NF1 mutation-driven neuronal hyperexcitability sets a threshold for tumorigenesis and therapeutic targeting of murine optic glioma.

BACKGROUND: With the recognition that noncancerous cells function as critical regulators of brain tumor growth, we recently demonstrated that neurons drive low-grade glioma initiation and progression. Using mouse models of neurofibromatosis type 1 (NF1)-associated optic pathway glioma (OPG), we showed that Nf1 mutation induces neuronal hyperexcitability and midkine expression, which activates an immune axis to support tumor growth, such that high-dose lamotrigine treatment reduces Nf1-OPG proliferation. Herein, we execute a series of complementary experiments to address several key knowledge gaps relevant to future clinical translation. METHODS: We leverage a collection of Nf1-mutant mice that spontaneously develop OPGs to alter both germline and retinal neuron-specific midkine expression. Nf1-mutant mice harboring several different NF1 patient-derived germline mutations were employed to evaluate neuronal excitability and midkine expression. Two distinct Nf1-OPG preclinical mouse models were used to assess lamotrigine effects on tumor progression and growth in vivo. RESULTS: We establish that neuronal midkine is both necessary and sufficient for Nf1-OPG growth, demonstrating an obligate relationship between germline Nf1 mutation, neuronal excitability, midkine production, and Nf1-OPG proliferation. We show anti-epileptic drug (lamotrigine) specificity in suppressing neuronal midkine production. Relevant to clinical translation, lamotrigine prevents Nf1-OPG progression and suppresses the growth of existing tumors for months following drug cessation. Importantly, lamotrigine abrogates tumor growth in two Nf1-OPG strains using pediatric epilepsy clinical dosing. CONCLUSIONS: Together, these findings establish midkine and neuronal hyperexcitability as targetable drivers of Nf1-OPG growth and support the use of lamotrigine as a potential chemoprevention or chemotherapy agent for children with NF1-OPG.

Reexamining the Minimum Sweat Rate Requirement for Sweat Chloride Testing.

BACKGROUND: Guidelines for sweat chloride testing endorse a minimum sweat rate for reporting results. Bilateral sweat collection is recommended, but if both sites fail to meet the minimum rate (quantity not sufficient, QNS), the test should be repeated. In this study, we examine the correlation between sweat rate and sweat chloride concentration ([Cl-]), assess the accuracy of specimens collected at suboptimal rates, and investigate the use of pooled bilateral specimens for chloride measurement. METHODS: Pearson correlation was employed to analyze the relationship between sweat rate and chloride concentration, [Cl-], in 674 macroduct collections. Weighted kappa was evaluated to determine cystic fibrosis (CF) diagnostic classification concordance for 18 tests with paired arms above vs below the minimum sweat rate. Deming regression was applied to compare [Cl-] from pooled bilateral specimens vs neat specimens in 27 collections with residual volume available after clinical testing. RESULTS: Pearson correlation of sweat rate vs [Cl-] was minimal (r = -0.0735) across specimens with varying rates and [Cl-]. There was substantial agreement in CF diagnostic classification between arms for bilateral collections with discordant sweat rates. Regression analysis of [Cl-] in pooled vs nonpooled specimens revealed a slope of 0.984 and an intercept of 0.796. CONCLUSIONS: Negligible correlation of sweat rate and [Cl-] suggests the minimum sweat rate for macroduct collectors may be overly stringent. Reporting of [Cl-] in specimens with ≥10 µL (rate ≥0.3 µL/min) may reduce QNS rates without compromising diagnostic accuracy. Preliminary data suggests pooling of bilateral collections may be a feasible option to achieve the required volume for testing.

A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

Regulation of PGC1α Downstream of the Insulin Signaling Pathway Plays a Role in the Hepatic Proteotoxicity of Mutant α1-Antitrypsin Deficiency Variant Z.

BACKGROUND & AIMS: Insulin signaling is known to regulate essential proteostasis mechanisms. METHODS: The analyses here examined effects of insulin signaling in the PiZ mouse model of α1-antitrypsin deficiency in which hepatocellular accumulation and proteotoxicity of the misfolded α1-antitrypsin Z variant (ATZ) causes liver fibrosis and cancer. RESULTS: We first studied the effects of breeding PiZ mice to liver-insulin-receptor knockout (LIRKO) mice (with hepatocyte-specific insulin-receptor gene disruption). The results showed decreased hepatic ATZ accumulation and liver fibrosis in PiZ x LIRKO vs PiZ mice, with reversal of those effects when we bred PiZ x LIRKO mice onto a FOXO1-deficient background. Increased intracellular degradation of ATZ mediated by autophagy was identified as the likely mechanism for diminished hepatic proteotoxicity in PiZ x LIRKO mice and the converse was responsible for enhanced toxicity in PiZ x LIRKO x FOXO1-KO animals. Transcriptomic studies showed major effects on oxidative phosphorylation and autophagy genes, and significant induction of peroxisome proliferator-activated-receptor-γ-coactivator-1α (PGC1α) expression in PiZ-LIRKO mice. Because PGC1α plays a key role in oxidative phosphorylation, we further investigated its effects on ATZ proteostasis in our ATZ-expressing mammalian cell model. The results showed PGC1α overexpression or activation enhances autophagic ATZ degradation. CONCLUSIONS: These data implicate suppression of autophagic ATZ degradation by down-regulation of PGC1α as one mechanism by which insulin signaling exacerbates hepatic proteotoxicity in PiZ mice, and identify PGC1α as a novel target for development of new human α1-antitrypsin deficiency liver disease therapies.

Fibroblast growth factor-23 and chronic allograft injury in pediatric renal transplant recipients: a Midwest Pediatric Nephrology Consortium study.

The chronic kidney disease-mineral bone disorder (CKD-MBD) produces fibroblast growth factor-23 (FGF-23) and related circulating pathogenic factors that are strongly associated with vascular injury and declining kidney function in native CKD. Similarly, chronic renal allograft injury (CRAI) is characterized by vascular injury and declining allograft function in transplant CKD. We hypothesized that circulating CKD-MBD factors could serve as non-invasive biomarkers of CRAI. We conducted a cross-sectional, multicenter case-control study. Cases (n = 31) had transplant function >20 mL/min/1.73 m(2) and biopsy-proven CRAI. Controls (n = 31) had transplant function >90 mL/min/1.73 m(2) and/or a biopsy with no detectable abnormality in the previous six months. We measured plasma CKD-MBD factors at a single time point using ELISA. Median (range) FGF23 levels were over twofold higher in CRAI vs. controls [106 (10-475) pg/mL vs. 45 (8-91) pg/mL; p < 0.001]. FGF23 levels were inversely correlated with transplant function (r(2) = -0.617, p < 0.001). Higher FGF23 levels were associated with increased odds of biopsy-proven CRAI after adjusting for transplant function, clinical, and demographic factors [OR (95% CI) 1.43 (1.23, 1.67)]. Relationships between additional CKD-MBD factors and CRAI were attenuated in multivariable models. Higher FGF23 levels were independently associated with biopsy-proven CRAI in children.

All plasma products are not created equal: Characterizing differences between plasma products.

BACKGROUND: Plasma can be manufactured by multiple methods. Few studies have compared quality parameters between plasma products that may affect efficacy and safety. METHODS: Four different plasma products were analyzed to include fresh frozen plasma (FFP), liquid plasma (LP), solvent detergent plasma (SDP), and a spray-dried, solvent detergent-treated plasma (SD-SDP) at multiple time points of storage. Parameters measured included red blood cell, platelet, and white blood cell counts; microparticle phenotypes; thrombin generation; and thrombelastography. These parameters were compared in 10 samples of each product. RESULTS: SDP and SD-SDP contained the smallest number of residual cells compared with FFP and LP. Platelets were the most common residual cell in all products and were highest in LP. FFP contained the greatest number of residual red blood cells. Total microparticle counts were elevated in LP and FFP compared with SDP and SD-SDP. Cell-derived microparticles in both LP and FFP were mostly platelet in origin. Microparticle counts in SDP and SD-SDP were negligible. Thrombelastography results demonstrated similar thrombin, fibrinogen, and platelet function on Day 28 LP compared with Day 5 thawed FFP. Thrombin generation assays revealed that the total, lag time to, and peak thrombin formation were higher in SDP and SD-SDP compared with FFP and LP. All parameters in FFP and LP products were characterized by a large degree of variability. CONCLUSION: The differences in cellular, microparticle, and functional hemostatic parameters measured between plasma products have the potential to affect efficacy and safety. Further study is needed to elucidate the potential immune effects of the cellular and microparticle differences noted as well as the clinical implications of altered thrombin generation kinetics in SD products.

Sweating the small stuff: adequacy and accuracy in sweat chloride determination.

OBJECTIVES: Sweat chloride testing is the gold standard for diagnosis of cystic fibrosis (CF). Our objectives were to: 1) describe variables that determine sweat rate; 2) determine the analytic and diagnostic capacity of sweat chloride analysis across the range of observed sweat rates; and 3) determine the biologic variability of sweat chloride concentration. METHODS: A retrospective analysis was performed using data from all sweat chloride tests performed at St. Louis Children's Hospital over a 21-month period. RESULTS: A total of 1397 sweat chloride tests (1155 sufficient [≥75 mg], 242 insufficient [<75 mg]), were performed on 904 individuals. The sweat weight collected from forearms was statistically greater than that collected from legs. There was a negligible correlation between sweat weight and chloride concentration (r=-0.06). The mean individual biologic CV calculated from individuals with two or more sweat collections ≥75 mg was 13.1% (95% CI: 11.3-14.9%; range 0-88%) yielding a reference change value of 36%. Using 60 mmol/L as the diagnostic chloride cutoff, 100% of CF cases were detected whether a minimum sweat weight of 75, 40, or 20 mg was required. CONCLUSIONS: 1) Collection of sweat from forearms is preferable to upper legs, particularly in very young infants; 2) sweat chloride concentrations are not highly dependent upon sweat rate; 3) a change in sweat chloride concentration exceeding 36% may be considered a clinically significant response to cystic fibrosis transmembrane receptor targeted therapy, and 4) sweat collections of less than 75 mg provide clinically accurate information.

Ketogenesis prevents diet-induced fatty liver injury and hyperglycemia.

Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately 1 billion individuals worldwide. However, the drivers of progressive steatohepatitis remain incompletely defined. Ketogenesis can dispose of much of the fat that enters the liver, and dysfunction in this pathway could promote the development of NAFLD. Here, we evaluated mice lacking mitochondrial 3-hydroxymethylglutaryl CoA synthase (HMGCS2) to determine the role of ketogenesis in preventing diet-induced steatohepatitis. Antisense oligonucleotide-induced loss of HMGCS2 in chow-fed adult mice caused mild hyperglycemia, increased hepatic gluconeogenesis from pyruvate, and augmented production of hundreds of hepatic metabolites, a suite of which indicated activation of the de novo lipogenesis pathway. High-fat diet feeding of mice with insufficient ketogenesis resulted in extensive hepatocyte injury and inflammation, decreased glycemia, deranged hepatic TCA cycle intermediate concentrations, and impaired hepatic gluconeogenesis due to sequestration of free coenzyme A (CoASH). Supplementation of the CoASH precursors pantothenic acid and cysteine normalized TCA intermediates and gluconeogenesis in the livers of ketogenesis-insufficient animals. Together, these findings indicate that ketogenesis is a critical regulator of hepatic acyl-CoA metabolism, glucose metabolism, and TCA cycle function in the absorptive state and suggest that ketogenesis may modulate fatty liver disease.

Sclerostin is positively associated with bone mineral density in men and women and negatively associated with carotid calcified atherosclerotic plaque in men from the African American-Diabetes Heart Study.

CONTEXT: Bone mineral density (BMD) and calcified atherosclerotic plaque (CP) demonstrate inverse relationships. Sclerostin, an endogenous regulator of the Wnt pathway and bone formation, has been associated with impaired osteoblast activation and may play a role in vascular calcification. OBJECTIVE: Our objective was to assess the relationships between sclerostin, BMD, and CP. DESIGN: Generalized linear models were fitted to test for associations between sclerostin, volumetric BMD (vBMD), and CP. PARTICIPANTS: A targeted population of 450 unrelated African Americans (AAs) with type 2 diabetes (T2D) was 56% female with mean/SD/median age of 55.4/9.5/55.0 years and a diabetes duration of 10.3/8.2/8.0 years. MAIN OUTCOME MEASURES: Plasma sclerostin, computed tomography-derived thoracic and lumbar vertebrae trabecular vBMD, coronary artery, carotid artery, and aortoiliac CP were measured. RESULTS: Plasma sclerostin was 1119/401/1040 pg/mL, thoracic vBMD was 206.3/52.4/204.8 mg/cm3, lumbar vBMD was 180.7/47.0/179.0 mg/cm3, coronary artery CP score was 284/648/13, carotid artery CP score was 46/132/0, and aortoiliac CP score was 1613/2910/282. Sclerostin levels were higher in men than women (P<.0001). Before and after adjusting for age, sex, body mass index, blood pressure, smoking, hemoglobin A1c, and low-density lipoprotein-cholesterol, plasma sclerostin levels were positively associated with thoracic and lumbar vertebrae vBMD (P<.0001). Sex-stratified analyses verified significant relationships in both men and women (both P<.001). Sclerostin was not associated with CP except for an inverse relationship with carotid CP in men (fully adjusted model, P=.03). CONCLUSIONS: In this cross-sectional study of AA men and women with T2D, circulating sclerostin was positively associated with vBMD in the spine in both sexes and inversely associated with carotid artery CP in men. Sclerostin may play a role in skeletal mineral metabolism in AA but fails to explain inverse relationships between BMD and CP.

Plasma Dickkopf1 (DKK1) concentrations negatively associate with atherosclerotic calcified plaque in African-Americans with type 2 diabetes.

BACKGROUND: Bone mineral density (BMD) and atherosclerotic arterial calcified plaque (CP) demonstrate inverse relationships through unknown mechanisms. Dickkopf-1 (DKK1) is an endogenous inhibitor of bone formation, and serum DKK1 has been associated with impaired osteoblast activation and susceptibility to bone loss. Plasma DKK1, BMD in the spine, and CP in three arterial beds were assessed in African-Americans (AAs) to determine relationships of serum DKK1 with atherosclerotic vascular calcification. METHODS: Plasma DKK1, computed tomography-derived trabecular volumetric BMD (vBMD) in thoracic and lumbar vertebrae, and coronary artery, carotid artery, and aortoiliac CP were measured in 450 unrelated AAs with type 2 diabetes. Generalized linear models were fitted to test for associations between DKK1, vBMD, and CP. RESULTS: Participants were 56% female with mean/SD/median age of 55.4/9.5/55.0 yr, diabetes duration of 10.3/8.2/8.0 yr, plasma DKK1 of 481.6/271.8/417 pg/ml, coronary artery CP mass score of 284/648/13, carotid artery CP mass score of 46/132/0, and aortoiliac CP mass score of 1613/2910/282. Adjusting for age, sex, body mass index, mean arterial blood pressure, smoking, hemoglobin A(1c), and low-density lipoprotein-cholesterol, DKK1 was inversely associated with coronary artery and aortoiliac CP [parameter estimates -0.0011 (P = 0.0137) and -0.0010 (P = 0.0214), respectively], with a trend for carotid artery CP (P = 0.1404). No associations were observed between DKK1 and vBMD in the thoracic or lumbar vertebrae. CONCLUSIONS: Plasma DKK1 levels were inversely associated with coronary artery and aortoiliac CP, but not vBMD, in this cross-sectional study of AAs with type 2 diabetes. DKK1 may play a role in vascular mineral metabolism in this clinical setting.

Prevalence of HIV infection among inpatients and outpatients in Department of Veterans Affairs health care systems: implications for screening programs for HIV.

OBJECTIVES: We sought to determine the prevalence of HIV in both inpatient and outpatient settings in 6 Department of Veterans Affairs (VA) health care sites. METHODS: We collected demographic data and data on comorbid conditions and then conducted blinded, anonymous HIV testing. We conducted a multivariate analysis to determine predictors of HIV infection. RESULTS: We tested 4500 outpatient blood specimens and 4205 inpatient blood specimens; 326 (3.7%) patients tested positive for HIV. Inpatient HIV prevalence ranged from 1.2% to 6.9%; outpatient HIV prevalence ranged from 0.9% to 8.9%. Having a history of hepatitis B or C infection, a sexually transmitted disease, or pneumonia also predicted HIV infection. The prevalence of previously undocumented HIV infection varied from 0.1% to 2.8% among outpatients and from 0.0% to 1.7% among inpatients. CONCLUSIONS: The prevalence of undocumented HIV infection was sufficiently high for routine voluntary screening to be cost effective in each of the 6 sites we evaluated. Many VA health care systems should consider expanded routine voluntary HIV screening.

Characterization of the association of tissue factor pathway inhibitor with human placenta.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is an endothelial-associated inhibitor of blood coagulation. Because the mechanism for attachment of TFPI to endothelium is not clear, we investigated its association with human placenta. METHODS AND RESULTS: Western blots demonstrate that treatment with phosphatidylinositol-specific phospholipase C (PIPLC) removes more placental TFPI than either PBS or heparin, a finding confirmed by immunohistochemistry. The amounts of heparin-releasable and PIPLC-releasable TFPI activity on placental endothelium were measured in placentas from 5 individuals. PIPLC removes >10-fold more TFPI activity from the placental fragments than 10 U/mL heparin and >100-fold more than 1 U/mL heparin. Pretreatment of the placental fragments with PIPLC increases the amount of heparin-releasable TFPI by approximately 3-fold. An antibody specific for the C-terminal region of TFPI recognizes PIPLC-releasable TFPI in Western blots. CONCLUSIONS: GPI-anchored TFPI is the predominant form on placental endothelium. Heparin-releasable TFPI likely represents only a small portion of the total TFPI on endothelium that remains attached to cell-surface glycosaminoglycans after cleavage of the GPI anchor by endogenous enzymes. The predominance of GPI-anchored TFPI suggests that heparin infusion does not significantly redistribute TFPI within the vasculature. The intact C-terminus in GPI-anchored TFPI indicates it is not directly attached to a GPI anchor. Rather, it most likely associates with endothelium by binding to a GPI-anchored protein.

Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency.

Determination of the reticulocyte hemoglobin content (CHr) provides an early measure of functional iron deficiency because reticulocytes are the earliest erythrocytes released into blood and circulate for only 1 to 2 days. The CHr in 78 patients undergoing bone marrow examination was measured to assess its clinical utility for the diagnosis of iron deficiency. Twenty-eight patients were iron deficient, based on the lack of stainable iron in the aspirate. The diagnostic power of CHr is limited in patients with high mean cellular volume (MCV) or red cell disorders such as thalassemia. However, when patients with MCV more than 100 fL are excluded, receiver operator curve analysis of CHr, ferritin, transferrin saturation, and MCV demonstrates that CHr has the highest overall sensitivity and specificity of these peripheral blood tests for predicting the absence of bone marrow iron stores.