Ryan M. Mitchell

A CSF biomarker panel for identification of patients with amyotrophic lateral sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with complicated pathogenesis that poses challenges with respect to diagnosis and monitoring of disease progression. Objectives: To identify a biomarker panel that elucidates ALS disease pathogenesis, distinguishes patients with ALS from neurologic disease controls, and correlates with ALS disease characteristics, and to determine the effect of HFE gene variants, a potential risk factor for sporadic ALS, on the biomarker profile. Methods: We obtained CSF samples by lumbar puncture from 41 patients with ALS and 33 neurologic disease controls. All patients were genotyped for HFE polymorphisms. We performed a multiplex cytokine and growth factor analysis and immunoassays for iron-related analytes. Classification statistics were generated using a support vector machine algorithm. Results: The groups of patients with ALS and neurologic disease controls were each associated with distinct profiles of biomarkers. Fourteen biomarkers differed between patients with ALS and the control group. The five proteins with the lowest p values differentiated patients with ALS from controls with 89.2% accuracy, 87.5% sensitivity, and 91.2% specificity. Expression of IL-8 was higher in those patients with lower levels of physical function. Expression of β2-microglobulin was higher in subjects carrying an H63D HFE allele, while expression of several markers was higher in subjects carrying a C282Y HFE allele. Conclusions: A CSF inflammatory profile associated with amyotrophic lateral sclerosis (ALS) pathogenesis may distinguish patients with ALS from neurologic disease controls, and may serve as a biomarker panel to aid in the diagnosis of ALS pending further validation. Some of these biomarkers differ by HFE genotype.

Plasma biomarkers associated with ALS and their relationship to iron homeostasis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with complicated pathogenesis with variable presentation and disease progression. There is a critical need for a panel of biomarkers to provide clinicians and researchers with additional information. In this study, multiplex immunoassays were used to screen a number of cytokines, growth factors, and iron‐related proteins. ALS patients had significantly higher plasma levels of L‐ferritin and lower concentrations of transferrin when compared to healthy controls and together classified a test group of subjects with 82% accuracy. Duration of ALS symptoms correlated positively with levels of monocyte chemoattractant protein 1 (MCP‐1) and negatively with levels of granulocyte‐macrophage colony stimulating factor (GM‐CSF). The biomarker profile suggests iron homeostasis is disrupted in ALS patients, and changes in ferritin and transferrin (Tf) appear to be indicators of ongoing inflammatory processes. The data demonstrate a plasma biomarker profile in ALS patients that may differ from published reports of cerebrospinal fluid biomarkers. Muscle Nerve, 2010

Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1

BackgroundPolymorphisms in the MHC class 1-like gene known as HFE have been proposed as genetic modifiers of neurodegenerative diseases that include neuroinflammation as part of the disease process. Variants of HFE are relatively common in the general population and are most commonly associated with iron overload, but can promote subclinical cellular iron loading even in the absence of clinically identified disease. The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways. We tested the hypothesis that the two most common HFE variants, H63D and C282Y, would affect cellular secretion of cytokines and trophic factors.MethodsWe screened a panel of cytokines and trophic factors using a multiplexed immunoassay in human neuroblastoma SH-SY5Y cells expressing different variants of HFE. The influence of cellular iron secretion on the potent chemokine monocyte chemoattractant protein-1 (MCP-1) was assessed using ferric ammonium citrate and the iron chelator, desferroxamine. Additionally, an antioxidant, Trolox, and an anti-inflammatory, minocycline, were tested for their effects on MCP-1 secretion in the presence of HFE variants.ResultsExpression of the HFE variants altered the labile iron pool in SH-SY5Y cells. Of the panel of cytokines and trophic factors analyzed, only the release of MCP-1 was affected by the HFE variants. We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status. A potential direct effect of HFE is considered because, despite having similar levels of intracellular iron, the association between HFE genotype and MCP-1 expression was different for the H63D and C282Y HFE variants. Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.ConclusionOur results demonstrate that HFE polymorphisms influence the synthesis and release of MCP-1. The mechanism of action involves cellular iron status but it appears there could be additional influences such as ER stress. Finally, these data demonstrate a pharmacogenetic effect of HFE polymorphisms on the ability of minocycline to inhibit MCP-1 secretion.

Biomarker-Based Predictive Models for Prognosis in Amyotrophic Lateral Sclerosis

IMPORTANCE Although median survival in amyotrophic lateral sclerosis (ALS) is 2 to 4 years, survival ranges from months to decades, creating prognostic uncertainty. Strategies to predict prognosis would benefit clinical management and outcomes assessments of clinical trials. OBJECTIVE To identify biomarkers in plasma and cerebrospinal fluid (CSF) of patients with ALS that can predict prognosis. DESIGN, PARTICIPANTS, AND SETTING We conducted a retrospective study of plasma (n = 29) and CSF (n = 33) biomarkers identified in samples collected between March 16, 2005, and August 22, 2007, from patients with ALS at an academic tertiary care center. Participants included patients who were undergoing diagnostic evaluation in the neurology outpatient clinic and were eventually identified as having definite, probable, laboratory-supported probable, or possible ALS as defined by revised El-Escorial criteria. All were white and none had a family history of ALS. Clinical information extended from initial presentation to death. Genotyping for hemochromatosis (HFE) gene status was performed. Multiplex and immunoassay analysis of plasma and CSF was used to measure levels of 35 biomarkers. Statistical modeling was used to identify biomarker panels that could predict total disease duration. MAIN OUTCOMES AND MEASURES Total disease duration, defined as the time from symptom onset to death, was the main outcome. The hypothesis being tested was formulated after data collection. RESULTS Multivariable models for total disease duration using biomarkers from plasma, CSF, and plasma and CSF combined incorporated 7, 6, and 6 biomarkers to achieve goodness-of-fit R2 values of 0.769, 0.617, and 0.962, respectively. After classification into prognostic categories, actual and predicted values achieved moderate to good agreement, with Cohen κ values of 0.526, 0.515, and 0.930 for plasma, CSF, and plasma and CSF combined models, respectively. Inflammatory biomarkers, including select interleukins, growth factors such as granulocyte colony-stimulating factor, and l-ferritin, had predictive value. CONCLUSIONS AND RELEVANCE This study provides proof-of-concept for a novel multivariable modeling strategy to predict ALS prognosis. These results support unbiased biomarker discovery efforts in larger patient cohorts with detailed longitudinal follow-up.

HFE polymorphisms affect cellular glutamate regulation

HFE gene variants are relatively common genetic variants in Caucasians. The H63D HFE genetic variant has been repeatedly associated with a number of neurodegenerative diseases. We developed neuroblastoma cell lines expressing different HFE polymorphisms to explore the mechanisms behind these associations. Here we tested the hypothesis that cells with the H63D variant have a phenotype that promotes glutamate toxicity. In support of this hypothesis, expression of H63D HFE is associated with increased calcium-induced glutamate secretion and decreased cellular glutamate uptake. The polymorphism-associated changes in glutamate secretion were mimicked by altering cellular iron. Additionally, intracellular calcium is altered in a genotype-specific manner which could further impact glutamate secretion. HFE-dependent effects on glutamate uptake were confirmed in astrocytoma cell lines with endogenous expression of HFE. The ability of minocycline and the antioxidant Trolox to increase glutamate uptake differed by HFE genotype and implicate oxidative stress in glutamate regulation. This study demonstrates HFE cellular effects that extend beyond iron regulation, and suggests that H63D HFE may promote glutamate toxicity.

HFE polymorphisms influence the response to chemotherapeutic agents via induction of p16INK4A

HFE is a protein that impacts cellular iron uptake. HFE gene variants are identified as risk factors or modifiers for multiple diseases. Using HFE stably transfected human neuroblastoma cells, we found that cells carrying the C282Y HFE variant do not differentiate when exposed to retinoic acid. Therefore, we hypothesized HFE variants would impact response to therapeutic agents. Both the human neuroblastoma and glioma cells that express the C282Y HFE variant are resistant to Temodar, geldanamycin and γ‐radiation. A gene array analysis revealed that p16INK4A (p16) expression was increased in association with C282Y expression. Decreasing p16 protein by siRNA resulted in increased vulnerability to all of the therapeutic agents suggesting that p16 is responsible for the resistance. Decreasing HFE expression by siRNA resulted in a 85% decrease in p16 expression in the neuroblastoma cells but not the astrocytoma cells. These data suggest a potential direct relationship between HFE and p16 that may be cell specific or mediated by different pathways in the different cell types. In conclusion, the C282Y HFE variant impacts the vulnerability of cancer cells to current treatment strategies apparently by increasing expression of p16. Although best known as a tumor suppressor, there are multiple reports that p16 is elevated in some forms of cancer. Given the frequency of the HFE gene variants, as high as 10% of the Caucasian population, these data provide compelling evidence that the C282Y HFE variant should be part of a pharmacogenetic strategy for evaluating treatment efficacy in cancer cells.

Fluorescence Identification of Head and Neck Squamous Cell Carcinoma and High-Risk Oral Dysplasia With BLZ-100, a Chlorotoxin-Indocyanine Green Conjugate

IMPORTANCE Surgical cure of head and neck squamous cell carcinoma (HNSCC) remains hampered by inadequately resected tumors and poor recognition of lesions with malignant potential. BLZ-100 is a chlorotoxin-based, tumor-targeting agent that has not yet been studied in HNSCC. OBJECTIVE To evaluate BLZ-100 uptake in models of HNSCC and oral dysplasia. DESIGN, SETTING, AND PARTICIPANTS This was an observational study (including sensitivity and specificity analysis) of BLZ-100 uptake in an orthotopic xenograft mouse model of HNSCC and a carcinogen-induced dysplasia model of hamster cheek pouches. INTERVENTIONS Various HNSCC xenografts were established in the tongues of NOD-scid IL2Rgammanull (NSG) mice. BLZ-100 was intravenously injected and fluorescence uptake was measured. To induce dysplasia, the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) was applied to the cheek pouch of Golden Syrian hamsters for 9 to16 weeks. BLZ-100 was subcutaneously injected, and fluorescence uptake was measured. MAIN OUTCOMES AND MEASURES The signal-to-background ratio (SBR) of BLZ-100 was measured in tumor xenografts. To calculate the sensitivity and specificity of BLZ-100 uptake, a digital grid was placed over tissue sections and correlative histologic sections to discretely measure fluorescence intensity and presence of tumor; a receiver operating characteristic (ROC) curve was then plotted. In the hamster dysplasia model, cheeks were graded according to dysplasia severity. The SBR of BLZ-100 was compared among dysplasia grades. RESULTS In HNSCC xenografts, BLZ-100 demonstrated a mean (SD) SBR of 2.51 (0.47). The ROC curve demonstrated an area under the curve (AUC) of 0.89; an SBR of 2.50 corresponded to 92% sensitivity and 74% specificity. When this analysis was focused on the tumor and nontumor interface, the AUC increased to 0.97; an SBR of 2.50 corresponded to 95% sensitivity and 91% specificity. DMBA treatment of hamster cheek pouches generated lesions representing all grades of dysplasia. The SBR of high-grade dysplasia was significantly greater than that of mild-to-moderate dysplasia (2.31 [0.71] vs 1.51 [0.34], P = .006). CONCLUSIONS AND RELEVANCE BLZ-100 is a sensitive and specific marker of HNSCC and can distinguish high-risk from low-risk dysplasia. BLZ-100 has the potential to serve as an intraoperative guide for tumor margin excision and identification of premalignant lesions.

H63D HFE polymorphisms are associated with increased disease duration and decreased muscle superoxide dismutase‐1 expression in amyotrophic lateral sclerosis patients

H63D HFE polymorphisms increase the risk of neurodegenerative disorders and, specifically, may increase amyotrophic lateral sclerosis (ALS) risk. Investigating the physiological alterations induced by H63D polymorphisms in ALS patients may elucidate mechanisms by which this genotype alters disease.

Hemostasis in Tonsillectomy

Tonsillectomy is a commonly performed procedure with an accepted risk of posttonsillectomy hemorrhage (PTH) approaching 5%, but catastrophic effects of hemorrhage are exceedingly rare. A variety of surgical techniques and hemostatic agents have been used to reduce the rate of hemorrhage, although none eliminate the risk. Numerous patient, surgical, and postoperative care factors have been studied for an association with PTH. The most consistent risk factors for PTH seem to be patient age and coagulopathies. Surgeon skill and surgical technique are most consistently associated with primary PTH.

Hearing Loss in Children With Craniofacial Microsomia.

Objective To evaluate the association between craniofacial phenotype and hearing loss in children with craniofacial microsomia. Design Retrospective cohort study. Setting Tertiary care childrens hospital. Patients Individuals with craniofacial microsomia. Main Outcome Measures Ear-specific audiograms and standardized phenotypic classification of facial characteristics. Results A total of 79 participants were included in the study. The mean age was 9 years (range, 1 to 23 years) and approximately 60% were boys. Facial anomalies were bilateral in 39 participants and unilateral in 40 participants (24 right, 16 left). Microtia (hypoplasia of the ear) was the most common feature (94%), followed by mandibular hypoplasia (76%), soft tissue deficiency (60%), orbital hypoplasia or displacement (53%), and facial nerve palsy (32%). Sixty-five individuals had hearing loss (12 bilateral and 53 unilateral). Hearing loss was conductive in 73% of affected ears, mixed in 10%, sensorineural in 1%, and indeterminate in 16%. Hypoplasia of the ear or mandible was frequently associated with ipsilateral hearing loss, although contralateral hearing loss occurred in 8% of hemifaces. Conclusions Hearing loss is strongly associated with malformations of the ipsilateral ear in craniofacial microsomia and is most commonly conductive. Hearing loss can occur contralaterally to the side with malformations in children with apparent hemifacial involvement. Children with craniofacial microsomia should receive early diagnostic hearing assessments.