RESUMEN
Lateral column deterioration and subsequent loss of function poses a challenge for limb preservation in patients with Charcot neuroarthropathy (CN). Application of "superconstructs" provides stability and clinical improvement to an often-ulcerated lateral foot. This study examines radiodensity in Hounsfield units (HU) to compare bone quality of lateral column fixation targets using computed tomography (CT) scans between patients with and without midfoot CN. A retrospective chart review identified control (nondiabetic, non-CN; n = 29) and midfoot CN (n = 21) groups. Patient demographics and medical history were collected. Two reviewers measured the mean HU of circular regions of interest centered on the fourth and fifth metatarsal heads as well as the anterior, middle, and posterior thirds of the calcaneus. Radiodensity was compared between groups, among calcaneal locations, Eichenholtz stages and Brodsky types. A p value ≤.05 was considered statistically significant. Age and body mass index were not significantly different between groups. The CN group exhibited greater HU than the control group at the metatarsal head and calcaneus (p < .001). The anterior calcaneus exhibited greater HU than the posterior calcaneus in the CN group (p = .02). The difference in HU was not statistically significant between Stages 0-1 and Stages 2-3 or midfoot Brodsky Types. Indirect bone density analysis revealed an increased density in CN compared to control patients with no significant difference between midfoot CN stages or types. The anterior calcaneus was the densest rearfoot bone among the CN patients, a result that may have implications in surgical fixation.
Asunto(s)
Artropatía Neurógena , Calcáneo , Pie Diabético , Huesos Metatarsianos , Humanos , Estudios Retrospectivos , Pie , Pie Diabético/cirugía , Huesos Metatarsianos/cirugía , Artropatía Neurógena/cirugíaRESUMEN
Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non-protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non-toxic inhibitor of protein glycosylation, and of N-butyl-deoxynojirimycin (NBdNM) a well-tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid-Sia) or sialylated N-glycosylated proteins (protein-Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of target-specific MGE, we found that in dopaminergic or sensory neurons >60% of Sia is lipid bound, and thus polysialic acid-neural cell adhesion molecule (PSA-NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell-specific sialylation processes for separate classes of membrane constituents.
Asunto(s)
Ácido N-Acetilneuramínico , Ácidos Siálicos , Humanos , Ácidos Siálicos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Glicoproteínas/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Glicosilación , LípidosRESUMEN
Charcot neuroarthropathy (CN) is a highly destructive, pathologic process with devastating consequences to foot structure and viability. The use of intramedullary fixation "superconstructs" allows for "re-bar" support of compromised bone and allows for some dynamic fixation. This study examines radiodensity in Hounsfield units (HU) to compare bone quality of medial column fixation targets using computed tomography scans between patients with and without midfoot CN. A retrospective chart review identified control (nondiabetic, non-CN; n = 29) and midfoot CN (n = 21) groups. Patient demographics and medical history were collected. Two reviewers measured the mean HU of a circular region of interest centered on the first metatarsal head and the anterior, middle, and posterior thirds of the talar body. Radiodensity was compared between groups, and among talar locations, Eichenholtz stages and Brodsky types, with statistical significance set at p ≤ .05. Age and body mass index were not significantly different between groups. The CN group maintained greater mean HU than the control group at the metatarsal head (p < .001), and talar body locations (p < .019). The difference in mean HU of these bones was not statistically significant between Stages 0 to 1 and Stages 2 to 3 or Brodsky Types 1 and 2. Mean HU differences among talus positions were not statistically significant. Indirect bone density analysis using HU showed an increased density in CN patients with no significant difference among talar body locations or midfoot Charcot stages and types. These results may assist in optimizing fixation length. Future studies may examine these densities in ankle CN.
Asunto(s)
Artropatía Neurógena , Pie Diabético , Huesos Metatarsianos , Artropatía Neurógena/diagnóstico por imagen , Artropatía Neurógena/cirugía , Pie , Humanos , Huesos Metatarsianos/diagnóstico por imagen , Huesos Metatarsianos/cirugía , Estudios RetrospectivosRESUMEN
Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3ß4, and α4ß2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2-3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4ß2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs.
Asunto(s)
Imidazolinas/farmacología , Neonicotinoides/farmacología , Agonistas Nicotínicos/farmacología , Nitrocompuestos/farmacología , Piridinas/farmacología , Receptores Nicotínicos/efectos de los fármacos , Alquenos/química , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Simulación del Acoplamiento Molecular , Neonicotinoides/metabolismo , Neuroblastoma/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nitrocompuestos/metabolismo , Oocitos , Plaguicidas/metabolismo , Plaguicidas/farmacología , Receptores Nicotínicos/metabolismo , Transducción de Señal/efectos de los fármacos , Xenopus laevisRESUMEN
Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+]i) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+]i signaling at 10-100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1-10 µM) blunted the signaling response of nicotine. The pesticides (at 3-30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Neonicotinoides/toxicidad , Plaguicidas/toxicidad , Receptores Nicotínicos/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Neuronas Dopaminérgicas/patología , Relación Dosis-Respuesta a Droga , Humanos , Neonicotinoides/administración & dosificación , Neuroblastoma/metabolismo , Técnicas de Placa-Clamp , Receptores Nicotínicos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
In cell biology, pharmacology and toxicology dose-response and concentration-response curves are frequently fitted to data with statistical methods. Such fits are used to derive quantitative measures (e.g. EC[Formula: see text] values) describing the relationship between the concentration of a compound or the strength of an intervention applied to cells and its effect on viability or function of these cells. Often, a reference, called negative control (or solvent control), is used to normalize the data. The negative control data sometimes deviate from the values measured for low (ineffective) test compound concentrations. In such cases, normalization of the data with respect to control values leads to biased estimates of the parameters of the concentration-response curve. Low quality estimates of effective concentrations can be the consequence. In a literature study, we found that this problem occurs in a large percentage of toxicological publications. We propose different strategies to tackle the problem, including complete omission of the controls. Data from a controlled simulation study indicate the best-suited problem solution for different data structure scenarios. This was further exemplified by a real concentration-response study. We provide the following recommendations how to handle deviating controls: (1) The log-logistic 4pLL model is a good default option. (2) When there are at least two concentrations in the no-effect range, low variances of the replicate measurements, and deviating controls, control values should be omitted before fitting the model. (3) When data are missing in the no-effect range, the Brain-Cousens model sometimes leads to better results than the default model.
Asunto(s)
Algoritmos , Técnicas In Vitro , Modelos Estadísticos , Línea Celular , Simulación por Computador , Células Hep G2 , Humanos , Modelos Biológicos , Distribución Normal , Proyectos de Investigación , Ácido Valproico/análisis , Ácido Valproico/toxicidadRESUMEN
Importance: Identifying infectious causes of subacute or chronic meningitis can be challenging. Enhanced, unbiased diagnostic approaches are needed. Objective: To present a case series of patients with diagnostically challenging subacute or chronic meningitis using metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) supported by a statistical framework generated from mNGS of control samples from the environment and from patients who were noninfectious. Design, Setting, and Participants: In this case series, mNGS data obtained from the CSF of 94 patients with noninfectious neuroinflammatory disorders and from 24 water and reagent control samples were used to develop and implement a weighted scoring metric based on z scores at the species and genus levels for both nucleotide and protein alignments to prioritize and rank the mNGS results. Total RNA was extracted for mNGS from the CSF of 7 participants with subacute or chronic meningitis who were recruited between September 2013 and March 2017 as part of a multicenter study of mNGS pathogen discovery among patients with suspected neuroinflammatory conditions. The neurologic infections identified by mNGS in these 7 participants represented a diverse array of pathogens. The patients were referred from the University of California, San Francisco Medical Center (n = 2), Zuckerberg San Francisco General Hospital and Trauma Center (n = 2), Cleveland Clinic (n = 1), University of Washington (n = 1), and Kaiser Permanente (n = 1). A weighted z score was used to filter out environmental contaminants and facilitate efficient data triage and analysis. Main Outcomes and Measures: Pathogens identified by mNGS and the ability of a statistical model to prioritize, rank, and simplify mNGS results. Results: The 7 participants ranged in age from 10 to 55 years, and 3 (43%) were female. A parasitic worm (Taenia solium, in 2 participants), a virus (HIV-1), and 4 fungi (Cryptococcus neoformans, Aspergillus oryzae, Histoplasma capsulatum, and Candida dubliniensis) were identified among the 7 participants by using mNGS. Evaluating mNGS data with a weighted z score-based scoring algorithm reduced the reported microbial taxa by a mean of 87% (range, 41%-99%) when taxa with a combined score of 0 or less were removed, effectively separating bona fide pathogen sequences from spurious environmental sequences so that, in each case, the causative pathogen was found within the top 2 scoring microbes identified using the algorithm. Conclusions and Relevance: Diverse microbial pathogens were identified by mNGS in the CSF of patients with diagnostically challenging subacute or chronic meningitis, including a case of subarachnoid neurocysticercosis that defied diagnosis for 1 year, the first reported case of CNS vasculitis caused by Aspergillus oryzae, and the fourth reported case of C dubliniensis meningitis. Prioritizing metagenomic data with a scoring algorithm greatly clarified data interpretation and highlighted the problem of attributing biological significance to organisms present in control samples used for metagenomic sequencing studies.
Asunto(s)
Meningitis/diagnóstico , Metagenoma/genética , Adolescente , Adulto , Animales , Aspergillus oryzae/genética , Candida/genética , Candidiasis/líquido cefalorraquídeo , Candidiasis/diagnóstico , Niño , Enfermedad Crónica , Cryptococcus neoformans/genética , Femenino , Infecciones por VIH/líquido cefalorraquídeo , Infecciones por VIH/diagnóstico , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histoplasma/genética , Histoplasmosis/líquido cefalorraquídeo , Histoplasmosis/diagnóstico , Humanos , Masculino , Meningitis/líquido cefalorraquídeo , Meningitis/microbiología , Meningitis Criptocócica/líquido cefalorraquídeo , Meningitis Criptocócica/diagnóstico , Metagenómica , Persona de Mediana Edad , Neuroaspergilosis/líquido cefalorraquídeo , Neuroaspergilosis/diagnóstico , Neurocisticercosis/líquido cefalorraquídeo , Neurocisticercosis/diagnóstico , Análisis de Secuencia de ARN/métodos , Taenia solium/genética , Adulto JovenRESUMEN
BACKGROUND: Congenital cancer is rare, and metastatic cancer of the infant at birth is even more unusual. Pregnancy management may be altered if fetal tumors are detected by prenatal ultrasound. CASE: We present a 29-week gestation with polyhydramnios and a fetus with a congenital malignant extrarenal rhabdoid tumor on the left neck and chest with generalized metastases. Cytogenetic analysis of the tumor cells revealed a trisomy 7. CONCLUSION: Prenatal ultrasound permits in utero detection of fetal tumors and identification of complications.