Inhaled Litsea glaucescens K. (Lauraceae) leaves' essential oil has anxiolytic and antidepressant-like activity in mice by BDNF pathway activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Litsea glaucescens K. (Lauraceae) is a small tree from the Mexican and Central American temperate forests, named as "Laurel". Its aromatic leaves are ordinarily consumed as condiments, but also are important in Mexican Traditional Medicine, and among the most important non wood forest products in this area. The leaves are currently used in a decoction for the relief of sadness by the Mazahua ethnic group. Interestingly, "Laurel" has a long history. It was named as "Ehecapahtli" (wind medicine) in pre-Columbian times and applied to heal maladies correlated to the Central Nervous System, among them depression, according to botanical texts written in the American Continent almost five centuries ago. AIM OF THE STUDY: Depression is the first cause of incapacity in the world, and society demands alternative treatments, including aromatherapy. We have previously demonstrated the antidepressant-like activity of L. glaucescens leaves' essential oil (LEO), as well as their monoterpenes linalool, and beta-pinene by intraperitoneal route in a mice behavioral model. Here we now examined if LEO and linalool exhibit this property and anxiolytic activity when administered to mice by inhalation. We also investigated if these effects occur by BDNF pathway activation in the brain. MATERIALS AND METHODS: The LEO was prepared by distillation with water steam and analyzed by gas chromatography-mass spectrometry (GC-MS). The monoterpenes linalool, eucalyptol and ß-pinene were identified and quantified. Antidepressant type properties were determined with the Forced Swim Test (FST) on mice previously exposed to LEO or linalool in an inhalation chamber. The spontaneous locomotor activity and the sedative effect were assessed with the Open Field Test (OFT), and the Exploratory Cylinder (EC), respectively. The anxiolytic properties were investigated with the Elevated Plus Maze Apparatus (EPM) and the Hole Board Test (HBT). All experiments were video documented. The mice were subjected to euthanasia, and the brain hippocampus and prefrontal cortex were dissected. RESULTS: The L. glaucescens essential oil (LEO) contains 31 compounds according to GC/MS, including eucalyptol, linalool and beta-pinene. The LEO has anxiolytic effect by inhalation in mice, as well as linalool, and ß-pinene, as indicated by OFT and EC tests. The LEO and imipramine have antidepressant like activity in mice as revealed by the FST; however, linalool and ketamine treatments didn't modify the time of immobility. The BDNF was increased in FST in mice treated with LEO in both areas of the brain as revealed by Western blot; but did not decrease the level of corticosterone in plasma. The OFT indicated that LEO and imipramine didn't reduce the spontaneous motor activity, while linalool and ketamine caused a significant decrease. CONCLUSION: Here we report by the first time that L. glaucescens leaves essential oil has anxiolytic effect by inhalation in mice, as well as linalool, and ß-pinene. This oil also maintains its antidepressant-like activity by this administration way, similarly to the previously determined intraperitoneally. Since inhalation is a common administration route for humans, our results suggest L. glaucescens essential oil deserve future investigation due to its potential application in aromatherapy.

Central nervous system activity of a Tabernaemontana arborea alkaloid extract involves serotonergic and opioidergic neurotransmission in murine models.

Tabernaemontana arborea (Apocynaceae) is a Mexican tree species known to contain ibogan type alkaloids. This study aimed at determining central nervous system-related activities of an alkaloid extract obtained from the root bark of T. arborea. A gas chromatography-mass spectrometry (GC-MS) analysis was performed to describe the alkaloid profile of the extract. A wide dosing range (0.1 to 56.2 mg/kg) of this extract was evaluated in different murine models. Electrical brain activity was examined by electroencephalography (EEG). The extract's effects on motor coordination, ambulatory activity, and memory were analyzed based on the rotarod, open field (OFT), and object recognition tests (ORT), respectively. Antidepressant and antinociceptive activities were determined using the forced swimming test (FST) and the formalin assay, respectively. In order to elucidate the underlying mechanisms of action, the 5-HT1A receptor antagonist WAY100635 (1 mg/kg) or the opioid receptor antagonist naloxone (1 mg/kg) was included in the latter experiments. GC-MS analysis (µg/mg extract) confirmed the presence of the monoterpenoid indole alkaloids (MIAs) voacangine (207.00), ibogaine (106.33), vobasine (72.81), coronaridine (30.72), and ibogamine (24.2) as principal constituents of the extract, which exhibited dose- and receptor-dependent antidepressant (0.1 to 1 mg/kg; 5-HT1A) and antinociceptive (30 and 56.2 mg/kg; opioid) effects, without altering motor coordination, ambulatory activity, and memory. EEG indicated CNS depressant activity at high doses (30 and 56.2 mg/kg). The root bark of T. arborea contains a mixture of alkaloids that may hold therapeutic value in pain relief and the treatment of psychiatric diseases without causing neurotoxic activity at effective doses.

Molecular Basis of Coronavirus Virulence and Vaccine Development.

Virus vaccines have to be immunogenic, sufficiently stable, safe, and suitable to induce long-lasting immunity. To meet these requirements, vaccine studies need to provide a comprehensive understanding of (i) the protective roles of antiviral B and T-cell-mediated immune responses, (ii) the complexity and plasticity of major viral antigens, and (iii) virus molecular biology and pathogenesis. There are many types of vaccines including subunit vaccines, whole-inactivated virus, vectored, and live-attenuated virus vaccines, each of which featuring specific advantages and limitations. While nonliving virus vaccines have clear advantages in being safe and stable, they may cause side effects and be less efficacious compared to live-attenuated virus vaccines. In most cases, the latter induce long-lasting immunity but they may require special safety measures to prevent reversion to highly virulent viruses following vaccination. The chapter summarizes the recent progress in the development of coronavirus (CoV) vaccines, focusing on two zoonotic CoVs, the severe acute respiratory syndrome CoV (SARS-CoV), and the Middle East respiratory syndrome CoV, both of which cause deadly disease and epidemics in humans. The development of attenuated virus vaccines to combat infections caused by highly pathogenic CoVs was largely based on the identification and characterization of viral virulence proteins that, for example, interfere with the innate and adaptive immune response or are involved in interactions with specific cell types, such as macrophages, dendritic and epithelial cells, and T lymphocytes, thereby modulating antiviral host responses and viral pathogenesis and potentially resulting in deleterious side effects following vaccination.

Concentración de interleuquina-6 en el vítreo de pacientes con desprendimiento de retina / Interleukin-6 concentrations in the vitreous body of patients with retinal detachment

OBJETIVO: Determinar los niveles de interleuquina-6 (IL-6) en el vítreo de pacientes con desprendimiento de retina (DdR). MATERIAL Y MÉTODO: Mediante vitrectomía vía pars plana, se recogieron muestras no diluidas de vítreo de 40 pacientes sin antecedentes de cirugía vítrea o intraocular previa, que fueron divididos en 2 grupos: A (n = 20) pacientes con DdR y B (n = 20) pacientes con membrana premacular y agujero macular. La concentración de IL-6 se determinó mediante radioinmunoensayo. RESULTADOS: La concentración vítrea de IL-6 en el grupo A fue 122,4 + -16 pg/mL (rango 91,5-620) y en el grupo B fue 46 +/- 23 pg/mL (rango 3-150) (p < 0,001). CONCLUSIONES: Estos resultados demuestran que la concentración vítrea de IL-6 está más elevada en los pacientes con DdR en comparación con el grupo control

OBJECTIVE: To measure interleuquin-6 (IL-6) levels in the vitreous body of patients with retinal detachment (RD). PATIENTS AND METHODS: Undiluted vitreous samples were obtained from 40 patients with no history of prior vitreous or intraocular surgery. Patients were divided into two groups: A (n = 20) patients with RD and B (n = 20) patients with pre-retinal macular membranes and macular holes. IL-6 was determined using radioimmunoassay. RESULTS: IL-6 vitreous concentration in group A was 122.4 + -16 pg/mL (range 91.5-620) and in group B was 46 +/- 23 pg/mL (range 3-150) (p < .001). CONCLUSIONS: These results show that the concentration of IL-6 in the vitreous body was significantly higher in patients with RD than in the control group

Fine-needle aspiration cytopathology in the diagnosis of Wilms tumor.

BACKGROUND/PURPOSE The International Society of Paediatric Oncology (SIOP) protocol for Wilms tumor (WT) includes preoperative chemotherapy as the initial approach. However, an inadequate treatment may be performed in case of histological misdiagnosis. We evaluated the impact of fine-needle aspiration cytopathology (FNAC) in the diagnosis of unilateral WT in our group of patients. MATERIALS AND METHODS A retrospective descriptive study of patients with diagnosis of unilateral WT who underwent FNAC prior to neoadjuvant chemotherapy between 1993 and 2009 was performed. We reviewed the cytological diagnosis obtained by ultrasound-guided FNAC and the histological correlation with the resected specimens. RESULTS FNAC was performed in 66 patients with unilateral WT. In 57 of the 58 patients with positive FNAC for WT, the final diagnosis was correct (PPV: 98.2%). In 8 cases with negative FNAC for WT, the final diagnosis was positive for WT in 3 patients (NPV: 62.5%). Sensitivity was 95% and specificity was 83.3%. No complications were found associated with the procedure, except for an episode of haematuria, which resolved spontaneously. CONCLUSIONS FNAC is a useful and feasible technique in children that may confirm the suspected diagnosis of unilateral WT, avoiding inadequate preoperative chemotherapy in case of a non-Wilms renal tumor.

Fine-needle aspiration cytopathology in the diagnosis of Wilms tumor

BACKGROUND/PURPOSE The International Society of Paediatric Oncology (SIOP) protocol for Wilms tumor (WT) includes preoperative chemotherapy as the initial approach. However, an inadequate treatment may be performed in case of histological misdiagnosis. We evaluated the impact of fine-needle aspiration cytopathology (FNAC) in the diagnosis of unilateral WT in our group of patients. MATERIALS AND METHODS A retrospective descriptive study of patients with diagnosis of unilateral WT who underwent FNAC prior to neoadjuvant chemotherapy between 1993 and 2009 was performed. We reviewed the cytological diagnosis obtained by ultrasound-guided FNAC and the histological correlation with the resected specimens. RESULTS FNAC was performed in 66 patients with unilateral WT. In 57 of the 58 patients with positive FNAC for WT, the final diagnosis was correct (PPV: 98.2%). In 8 cases with negative FNAC for WT, the final diagnosis was positive for WT in 3 patients (NPV: 62.5%). Sensitivity was 95% and specificity was 83.3%. No complications were found associated with the procedure, except for an episode of haematuria, which resolved spontaneously. CONCLUSIONS FNAC is a useful and feasible technique in children that may confirm the suspected diagnosis of unilateral WT, avoiding inadequate preoperative chemotherapy in case of a non-Wilms renal tumor (AU)

Etiología y Resistencias a los antibióticos en las infecciones de la localización quirúrgica tras cirugía abdominal / Etiology and antimicrobial resistance of surgical site infections after abdominal surgery

Las infecciones de la localización quirúrgica (ILQ) tras cirugía abdominal son una complicación frecuente. La resistencia de los microorganismos a los antimicrobianos ha aumentado progresivamente en los últimos años. El objetivo es describir la etiología de las ILQ, la frecuencia de resistencias de los microorganismos, y caracterizar los factores que se asocian a las ILQ por microorganismos resistentes (MR). MÉTODOS. Encuestas anuales de prevalencia de infecciones en los pacientes hospitalizados durante el periodo 1999-2005. Se seleccionaron todas las ILQ presentes en los pacientes que habían recibido cirugía abdominal. Se recogieron características demográficas de los pacientes, datos microbiológicos, y de potenciales factores de riesgo. RESULTADOS. Se identificaron un total de 3.564 ILQ en las que se aislaron 3.590 microorganismos. Los más frecuentes fueron: Escherichia coli (25,9%), Enterococcus faecalis (9,4%), Pseudomonas aeruginosa (7,0%) y Staphylococcus aureus (6,5%). Las resistencias a cefalosporinas de tercera generación oscilan entre el 2,5% para K. pneumoniae y el 30% para Enterobacter sp., y para las fluorquinolonas entre el 3,8% de K. pneumoniae y el 16,1% para E. coli (25% en el año 2005). S aureus es resistente a la oxacilina en el 38,2% de los aislados. Los principales factores asociados a infecciones por MR son el año de la encuesta, el tamaño del hospital, y el número de factores de riesgo intrínseco del paciente. CONCLUSIONES. Los microorganismos implicados en las ILQ han aumentado progresivamente sus resistencias antimicrobianas durante el periodo 1999 a 2005. El grado de resistencia a los antimicrobianos está condicionado por el nivel de riesgo intrínseco de los pacientes y el tamaño del hospital (AU)

Surgical site infection (SSI) is a common complication after abdominal surgery. Microorganism resistance to antimicrobials has been increasing progressively during the last years. The objective is to describe the aetiology of SSI, the frequency of antimicrobial resistance and to characterize the factors associated to SSI caused by resistant microorganims (RM).METHODS. A series of yearly prevalence surveys of hospitalized patients during the period 1999-2005 in Spain. Selection of all SSI developed by patients operated on by abdominal procedures. Information recorded included demographic characteristic of patients, microbiological data, and potential risk factors. RESULTS. A total 3,590 microorganisms corresponding to a 3,564 SSI were identified. The most frequent microorganisms isolated were Escherichia coli (25.9%), Enterococcus faecalis (9.4%), Pseudomonas aeruginosa (7.0%) and Staphylococcus aureus (6.5 %). Third generation cephalosporin resistance ranged from 2.5% (K. pneumoniae) and 30% (Enterobacter sp.). Fluorquinolone resistance ranged from 3.8% (K. pneumoniae) and 16.1% (E. coli) (25% for the year 2005). Methicillin resistant S. aureus reached 38% of isolates. The main factors associated to MR infections were year of the survey, size of the hospital and the number of intrinsic risk factors of the patients. CONCLUSIONS Microorganisms involved in SSI have been progressively increasing antimicrobial resistance during the period 1999 through 2005. Patient intrinsic risk and the size of the hospital condition the level of antimicrobial resistance (AU)

Analysis of the expression of HLA class I, proinflammatory cytokines and chemokines in primary tumors from patients with localized and metastatic renal cell carcinoma.

Changes in the human leukocyte antigen (HLA) class I expression and cytokine and chemokine production both by cancer cells and by normal surrounding tissue are believed to be responsible for immune escape and tumor progression. In this study, we compared the tumor expression levels of HLA heavy chain (HLAhc), beta-2-microglobulin (beta2m), chemokines (Interferon-gamma-inducible Protein-10 (IP-10), Interferon-inducible T-cell Alpha-Chemoattractant (I-TAC), Stromal cell-Derived Factor-1 (SDF-1), Macrophage Inflammatory Protein-1-alpha (MIP-1-alpha) and Regulated upon Activation, Normally T-Expressed, and presumably Secreted (RANTES)) and cytokines (Vascular Endothelial Growth Factor (VEGF), Interferon-gamma (IFN-gamma), Interleukin-10 (IL-10), Tumor Growth Factor-beta (TGB-beta)) in primary tumors and adjacent normal tissues from patients with localized and metastatic renal cell carcinoma (RCC) using a quantitative real-time polymerase chain reaction technique. We report that the expression of HLAhc, beta2m and the studied cytokines and chemokines (except for SDF-1) was significantly higher in the tumor (29 samples) than in the normal tissue (14 samples). When we compared the tumor expression levels between patients with localized RCC and patients with advanced metastatic stage, we found that the messenger RNA expression levels of HLAhc and beta2m were much lower in patients with metastatic RCC (6 cases) than in patients with localized cancer (23 cases), with levels similar to those in normal tissue. This was also confirmed on a protein level by immunohistological labeling of tumor tissues. Thirty-nine percent of the analyzed RCC tumors showed partial loss of HLA class I molecules, while 6% of the tumors showed HLA class I total loss. The expression of IP-10, SDF-1 and VEGF-c was also significantly lower in patients with advanced tumor, while the IFN-gamma expression in metastatic RCC was not detectable. Our findings show that primary RCC tumors are characterized by a high expression of HLAhc and a presence of proinflammatory mediators and chemokines. We also observed that disease progression and development of metastasis in RCC are associated with decreased expression of HLAhc, beta2m, IP-10, SDF-1 and IFN-gamma. This microenvironment may suppress the cytotoxic response, creating conditions that favor tumor escape and cancer progression.

Ictus isquémico tras infección por varicela / Ischemic stroke after varicella infection

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Multiple mechanisms of immune evasion can coexist in melanoma tumor cell lines derived from the same patient.

Progressive tumor growth may be associated with suppression of the immune response. Many different mechanisms may contribute to immune evasion. We investigated some of these mechanisms in melanoma cells lines generated from two patients. These cell lines show a complex pattern of altered HLA expression; however, the resulting phenotype did not satisfactorily explain the simultaneous evasion of T and NK cell cytotoxicity. Two additional alterations have now been detected in these melanoma cell lines: (1) resistance to FAS-induced apoptosis caused by defective FAS gene expression, and (2) constitutive expression of immunosuppressive cytokines. Our results show that several of the major mechanisms for immune evasion may coexist in a single tumor. This suggests that tumor progression may give rise to an extremely resistant phenotype, which may be an impediment to some immunotherapeutic strategies. We hypothesize that the simultaneous presence of several mechanisms involved in tumor immune evasion must be the result of progressive selection of characteristics that are advantageous for tumor survival in a competent host. Our findings do not support the possibility that FASL expression is a common mechanism of evasion of immune response in melanoma cells.

Microsatellite instability analysis in tumors with different mechanisms for total loss of HLA expression.

Down-regulation of the expression of major histocompatibility complex molecules is a frequent event that is associated with the poor immunogenicity of tumor cells. Acquired resistance to T-cell-based immunotherapy has been associated with loss of functional beta2-microglobulin expression. This anomaly appears to be particularly relevant in tumors exhibiting a defect in DNA-mismatch repair, and induces structural abnormalities in HLA cell-surface expression that are not reversible by cytokine treatment. We examined HLA expression in 118 melanoma, colon or larynx tumors to identify total loss of HLA class I expression with or without somatic beta2-microglobulin gene mutation. Microsatellite instability was investigated in these tumors to determine whether a replication error phenotype (RER+) implied a particular alteration in HLA phenotype. A total of 7.6% of the tumors showed the RER+ phenotype, and 12.7% were HLA-ABC-negative. In the RER+ group, only one tumor was HLA-ABC-negative and no beta2-microglobulin mutation was identified. In contrast, in the HLA-ABC-negative group, only one tumor showed microsatellite instability. None of the three melanomas that contained beta2-microglobulin mutation exhibited the mutator phenotype. These findings suggest that beta2-microglobulin mutation in human melanoma tumors may arise through a mechanism that does not necessarily involve microsatellite instability. Our results also indicate that somatic mutations of the beta2-microglobulin gene are not the main mechanism of total loss of HLA expression.

Looking for HLA-G expression in human tumours.

Tumour and virus infected cells escape CTLs responses by losing some or all HLA class I molecules. However the NK escape mechanism that uses the HLA-A, -B, and -C tumour deficient variants is unknown. To determine whether HLA-G is expressed on tumour cells and thus favours tumour escape by abolishing NK lysis, we studied HLA-G in a large panel of human tumour tissues and human tumour cell lines of different origin that were previously characterized for HLA-A, -B, and -C expression. We studied HLA-G mRNA transcripts using RT-PCR, and HLA-G1 expression by FACS and immunohistochemical techniques. We found several mRNA transcripts of HLA-G isoforms in most of the samples studied. However, we detected no cell surface expression of HLA-G1 using two specific monoclonal antibodies (mAbs) (87G and 01G). We cannot, however, exclude the possibility that some isoforms other than HLA-G1 may be expressed in some tumours.

Chromosome loss is the most frequent mechanism contributing to HLA haplotype loss in human tumors.

Loss of heterozygosity (LOH) in the short arm of chromosome 6 (6p) was detected in samples obtained from colon (13.8%), larynx (17.6%) and melanoma (15.3%) tumors. The parallel study of HLA-antigen expression in tumor tissues using locus- and polymorphic-specific antibodies in combination with LOH microsatellite analysis on 6p allowed us to establish that LOH in chromosome 6 is a representative phenomenon in most tumor cells present in a given tumor tissue. In most cases, specific HLA alleles had been lost in a predominant population of tumor cells, indicating that LOH is a non-irrelevant mutation that probably confers a selective advantage for survival of the mutant cell. We also demonstrate that LOH frequently occurred through chromosome loss rather than somatic recombination. LOH at all loci studied on the p and q arms of chromosome 6 was observed in at least 56.2% (9/17) cases. This HLA-associated microsatellite analysis was a useful tool for classifying tumors as LOH-positive or -negative, and therefore to consider a patient as a potential non-responder or responder in a vaccination trial.

Differential effect on U937 cell differentiation by targeting transcriptional factors implicated in tissue- or stage-specific induced integrin expression.

The inhibition of transcription factor functions was used to define their role in phorbol ester-induced cellular differentiation of a monocytic cell line, U937. We demonstrate a differential effect on cell adhesion and differentiation: antisense or competitive binding with double-stranded oligonucleotides antagonized the functions of AP-1, NF-kappaB, and PU.1 transcriptional factors. In the presence of phorbol 12-myristate 13-acetate (PMA), U937 cells attached to the plastic surface and cells were characterized by marked expression of beta2-integrin molecules on the cell surface. We show that the in vivo differentiation of U937 cells appears to occur normally in the absence of AP-1 activity. In contrast, the addition to the cell culture of phosphorothioate oligonucleotides that contained the NF-kappaB or PU.1 binding sites significantly inhibited U937 differentiation. The absence of NF-kappaB led to pleiotropic effects with a clear reduction in the expression of integrin and other lineage-specific myeloid antigens on the cell surface. In contrast, the absence of PU.1 had a more restricted effect on integrin expresion on the cell surface, probably as a result of blockage of CD18 gene expression.

Unresponsiveness to interferon associated with STAT1 protein deficiency in a gastric adenocarcinoma cell line.

HC class I expression can be up-regulated by interferons (IFN) and other cytokines. Both IFNalpha and IFNgamma have been shown to exert their effects via a recently discovered signalling pathway by inducing tyrosine phosphorylation of their receptors. Receptors for interferons and other cytokines signal through the action of associated protein tyrosine kinases of the JAK family (Janus kinase) and latent cytoplasmic transcriptional activators from the STAT family (signal transducers and activators of transcription). Here we report a gastric adenocarcinoma cell line, AGS, that is defective in its response to either IFNalpha or IFNgamma. AGS cells display selective alterations only in MHC class I inducibility and not in constitutive MHC class I expression. In nuclear extracts of AGS cells, no binding activity to interferon-responsive elements (GAS/ISRE) was observed. We found that AGS cells showed an extremely low level of STAT1 expression, which may be responsible for the absence of biological response to IFN. Because STAT1-deficient cells are highly sensitive to infection by virus, the absence of these proteins may also contribute to the tumor phenotype, giving the tumor a selective advantage, by inhibiting cell growth suppression mediated by IFN and abetting escape from the T cell antitumor response.

In vivo and in vitro generation of a new altered HLA phenotype in melanoma-tumour-cell variants expressing a single HLA-class-I allele.

A new HLA-class-I altered phenotype is described in melanoma. This phenotype is the result of a combination of HLA-B-locus down-regulation and HLA-haplotype loss. The alteration was found in 2 melanoma cell lines generated from 2 patients; one was derived from an in vivo lesion (FM37) and the other was obtained after in vitro immunoselection (R22.2). The R22.2 cell line was isolated from FM55P, a cell line derived from a primary melanoma, after in vitro treatment with a heterologous HLA-A2-restricted cytotoxic-T-lymphocyte (CTL) clone. Two additional cell lines from patient 55 were obtained from 2 s.c. metastases (FM55M1 and FM55M2). Iso-electric focusing and flow-cytometric studies showed a significant reduction in the expression of both HLA-B alleles in all cell lines studied. The expression of HLA-B-locus products recovered completely after IFN-gamma treatment of FM55P, M1 and M2. In contrast, FM37 and R22.2 tumour cells showed an additional HLA defect: the absence of one HLA haplotype. Simple tandem-repeat polymorphism markers spanning chromosome 6 showed that DNA from the 2 samples (FM37 and R22.2) showed loss of heterozygosity (LOH). In both cases, homozygosity was observed on 6p, which maps the HLA region, the final consequence being a tumour cell that expressed a single HLA-class-I allele (HLA-A3 and HLA-A1 respectively). FM37 cells may thus reflect the in vivo counterpart of resistance to lysis by HLA-A2-restricted tumour-infiltrating lymphocytes.