Thiosulfinate-Enriched Allium sativum Extract Exhibits Differential Effects between Healthy and Sepsis Patients: The Implication of HIF-1α.

Garlic (Allium sativum) has historically been associated with antioxidant, immunomodulatory, and microbiocidal properties, mainly due to its richness in thiosulfates and sulfur-containing phytoconstituents. Sepsis patients could benefit from these properties because it involves both inflammatory and refractory processes. We evaluated the effects of thiosulfinate-enriched Allium sativum extract (TASE) on the immune response to bacterial lipopolysaccharide (LPS) by monocytes from healthy volunteers (HVs) and patients with sepsis. We also explored the TASE effects in HIF-1α, described as the key transcription factor leading to endotoxin tolerance in sepsis monocytes through IRAK-M expression. Our results showed TASE reduced the LPS-triggered reactive oxygen species (ROS) production in monocytes from both patients with sepsis and HVs. Moreover, this extract significantly reduced tumor necrosis factor (TNF)-α, interleukin-1ß, and interleukin-6 production in LPS-stimulated monocytes from HVs. However, TASE enhanced the inflammatory response in monocytes from patients with sepsis along with increased expression of human leukocyte antigen-DR. Curiously, these dual effects of TASE on immune response were also found when the HV cohort was divided into low- and high-LPS responders. Although TASE enhanced TNFα production in the LPS-low responders, it decreased the inflammatory response in the LPS-high responders. Furthermore, TASE decreased the HIF-1α pathway-associated genes IRAK-M, VEGFA and PD-L1 in sepsis cells, suggesting HIF-1α inhibition by TASE leads to higher cytokine production in these cells as a consequence of IRAK-M downregulation. The suppression of this pathway by TASE was confirmed in vitro with the prolyl hydroxylase inhibitor dimethyloxalylglycine. Our data revealed TASE's dual effect on monocyte response according to status/phenotype and suggested the HIF-1α suppression as the possible underlying mechanism.

Black Garlic and Thiosulfinate-Enriched Extracts as Adjuvants to Ceftriaxone Treatment in a Rat Peritonitis Model of Sepsis.

To date, there have been no new drugs or adjuvants able to decrease both morbidity and mortality in the context of sepsis and septic shock. Our objective was to evaluate the use of thiosulfinate-enriched Allium sativum and black garlic extracts as adjuvants in the management of sepsis. An experimental in vivo study was carried out with male Sprague-Dawley® rats. Animals were randomized in four treatment groups: antibiotic (ceftriaxone) treatment (group I), ceftriaxone plus thiosulfinate-enriched extract (TASE, group II), ceftriaxone plus thiosulfinate-enriched extract and black garlic extracts (TASE + BGE, group III), and ceftriaxone plus black garlic extract (BGE, group IV). All animals were housed and inoculated with 1 × 1010 CFU/15 mL of intraperitoneal Escherichia coli ATCC 25922. Subsequently, they received a daily treatment according to each group for 7 days. Clinical, analytical, microbiological, and histopathological parameters were evaluated. Statistically significant clinical improvement was observed in rats receiving garlic extracts in weight (groups II and III), ocular secretions, and piloerection (group IV). Moreover, less liver edema, vacuolization, and inflammation were observed in groups receiving adjuvant support (groups II, III, and IV). When comparing interleukins 24 h after bacteria inoculum, we found statistically significant differences in TNF-alpha levels in groups receiving BGE (groups III and IV, p ≤ 0.05). Blood and peritoneal liquid cultures were also analyzed, and we detected a certain level of Enterococcus faecalis in peritoneal cultures from all treatment groups and less bacteria presence in blood cultures in rats receiving garlic extracts (groups II, III, and IV). In conclusion, TASE and BGE could be promising nutraceutical or medicinal agents as coadjuvants in the treatment of sepsis because of its effects in modulating the inflammatory response.

In Vitro Antioxidant and Pancreatic Anticancer Activity of Novel 5-Fluorouracil-Coumarin Conjugates.

Molecular hybridization consists of the combination of two or more non-identical pharmacophores in a single molecule. It has emerged as a promising strategy that allows the design of molecular frameworks with enhanced activity and affinity compared to their parent drugs. In this work, two novel hybrids that combine the well-known anticancer chemotherapeutic agent 5-fluorouracil with antioxidant coumarin derivatives have been synthesized and characterized by means of a copper-catalyzed azide-alkyne cycloaddition (CuAAC). The conjugates showed good antioxidant properties and a high tendency to aggregate and form stable nanoparticles in aqueous media, with regular shape and uniform size. These materials have proven to be preferential cytotoxic agents in vitro against human pancreatic cancer cells PANC-1, with an activity superior to free 5-fluorouracil. These results open up the possibility of exploiting the synergistic combination between 5-fluorouracil and coumarin derivatives and warrant further investigation of these hybrids as promising pancreatic anticancer agents.

Clinical features and mortality of COVID-19 patients admitted to ICU according to SOFA score.

The Sequential Organ Failure Assessment (SOFA) could function as an effective risk stratification tool in the admission of critically ill patients with COVID-19 and would allow stratification based on a risk assessment. We aimed to examine whether the SOFA score is useful to define 2 severity profiles in COVID-19 patients admitted to ICU: mild with SOFA < 5, and severe with SOFA ≥ 5. A retrospective cohort, multicenter study was conducted from February 11 to May 11, 2020. We analyzed patients admitted to all ICUs of the 14 public hospitals of the Castilla-La Mancha Health Service at the beginning of the pandemic and with SARS-CoV-2 infection. Patients were divided in 2 groups according to the level of severity by SOFA at admission to the ICU. Cox regression was used to evaluate factors associated with survival and Kaplan-Meier test to examine survival probability. In total, 405 patients with a complete SOFA panel were recruited in the 14 participating ICUs. SOFA <5 group showed that age above 60 years and D-dimer above 1000 ng/mL were risk factors associated with lower survival. In SOFA ≥ 5 it was found that high blood pressure was a risk factor associated with shorter survival. Kaplan-Meier showed lower survival in SOFA ≥ 5 in combination with high blood pressure, time since viral symptom onset to admission in ICU < 7 days, D-dimer ≥1000 ng/mL and respiratory pathology. However, SOFA < 5 showed only higher age (≥60 years) associated with lower survival. Age over 60 years and D-dimer over 1000 ng/mL were risk factors reflecting lower survival in patients with SOFA < 5. Moreover, SOFA ≥ 5 patients within a week after COVID-19 onset and comorbidities such as high blood pressure and previous respiratory pathology showed lower survival.

Aprotinin treatment against SARS-CoV-2: A randomized phase III study to evaluate the safety and efficacy of a pan-protease inhibitor for moderate COVID-19.

BACKGROUND: SARS-CoV-2 virus requires host proteases to cleave its spike protein to bind to its ACE2 target through a two-step furin-mediated entry mechanism. Aprotinin is a broad-spectrum protease inhibitor that has been employed as antiviral drug for other human respiratory viruses. Also, it has important anti-inflammatory properties for inhibiting the innate immunity contact system. METHODS: This was a multicentre, double-blind, randomized trial performed in four Spanish hospitals comparing standard treatment versus standard treatment + aprotinin for patients with COVID-19 between 20 May 2020 and 20 October 2021. The primary efficacy outcomes were length of hospital stay and ICU admission. The secondary endpoints were each of the primary efficacy outcomes and a composite of oxygen therapy, analytical parameters and death. Safety outcomes included adverse reactions to treatment during a 30-day follow-up period. Treatment was given for 11 days or till discharge. RESULTS: With almost identical analytical profiles, significant differences were observed in treatment time, which was 2 days lower in the aprotinin group (p = .002), and length of hospital admission, which was 5 days shorter in the aprotinin group (p = .003). The incidence of discharge was 2.19 times higher (HR: 2.188 [1.182-4.047]) in the aprotinin group than in the placebo group (p = .013). In addition, the aprotinin-treated group required less oxygen therapy and had no adverse reactions or side effects. CONCLUSION: Inhaled aprotinin may improve standard treatment and clinical outcomes in hospitalized patients with COVID-19, resulting in a shorter treatment time and hospitalization compared with the placebo group. The administration of aprotinin was safe.

Análisis de los pacientes menores de edad con sospecha de COVID-19 en un servicio de urgencias y su impacto en el estudio de contactos / Pediatric patients with suspected COVID-19 in an emergency department: analysis of characteristics and transmission to contacts

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Characteristics and Risk Factors Associated With Mortality in a Multicenter Spanish Cohort of Patients With COVID-19 Pneumonia.

INTRODUCTION: Spain is one of the countries with the highest number of COVID-19 patients. Unfortunately, few data for regions are available. OBJECTIVES: This study aimed to describe the characteristics and independent risk factors associated with COVID-19 mortality in Castilla-La Mancha, Spain. METHODS: Cohort and multicenter study in all 14 public hospitals of the Castilla-La Mancha Health Service. Baseline characteristics, preexisting comorbidities, symptoms, clinical features and treatments were included. Multivariable logistic regression was used to evaluate factors associated with death and Kaplan-Meier test to examine survival probability. Statistical significance was considered with p < 0.05 (95% CI). SPSS (version 24.0 for Windows) and R 4.0.2 (R Statistics) software were used. RESULTS: The cohort comprised 12,126 patients sequentially attended between February 11 and May 11, 2020. The mean age of patients was 66.4 years; 5667 (46.7%) were women. Six protective factors against exitus were defined: female sex, anosmia, cough, chloroquine and azithromycin. The risk factors were: age over 50, obesity, cardiac pathology, fever, dyspnea, lung infiltrates, lymphopenia, D-dimer above 1000 ng/mL, and mechanical ventilation requirement. Survival analysis showed higher survival rate in women (75.7%) than men (72.1%). Cumulative survival was 87.5% for non-hospitalized patients, 70.2% for patients admitted to hospital and 61.2% in ICU patients. Additionally, survival probability decreased with increasing age range. CONCLUSION: Determination of protective or death-promoting factors could be useful to stratify patients by severity criteria and to improve COVID-19 care management.

INTRODUCCIÓN: España es uno de los países con mayor número de pacientes con COVID-19. Desafortunadamente, se dispone de pocos datos por regiones. OBJETIVOS: Describir las características y los factores de riesgo independientes asociados a mortalidad por COVID-19 en Castilla-La Mancha, España. MÉTODOS: Estudio de cohorte, multicéntrico de los 14 hospitales públicos de Castilla-La Mancha. Se evaluaron las características clínicas, comorbilidades preexistentes, síntomas y tratamientos. Se utilizó una regresión logística multivariable para evaluar los factores asociados a muerte y Kaplan-Meier para medir supervivencia. Se consideró significación estadística con p < 0,05 (IC 95%). Se utilizaron los programas SPSS (versión 24.0 para Windows) y R 4.0.2 (R Statistics). RESULTADOS: Se estudiaron 12.126 pacientes atendidos secuencialmente entre el 11 de febrero y el 11 de mayo de 2020. La edad media fue de 66,4 años; 5.667 (46,7%) fueron mujeres. Se definieron seis factores protectores contra el exitus: sexo femenino, anosmia, tos, cloroquina y azitromicina. Los factores de riesgo fueron: edad superior a 50, obesidad, patología cardíaca, fiebre, disnea, infiltrados pulmonares, linfopenia, dímero-D > 1.000 ng/mL y necesidad de ventilación mecánica. Se observó mayor tasa de supervivencia en mujeres (75,7%) que en hombres (72,1%). La supervivencia acumulada fue del 87,5% para pacientes no hospitalizados, 70,2% para admitidos en planta hospitalaria y 61,2% en la Unidad de Cuidados Intensivos (UCI). Además, la probabilidad de supervivencia disminuyó con el aumento del rango de edad. CONCLUSIÓN: La determinación de los factores protectores o favorecedores de muerte podría ser útil para estratificar pacientes por criterios de gravedad y mejorar la atención frente a la COVID-19.

Characterization of Permeability Barrier Dysfunction in a Murine Model of Cutaneous Field Cancerization Following Chronic UV-B Irradiation: Implications for the Pathogenesis of Skin Cancer.

Chronic ultraviolet B (UV-B) irradiation is known to be one of the most important hazards acting on the skin and poses a risk of developing photoaging, skin with cutaneous field cancerization (CFC), actinic keratosis (AKs), and squamous cell carcinomas (SCCs). Most of the UV-B light is absorbed in the epidermis, affecting the outermost cell layers, the stratum corneum, and the stratum granulosum, which protects against this radiation and tries to maintain the permeability barrier. In the present work, we show an impairment in the transepidermal water loss, stratum corneum hydration, and surface pH after chronic UV-B light exposure in an immunologically intact mouse model (SKH1 aged mice) of skin with CFC. Macroscopic lesions of AKs and SCCs may develop synchronically or over time on the same cutaneous surface due to both the presence of subclinical AKs and in situ SCC, but also the accumulation of different mutations in keratinocytes. Focusing on skin with CFC, yet without the pathological criteria of AKs or SCC, the presence of p53 immunopositive patches (PIPs) within the epidermis is associated with these UV-B-induced mutations. Reactive epidermis to chronic UV-B exposure correlated with a marked hyperkeratotic hyperplasia, hypergranulosis, and induction of keratinocyte hyperproliferation, while expressing an upregulation of filaggrin, loricrin, and involucrin immunostaining. However, incidental AKs and in situ SCC might show neither hypergranulosis nor upregulation of differentiation markers in the upper epidermis. Despite the overexpression of filaggrin, loricrin, involucrin, lipid enzymes, and ATP-binding cassette subfamily A member 12 (ABCA12) after chronic UV-B irradiation, the permeability barrier, stratum corneum hydration, and surface pH were severely compromised in the skin with CFC. We interpret these results as an attempt to restore the permeability barrier homeostasis by the reactive epidermis, which fails due to ultrastructural losses in stratum corneum integrity, higher pH on skin surface, abundant mast cells in the dermis, and the common presence of incidental AKs and in situ SCC. As far as we know, this is the first time that the permeability barrier has been studied in the skin with CFC in a murine model of SCC induced after chronic UV-B irradiation at high doses. The impairment in the permeability barrier and the consequent keratinocyte hyperproliferation in the skin of CFC might play a role in the physiopathology of AKs and SCCs.

Clusterin overexpression as a potential neuroprotective response to the pathological effects of high fat dieting on the brain reward system.

High-fat diets (HFDs) can lead to pathological changes in the brain underlying several behavioral disturbances (e.g., reward deficiency). To further increase our knowledge of these associations, we studied the sucrose reward and the brain expression of clusterin, a protein that is overexpressed after several kind of brain damaging conditions. C57BL/6J male mice were differentially fed on an HFD or standard chow for 41 days and underwent 11 sucrose place conditioning sessions followed by 4 extinction sessions to monitor the effects of HFD on sucrose reward by means of free choice tests. We quantified clusterin expression by immunochemistry in the nucleus accumbens, dorsal striatum and cingulate cortex. HFD tended to provoke a transient potentiation in the acquisition of sucrose-conditioned place preference, but this effect was followed by a much more consistent reduction in sucrose preference, which spontaneously disappeared after 31 days of an HFD with no need for extinction learning. The HFD mice showed higher clusterin expression in the nucleus accumbens but not in the other brain areas studied. The results confirm that HFDs strongly influence the rewarding properties of palatable foods and suggest a direct connection with neurotoxic alterations in the brain reward system tagged by clusterin overexpression.

Characteristics and Risk Factors Associated With Mortality in a Multicenter Spanish Cohort of Patients With COVID-19 Pneumonia / Características y factores de riesgo asociados a mortalidad en una cohorte multicéntrica española de pacientes con neumonía por COVID-19

INTRODUCTION: Spain is one of the countries with the highest number of COVID-19 patients. Unfortunately, few data for regions are available. OBJECTIVES: This study aimed to describe the characteristics and independent risk factors associated with COVID-19 mortality in Castilla-La Mancha, Spain. METHODS: Cohort and multicenter study in all 14 public hospitals of the Castilla-La Mancha Health Service. Baseline characteristics, preexisting comorbidities, symptoms, clinical features and treatments were included. Multivariable logistic regression was used to evaluate factors associated with death and Kaplan-Meier test to examine survival probability. Statistical significance was considered with p < 0.05 (95% CI). SPSS (version 24.0 for Windows) and R 4.0.2 (R Statistics) software were used. RESULTS: The cohort comprised 12,126 patients sequentially attended between February 11 and May 11, 2020. The mean age of patients was 66.4 years; 5667 (46.7%) were women. Six protective factors against exitus were defined: female sex, anosmia, cough, chloroquine and azithromycin. The risk factors were: age over 50, obesity, cardiac pathology, fever, dyspnea, lung infiltrates, lymphopenia, D-dimer above 1000 ng/mL, and mechanical ventilation requirement. Survival analysis showed higher survival rate in women (75.7%) than men (72.1%). Cumulative survival was 87.5% for non-hospitalized patients, 70.2% for patients admitted to hospital and 61.2% in ICU patients. Additionally, survival probability decreased with increasing age range. CONCLUSION: Determination of protective or death-promoting factors could be useful to stratify patients by severity criteria and to improve COVID-19 care management

INTRODUCCIÓN: España es uno de los países con mayor número de pacientes con COVID-19. Desafortunadamente, se dispone de pocos datos por regiones. OBJETIVOS: Describir las características y los factores de riesgo independientes asociados a mortalidad por COVID-19 en Castilla-La Mancha, España. MÉTODOS: Estudio de cohorte, multicéntrico de los 14 hospitales públicos de Castilla-La Mancha. Se evaluaron las características clínicas, comorbilidades preexistentes, síntomas y tratamientos. Se utilizó una regresión logística multivariable para evaluar los factores asociados a muerte y Kaplan-Meier para medir supervivencia. Se consideró significación estadística con p < 0,05 (IC 95%). Se utilizaron los programas SPSS (versión 24.0 para Windows) y R 4.0.2 (R Statistics). RESULTADOS: Se estudiaron 12.126 pacientes atendidos secuencialmente entre el 11 de febrero y el 11 de mayo de 2020. La edad media fue de 66,4 años; 5.667 (46,7%) fueron mujeres. Se definieron seis factores protectores contra el exitus: sexo femenino, anosmia, tos, cloroquina y azitromicina. Los factores de riesgo fueron: edad superior a 50, obesidad, patología cardíaca, fiebre, disnea, infiltrados pulmonares, linfopenia, dímero-D > 1.000 ng/mL y necesidad de ventilación mecánica. Se observó mayor tasa de supervivencia en mujeres (75,7%) que en hombres (72,1%). La supervivencia acumulada fue del 87,5% para pacientes no hospitalizados, 70,2% para admitidos en planta hospitalaria y 61,2% en la Unidad de Cuidados Intensivos (UCI). Además, la probabilidad de supervivencia disminuyó con el aumento del rango de edad. CONCLUSIÓN: La determinación de los factores protectores o favorecedores de muerte podría ser útil para estratificar pacientes por criterios de gravedad y mejorar la atención frente a la COVID-19

Influence of biomedical education on health and eating habits of university students in Spain.

OBJECTIVE: The aim of this study was to explore the influence of an enrolled degree course on health and eating habits in a population of Spanish university students (17-26 y of age). METHODS: A cross-sectional observational study was carried out with 648 students. Volunteers were stratified into biomedical (medicine and nursing, 48%) and non-biomedical students (other fields of study, 52%). Data were collected using previously self-reported questionnaires focused on anthropometric and sociodemographic profile, lifestyle practices, body image perception, health consciousness, eating habits, physical activity, and food addiction. Mann-Whitney U tests and Pearson's χ2 tests were applied to identify associations between the two groups. RESULTS: Self-reported body mass index was higher for the non-biomedical group (22.1 ± 3.1 versus 23 ± 5 kg/m2; P < 0.05), which also reported less regularity in taking meals (91 versus 95%; P < 0.05), eating fewer colored vegetables and fruits (65 versus 77%; P < 0.001) and a higher alcohol intake (27 versus 20%; P < 0.001). In contrast, the proportion of students that showed more interest in the diet-health duality (92 versus 85%; P < 0.001) and a desire to adopt healthier habits (80 versus 78%; P < 0.05) was larger in the biomedical group. Dietary habits, obtained by means of a food frequency questionnaire, suggested that biomedical students make healthier food choices. Additionally, the group of biomedical students took more walks per week (5.8 ± 1.8 versus 5.5 ± 1.9; P < 0.05). CONCLUSIONS: Healthier lifestyle factors cluster into the biomedical group in various components of the study, except food addiction where no differences were observed. The data presented here suggest the necessity to develop health promotion strategies targeting university students.

Topical Applications of Thiosulfinate-Enriched Allium sativum Extract Accelerates Acute Cutaneous Wound Healing in Murine Model.

OBJECTIVE: To determine whether topical applications of thiosulfinate-enriched Allium sativum extract (TASE) can accelerate acute cutaneous wound healing (WH) in a murine model. METHODS: Keratinocyte viability and in vitro wound closure were assessed in keratinocyte cultures. Effects of topical TASE (0.5 µg/mL of allicin in 97% ethanol) on acute cutaneous WH were determined in a murine model of acute cutaneous wound. Twelve mice were alternately assigned to the vehicle- and TASE-treated groups (n=6 per group). Expression levels of mRNA for keratinocyte differentiation marker-related proteins (filaggrin, loricrin and involucrin) and lipid synthetic enzymes (elongation of very long chain fatty acids protein 4 (ELOVL4), fatty acid synthase (FA2H), 3-hydroxy- 3-methyl-glutaryl-coenzyme A reductase (HMGCoA), and serine palmitoyltransferase (SPT)) were assessed using real-time quantitative polymerase chain reaction on day 3 and 8 after wounding, while transepidermal water loss (TEWL) rates were measured in wounded areas. RESULTS: TASE accelerated WH both in vivo (40% vs. 22% reduction in wound area, P<0.01) and in vitro (90% vs. 65% reduction in wound area, P<0.01). Moreover, topical applications of TASE upregulated the expression levels of epidermal mRNA for ELOVL4, HMGCoA, SPT, filaggrin, loricrin and involucrin (P<0.05 vs. vehicle-treated controls) on day 3 after wounding. Likewise, TASE significantly lowered TEWL rates in comparison with vehicle alone on day 8 (33.06±2.09 g/(m2·h) vs. 24.60±2.04 g/(m2·h), P<0.01). CONCLUSIONS: Topical applications of TASE stimulated keratinocyte proliferation and formation of epidermal permeability barrier function, leading to acceleration of acute cutaneous WH. Topical products containing TASE could be used to manage acute cutaneous WH.

Cost Effective Use of a Thiosulfinate-Enriched Allium sativum Extract in Combination with Chemotherapy in Colon Cancer.

In this work, we sought to investigate the effects of a thiosulfinate-enriched garlic extract, co-administered with 5-fluorouracil (5-FU) or oxaliplatin chemotherapy, on the viability of colon cancer cells (Caco-2 and HT-29). We also addressed the economic feasibility of a new combined treatment of this thiosulfinate-enriched garlic extract, with oxaliplatin that could reduce the dosage and costs of a monotherapy. The thiosulfinate-enriched garlic extract not only enhanced the impact of 5-FU and oxaliplatin (500 µM) in decreasing Caco-2 and HT-29 viability, but also showed a higher effect than standard 5-FU and oxaliplatin chemotherapy as anti-cancer agents. These results provided evidences for the combination of lyophilized garlic extract and 5-FU or oxaliplatin as a novel chemotherapy regimen in colon cancer cells that may also reduce the clinical therapy costs.

Desarrollo de modelo experimental animal de peritonitis bacteriana / Development of animal experimental model for bacterial peritonitis

OBJETIVO: Desarrollar un modelo de sepsis abdominal en animal de experimentación. MATERIAL Y MÉTODOS: Se utilizan ratas Sprague-Dawley(R), machos de 5 semanas con pesos entre 270-280 g en el momento de la inoculación (N=39). Inicialmente se realiza un estudio piloto (N=9), distribuyéndolas en 3 grupos (3/3/3) con inóculo de 1cc de Escherichia coli ATCC 25922 intraperitoneal en concentraciones de 10E8, 10E9 y 10E10 UFC. En un segundo estudio (N=6) con distribución en dos grupos (3/3) se utilizan 1cc una concentración de E. coli 10E10 UFC que se diluyen en 10 y 15 ml de agua destilada para su inoculación. Por último se inicia un ensayo experimental con aleatorización de 24 ratas en tres grupos de tratamiento tras la infección intraperitoneal: Grupo I con suero fisiológico (N=6), Grupo II con antibiótico (ceftriaxona) (N=9), Grupo III con antibiótico más adyuvante (ceftriaxona más alicina) (N=9). Se realizan muestras microbiológicas de sangre y líquido peritoneal, así como estudio histopatológico de órganos intraperitoneales (hígado, diafragma y peritoneo). RESULTADOS: Se observa muerte en el 100% de las ratas infectadas con la concentración de E. coli 10E10 UFC con la dilución de 15 ml de agua destilada y sin antibiótico. El hemocultivo y cultivo de líquido peritoneal es positivo a la misma cepa en todas ellas. Se observa la formación de abscesos en la superficie del hígado e infiltración por polimorfonucleares en los tejidos. CONCLUSIÓN: Se establece que la dosis letal de E. coli es 10E10 UFC diluida en 15 ml agua destilada en inyección intraperitoneal

OBJECTIVE: The aim of the study was to develop a model of abdominal sepsis in the experimental animal. MATERIAL AND METHODS: Sprague-Dawley male rats of 5 weeks (N=39) were used. Initially, a pilot study (N = 9) was performed and distributed in 3 groups with 1cc inoculum of Escherichia coli ATCC 25922 intraperitoneally at concentrations of 10E8, 10E9 and 10E10 CFU. Subsequently, concentrations of 10E10 CFU are used in two groups of 3 rats with dilutions of 10 cc and 15 cc of distilled water respectively. Finally, a randomized trial of 24 rats was started in three treatment groups after intraperitoneal infection: Group I with physiological serum (N = 6), Group II with ceftriaxone (N = 9), Group III with ceftriaxone plus allicin (N = 9). Microbiological samples of blood and peritoneal fluid were made, as well as histopathological study of intraperitoneal organs (liver, diaphragm and peritoneum). RESULTS: Death of 100% of the rats infected with 10E10 E. coli UFC concentration with the dilution of 15 ml of distilled water and without antibiotic was oberved. The blood culture and peritoneal fluid culture was positive for the same strain in all of them. The formation of abscesses on the liver surface and polymorphonuclear infiltration in tissues were observed. CONCLUSION: The lethal dose of E. coli is 10E10 CFU diluted in 15 cc distilled water by intraperitoneal injection

A high-fat diet combined with food deprivation increases food seeking and the expression of candidate biomarkers of addiction.

A mouse model has been developed to study the effect of dietary fat combined with food deprivation periods on palatable food seeking and on the expression of three potential addiction biomarkers in the nucleus accumbens: fumarate hydratase (FH), ATP synthase subunit alpha (ATP5a1) and transketolase (TKT). Forty C57BL/6 J male mice, four-week old, were fed either with a high-fat (HF) diet or standard diet along the experiment. After 3 weeks of differential feeding, animals underwent a two-week training period of two daily sessions where visual cues were paired either to palatable food (chocolate cereals) or no food at all. This training was prolonged one more week with similar, one daily sessions preceded by 12 hours of food deprivation. A behavioural test was finally conducted where mice were confined for 30 minutes either in food unpaired compartments or in compartments previously paired with cereals, but now with empty food trays. Total activity during this behavioural test and serum corticosterone levels right after it were similar in all experimental groups. Mice tested in food-paired compartments showed a marked preference for the empty food tray that gradually disappeared in standard diet-fed individuals but persisted in HF-fed mice. HF-fed mice also overexpressed FH, ATP5a1 and TKT, which positively correlated with the persistence of preference for the empty food tray. It is suggested that HF diets combined with food deprivation may enhance food seeking behaviours while upregulating FH/ATP5a1/TKT, which are further envisaged as biomarkers of addiction.

Imaging local estrogen production in single living cells with recombinant fluorescent indicators.

Estrogens are steroid hormones with many systemic effects in addition to development and maintenance of the female reproductive system, and ligands of estrogen receptors are of clinical importance because of their use as oral contraceptive, hormone replacement and antitumoral therapy. In addition, tumoral tissues have been found to express aromatase and other steroidogenic enzymes synthesizing estradiol. To aid in the understanding of these processes, we have developed assays to image the local production of estrogens in isolated living mammalian cells. We constructed biosensors based on estrogen receptor α ligand binding domain and fluorescent proteins by following two approaches. First, the ligand binding domain and a short fragment of steroid receptor coactivator-1 were appended to a circularly permuted yellow fluorescent protein to construct an excitation ratio estrogen indicator. In the second strategy, we constructed emission ratio sensors based on fluorescence resonance energy transfer, containing the ligand binding domain flanked by donor and acceptor fluorescent proteins. Estrogens altered the fluorescence signal of cells transfected with the indicators in a dose-dependent manner. We imaged local estrogen production in adrenocortical H295 cells expressing aromatase and transfected with the fluorescent sensors. In addition, paracrine detection was observed in HeLa cells harboring the indicators and co-cultured with H295 cells. This imaging approach may allow detection of physiological levels of these hormones in suitable animal models.

mTOR/S6 kinase pathway contributes to astrocyte survival during ischemia.

Neurons are highly dependent on astrocyte survival during brain damage. To identify genes involved in astrocyte function during ischemia, we performed mRNA differential display in astrocytes after oxygen and glucose deprivation (OGD). We detected a robust down-regulation of S6 kinase 1 (S6K1) mRNA that was accompanied by a sharp decrease in protein levels and activity. OGD-induced apoptosis was increased by the combined deletion of S6K1 and S6K2 genes, as well as by treatment with rapamycin that inhibits S6K1 activity by acting on the upstream regulator mTOR (mammalian target of rapamycin). Astrocytes lacking S6K1 and S6K2 (S6K1;S6K2-/-) displayed a defect in BAD phosphorylation and in the expression of the anti-apoptotic factors Bcl-2 and Bcl-xL. Furthermore reactive oxygen species were increased while translation recovery was impaired in S6K-deficient astrocytes following OGD. Rescue of either S6K1 or S6K2 expression by adenoviral infection revealed that protective functions were specifically mediated by S6K1, because this isoform selectively promoted resistance to OGD and reduction of ROS levels. Finally, "in vivo" effects of S6K suppression were analyzed in the permanent middle cerebral artery occlusion model of ischemia, in which absence of S6K expression increased mortality and infarct volume. In summary, this article uncovers a protective role for astrocyte S6K1 against brain ischemia, indicating a functional pathway that senses nutrient and oxygen levels and may be beneficial for neuronal survival.