Inmunosenescencia: el rol de la edad en la esclerosis múltiple / Immunosenescence: the role of age in multiple sclerosis

Introducción: Junto con el envejecimiento de la población general, la prevalencia de ancianos con esclerosis múltiple (EM) se encuentra en aumento. El sistema inmunológico sufre profundos cambios a lo largo de la vida, por lo que parece imprescindible conocer qué diferencias presentan respecto a pacientes más jóvenes. Desarrollo: La inmunosenescencia, definida como la alteración del sistema inmunológico en relación con el envejecimiento natural, juega un papel esencial en la tolerancia, efectos adversos y respuesta a los tratamientos modificadores de la enfermedad. Entre las principales características de este fenómeno, la involución que sufre el timo es la más destacable. Este hecho genera una reducción en la producción de células T vírgenes. Además, se observa una ratio de linfocitos CD4 + /CD8 + invertido, alteraciones severas en el funcionamiento de las células NK o una disminución en la capacidad de reparación tisular cerebral. Conclusiones: El número de personas de edad avanzada con EM se encuentra en aumento en coincidencia con el envejecimiento de la población general y gracias al avance en los tratamientos modificadores de la enfermedad, así como a la mejora en la asistencia sociosanitaria de estos pacientes. El envejecimiento del sistema inmunitario conlleva un mayor riesgo de infecciones, tumores y enfermedades autoinmunes en los ancianos. En la EM, además, tiene lugar una aceleración en la neurodegeneración por la pérdida de capacidad de remielinización del sistema nervioso. Conocer los cambios que tienen lugar en el sistema inmunológico de la población de edad avanzada es esencial para mejorar la atención de este grupo de pacientes cada vez más prevalente.(AU)

Introduction: The number of elderly people with multiple sclerosis (MS) has increased in line with population ageing. As the immune system presents profound changes over an individual's lifetime, it is important to understand the differences between these patients and younger patients. Development: Immunosenescence, defined as age-related alterations naturally occurring in the immune system, particularly influences tolerance, response, and adverse effects of disease-modifying treatments for MS. Thymic involution is the most noteworthy characteristic of this phenomenon. This process leads to a reduction in the number of virgin T cells. Other effects include an inverted CD4 + /CD8 + cell ratio, severe alterations in NK cell functioning, and reduced tissue repair capacity in the brain. Conclusions: The number of older people with MS is increasing due to population ageing, advances in disease-modifying treatments, and improved health and social care of these patients. Ageing of the immune system increases the risk of infections, tumours, and autoimmune diseases in elderly individuals. Furthermore, neurodegeneration is accelerated in patients with MS due to the nervous system's loss of remyelination capacity. Understanding of the changes affecting the immune system in the elderly population is essential to improving the care provided to this ever-growing patient group.(AU)

Immunosenescence: the role of age in multiple sclerosis.

INTRODUCTION: The number of elderly people with multiple sclerosis (MS) has increased in line with population ageing. As the immune system presents profound changes over an individual's lifetime, it is important to understand the differences between these patients and younger patients. DEVELOPMENT: Immunosenescence, defined as age-related alterations naturally occurring in the immune system, particularly influences tolerance, response, and adverse effects of disease-modifying treatments for MS. Thymic involution is the most noteworthy characteristic of this phenomenon. This process leads to a reduction in the number of virgin T cells. Other effects include an inverted CD4+/CD8+ cell ratio, severe alterations in NK cell functioning, and reduced tissue repair capacity in the brain. CONCLUSIONS: The number of older people with MS is increasing due to population ageing, advances in disease-modifying treatments, and improved health and social care of these patients. Ageing of the immune system increases the risk of infections, tumours, and autoimmune diseases in elderly individuals. Furthermore, neurodegeneration is accelerated in patients with MS due to the nervous system's loss of remyelination capacity. Understanding of the changes affecting the immune system in the elderly population is essential to improving the care provided to this ever-growing patient group.

Oral and general health conditions involved in periodontal status during pregnancy: a prospective cohort study.

PURPOSE: Pregnancy is a period in a woman's life that has important consequences on oral health, particularly for gingival health. Present study aims to identify women at higher risk of developing periodontal disease (gingivitis and periodontitis) during late pregnancy and evaluate how this condition evolves during this period. METHODS: Prospective cohort study was designed with pregnant women who were assessed during the first and third trimesters of gestation in a southern Spanish public hospital. Data regarding gingival and periodontal health, oral hygiene, and overall health status (obesity and diabetes mellitus) were collected. Reporting followed STROBE checklist. RESULTS: Significantly higher number of women had the periodontal and gingival disease in the third trimester of gestation compared with in early pregnancy. In the third trimester of gestation, 42 (28.6%) and 63 (42.9%) of women presented symptoms of periodontal disease and gingival disease, respectively. Obesity (OR 2.834; 95%CI 0.919-8.741), worse oral hygiene during the first trimester of gestation (OR: 4.031; 95%CI 2.12-7.65), and periodontal disease during early pregnancy (OR: 15.104; 95%CI 3.60-63.36) most effectively predicted periodontal disease during late pregnancy. CONCLUSIONS: Pregnancy is associated with exacerbated periodontal and gingival disease symptoms throughout the different trimesters of gestation. Obesity and oral hygiene during early pregnancy were the risk factors that most contributed to the aforementioned changes in periodontal disease.

Involvement of progesterone and estrogen receptors in the ram sperm acrosome reaction.

The steroid hormones 17-ß estradiol (E2) and progesterone (P4) can regulate capacitation, hyperactive motility, and the acrosome reaction (AR) during the sperm transit through the female tract. Moreover, exogenous P4 and E2 can induce the AR in ovine spermatozoa, and progesterone receptor (PR) and estrogen receptors (ERα and ERß) are present in these cells. Thus, to investigate whether the effects both steroid hormones in ram sperm capacitation and AR are receptor-mediated, we incubated them with receptor agonists (tanaproget 1 µM and 5 µM for PR or resveratrol 5 µM and 10 µM for ER) or antagonists (mifepristone 4 µM and 40 µM for PR or tamoxifen 5 µM and 10 µM for ER) in capacitating conditions. The addition of receptor modulators did not affect sperm viability or total motility, although changes in progressive motility were detected. The incubation with both receptor agonists increased the percentage of acrosome-reacted spermatozoa, evaluated by chlortetracycline staining, when compared with the capacitated nontreated sample (Cap-C, P < 0.001). Moreover, the ER agonist resveratrol 10 µM provoked a greater AR than E2 (P < 0.01). Furthermore, the incubation with the receptor antagonists prevented the induction of the AR by P4 or E2, as the antagonists-treated spermatozoa presented a similar CTC pattern to that of Cap-C. In conclusion, these results confirm that P4 and E2 can induce the AR in ram spermatozoa and that this effect is receptor-mediated.

Immunosenescence: the role of age in multiple sclerosis. / Inmunosenescencia: el rol de la edad en la esclerosis múltiple.

INTRODUCTION: The number of elderly people with multiple sclerosis (MS) has increased in line with population ageing. As the immune system presents profound changes over an individual's lifetime, it is important to understand the differences between these patients and younger patients. DEVELOPMENT: Immunosenescence, defined as age-related alterations naturally occurring in the immune system, particularly influences tolerance, response, and adverse effects of disease-modifying treatments for MS. Thymic involution is the most noteworthy characteristic of this phenomenon. This process leads to a reduction in the number of virgin T cells. Other effects include an inverted CD4 + /CD8 + cell ratio, severe alterations in NK cell functioning, and reduced tissue repair capacity in the brain. CONCLUSIONS: The number of older people with MS is increasing due to population ageing, advances in disease-modifying treatments, and improved health and social care of these patients. Ageing of the immune system increases the risk of infections, tumours, and autoimmune diseases in elderly individuals. Furthermore, neurodegeneration is accelerated in patients with MS due to the nervous system's loss of remyelination capacity. Understanding of the changes affecting the immune system in the elderly population is essential to improving the care provided to this ever-growing patient group.

Underlying molecular mechanism in the modulation of the ram sperm acrosome reaction by progesterone and 17ß-estradiol.

Steroid hormones progesterone (P4) and 17ß-estradiol (E2) not only have important functions in regulation of reproductive processes in mammals but also have direct effects on spermatozoa. There can be induction of the acrosome reaction in ram spermatozoa by P4 and E2 and, in the present study, there was further investigation of mechanisms underlying this effect. In a medium containing agents that increase cAMP, the presence of both P4 and E2 led to changes in the localization of proteins phosphorylated in tyrosine residues evaluated by indirect immunofluorescence. The inclusion of P4 at 1 µM in the media induced an increase in Ca2+i and mobilization in the area of the acrosome (Fluo-4 and Rhod-5 staining, respectively), an increase in ROS (H2DCFDA staining) and a substantial disruption of the acrosome (evaluated using RCA), while E2 did not have these effects. There were no effects on cAMP concentrations or PKA activity with inclusion of these hormones in the media. The inclusion of P4 at 100 pM in the media led to changes in values for sperm kinematic variables which could indicate there was an inhibition of the hyperactivation caused by agents that induce an increase in cAMP concentrations. In conclusion, results from the present study indicate that P4 and E2 promote mechanisms regulating the acrosome reaction in ram spermatozoa, however, these effects on mechanisms are different for the two hormones, and for E2, require further clarification.

Vitamin D, preeclampsia and prematurity: A systematic review and meta-analysis of observational and interventional studies.

BACKGROUND: Vitamin D has important functions outside of bone metabolism. Deficiency has been associated with several adverse outcomes during pregnancy such as preeclampsia and prematurity. There is an increasing body of literature on this topic with studies performed to date having produced contradictory results. OBJECTIVE: To synthesize the literature about vitamin D deficiency and its association with preeclampsia and prematurity in order to determine if maternal vitamin D insufficiency and/or deficiency during pregnancy is associated with the prevalence of preeclampsia and prematurity. DESIGN: A systematic review and meta-analysis of observational and interventional studies. METHODS: Two independent researchers reviewed the included studies according to PRISMA reporting guidelines. A protocol for this review was registered in PROSPERO with the registration number: "CRD42019136318". Three electronic databases (PubMed, ScienceDirect and Web of Science); were searched in order to identify eligible studies. Observational and interventional studies were selected which had been published in the last 6 years, and analysed the association between maternal vitamin D concentrations during pregnancy and the development of preeclampsia and/or preterm birth. Data were extracted and presented in tables and figures. Fixed and random-effects meta-analyses were performed on the studies which provided enough sample data to calculate odds ratios. Results from both statistical methods were compared. Meta-analysis cut-off points for vitamin D insufficiency and deficiency were defined as <75nmol/L and <50nmol/L, respectively. RESULTS: Fifty-five studies met the inclusion criteria. Fixed-effects meta-analysis of the interventional studies indicated that vitamin D supplementation acts as a prevention factor for preeclampsia and prematurity. Fixed-effects meta-analysis of observational studies concluded that vitamin D insufficiency and deficiency are associated with a higher risk of developing preeclampsia. However, prematurity and vitamin D were only associated when maternal vitamin D concentrations was <75 nmol/L. Random-effects meta-analysis found no significant association between vitamin D, preeclampsia and prematurity in either observational or interventional studies. CONCLUSION: Higher vitamin D concentrations during pregnancy could be associated with a decreased risk of preeclampsia and prematurity but statistical significance of associations depends on the study design used. Well-designed clinical trials with vitamin D supplementation are needed in order to better define associations.

Micro ad-hoc Health Social Networks (uHSN). Design and evaluation of a social-based solution for patient support.

OBJECTIVE: To contribute the design, development, and assessment of a new concept: Micro ad hoc Health Social Networks (uHSN), to create a social-based solution for supporting patients with chronic disease. DESIGN: After in-depth fieldwork and intensive co-design over a 4-year project following Community-Based Participatory Research (CBPR), this paper contributes a new paradigm of uHSN, defining two interaction areas (the "backstage", the sphere invisible to the final user, where processes that build services take place; and the "onstage", the visible part that includes the patients and relatives), and describes a new transversal concept, i.e., "network spaces segments," to provide timely interaction among all involved profiles and guaranteeing qualitative relationships. This proposal is applicable to any service design project and to all types of work areas; in the present work, it served as a social-based solution for supporting patients with chronic disease in two real-life health scenarios: a Parkinson disease patient association and a Stroke rehabilitation service in a hospital. These two scenarios included the following main features: thematic (related to the specific disease), private, and secure (only for the patient, relatives, healthcare professional, therapist, carer), with defined specific objectives (around patient support), small size (from tens to hundreds of users), ability to integrate innovative services (e.g., connection to hospital information service or to health sensors), supported by local therapeutic associations, and clustered with preconfigured relationships among users based in network groups. MEASUREMENTS: Using a mixed qualitative and quantitative approach for 6 months, the performance of the uHSN was assessed in the two environments: a hospital rehabilitation unit working with Stroke patients, and a Parkinson disease association providing physiotherapy, occupational therapy, psychological support, speech therapy, and social services. We describe the proposed methods for evaluating the uHSN quantitatively and qualitatively, and how the scientific community can replicate and/or integrate this contribution in its research. RESULTS: The uHSN overcomes the main limitations of traditional HSNs in the main areas recommended in the literature: privacy, security, transparency, system ecology, Quality of Service (QoS), and technology enhancement. The qualitative and quantitative research demonstrated its viability and replicability in four key points: user acceptance, productivity improvement, QoS enhancement, and fostering of social relations. It also meets the expectation of connecting health and social worlds, supporting distance rehabilitation, improving professionals' efficiency, expanding users' social capital, improving information quality and immediacy, and enhancing perceived peer/social/emotional support. The scientific contributions of the present paper are the first step not only in customizing health solutions that empower patients, their families, and healthcare professionals, but also in transferring this new paradigm to other scientific, professional, and social environments to create new opportunities.

Modificaciones epigenéticas en neurología: alteraciones en la metilación del ADN en la esclerosis múltiple / Epigenetic changes in neurology: DNA methylation in multiple sclerosis

Introducción: La epigenética se define como el estudio de los mecanismos que regulan la expresión génica sin modificar la secuencia de ADN, siendo entre ellos el más conocido la metilación del ADN. La esclerosis múltiple (EM) es una enfermedad de etiología no del todo conocida, en la que se plantea que la participación de factores ambientales sobre individuos con una determinada predisposición genética, pueden resultar claves para el desarrollo de la enfermedad. Es en esta intersección entre la predisposición genética y los factores ambientales donde la metilación del ADN puede desempeñar un papel patogénico. Desarrollo: Realizamos una revisión bibliográfica de los efectos que los factores de riesgo ambiental para el desarrollo de EM pueden ejercer sobre los distintos mecanismos epigenéticos, así como la implicación que presentan dichas modificaciones en el desarrollo de la enfermedad. Conclusión: El conocimiento de las modificaciones epigenéticas involucradas en la patogenia de la EM abre una nueva vía de investigación para la identificación de potenciales biomarcadores, así como para la búsqueda de nuevas dianas terapéuticas (AU)

Introduction: Epigenetics is defined as the study of the mechanisms that regulate gene expression without altering the underlying DNA sequence. The best known is DNA methylation. Multiple Sclerosis (MS) is a disease with no entirely known etiology, in which it is stated that the involvement of environmental factors on people with a genetic predisposition, may be key to the development of the disease. It is at this intersection between genetic predisposition and environmental factors where DNA methylation may play a pathogenic role. Development: A literature review of the effects of environmental risk factors for the development of MS can have on the different epigenetic mechanisms as well as the implication that such changes have on the development of the disease. Conclusion: Knowledge of epigenetic modifications involved in the pathogenesis of MS, opens a new avenue of research for identification of potential biomarkers, as well as finding new therapeutic targets (AU)

Steroid hormone receptors and direct effects of steroid hormones on ram spermatozoa.

This study was based on the assumption that steroid hormones present in the female genital tract may have a rapid effect on ram spermatozoa by interaction with specific surface receptors. We demonstrate the presence of progesterone (PR) and estrogen (ER) receptors in ram spermatozoa, their localization changes during in vitro capacitation and the actions of progesterone (P4) and 17ß-estradiol (E2) on ram sperm functionality. Immunolocalization assays revealed the presence of PR mainly at the equatorial region of ram spermatozoa. Western blot analyses showed three bands in ram sperm protein extracts of 40-45 kDa, compatible with those reported for PR in the human sperm membrane, and both classical estrogen receptors (66 kDa, ERα and 55 kDa, ERß). ERα was located in the postacrosomal region of all the spermatozoa and ERß on the apical region of 63.7% of the cells. The presence of ERß was correlated with the percentage of non-capacitated spermatozoa evaluated by chlortetracycline staining (R = 0.848, P < 0.001). This significantly decreased after in vitro capacitation and nearly disappeared when acrosome reaction was induced. The addition of P4 and E2 before in vitro capacitation resulted in a higher (P < 0.001) acrosome-reacted sperm rate compared with the control (13.0%), noticeably greater after 3 h and when added to a high-cAMP medium (37.3% and 47.0% with E2 and P4, respectively). In conclusion, the results of this study demonstrate for the first time that ovine spermatozoa have progesterone and estrogen receptors and that both steroid hormones are related with the induction of the acrosome reaction.

Profile and reproductive roles of seminal plasma melatonin of boar ejaculates used in artificial insemination programs.

Melatonin (MLT) is present in seminal plasma (SP) of mammalian species, including pigs, and it is credited with antioxidant properties. This study aims to identify the sources of variation and the role of boar SP MLT on sperm quality and functionality and in vivo fertilizing ability of liquid-stored semen doses used in AI programs. The SP MLT was measured using an ELISA kit in a total of 219 ejaculates collected from 76 boars, and reproductive records of 5,318 AI sows were recorded. Sperm quality was assessed according to motility (computer-aided sperm analysis) and viability (cytometry evaluation). Sperm functionality was assessed according to the cytometric determination of intracellular HO generation, total and mitochondrial O production, and lipid peroxidation in liquid AI semen samples stored at 17°C over 144 h. The concentration of SP MLT differed among seasons ( < 0.01) and day length periods ( < 0.001) of the year, demonstrating that the ejaculates collected during the increasing day length period (9.80 ± 1.38 pg/mL, range: 2.75-21.94) had lower SP MLT concentrations than those collected during the decreasing day length period (16.32 ± 1.67 pg/mL, range: 5.02-35.61). The SP MLT also differed ( < 0.001) among boars, among ejaculates within boar, and among portions within the ejaculate, demonstrating that SP from the first 10 mL of sperm-rich ejaculate fraction (SRF) exhibited lower MLT concentrations than post-SRF. The SP MLT was negatively related ( < 0.001) to mitochondrial O production in viable sperm. The SP MLT did not differ among AI boars ( = 14) hierarchically grouped according to high and low fertility outcomes. In conclusion, SP MLT concentration in AI boars varies depending on the season of ejaculate collection and differs among boars, ejaculates within boar, and portions within ejaculate. The SP MLT may act at the mitochondrial level of sperm by reducing the generation of O. However, this antioxidant role of SP MLT was not reflected in sperm quality or in vivo fertility outcomes of AI semen doses.

Epigenetic changes in neurology: DNA methylation in multiple sclerosis. / Modificaciones epigenéticas en neurología: alteraciones en la metilación del ADN en la esclerosis múltiple.

INTRODUCTION: Epigenetics is defined as the study of the mechanisms that regulate gene expression without altering the underlying DNA sequence. The best known is DNA methylation. Multiple Sclerosis (MS) is a disease with no entirely known etiology, in which it is stated that the involvement of environmental factors on people with a genetic predisposition, may be key to the development of the disease. It is at this intersection between genetic predisposition and environmental factors where DNA methylation may play a pathogenic role. DEVELOPMENT: A literature review of the effects of environmental risk factors for the development of MS can have on the different epigenetic mechanisms as well as the implication that such changes have on the development of the disease. CONCLUSION: Knowledge of epigenetic modifications involved in the pathogenesis of MS, opens a new avenue of research for identification of potential biomarkers, as well as finding new therapeutic targets.

Repeated sugammadex reversal of muscle relaxation during lumbar spine surgery with intraoperative neurophysiological multimodal monitoring / Reversión repetida del bloqueo neuromuscular con sugammadex en cirugía del raquis lumbar, con monitorización neurofisiológica multimodal intraoperatoria

Intraoperative neurophysiological monitoring during spine surgery is usually acomplished avoiding muscle relaxants. A case of intraoperative sugammadex partial reversal of the neuromuscular blockade allowing adequate monitoring during spine surgery is presented. A 38 year-old man was scheduled for discectomy and vertebral arthrodesis throughout anterior and posterior approaches. Anesthesia consisted of total intravenous anesthesia plus rocuronium. Intraoperatively monitoring was needed, and the muscle relaxant reverted twice with low dose sugammadex in order to obtain adequate responses. The doses of sugammadex used were conservatively selected (0.1mg/kg boluses increases, total dose needed 0.4mg/kg). Both motor evoqued potentials, and electromyographic responses were deemed adequate by the neurophysiologist. If muscle relaxation was needed in the context described, this approach could be useful to prevent neurological sequelae. This is the first study using very low dose sugammadex to reverse rocuronium intraoperatively and to re-establish the neuromuscular blockade (AU)

La monitorización neurofisiológica intraoperatoria en cirugía de raquis se suele llevar a cabo evitando los bloqueantes neuromusculares. Presentamos el caso de un paciente en el que se empleó reversión parcial del bloqueo neuromuscular que permitió la monitorización intraoperatoria con respuestas adecuadas. Un paciente varón de 38 años fue sometido a discectomía y artrodesis mediante abordajes consecutivos anterior y posterior. Se procedió con anestesia intravenosa y rocuronio como bloqueante neuromuscular. Se precisó monitorización intraoperatoria y el bloqueante neuromuscular fue revertido por 2 veces con dosis bajas de sugammadex para obtener las respuestas adecuadas. Las dosis de sugammadex usadas fueron seleccionadas de forma conservadora (bolos incrementales de 0,1mg/kg, dosis total requerida 0,4mg/kg). Tanto los potenciales evocados motores como las respuestas electromiográficas fueron juzgadas adecuadas por el neurofisiólogo. Si se precisa relajación muscular en el contexto descrito, esta aproximación podría ser útil para contribuir a la prevención de secuelas neurológicas. Este es el primer estudio en el que se emplean dosis muy bajas de sugammadex intraoperatoriamente para revertir rocuronio y restablecer, consecutivamente, el bloqueo neuromuscular (AU)

Repeated sugammadex reversal of muscle relaxation during lumbar spine surgery with intraoperative neurophysiological multimodal monitoring.

Intraoperative neurophysiological monitoring during spine surgery is usually acomplished avoiding muscle relaxants. A case of intraoperative sugammadex partial reversal of the neuromuscular blockade allowing adequate monitoring during spine surgery is presented. A 38 year-old man was scheduled for discectomy and vertebral arthrodesis throughout anterior and posterior approaches. Anesthesia consisted of total intravenous anesthesia plus rocuronium. Intraoperatively monitoring was needed, and the muscle relaxant reverted twice with low dose sugammadex in order to obtain adequate responses. The doses of sugammadex used were conservatively selected (0.1mg/kg boluses increases, total dose needed 0.4mg/kg). Both motor evoqued potentials, and electromyographic responses were deemed adequate by the neurophysiologist. If muscle relaxation was needed in the context described, this approach could be useful to prevent neurological sequelae. This is the first study using very low dose sugammadex to reverse rocuronium intraoperatively and to re-establish the neuromuscular blockade.

Expression, cellular localization, and involvement of the pentose phosphate pathway enzymes in the regulation of ram sperm capacitation.

Spermatozoa require substantially more ATP than other cells, not only for sustaining sperm motility but also for regulating protein phosphorylation during capacitation. In this study, we have reported for the first time the presence of the two key enzymes of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in ovine spermatozoa by indirect immunofluorescence, Western blotting, in-gel activity, and reverse transcription polymerase chain reaction analysis. We found that the activity of both enzymes significantly increased after in vitro capacitation in the presence of high-cAMP levels, with a concomitant increase in protein tyrosine phosphorylation and in the proportion of sperm-capacitated pattern assessed by the chlortetracycline staining. These results suggest that PPP is related with the progress of capacitation and that a relationship between calcium compartmentalization, protein tyrosine phosphorylation and PPP seems to exist. This is the first report that shows a connection between the PPP, cAMP/PKA signaling pathways and sperm capacitation. These findings can be of high-biological importance to improve our knowledge of the biochemical mechanisms involved in the acquisition of mammalian sperm functional competence and, ultimately, fertility.

Recurrent acute thermal lesion induces esophageal hyperproliferative premalignant lesions in mice esophagus.

Hot beverage consumption is a risk factor for esophageal squamous cell carcinoma, but the underlying mechanisms are still unknown. We developed an experimental mouse model to understand the mechanism of thermal lesion to esophageal carcinogenesis. Female BALB/c mice were treated by gavage with water at different temperatures three times a week and nitrosamines in the drinking water. Water at 70°C, but not at lower temperatures, initially induced an esophageal necrosis that healed and became resistant to necrosis after further administrations. However, when 70°C water was associated with N-nitrosodiethylamine at doses above 1 ppm, there was interference in epithelial regeneration, allowing recurrent thermal injury and inflammation. Recurrent thermal injury resulted in hyper proliferative premalignant lesions being induced earlier (at 4 weeks) and at a higher frequency (4-fold increase at 16 weeks) when compared to mice treated with NDEA only. Ki-67 immunostaining revealed that recurrent thermal injury induced basal cell proliferation resulting in the expansion of epithelial basal cells, confirmed by the increase in cytokeratin 14 positive cells with concomitant reduction of differentiated cytokeratin 5 positive cells. We conclude that recurrent thermal lesion may act as a tumor promoter though a strong proliferation stimulus of esophageal epithelial basal cells.

Evidence of melatonin synthesis in the ram reproductive tract.

Melatonin is a ubiquitous molecule found in a wide range of fluids, one of them being ram seminal plasma, in which it can reach higher concentrations than those found in blood, suggesting an extrapineal secretion by the reproductive tract. In order to identify the source of the melatonin found in ram seminal plasma, we first tried to determine whether the melatonin levels were maintained during the day. For this purpose, melatonin concentrations were measured in seminal plasma obtained from first ejaculates of six rams at 6:00 a.m. in total darkness, at 10:00 a.m. and at 14:00 p.m. The melatonin concentration was higher (p < 0.05) in ejaculates collected at 6:00 a.m. than at 10:00 and 14:00. There was no statistical difference between the latter. To further corroborate an extrapineal secretion of melatonin, the presence of the two key enzymes involved in melatonin synthesis, arylalkylamine-N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT) was analyzed by RT-PCR, q-PCR and Western-blot in ram testes, epididymis, and accessory glands. The RT-PCR showed the presence of the m-RNA codifying both AANAT and ASTM in all the tissues under study, but the q-PCR and Western-blot revealed that gene expression of these enzymes was significantly higher in the testis (p < 0.05). Immunohistochemistry confirmed the presence of AANAT and ASMT in the testis and revealed that they were found in the Leydig cells, spermatocytes, and spermatids. Also, measurable levels of melatonin were found in testicular tissue and the tail of the epididymis. In conclusion, our study indicates that the testes are one of the likely sources of the high levels of melatonin found in ram seminal plasma, at least during the day.

Incidence and risk conditions of ischemic stroke in older adults.

OBJECTIVE: The objective of this study was to investigate incidence and mortality from ischemic stroke in older adults with specific underlying chronic conditions, evaluating the influence of these conditions in developing stroke. MATERIALS & METHODS: Population-based cohort study involving 27,204 individuals ≥60 years old in Southern Catalonia, Spain. All cases of hospitalization from ischemic stroke (confirmed by neuro-imaging) were collected from 01/12/2008 until 30/11/2011. Incidence rates and 30-day mortality were estimated according to age, sex, chronic illnesses, and underlying conditions. Multivariable Cox regression analysis was used to calculate Hazards Ratio (HR) and estimate the association between baseline conditions and risk of developing stroke. RESULTS: Mean incidence rate reached 453 cases per 100,000 person-years. Maximum rates appeared among individuals with history of prior stroke (2926 per 100,000), atrial fibrillation (1815 per 100,000), coronary artery disease (1104 per 100,000), nursing-home residence (1014 per 100,000), and advanced age ≥80 years (1006 per 100,000). Thirty-day mortality was 13% overall, reaching 21% among patients over 80 years. Age [HR: 1.06; 95% confidence interval (CI): 1.04-1.07], history of prior stroke (HR: 5.08; 95% CI: 3.96-6.51), history of coronary artery disease (HR: 1.65; 95% CI: 1.21-2.25), atrial fibrillation (HR: 2.96; 95% CI: 2.30-3.81), diabetes mellitus (HR: 1.55; 95% CI: 1.23-1.95), and smoking (HR: 1.64; 95% CI: 1.15-2.34) emerged independently associated with an increased risk of ischemic stroke. CONCLUSION: Incidence and mortality from ischemic stroke remains considerable. Apart from age and history of atherosclerosis (prior stroke or coronary artery disease), atrial fibrillation, diabetes, and smoking were the underlying conditions most strongly associated with an increased risk.

Cleaved PARP-1, an Apoptotic Marker, can be Detected in Ram Spermatozoa.

The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly-ADP-ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly-ADP-ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 µm) or betulinic acid (200 µm). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p < 0.001), and a 12-h incubation increased cPARP similarly in both fractions (p < 0.001). cPARP seems to be an early marker of apoptosis in ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between-male differences on sperm fertility.

Intracellular calcium movements of boar spermatozoa during 'in vitro' capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model.

This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.