Administering copper reduces hyper-excitability generated by penicillin G in motor cortex neurons from rat brain slices.

Recording synaptic activity of layer III neurons from motor cortex slices, which was provoked by stimulating layer IV, generated synaptic responses of the field potential (FP) that went from mean 100 µV to 600 µV when the stimulus was increased up to twice the threshold. Administering 100 µM or 200 µM of copper, increased the responses to mean 800 µV and 820 µV, respectively. The response to 200 µM was not significantly greater than that to 100 µM. However, all FP responses were significantly lower to a copper concentration of 500 µM. The basal FP was slowly restored by removing the copper with Krebs-Ringer(K-R), resulting in similar characteristics to those observed before copper administration. Then, neurons were perfused with penicillin (2000 UI) to increase cortical excitability and to assess the depressing effect of high concentrations of copper. Administering 500 µM of copper significantly reduced the activity generated by penicillin, while removing by wash(K-R) penicillin and copper generated FP responses similar to those obtained at baseline. Our data indicate that depending on the concentration, copper can behave as an activating or blocking agent for cortical activity.

Myasthenia gravis: descriptive analysis of life-threatening events in a recent nationwide registry.

BACKGROUND AND PURPOSE: Myasthenia gravis (MG) may become life-threatening if patients have respiratory insufficiency or dysphagia. This study aimed to determine the incidence, demographic characteristics, risk factors, response to treatment and outcome of these life-threatening events (LTEs) in a recent, population-based sample of MG patients. METHODS: A retrospective analysis of MG patients who presented with an LTE between 2000 and 2013 was performed. Participants were identified from a neuromuscular diseases registry in Spain that includes 648 patients with MG (NMD-ES). RESULTS: Sixty-two (9.56%) patients had an LTE. Thirty-two were classified as class V according to the MG Foundation of America, and 30 as class IVB. Fifty per cent were previously diagnosed with MG and median duration of the disease before the LTE was 24 months (3-406). The most common related factor was infection (n = 18). All patients received intravenous human immunoglobulin; 11 had a second infusion and six had plasma exchange. Median time to feeding tube removal was 13 days (1-434). Median time to weaning from ventilation was 12 days (3-176), and it was significantly shorter in late onset MG (≥50 years) (P = 0.019). LTEs improved <2 weeks in 55.8% but did not improve until after 1 month in 20% of patients. Four patients died. No other factors influenced mortality or duration of LTEs. CONCLUSIONS: The percentage of LTEs in MG patients was low, particularly amongst those previously diagnosed and treated for the disease. The significant percentage of treatment-resistant LTEs indicates that more effective treatment approaches are needed for this vulnerable sub-population.

[Unicompartmental knee prosthesis]. / Prótesis unicompartimental de rodilla.

In a patient with severe unicompartmental knee osteoarthritis where conservative treatments have been exhausted, with painful symptoms located on the affected side and with a reducible axis, the unicompartmental knee prosthesis (UKP) is the first option for our work group. Within the study to confirm the diagnosis and plan the surgery, weight-bearing knee x-rays, Rosenberg x-rays, and teleradiographs of the lower extremities stand out. The objective of surgery is to replace the affected area, restoring the anatomy with an adequate balance of soft tissues. Regarding alignment, the challenge is not to overload the opposite side or that of the prosthesis. There are mobile and fixed plates and although the clinical and survival results are similar, in recent years with the incorporation of robotic surgery, the balance has tipped towards the use of fixed plates. The clinical and functional results are better and there are fewer complications than when total knee prostheses (TKP) are used in the same type of patients. The survival studied in registries is lower than for TKP, but when used in high-flow centers where the percentage of UKP is close to a third of the total with strict patient selection, the duration is as good as in PTR.

En un paciente con artrosis unicompartimental de rodilla grave, donde se agotaron los tratamientos conservadores que tiene clínica dolorosa localizada en el lado afectado y con alteración del eje reductible, la prótesis unicompartimenal de rodilla (PUR) es la primera opción para nuestro grupo de trabajo. Dentro del estudio para confirmar el diagnóstico y planificar la cirugía destacan las radiografías de rodillas con carga, radiografía de Rosenberg y telerradiografías de extremidades inferiores. El objetivo de la cirugía es reemplazar la zona afectada, restituyendo la anatomía con un adecuado balance de partes blandas. Respecto al alineamiento el desafío es no sobrecargar el lado contrario ni tampoco el de la prótesis. Existen de platillo móvil y fijo y aunque los resultados clínicos y de supervivencia son semejantes, en los últimos años con la incorporación de la cirugía robótica, la balanza se ha inclinado para el uso de los platillos fijos. En pacientes con artrosis unicompartimental los resultados clínicos y funcionales son mejores con PUR y tiene menos complicaciones que cuando se usan prótesis totales (PTR). La supervivencia estudiada en registros es menor que para PTR, pero cuando se usa en centros de alto flujo en que el porcentaje de PUR es cercano a un tercio del total con una estricta selección de pacientes, la duración es tan buena como en la PTR.

Dexamethasone modulates expression of genes involved in the innate immune system, growth and stress and increases susceptibility to bacterial disease in Senegalese sole (Solea senegalensis Kaup, 1858).

Cortisol, the main glucocorticoid in fish, undertakes pleiotropic biological effects in response to stressors to maintain homeostasis. It can exert several actions on the immune system, growth and cellular metabolism, establishing a fine-tune regulation stress response and cross-talk interactions with other regulatory pathways. In this study, we investigated a causal relationship between high levels of glucocorticoids and susceptibility to pathogens and modification of gene expression profiles in Senegalese sole. For this purpose, we carried out two experiments using post-metamorphic individuals (21 days after hatching) that were exposed to dexamethasone (DXM), a potent glucocorticoid, in order to mimic cortisol effects. We quantified transcript levels of a wide set of genes involved in innate immune system (g-type lysozyme and hepcidin (hamp1)), HPI axis (crf, crfbp, pomcα, pomcß, gr1 and gr2), HPT axis (tgb), cellular stress defense system (hsp70 and hsp90aa), GH/IGF axis (igf-I and igf-Ir) and the neuropeptide trh. Short-term exposure to 0.1, 1 and 10 ppm DXM provoked a reduction of pomcß transcripts and an increase of crfbp mRNAs in a dose-dependent manner at 48 and 72 h after treatment. Moreover, g-type lysozyme transcript levels decreased significantly at 72 h whereas hamp1 mRNA levels increased at 48 h after exposure. Long-term DXM treatment (10 ppm DXM) affected negatively weight of soles (~20% lower than controls). Moreover, reduced mRNA levels were observed for pomcß after 1 week and igf-I and hamp1 after 2 weeks. In contrast, crfbp and crf increased mRNA levels after 2 weeks. hsp70 exhibited a dual response increasing transcript levels at 1 week after treatment and reducing thereafter. No significant changes in gene expression were observed at any time during this study for tgb, trh, hsp90aa, pomcα, gr1 and gr2. Finally, a challenge experiment using the pathogen Photobacterium damselae subsp piscicida confirmed earlier and higher mortalities in DXM-treated animals. Taken together, these data indicate that a prolonged exposure to DXM increases the susceptibility to pathogens and reduces growth. Moreover, DXM can trigger a wide cellular response modulating the expression of genes involved in the innate immune system, HPI and GH/IGF axes as well as cellular stress defense. These results are highly valuable to evaluate responses associated to aquaculture stressful conditions and discriminate specific glucocorticoid-mediated effects.

A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

AIMS: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. METHODS AND RESULTS: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). CONCLUSIONS: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.

Detection of infectious pancreatic necrosis virus (IPNV) from asymptomatic redbanded seabream, Pagrus auriga Valenciennes, and common seabream, Pagrus pagrus (L.), using a non-destructive procedure.

A non-destructive procedure based on nested RT-PCR and dot-blot hybridization has been developed for the detection of asymptomatic IPNV-carrier fish. The pair of primers designed for RT-PCR amplified a 599-bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT-PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191-bp probe was designed for hybridization studies used in combination with the nested RT-PCR. The application of the nested RT-PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV-carriers, respectively. The combination of nested RT-PCR and dot-blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.

GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein.

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1, 979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

Spanish multidisciplinary clinical practice guidelines for Anderson-Fabry Disease in Adults. I. Method and recommendations. / Guía de práctica clínica multidisciplinar española sobre la enfermedad de Anderson-Fabry en adultos. I: Método y recomendaciones.

Anderson-Fabry disease is a severe progressive multisystem condition of genetic origin that affects men and women, reducing their life expectancy and quality of life. The considerable variability in its clinical expression, the difficulties in diagnosing the condition and the current availability of several alternatives for its treatment represent a considerable challenge that justifies the development of evidence-based clinical practice guidelines that can help health professionals in the decision-making process for managing these patients. To develop these guidelines, we conducted a systematic search of the main reference databases using strategies adapted to each of the 32 clinical questions considered. We prepared documents to synthesise the evidence and assess its quality for each of the questions. The methodology employed is based on the Spanish methodology manual for preparing clinical practice guidelines, incorporating the GRADE methodology in the assessment of the scientific evidence and the preparation of the recommendations, considering the quality of the evidence, the risk-benefit balance, patient values and preferences, equity and use of resources. For the definitive preparation of the recommendations, we conducted a structured consensus process based on the Delphi-RAND methodology in 2 rounds, with an expert panel proposed by various scientific societies, research centres and patient associations. Ultimately, we developed 92 specific recommendations for managing Fabry disease.

Prótesis unicompartimental de rodilla / Unicompartmental knee prosthesis

Resumen: En un paciente con artrosis unicompartimental de rodilla grave, donde se agotaron los tratamientos conservadores que tiene clínica dolorosa localizada en el lado afectado y con alteración del eje reductible, la prótesis unicompartimenal de rodilla (PUR) es la primera opción para nuestro grupo de trabajo. Dentro del estudio para confirmar el diagnóstico y planificar la cirugía destacan las radiografías de rodillas con carga, radiografía de Rosenberg y telerradiografías de extremidades inferiores. El objetivo de la cirugía es reemplazar la zona afectada, restituyendo la anatomía con un adecuado balance de partes blandas. Respecto al alineamiento el desafío es no sobrecargar el lado contrario ni tampoco el de la prótesis. Existen de platillo móvil y fijo y aunque los resultados clínicos y de supervivencia son semejantes, en los últimos años con la incorporación de la cirugía robótica, la balanza se ha inclinado para el uso de los platillos fijos. En pacientes con artrosis unicompartimental los resultados clínicos y funcionales son mejores con PUR y tiene menos complicaciones que cuando se usan prótesis totales (PTR). La supervivencia estudiada en registros es menor que para PTR, pero cuando se usa en centros de alto flujo en que el porcentaje de PUR es cercano a un tercio del total con una estricta selección de pacientes, la duración es tan buena como en la PTR.

Abstract: In a patient with severe unicompartmental knee osteoarthritis where conservative treatments have been exhausted, with painful symptoms located on the affected side and with a reducible axis, the unicompartmental knee prosthesis (UKP) is the first option for our work group. Within the study to confirm the diagnosis and plan the surgery, weight-bearing knee x-rays, Rosenberg x-rays, and teleradiographs of the lower extremities stand out. The objective of surgery is to replace the affected area, restoring the anatomy with an adequate balance of soft tissues. Regarding alignment, the challenge is not to overload the opposite side or that of the prosthesis. There are mobile and fixed plates and although the clinical and survival results are similar, in recent years with the incorporation of robotic surgery, the balance has tipped towards the use of fixed plates. The clinical and functional results are better and there are fewer complications than when total knee prostheses (TKP) are used in the same type of patients. The survival studied in registries is lower than for TKP, but when used in high-flow centers where the percentage of UKP is close to a third of the total with strict patient selection, the duration is as good as in PTR.

UHPLC-MS and MALDI-MS study of aluminum phthalocyanine chloride and development of a bioanalytical method for its quantification in nanoemulsions and biological matrices.

Metal phthalocyanines are promising components in photodynamic therapy. Aluminum phthalocyanine chloride (AlClPc) has been used to treat oral cancer in mice, human carious tissue, lung cancer cells and other conditions. To overcome the high hydrophobicity of AlClPc, phthalocyanine is often encapsulated in nanoformulations. Despite increased usage, little is known about the pharmacokinetics and biodistribution of AlClPc. The aim of this study was the development and validation of a UHPLC-MS method for the determination of AlClPc in solution after extraction from nanoformulations and biological matrices such as plasma and tissue. The described method has been assayed as to selectivity, linearity, limits of detection and quantification, precision and recovery. The present study is the first to describe the behavior of AlClPc in biological matrices with mass spectrometry as well as the first to describe the chromatographic behavior of AlClPc contaminants. Molecular mass analysis identified dechlorination of AlClPc by both LC/MS and MALDI-MS and an adduct formation in LC/MS. The parameters observed indicated that the method has applicability and robustness for use in biodistribution studies.

Viral infections as a cause of cancer (review).

In order to promote carcinogenesis multiple factors must be orchestrated. The alteration of the cellular genome after a carcinogenic exposure may result in malignancy if apoptosis is prevented and the immune surveillance fails to eliminate the transformed cell. Infectious agents may exert these properties and transform a host cell. Viruses associated with human cancer are known as 'tumor viruses'. Most of them are capable of integrating into the host genome and have the ability to immortalize the target cell in order to allow their own replication. The infected cell expresses the viral genes, which are able to induce cell growth, proliferation and prevent apoptosis. This review focuses on Epstein-Barr virus, human papilloma virus, hepatitis C virus, hepatitis B virus, human herpes virus 8 and human T-cell leukemia virus, since they have been already established as causative agents of human cancer. An understanding of the viral replication mechanism may provide new targets for the development of specified viral therapy that may have an impact not only on viral infections but in human cancer as well.

Interference of chronically ingested copper in long-term potentiation (LTP) of rat hippocampus.

The objective of our study was to find the evidence of copper interaction in LTP, motivated by copper involvement in neurodegenerative illness, like Parkinson, Alzheimer and Amyotrophic Lateral Sclerosis, and we initiated the study of this element in the LTP. For this purpose we used hippocampus slices of rats chronically consuming copper dissolved in water (CuDR; n=26) and non-copper-consuming rats (CR; n=20). The CuDR rats received 8--10 mg/day during 20--25 days. Electrophysiological tests showed absence of LTP in CuDR slices, contrary to CR slices. The stimulus-response test applied before and after LTP showed significant increases of synaptic potential in the CR group. This did not occur in the CuDR group, except for the initial values, which probably seem associated to an early action of copper. The paired-pulse (PP) test, applied to CR and CuDR prior to tetanic stimulation, showed a significant reduction in PP, for the 20-, 30- and 50-ms intervals in CuDR. At the end of the experiments, copper concentration was 54.2 times higher in CuDR slices, compared to the concentration present in CR slices. Our results show that copper reduces synaptic sensibility and also the facilitation capability. These effects represent a significant disturbance in the plasticity phenomenon associated with learning and memory.

Smoking during pregnancy and lactation and its effects on breast-milk volume.

The influence of cigarette smoking on daily breast-milk volume was measured by the dose-to-mother deuterium-dilution method in 10 smoking and 10 nonsmoking mothers. After administration of deuterium to the mother, breast milk and infant saliva were sampled over 14 d and analyzed by mass spectrometry. Nonsmoking mothers had a significantly greater breast-milk volume than did smokers [961 +/- 120 vs 693 +/- 110 g/d, mean +/- SD; t = 5.21, P less than 0.0001). Growth rates of the infants were also measured. Weight increase of infants of non-smoking mothers was 550 +/- 130 g whereas of infants of smoking mothers it was only 340 +/- 170 g (t = 3.11, P less than 0.01). These results indicate that cigarette smoking has a negative influence on breast-milk volume whereas the lower infant-growth rates of the smoking mothers suggest also that their breast-milk output was insufficient to support the energy requirements of their infants.

Repeated tail pinch leads to desensitization of postsynaptic alpha2-adrenoceptors which modulate the jaw-opening reflex in the rat.

There are few in vivo studies which have investigated the modulation of central postsynaptic alpha2-adrenoceptors functionality provoked by stress. We assessed in the rat the effects of either single or repeated tail pinch on clonidine-induced inhibition of the jaw-opening reflex (JOR) via activation of postsynaptic central alpha2-adrenoceptors. At the end of each experimental period, the progressive inhibition of the digastric electromyographic responses elicited by orofacial electrical stimulation after the i.v. administration of cumulative doses (x3.3) of clonidine (0.1-10000 microg/kg) was recorded. Single tail pinch did not significantly modify the ability of the agonist to inhibit the JOR, although there was a tendency to decrease the basal amplitude of the reflex (a 40% reduction) immediately after exposure to the single stressor. However, the dose-response curve for clonidine-induced inhibition of the JOR was clearly shifted to the right in rats exposed to repeated tail pinch (ED50 was increased by 152%, P < 0.0001) when compared with the unstressed control group, without affecting the slope of the inhibitory function and the estimated maximum effect for the agonist. These results show that repeated stress leads to a subsensitivity of the alpha2-adrenoceptors which modulate the JOR, suggesting the development of adaptive mechanisms in postsynaptic alpha2-adrenoceptors in response to stress.

Neurological and behavioral effects of a unilateral frontal cortical lesion in fetal kittens. II. Visual system tests, and proposing an "optimal developmental period" for lesion effects.

Nine fetal kittens sustained removal of the left frontal cortex during the last third of gestation (E 43-55) and were compared to animals sustaining a similar lesion postnatally (P 8-14) as well as to intact littermates. Beginning after 6 months of age, the animals' visual field and depth perception were assessed. In addition, pupil size as well as eye alignment were measured. On two visual field tests the fetal-lesioned cats showed test dependent decrements for some angles of vision. In terms of depth perception, only the prenatal-lesioned animals showed a higher binocular threshold; they also showed ocular misalignment which may have contributed to their depth perception impairment. Moreover, these animals had a larger ipsilateral pupil. The neonatal-lesioned animals were like normal cats for all tests and measurements. We conclude that, as for the tests reported in the preceding paper, the outcome for visual related behaviors of a prenatal frontal cortical lesion in the cat is also worse than that of a similar lesion sustained neonatally. Dysgenetic anatomical changes of the visual system induced indirectly by the frontal lesion are proposed as a possible explanation for these age-at-lesion differences. Based on the present work as well as on the literature, we propose the existence of an "optimal developmental period" for the best behavioral and anatomical outcome of perinatal brain lesions. We argue that this concept fits contemporary data and can better explain the different age-at-lesion effects of brain injury across animals species than the "Kennard Principle" (or "infant-lesion effect").

Simultaneous unitary neuronal activity in both superior colliculi and its relation to eye movements in the cat.

The analysis of simultaneous unitary neuronal activity related to eye movement and recorded in both superior colliculi has shown a mirror-functioning image. Increase of the frequency discharge in a collicular unit, was associated with a decrease of the frequency discharge in the contralateral superior colliculus unit. This unitary neuronal reciprocal behaviour was observed each time a horizontal or oblique eye movement was produced. It is possible that this reciprocal functioning between neurones in both superior colliculi could exert an important influence on oculomotor brainstem structures. These results give a better idea of the role played by both superior colliculi in the control of conjugate eye movements.

Study of the morphological, electrophysiological and behavioral effects of unilateral kainic acid injection into the cat's substantia nigra.

Morphological, electrophysiological and behavioral studies were carried out in cats after unilateral kainic acid injection in the substantia nigra. A forced contralateral head turning and compulsive circling was observed after surgery. Fifteen days after, when asymmetry disappeared, apomorphine induced an ipsilateral head and body turning, that was blocked by haloperidol. The percentage of turning, after electrical stimulation in the superior colliculus or pulvinar-lateralis posterior complex, was affected by substantia nigra lesion. This work demonstrates that the nigro-pulvinar-lateral posterior and the nigrotectal projection modulate the capability of electrical stimulation of the target structures to elicit turning, and after unilateral substantia nigra lesion, two opposite directions of asymmetry appear, which are time-dependent and modulated by different neurotransmitters.