Long-term use of prescription opioids for non-cancer pain and mortality: a population-based, propensity-weighted cohort study.

OBJECTIVE: The objective of this study was to identify factors associated with long-term opioid use and to assess the association between long-term use and death. STUDY DESIGN: Retrospective cohort study combining several population-wide databases and covering a population of five million inhabitants, including all adults who were initiated on opioid treatment from 2014 to 2018 for non-cancer pain. METHODS: We used logistic regression models to identify factors associated with chronic opioid use and carried out survival analyses using multivariable Cox regression modelling for all-cause mortality during follow-up using inverse probability of treatment weighting (IPTW) and propensity scores based on the probability of using opioids chronically. RESULTS: Among 760,006 patients, 82,423 (10.85%) used opioids for 90 days or more after initiation. Initial therapy characteristics associated with higher risk for long-term use were initiating with long- and short-acting opioids (when compared to tramadol, odds ratio [OR]: 2.63, 95% confidence interval [CI]: 2.57, 2.69 and OR: 1.60, 95%CI: 1.46, 1.76, respectively), using higher daily doses (when compared to 50 morphine milligramme equivalent [MME] or less, prescribing 50 to 89 daily MME, OR: 1.76, 95%CI: 1.65, 1.87; 90 to 119 daily MME, OR: 2.44, 95%CI: 1.99, 3.01; and more than 120 daily MME, OR: 1.77, 95%CI: 1.64, 1.91), and overlapping with gabapentinoids (OR: 2.26, 95%CI: 2.20, 2.32), benzodiazepines (OR: 1.32, 95%CI: 1.30, 1.35), and antipsychotics (OR: 1.21, 95%CI: 1.16, 1.26). After IPTW, chronic opioid use was associated with higher risk of all-cause mortality when compared to short-term use (Hazard Ratio (HR): 1.37, 95%CI: 1.32, 1.42). Sensitivity analyses provided similar results. CONCLUSION: These findings may help healthcare managers to identify and address patients at higher risk of long-term use and riskier prescription patterns.

Metallothionein expression in the central nervous system in response to chronic heavy metal exposure: possible neuroprotective mechanism.

It has been reported that volcanoes release several tonnes of mercury per year among other heavy metals through eruptions, fumaroles, or diffuse soil degassing. Since a high percentage of the world's population lives in the vicinity of an active volcano, the aim of this study is to evaluate the accumulation of these metals in the central nervous system and the presence of cellular mechanisms of heavy metal detoxification such as metallothioneins. To carry out this study, wild mice (Mus musculus) chronically exposed to an active volcanic environment were captured in Furnas village (Azores, Portugal) and compared with those trapped in a reference area (Rabo de Peixe, Azores, Portugal). On the one hand, the heavy metal load has been evaluated by analyzing brain and cerebellum using ICP-MS and a mercury analyzer and on the other hand, the presence of metallothionein 2A has been studied by immunofluorescence assays. Our results show a higher load of metals such as mercury, cadmium and lead in the central nervous system of exposed mice compared to non-exposed individuals and, in addition, a higher immunoreactivity for metallothionein 2A in different areas of the cerebrum and cerebellum indicating a possible neuroprotection process.

The Health Hazards of Volcanoes: First Evidence of Neuroinflammation in the Hippocampus of Mice Exposed to Active Volcanic Surroundings.

Neuroinflammation is a process related to the onset of neurodegenerative diseases; one of the hallmarks of this process is microglial reactivation and the secretion by these cells of proinflammatory cytokines such as TNFα. Numerous studies report the relationship between neuroinflammatory processes and exposure to anthropogenic air pollutants, but few refer to natural pollutants. Volcanoes are highly inhabited natural sources of environmental pollution that induce changes in the nervous system, such as reactive astrogliosis or the blood-brain barrier breakdown in exposed individuals; however, no neuroinflammatory event has been yet defined. To this purpose, we studied resting microglia, reactive microglia, and TNFα production in the brains of mice chronically exposed to an active volcanic environment on the island of São Miguel (Azores, Portugal). For the first time, we demonstrate a proliferation of microglial cells and an increase in reactive microglia, as well an increase in TNFα secretion, in the central nervous system of individuals exposed to volcanogenic pollutants.

Chronic exposure to volcanic gaseous elemental mercury: using wild Mus musculus to unveil its uptake and fate.

Volcanoes are a natural source of gaseous elemental mercury (GEM) (Hg0). Monitoring GEM releases of volcanic origin has been widely studied; however, few studies have been performed about the biomonitoring of species exposed to GEM, rendering an unknown risk to the worldwide populations living in the vicinity of an active volcano. In this pilot study, we used Mus musculus as a bioindicator species to understand to what extent lungs are the main route of mercury uptake in populations chronically exposed to active volcanic environments. Autometallographic silver protocol was used to detect mercury deposits in the histological lung slides. Abundant mercury deposits were found in the lungs of specimens captured at the site with volcanic activity (Furnas Village, S. Miguel Island-Azores). The presence of mercury in the lungs could represent not only hazardous effects to the lung itself but also to other tissues and organs, such as brain and kidneys. This study confirms that the main uptake route for GEM is the lungs and that, even at very low concentrations in the environment, a chronic exposure to Hg0 results in its bioaccumulation in the lung tissue. These results reinforce that biomonitoring studies should be combined with monitoring classical approaches in order to better characterize the risks of exposure to Hg0 in volcanic environments.

First record on mercury accumulation in mice brain living in active volcanic environments: a cytochemical approach.

The health effects of mercury vapor exposure on the brain in volcanic areas have not been previously addressed in the literature. However, 10% of the worldwide population inhabits in the vicinity of an active volcano, which are natural sources of elemental mercury emission. To evaluate the presence of mercury compounds in the brain after chronic exposure to volcanogenic mercury vapor, a histochemical study, using autometallographic silver, was carried out to compare the brain of mice chronically exposed to an active volcanic environment (Furnas village, Azores, Portugal) with those not exposed (Rabo de Peixe village, Azores, Portugal). Results demonstrated several mercury deposits in blood vessels, white matter and some cells of the hippocampus in the brain of chronically exposed mice. Our results highlight that chronic exposure to an active volcanic environment results in brain mercury accumulation, raising an alert regarding potential human health risks. These findings support the hypothesis that mercury exposure can be a risk factor in causing neurodegenerative diseases in the inhabitants of volcanically active areas.

Exploring polyamine metabolism of Alternaria alternata to target new substances to control the fungal infection.

Polyamines are essential for all living organisms as they are involved in several vital cell functions. The biosynthetic pathway of polyamines and its regulation is well established and, in this sense, the ornithine descarboxylase (ODC) enzyme acts as one of the controlling factors of the entire pathway. In this work we assessed the inhibition of the ODC with D, l-α-difluoromethylornithine (DFMO) on Alternaria alternata and we observed that fungal growth and mycotoxin production were reduced. This inhibition was not completely restored by the addition of exogenous putrescine. Actually, increasing concentrations of putrescine on the growth media negatively affected mycotoxin production, which was corroborated by the downregulation of pksJ and altR, both genes involved in mycotoxin biosynthesis. We also studied the polyamine metabolism of A. alternata with the goal of finding new targets that compromise its growth and its mycotoxin production capacity. In this sense, we tested two different polyamine analogs, AMXT-2455 and AMXT-3016, and we observed that they partially controlled A. alternata's viability in vitro and in vivo using tomato plants. Finding strategies to design new fungicide substances is becoming a matter of interest as resistance problems are emerging.

Human Mesenchymal Stem Cells Growth and Osteogenic Differentiation on Piezoelectric Poly(vinylidene fluoride) Microsphere Substrates.

The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC) fate when cultured in supports with varying topography. Poly(vinylidene fluoride) (PVDF) culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM). Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride) is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.

Cultural adaptation and validation of a peninsular Spanish version of the MSTCQ© (Multiple Sclerosis Treatment Concerns Questionnaire). / Adaptación cultural y validación al español de España del MSTCQ© (Multiple Sclerosis Treatment Concerns Questionnaire).

INTRODUCTION: Although subcutaneous treatments for multiple sclerosis (MS) have been shown to be effective, adverse reactions and pain may adversely affect treatment satisfaction and adherence. This study presents an adapted and validated Spanish version of the Multiple Sclerosis Treatment Concerns Questionnaire© (MSTCQ), which evaluates satisfaction with the injection device (ID) across 4 domains: injection system (A), side effects (B) (flu-like symptoms, reactions, and satisfaction), experience with treatment (C) and benefits (D). METHODS: Two study phases: 1) Cultural adaptation process with input from experts (n=6) and patients (n=30). 2) Validation obtained by means of an observational, cross-sectional, multi-centre study evaluating 143 adult MS patients using an ID. Tools employed: MSTCQ©, Patient-Reported Indices for Multiple Sclerosis (PRIMUS©), and Treatment Satisfaction Questionnaire for Medication (TSQM©). Psychometric properties: Feasibility (percentage of valid cases and floor/ceiling effects); Reliability (Cronbach α) and test-retest correlation (n=41, intraclass correlation coefficient, ICC); and construct validity (factor analysis of domains A and B) and convergent validity (Spearman rank-order correlation for MSTCQ© vs TSQM©). RESULTS: Mean age (SD) was 41.94 (10.47) years, 63% of the group were women, and 88.11% presented relapsing-remitting MS. Mean (SD) EDSS score was 2.68 (1.82) points. MSTCQ© completion was high (0%-2.80% missing data). Internal consistency was high at α=0.89 for the total score (A+B) and α=0.76, 0.89, and 0.92 for domains A, B, and C, respectively. The version demonstrated excellent test-retest reliability for the total (ICC=0.98) and for domains A, B, and C: ICC=0.82, 0.97, and 0.89, respectively. Factor analysis corroborated the internal structure of the original questionnaire. The association between total and domain scores on both the MSTCQ© and the TSQM© was moderately strong (Rho=0.42-0.74) and significant (P<.05 and P<.01). CONCLUSION: The Spanish version of MSTCQ© demonstrates appropriate psychometric properties.

Eyelid myokymia in patients with migraine taking topiramate.

OBJECTIVE: Although ocular side effects of topiramate are common, neuroophthalmologic manifestations such as blepharospasm, myokymia and oculogyric crisis are scarcely reported. METHODS: We present a serie of 8 patients with migraine who developed eyelid myokymia after treatment with topiramate. We reviewed all patients with migraine treated with topiramate attending the headache outpatient clinic of our hospital from January 2008 to December 2012. RESULTS: During the study period, a total of 140 patients with migraine were treated with topiramate in our headache clinic. Eight presented eyelid myokymia after beginning treatment with topiramate (5,7%). Topiramate was stopped and myokymia disappeared in all patients, it was prescribed again and eyelid myokymia reappeared with their previous characteristics in all patients. CONCLUSIONS: Eyelid myokymia is an underreported side-effect of topiramate in patients with migraine, of unknown cause, so that in future, further studies are need to examine whether patients with migraine are predisposed or not to this adverse effect.

Targeting Fusarium graminearum control via polyamine enzyme inhibitors and polyamine analogs.

Fusarium graminearum not only reduces yield and seed quality but also constitutes a risk to public or animal health owing to its ability to contaminate grains with mycotoxins. Resistance problems are emerging and control strategies based on new targets are needed. Polyamines have a key role in growth, development and differentiation. In this work, the possibility of using polyamine metabolism as a target to control F. graminearum has been assessed. It was found that putrescine induces mycotoxin production, correlating with an over expression of TRI5 and TRI6 genes. In addition, a homolog of the Saccharomyces cerevisiae TPO4 involved in putrescine excretion was up-regulated as putrescine concentration increased while DUR3 and SAM3 homologues, involved in putrescine uptake, were down-regulated. When 2.5 mM D, l-α-difluoromethylornithine (DFMO) was added to the medium, DON production decreased from 3.2 to 0.06 ng/mm(2) of colony and growth was lowered by up to 70 per cent. However, exogenous putrescine could overcome DFMO effects. Five polyamine transport inhibitors were also tested against F. graminearum. AMXT-1505 was able to completely inhibit in vitro growth and DON production. Additionally, AMXT-1505 blocked F. graminearum growth in inoculated wheat spikes reducing DON mycotoxin contamination from 76.87 µg/g to 0.62 µg/g.

Migraine education brochures and patient-perceived satisfaction.

INTRODUCTION: Brochures are commonly used as educational tools in daily neurological practice. They are provided to increase the general population's knowledge of a specific disease and also to combat sources of erroneous information. Surveys are the most commonly used method of ascertaining user satisfaction with services received. OBJECTIVES: This study will assess patient-perceived satisfaction and provide feedback to measure the comprehensibility and overall utility of an educational brochure on migraine. MATERIAL AND METHODS: Open prospective multicentre study of a group of patients diagnosed with migraine in neurology clinics in Alicante province. During the initial visit, each patient received a migraine brochure prepared by the Valencian Society of Neurology's study group for headaches (CEFALIC). During a follow-up visit, they were then asked to fill out a personal survey on the overall quality of the information in the brochure. RESULTS: We included a total of 257 patients diagnosed with migraine (83% episodic migraine; 17% chronic migraine); mean age was 37.6 years. Two hundred seven patients confirmed having read the brochure (80.5%); 50 patients (19.5%) either forgot to read it or had no interest in doing so. The brochure seemed interesting and easy to understand according to 90% of the patients. Seventy-six per cent of the respondents stated that reading the brochure increased their overall knowledge of migraine, while 50% of the patients found the brochure useful for improving migraine control. CONCLUSIONS: Patients found the migraine educational brochure to be comprehensible, a means of increasing overall knowledge of the disease, and useful for increasing control over migraines. Evaluations of the educational brochures that we provide to our patients with migraine should be studied to discover the causes of dissatisfaction, determine the level of quality of service, and investigate potential areas for improvement.

Osteogenic differentiation of human mesenchymal stem cells on electroactive substrates.

This study investigates the effect of electroactivity and electrical charge distribution on the biological response of human bone marrow stem cells (hBMSCs) cultured in monolayer on flat poly(vinylidene fluoride), PVDF, substrates. Differences in cell behaviour, including proliferation, expression of multipotency markers CD90, CD105 and CD73, and expression of genes characteristic of different mesenchymal lineages, were observed both during expansion in basal medium before reaching confluence and in confluent cultures in osteogenic induction medium. The crystallisation of PVDF in the electrically neutral α-phase or in the electroactive phase ß, both unpoled and poled, has been found to have an important influence on the biological response. In addition, the presence of a permanent positive or negative surface electrical charge distribution in phase ß substrates has also shown a significant effect on cell behaviour.

Switching from natalizumab to fingolimod: an observational study.

BACKGROUND: Multiple sclerosis patients who discontinue using natalizumab are at risk of a rebound in disease activity. However, the optimal alternative therapy is not currently known. AIMS OF THE STUDY: We report on clinical and MRI data and patient safety in a group of relapsing-remitting multiple sclerosis patients who tested seropositive for the JC virus and who have switched from natalizumab to fingolimod because of concerns regarding PML risks. METHODS: The test for JC virus antibodies was performed in 18 relapsing-remitting multiple sclerosis patients who were being treated with natalizumab for more than 1 year. Eight seropositive patients switched to fingolimod while the seronegative patients continued with natalizumab. RESULTS: After switching to fingolimod, five of eight patients (63%) experienced clinical relapses, and MRI activity was detected in six of eight patients (75%). Neither clinical relapses nor MRI activity was observed in the patients who continued with natalizumab. No serious adverse effects were detected. CONCLUSIONS: Natalizumab is an effective treatment for relapsing-remitting multiple sclerosis, but its discontinuation continues to be a complex problem. All of the therapies tried thus far, including fingolimod, have been unable to control the reactivation of the disease. Further studies addressing alternative therapies after natalizumab discontinuation are necessary.

Capture-recapture as a potentially useful procedure for assessing prevalence of multiple sclerosis: methodologic exercise using Portuguese data.

BACKGROUND: Capture-recapture methods (CRMs) are well established in epidemiologic surveillance and considered useful for the task of correcting for case-finding limitations in multiple sclerosis (MS) prevalence surveys. To date, however, CRMs have been exclusively applied to crude prevalence figures. This study therefore sought to explore an age-specific application of this method to an urban Portuguese population of 229,342. METHODS: We used a CRM to correct for the age-specific prevalence of MS obtained from two data sources, i.e. general practitioners in three primary-care districts and a neurology unit at the referral hospital. The corrected figures were adjusted for age using the European standard population as reference. RESULTS: When applied to 95 MS patients, the CRM impact was highest at ages 50-59 years, with a 110% increase in cases where the corrected prevalence was highest, i.e. 181.8 (95% CI 75.7-287.9) per 100,000, and lowest, nil, at ages ≥70 years, with an unchanged corrected prevalence of 13.8. The crude prevalence of 41.4 per 100,000 increased by 36% to 56.20 per 100,000 when it was CRM- and age-adjusted. Source independence was poor. CONCLUSIONS: CRMs can be differentially applied to MS counts. Valid comparisons may require simultaneous adjustment for age and other variables, such as diagnostic delay and diagnostic criteria. CRM applications to crude figures and dependent sources should be approached with caution.

Occurrence of Volcanogenic Inorganic Mercury in Wild Mice Spinal Cord: Potential Health Implications.

Mercury accumulation has been proposed as a toxic factor that causes neurodegenerative diseases. However, the hazardous health effects of gaseous elemental mercury exposure on the spinal cord in volcanic areas have not been reported previously in the literature. To evaluate the presence of volcanogenic inorganic mercury in the spinal cord, a study was carried out in São Miguel island (Azores, Portugal) by comparing the spinal cord of mice exposed chronically to an active volcanic environment (Furnas village) with individuals not exposed (Rabo de Peixe village), through the autometallographic silver enhancement histochemical method. Moreover, a morphometric and quantification analysis of the axons was carried out. Results exhibited mercury deposits at the lumbar level of the spinal cord in the specimens captured at the site with volcanic activity (Furnas village). A decrease in axon calibre and axonal atrophy was also observed in these specimens. Given that these are relevant hallmarks in the neurodegenerative pathologies, our results highlight the importance of the surveillance of the health of populations chronically exposed to active volcanic environments.

Response to creatine analogs in fibroblasts and patients with creatine transporter deficiency.

Creatine transporter (CRTR) deficiency is one of the most frequent causes of X-linked mental retardation. The lack of an effective treatment for this disease, in contrast to creatine (Cr) biosynthesis disorders that respond to Cr monohydrate (CM), led us to analyze the efficacy of a lipophilic molecule derived from Cr, creatine ethyl ester (CEE), in fibroblasts and patients with CRTR deficiency. CM and CEE uptake studies were performed in six controls and four fibroblast cell lines from patients. We found a significant increase in Cr uptake after 72 h of incubation with CEE (500 micromol/L) in patients and control fibroblasts compared to incubation with CM. Subsequently, we assayed the clinical effect of CEE administration in four patients with CRTR deficiency. After 1 year of treatment, a lack of significant improvement in neuropsychological assessment or changes in Cr level in brain (1)H MRS was observed, and CEE was discontinued. In conclusion, this 12-month trial with CEE did not increase the brain concentration of Cr. Our in vitro data lend support to the idea of a certain passive transport of CEE in both pathological and control cells, although more lipophilic molecules or other cell systems that mimic the BBB should be used for a better approach to the in vivo system.

Creatine transporter deficiency in two adult patients with static encephalopathy.

Creatine transporter deficiency is a recently identified X-linked inborn error of metabolism. The natural course of the disease is not well delineated since clinical data from adult patients have scarcely been reported. A progressive course of the disease has been noted in a few described cases. We report the first two Spanish adult patients with creatine transporter deficiency and compare their clinical phenotype and the evolution of the disease with those of other published cases. The two brothers were identified in a study of a cohort of 610 mentally handicapped male patients. The disease was detected by biochemical studies and confirmed by DNA studies. The most significant clinical features were mental retardation, epilepsy and autistic behaviour, and these symptoms did not worsen, in contrast to other reports. They did not present gastrointestinal problems or movement disorders. Creatine transporter deficiency could be an underdiagnosed metabolic disorder and should be considered in adult patients with mental retardation. Clinical presentation of this disorder showed marked differences among adult patients and the course of the disease was static in our cases. Detection of additional adult patients might allow better understanding of the phenotypic outcome at a later age.

[Epidemiology of multiple sclerosis in Spain]. / Epidemiologia de la esclerosis multiple en España.

INTRODUCTION: Multiple sclerosis is a chronic autoimmune, inflammatory and neurodegenerative disease of the central nervous system and the most common non-traumatic disabling neurological disease in young adults. In the latest decades, multiple sclerosis is increasing worldwide, especially in women. The latitudinal distribution has been progressively attenuated. AIM: To review the epidemiological studies of multiple sclerosis in Spain to verify if this worldwide trend also occurs in Spain. DEVELOPMENT: We searched PubMed and Teseo databases using the search terms «epidemiology¼, «prevalence¼, «incidence¼, «multiple sclerosis¼ and «Spain¼. We selected articles published in Spanish and English between 1968 and 2018. CONCLUSIONS: Recent epidemiological studies confirm that Spain is a medium-high risk area for MS. The prevalence of MS has increased significantly throughout Spain in the latest years, especially in women, and recent studies show prevalence as high as 80-180 cases per 100,000.

TITLE: Epidemiologia de la esclerosis multiple en España.Introduccion. La esclerosis multiple es una enfermedad cronica autoinmune, inflamatoria y degenerativa del sistema nervioso central, y es el trastorno neurologico discapacitante no traumatico mas comun en adultos jovenes. Los estudios de prevalencia mas recientes indican que la frecuencia de la enfermedad ha aumentado en el mundo en las ultimas decadas, que dicho incremento de la prevalencia ocurre fundamentalmente a expensas de un mayor numero de casos de mujeres con formas remitentes, y que el gradiente latitudinal de la incidencia de la enfermedad se viene atenuando. Objetivo. Revisar los estudios sobre epidemiologia de esclerosis multiple en España para verificar si las tendencias mundiales se confirman en nuestro pais. Desarrollo. Busqueda bibliografica en las bases de datos PubMed y Teseo usando como palabras clave «epidemiology¼, «prevalence¼ e «incidence¼, cruzandolas con los terminos «multiple sclerosis¼ y «Spain¼; se realiza una seleccion inicial por titulo y resumen, en castellano e ingles, entre los años 1968 y 2018. Conclusiones. Un buen numero de estudios epidemiologicos recientes en España confirman que es una region de prevalencia media-alta de la enfermedad a lo largo de su geografia. Las cifras de prevalencia aumentan progresivamente a lo largo de las ultimas decadas hasta alcanzar en la actualidad 80-180 casos por 100.000 habitantes, y ello ha ocurrido a expensas de una mayor frecuencia de la enfermedad en las mujeres.

Arginine supplementation in four patients with X-linked creatine transporter defect.

BACKGROUND: Treatment with oral creatine monohydrate has not shown efficacy in patients with creatine transporter deficiency (CRTR-D). Another therapeutic option proposed is L-arginine, the substrate for the enzyme L-arginine:glycine amidinotransferase (AGAT). We evaluate clinical characteristics and cerebral creatine replenishment after L-arginine therapy in four patients with CRTR-D. PATIENTS AND METHODS: Four boys with genetically confirmed diagnosis of CRTR-D (ages 9-16 years) were supplemented with L-arginine (0.4 g/kg per day) for a period of 9 months. Treatment efficacy was evaluated by clinical and neuropsychological assessment and determination of creatine signals by brain proton magnetic resonance spectroscopy ((1)H-MRS). RESULTS: Epileptic seizures remained well controlled with antiepileptic drugs in three cases, both before and after L-arginine supplementation. Vineland Adaptive Behaviour Scale did not show any change in communication, daily living skills, socialization or motor skills, and a lack of improvement in brain (1)H-MRS follow-up was observed. L-Arginine was discontinued at the end of the observation period. CONCLUSIONS: Nine months of L-arginine supplementation did not show effectiveness in the four patients affected with CRTR-D in this protocol.