CI 1.91 to 3.34) postinjection. Patient recollection of preinjection symptoms was significantly more severe than actual at the 2-week time point (MD = 1.39, 95% CI 4.82 to 6.08). The magnitude of recall bias was mild and exceeded the minimal clinically important difference (MCID). No significant recall bias was noted on patient recollection of postinjection symptoms at 4 hours (MD = 0.41, 95% CI -1.05 to 0.23). Patient recollection of symptoms was also significantly more severe than actual at 24 hours (MD = 0.63, 95% CI -1.17 to -0.07), mild magnitude of bias that did not exceed MCID. Linear regression models for differences between actual and recalled pain scores reveal that for recall at 4 hours postinjection, older patients were better at recalling pain. LIMITATIONS: Baseline pain scores were completed in person, in front of a provider. The short-term pain scores were completed while at home, and then recalled scores were obtained by phone call encounter. Telephone surveys can lead to interview bias. All patients received incentive for completion of study. It is unclear if patient incentives have any impact on patient recall. Patients were contacted 2 weeks postinjection; this time point is standard at our institution, but could vary depending on practice location. Lastly, the enrolled patients did not all share the same indication for injection, and pain was not stratified between back and leg pain. CONCLUSIONS: Relying on patient recollection does not provide an accurate measure of preinjection status after lumbar epidural steroid injection, although patients did recall their 4-hour postinjection status. These findings support previous studies indicating that relying on patient recollection does not provide an accurate measure of preintervention symptoms. Patient recollection of postintervention symptoms, however, may have some clinical utility and requires further study.

A long-acting tumor necrosis factor alpha-binding protein demonstrates activity in both in vitro and in vivo models of endometriosis.

Endometriosis is characterized by the presence of elevated proinflammatory cytokines such as tumor necrosis factor (TNF) alpha in the peritoneal cavity. Blocking interaction of TNFalpha with its receptor by the addition of excess TNFalpha-binding protein (TBP)-1 (a soluble form of TNF receptor-1) was effective in animal models of endometriosis. Recently, a novel, high-affinity inhibitor of TNFalpha, TNF-soluble high-affinity receptor complex (TNF-SHARC), was created by fusing TBP to both the alpha and beta subunits of inactive human chorionic gonadotropin. This dimeric protein was effective in inhibiting collagen-induced arthritis in mice. In the present study, the efficacy of TNF-SHARC in cellular and in vivo models of endometriosis was examined. TBP and TNF-SHARC dose-dependently inhibited TNFalpha-induced secretion of interleukin (IL)-6, IL-8, granulocyte macrophage-colony-stimulating factor, and monocyte chemoattractant protein-1 in immortalized human endometriotic cells. An in vivo mouse model of experimentally induced endometriosis using cycling C57BL/6 mice was established. Antide treatment (0.5 mg/kg), used as positive control, initiated 7 days after the establishment of the disease, reduced the weight of the lesions compared with control. TNF-SHARC at 3 mg/kg was not effective in inhibiting the disease, whereas at 9 mg/kg there was reduction in the lesion weight. In addition, antide and TNF-SHARC treatment in vivo increased in vitro natural killer cell activity compared with untreated animals. Thus, we provide evidence for supporting the development of TNF-SHARC as a therapeutic candidate for treating endometriosis in human.

Tumor necrosis factor-alpha regulates inflammatory and mesenchymal responses via mitogen-activated protein kinase kinase, p38, and nuclear factor kappaB in human endometriotic epithelial cells.

Tumor necrosis factor (TNF)-alpha is central to the endometriotic disease process. TNF-alpha receptor signaling regulates epithelial cell secretion of inflammation and invasion mediators. Because epithelial cells are a disease-inducing component of the endometriotic lesion, we explored the response of 12Z immortalized human epithelial endometriotic cells to TNF-alpha. This report reveals the impact of disruption of established TNF-alpha-induced signaling cascades on the expression of biomarkers of inflammation and epithelial-mesenchymal transition (EMT) from endometriotic epithelial cells. Note that we show the molecular potential of soluble TNF-R1 [TNF binding protein (TBP)] and a panel of small molecule kinase inhibitors to block endometriotic gene expression directly. The TNF-alpha receptor is demonstrated to signal through IkappaB kinase complex (IKK) 2 > IkappaB > nuclear factor kappaB, extracellular signal-regulated kinase > mitogen-activated protein kinase kinase (MEK), p38, and phosphatidylinositol 3-kinase (PI3K) > Akt1/2. TNF-alpha induces the expression of transcripts for inflammatory mediators interleukin (IL)-6, IL-8, regulated on activation normal T cell expressed and secreted, TNF-alpha, granulocyte macrophage-colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein (MCP)-1 and also invasion mediators matrix metalloproteinase (MMP)-7, MMP-9, and intracellular adhesion molecule-1. Indeed, TBP inhibits the TNF-alpha-induced expression of all the above endometriotic genes in 12Z endometriotic epithelial cells. The secretion of IL-6, IL-8, GMCSF, and MCP-1 by TNF-alpha is blocked by TBP. Interestingly, MEK, p38, and IKK inhibitors block TNF-alpha-induced IL-8, IL-6, and GM-CSF secretion and 12z invasion, whereas the PI3K inhibitors do not. The only inhibitor to block MCP-1 expression is the p38 inhibitor. Last, TBP, MEK inhibitor, or p38 inhibitor also block cell surface expression of N-cadherin, a marker of mesenchymal cells. Taken together, these results demonstrate that interruption of TNF-alpha-induced signaling pathways in human endometriotic epithelial cells results in decreased expression and secretion of biomarkers for inflammation, EMT, and disease progression.

Pharmacological inhibition of phosphodiesterase 4 triggers ovulation in follicle-stimulating hormone-primed rats.

Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides to render them biologically inactive. As such, these enzymes are critical regulators of signal transduction pathways that use cyclic nucleotides as second messengers. PDE4 is one such member that has been identified in ovarian tissue and purported to have a role in the regulation of gonadotropin action. In the present study, selective PDE4 inhibitors enhanced intracellular signaling in a human LH receptor-expressing granulosa cell line. In vivo, PDE4 inhibition in FSH-primed rats resulted in ovulation, indicating that the PDE4 inhibitors can substitute for LH and human chorionic gonadotropin (hCG) in this process. Moreover, when coadministered with a subeffective dose of hCG, PDE4 inhibitors acted synergistically to enhance the ovulation response. Inhibitors of PDE3 or PDE5 had no ovulatory effect under similar conditions. Oocytes that were ovulated after PDE4 inhibition could be fertilized in vitro at a rate similar to that of oocytes from hCG-induced ovulation. Moreover, such oocytes were fully capable of being fertilized in vivo and developing into normal live pups. These results indicate that small molecule PDE4 inhibitors may be orally active alternatives to hCG as part of a fertility treatment regimen.

Association of epigenetic inactivation of RASSF1A with poor outcome in human neuroblastoma.

PURPOSE: To investigate the prevalence and potential clinical significance of epigenetic aberrations in neuroblastoma (NB). EXPERIMENTAL DESIGN: The methylation status of 11 genes that are frequently epigenetically inactivated in adult cancers was assayed in 13 NB cell lines. The prevalence of RASSF1A and TSP-1 methylation was also analyzed in 56 NBs and 5 ganglioneuromas by methylation-specific PCR. Associations between the methylation status of RASSF1A and TSP-1 and patient age, tumor stage, tumor MYCN status, and patient survival were evaluated. RESULTS: Epigenetic changes were detected in all 13 NB cell lines, although the pattern of gene methylation varied. The putative tumor suppressor gene RASSF1A was methylated in all 13 cell lines, and TSP-1 and CASP8 were methylated in 11 of 13 cell lines. Epigenetic changes of DAPK and FAS were detected in only small numbers of cell lines, whereas none of the cell lines had methylation of p16, p21, p73, RAR-beta2, SPARC, or TIMP-3. RASSF1A was also methylated in 70% of the primary NB tumors tested, and TSP-1 methylation was detected in 55% of the tumors. RASSF1A methylation was significantly associated with age >1 year (P < 0.01), high-risk disease (P < 0.016), and poor survival (P < 0.001). In contrast, no association between TSP-1 methylation and prognostic factors or survival was observed. CONCLUSIONS: Our results suggest that epigenetic inactivation of RASSF1A may contribute to the clinically aggressive phenotype of high-risk NB.

Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment.

Alport syndrome is a genetic disease of collagen IV (α3, 4, 5) resulting in renal failure. This study was designed to investigate sex-phenotype correlations and evaluate the contribution of macrophage infiltration to disease progression using Col4a3 knock out (Col4a3KO) mice, an established genetic model of autosomal recessive Alport syndrome. No sex differences in the evolution of body mass loss, renal pathology, biomarkers of tubular damage KIM-1 and NGAL, or deterioration of kidney function were observed during the life span of Col4a3KO mice. These findings confirm that, similar to human autosomal recessive Alport syndrome, female and male Col4a3KO mice develop renal failure at the same age and with similar severity. The specific contribution of macrophage infiltration to Alport disease, one of the prominent features of the disease in human and Col4a3KO mice, remains unknown. This study shows that depletion of kidney macrophages in Col4a3KO male mice by administration of clodronate liposomes, prior to clinical onset of disease and throughout the study period, does not protect the mice from renal failure and interstitial fibrosis, nor delay disease progression. These results suggest that therapy targeting macrophage recruitment to kidney is unlikely to be effective as treatment of Alport syndrome.

Random postoperative day-3 cortisol concentration as a predictor of hypothalamic-pituitary-adrenal axis integrity after transsphenoidal surgery.

OBJECTIVE: To determine whether a random postoperative day-3 cortisol value of 10 µg/dL or greater is predictive of adrenal sufficiency 3 to 10 weeks after transsphenoidal surgery (TSS) and during long-term clinical follow-up. METHODS: We retrospectively reviewed the case records of patients who underwent TSS at our institution between 1991 and 2008. Inclusion criteria were as follows: random cortisol measured on the morning of postoperative day 3, adrenal dynamic testing performed 3 to 10 weeks after TSS, and clinical assessment of the hypothalamic-pituitary-adrenal (HPA) axis at least 6 months after TSS. RESULTS: A total of 466 patients underwent TSS at our institution during the study period. Eighty-three patients met study inclusion criteria. Sensitivity of a random postoperative day-3 serum cortisol value of 10 µg/dL or greater for the prediction of adrenal sufficiency at a median follow-up of 42 days was 64.81% (95% confidence interval, 50.6%-77.32%), with an odds ratio of 3.1 (95% confidence interval, 1.08-8.58). Specificity was 62.1% (95% confidence interval, 42.3%-79.3%). At a median follow-up of 500 days, only 2 patients with a postoperative day-3 cortisol value of 10 µg/dL or greater required hydrocortisone replacement, both of whom had multiple anterior pituitary hormone deficiencies and evidence of pituitary dysfunction during the perioperative period. CONCLUSIONS: In the appropriate clinical context, a postoperative day-3 cortisol value of 10 µg/dL or greater accurately predicts the integrity of the HPA axis. The final decision regarding corticosteroid replacement should be personalized, considering the postoperative day-3 cortisol level, the clinical context in which the measurement was obtained, and any evidence of concomitant pituitary dysfunction in the perioperative period.

Cytokines and growth factors inhibit tumor necrosis factor alpha-induced up-regulation of fibronectin binding on bovine endometrial cells.

OBJECTIVE: To design a high-throughput cell assay to identify molecules modulating adhesion induced by tumor necrosis factor alpha (TNF-alpha) of endometrial cells to mesothelium. DESIGN: Prospective study. SETTING: Biotech company. PATIENT(S): Bovine endometrial (BEND) and human mesothelial cells. INTERVENTION(S): Endometrial cells were treated with TNF-alpha and different proteins. MAIN OUTCOME MEASURE(S): TNF-alpha increased binding of fibronectin-coated fluorescein isothiocyanate (FITC) beads. The ability of various proteins to inhibit TNF-alpha-induced fibronectin-bead binding was measured. RESULT(S): Treatment of BEND cells with TNF-alpha increased binding of fibronectin-coated beads. Addition of TNF-alpha-binding protein abrogated the effect of TNF-alpha in a dose-dependent manner. The initial screen of 1014 proteins identified interferon-alpha2 (IFN-alpha2), inteleukin-17 (IL-17), transforming growth factor beta (TGF-beta), and platelet-derived growth factor (PDGF) as inhibiting TNF-alpha-induced bead binding. Interferon-alpha2, IL-17, and TGF-beta inhibited bead-binding with an IC50 (ng/mL, mean +/- SD) of 0.15 +/- 0.11, 0.098 +/- 0.008, and 5.91 +/- 0.72, respectively. All three isoforms of PDGF (AA, AB, and BB) were also found to inhibit TNF-alpha-induced bead-binding, with IC50s (ng/mL) of 1.8 +/- 0.45, 10.0 +/- 1.49, and 1.72 +/- 0.73, respectively. CONCLUSION(S): We describe a novel high-throughput cell-based assay for endometrial cell binding to fibronectin. We show that IFN-alpha, IL-17, TGF-beta, and PDGF have inhibitory actions on adhesion of endometrial cells to fibronectin.

Leukemia inhibitory factor induces cumulus expansion in immature human and mouse oocytes and improves mouse two-cell rate and delivery rates when it is present during mouse in vitro oocyte maturation.

OBJECTIVE: To examine the role of leukemia inhibitory factor (LIF) during in vitro maturation (IVM) on human and mice cumulus expansion and mice oocyte competence by in vitro fertilization (IVF), culture, and embryo transfer (ET). DESIGN: Prospective animal and human study. SETTING: Serono laboratories and IVF clinic. PATIENT(S): Healthy women volunteers and 8-week-old female mice. INTERVENTION(S): Cumulus compacted human and mice oocytes were matured in IVM media with and without recombinant follicle-stimulating hormone (FSH) and with and without LIF. Mice IVM oocytes with and without 0.2 IU/mL of recombinant FSH; or with and without recombinant FSH + LIF (0.1, 1.0, 1000.0 ng/mL) and ovulated oocytes were in vitro fertilized and cultured. We transferred 395 blastocysts to the uterine horn of 2.5-day pseudopregnant female mice. MAIN OUTCOME MEASURE(S): Cumulus expansion in human and mice oocytes, and two-cell rate, blastocyst rate, and delivered rate of live pups in mice. RESULT(S): In human and mouse oocytes, LIF induced cumulus expansion. When 1000 ng/mL of LIF was added in combination with recombinant FSH, a statistically significant increase in cleavage rate, embryo development rate, and birth rate was observed when compared with oocytes matured with FSH alone. CONCLUSION(S): Leukemia inhibitory factor induced cumulus expansion similarly in human and mouse cumulus-oocyte complexes, and recombinant FSH plus LIF supplementation during mouse IVM significantly improved oocyte competence as measured by cleavage rate, blastocyst development, and birth rate.