An immunomagnetic separation/loop-mediated isothermal amplification method for rapid direct detection of thermotolerant Campylobacter spp. during poultry production.

AIMS: To develop a rapid test for thermotolerant Campylobacter in poultry faeces. METHODS AND RESULTS: The reported method is based on immunomagnetic separation and loop-mediated isothermal DNA amplification (IMS/LAMP). This LAMP assay is specific (demonstrated using 10 Campylobacter strains and 13 non-Campylobacter bacterial species) and sensitive (95% probability of detecting 22 genome copies). A competitive internal amplification control (IAC) has been incorporated to give unambiguous determination of negative results. Immunoseparation of Campylobacter allows direct LAMP detection from poultry boot swab samples in 90 min without enrichment or DNA purification (74% probability of detecting 10(4) CFU ml(-1) of a boot swab suspension). The analysis of 17 samples from commercial turkey farms showed 100% correlation with parallel results obtained by standard microbiological methods. CONCLUSIONS: A rapid test has been developed for direct detection of thermotolerant Campylobacter spp. in boot swab samples, thus bypassing culture enrichment or DNA extraction. The test has potential to be carried out by farm personnel on site. SIGNIFICANCE AND IMPACT OF THE STUDY: The method offers an inexpensive approach to monitor poultry infection in near real time, assisting flock management and controls to prevent introduction of Campylobacter into the food chain.

FXR-dependent and -independent interaction of glucocorticoids with the regulatory pathways involved in the control of bile acid handling by the liver.

Treatment with glucocorticoids (GCs) may cause adverse effects, including cholestasis. The ability of dexamethasone, prednisolone and budesonide to affect the liver handling of bile acids (BAs) has been investigated. In rats treated with GCs for 4 days, altered serum and bile BA levels, changed conjugation pattern, and delayed and decreased ability to conjugate/secrete exogenously administered deoxycholate, were found using HPLC-MS/MS. RT-QPCR analyses revealed that GC treatment also induced a down-regulation of liver nuclear receptors (Fxr, Gr and Shp), transporters (Ntcp, Mrp4 and Bcrp) and enzymes (Cyp7a1 and Baat), whereas Bsep, Mrp2 and Cyp27a1 were up-regulated. Human HepG2 and Alexander cell lines were used as in vitro models of liver cells with and without constitutive FXR expression, respectively. In HepG2 cells, GCs induced a decreased expression of FXR and SHP, and inhibited the regulatory effect of GW4064 on FXR-target genes. In Alexander cells, only when they were transfected with FXR+RXR, GW4064 caused up-regulation of SHP and OSTß, and a down-regulation of CYP27A1. GCs had the opposite effect on these genes, both in the absence and in the presence of FXR expression. Co-transfection of Alexander cells with IR-1-Luc and FXR+RXR revealed that GCs did not inhibit but moderately enhanced FXR activity. Moreover, GCs have a synergistic effect on GW4064-induced FXR activation, whereas chenodeoxycholate and GW4064 have an additive effect. In conclusion, GCs are able to directly or indirectly activate FXR but they also antagonize, through FXR-independent mechanisms, the expression of FXR and FXR target genes involved in the hepatic handling of BAs.

Diferencias histomorfológicas entre tejido gingival clínicamente sano de niños y adultos jóvenes / Differences between histomorphologic clinically healthy gingival tissue of children and young adults

Existe controversia con respecto a la influencia del factor histomorfológico en la menor severidad en la respuesta inflamatoria del tejido gingival de la población infantil con respecto a la población adulta ante la biopelícula dental. El objetivo de ésta investigación fue identificar las diferencias histomorfológicas entre el tejido gingival clínicamente sano de niños y adultos jóvenes. Se realizaron exodoncias de dientes temporales y permanentes sanos con su tejido gingival adherido a niños de 6 a 10 y adultos de 18 a 25 años de edad, para posterior análisis histomorfológico bajo microscopía de luz. El área total del epitelio de unión fue mayor en los niños presentando mayor grosor y número de capas en la zona apical. Se observó además, infiltrado celular inflamatorio adyacente al epitelio de unión, mayor cantidad de vasos, predominio de densidad laxa en las fibras del tejido conectivo y paraqueratinización del epitelio bucal. No se encontraron diferencias en la amplitud de los tejidos ni en su espesor con respecto a los epitelios bucal y surcular, tampoco en las dimensiones del tejido conectivo ni en el grosor de la capa córnea. El presente estudio confirmó la existencia de diferencias histomorfológicas entre los tejidos gingivales de niños y adultos jóvenes en condiciones de salud clínica, resaltando la importancia del factor histomorfológico, como uno de los parámetros que podrían influir en la respuesta de los tejidos gingivales a la biopelícula dental a diferentes de edades

The effect of the histomorphologic features in the minor severity of the inflammatory response in gingival tissues against the dental biofilm in childhood is matter of controversy. The objective was to identify the differences between the histomorphologic characteristics in healthy gingival tissues of children and young adults. Extraction of healthy temporal and permanent teeth and its gingival tissues were carried out in children of 6 -10 years old and adults of 18 - 25 years old, the histological samples were processed and analyzed using light microscopy. Children showed a bigger area of the junctional epithelium, more thickness of the apical area as well as a high number of union layers; presence of inflammatory cell infiltrated around the junctional ephytelium, increased number of blood vessels, predominance of laxe density of the connective tissue and pharaqueratinization of the oral ephytelium. There were no differences in the width and thickness of the oral epithelium and surcular tissues, the width of the connective tissue and the thickness of the stratum corneum did not showed differences. The present study confirmed the occurrence of histomorphologic differences in healthy gingival tissues between children and young adults, highlighting the importance of this parameter as one of the characteristics that could influence the gingival response to the dental biofilm at different age

Up-regulation of FXR isoforms is not required for stimulation of the expression of genes involved in the lack of response of colon cancer to chemotherapy.

Several mechanisms are involved in the poor response of colorectal adenocarcinoma (CRAC) to pharmacological treatment. Since preliminary evidences have suggested that the enhanced expression of farnesoid X receptor (FXR) results in the stimulation of chemoresistance, we investigated whether FXR up-regulation is required for the expression of genes that characterize the multidrug resistance (MDR) phenotype of CRAC. Samples of tumours and adjacent healthy tissues were collected from naive patients. Using Taqman Low-Density Arrays, the abundance of mRNA of 87 genes involved in MDR was determined. Relevant changes were re-evaluated by conventional RT-QPCR. In healthy tissue the major FXR isoforms were FXRα2(+/-) (80%). In tumours this predominance persisted (91%) but was accompanied by a consistent reduction (3-fold) in total FXR mRNA. A lower FXR expression was confirmed by immunostaining, in spite of which there was a significant change in the expression of MDR genes. Pharmacological challenge was simulated "in vitro" using human CRAC cells (LS174T cells). Short-term (72h) treatment with cisplatin slightly increased the almost negligible expression of FXR in wild-type LS174T cells, whereas long-term (months) treatment induced a cisplatin-resistant phenotype (LS174T/R cells), which was accompanied by a 350-fold up-regulation of FXR, mainly FXRα1(+/-). However, the changed expression of MDR genes in LS174T/R cells was not markedly affected by incubation with the FXR antagonist Z-guggulsterone. In conclusion, although the enhanced expression of FXR may be involved in the stimulation of chemoresistance that occurs during pharmacological treatment, FXR up-regulation is not required for the presence of the MDR phenotype characteristic of CRAC.

No correlation between the expression of FXR and genes involved in multidrug resistance phenotype of primary liver tumors.

Farnesoid X receptor (FXR) has been recently reported to enhance chemoresistance through bile acid-independent mechanisms. Thus, FXR transfection plus activation with GW4064 resulted in reduced sensitivity to cisplatin-induced toxicity. This is interesting because primary tumors of the liver, an organ where FXR is expressed, exhibit marked refractoriness to pharmacological treatment. Here we have determined whether FXR is upregulated in hepatocellular carcinoma (HCC), cholangiocarcinoma (CGC) and hepatoblastoma (HPB) and whether this is related with the expression of genes involved in mechanisms of chemoresistance. Using RT-QPCR and Taqman low density arrays we have analyzed biopsies from healthy livers or surgically removed tumors from naive patients and cell lines derived from HCC (SK-HEP-1, Alexander and Huh7), CGC (TFK1) and HPB (HepG2), before and after exposure to cisplatin at IC50 for 72 h. In liver tumors FXR expression was not enhanced but significantly decreased (healthy liver > HCC > HPB ≈ CGC). Except for CGC, this was not accompanied by changes in the proportions of FXR isoforms. Changes in 36 genes involved in drug uptake/efflux and metabolism, expression/function of molecular targets, and survival/apoptosis balance were found. Changes affecting SLC22A1, CYP2A1 and BIRC5 were shared by HCC, CGC and HPB. Similarity in gene expression profiles between cell lines and parent tumors was found. Pharmacological challenge with cisplatin induced changes that increased this resemblance. This was not dependent upon FXR expression. Thus, although FXR may play a role in inducing chemoresistance under certain circumstances, its upregulation does not seem to be involved in the multidrug resistance phenotype characteristic of HCC, CGC and HPB.

Molecular bases of liver cancer refractoriness to pharmacological treatment.

Hepatocellular carcinoma and cholangiocarcinoma are the two most important primary malignancies of the liver. These are among the tumours with the lowest response to pharmacological treatment based on currently available drugs. This is due either to the existence of refractoriness of the initial tumour or to the ability of cancer cells to develop chemoresistance during treatment. Liver cancers share some of the mechanisms responsible for drug refractoriness with other types of tumours, such as a reduction in drug uptake; enhanced drug export; intracellular inactivation of the active agent; alteration of the molecular target; an increase in the activity of the target route to be inhibited, or the appearance or stimulation of alternative routes; enhanced repair of drug-induced modifications in the target molecules, and the activation/ inhibition of intracellular signalling pathways, all of which lead to a negative balance between the apoptosis/survival of tumour cells. The aim of the present article is to review how these mechanisms of chemoresistance affect the different families of drugs that are being or have been used to treat hepatocellular carcinoma and cholangiocarcinoma. A better understanding of the molecular bases of drug refractoriness is needed in order to develop novel drugs or pharmacological strategies aimed at overcoming resistance to anticancer agents.

Overview of the molecular bases of resistance to chemotherapy in liver and gastrointestinal tumours.

Primary malignancies of the liver and the gastrointestinal tract constitute one of the main health problems worldwide. Together, these types of tumour are the first cause of death due to cancer, followed by lung and breast cancer respectively. One important limitation in the treatment of these tumours is that, with a few exceptions, they exhibit marked resistance to currently available drugs. Moreover, most of them develop chemoresistance during treatment. The mechanisms responsible for drug refractoriness in gastrointestinal tumours include a reduction in drug uptake; enhanced drug export; intracellular inactivation of the effective agent; alteration of the molecular target; an increase in the activity of the target route to be inhibited or the appearance or stimulation of alternative routes; enhanced repair of drug-induced modifications in the target molecules, and the activation/inhibition of intracellular signalling pathways, which leads to a negative balance between the apoptosis/survival of tumour cells. A better understanding of these mechanisms is needed in order to develop accurate tests to predict the lack of response to chemotherapy and novel approaches aimed at overcoming resistance to anticancer agents. The purpose of the present review is to offer an updated overview of the molecular mechanisms of resistance to cytostatic drugs in the most frequent types of primary malignant tumour affecting the liver and gastrointestinal tract.

[Bilateral anterior opercular syndrome as a manifestation of a non-convulsive epileptic state]. / Síndrome opercular anterior como manifestación de estado epiléptico no convulsivo.

INTRODUCTION: Foix-Chavany-Marie syndrome, or bilateral anterior opercular syndrome, is characterised by facio-pharyngo-glosso-masticatory diplegia with 'automatic-voluntary dissociation', which consists in the abolition of voluntary movements while involuntary movements and reflexes are preserved. It is produced by bilateral involvement of the anterior or frontal opercular region. In adults it is related to ischemic lesions. In childhood it presents congenitally in perisylvian dysplasias and as an acquired disorder in encephalitis or can be episodic in symptomatic or idiopathic epilepsies such as benign rolandic epilepsy. CASE REPORT: A 13-year-old patient who presented, over five straight days, four episodes of facial dysplegia, anarthria, dysphagia, drooling, paralysis of the upper limbs, while involuntary facial expression was normal and the corneal, cough and gag reflexes were preserved. The first three come to an end spontaneously at 2, 4 and 20 hours, respectively; the fourth episode concluded an hour and a half after onset, following administration of intravenous phenytoin for 5 minutes. Computerised axial tomography and magnetic resonance images of the brain, as well as the interictal electroencephalograms (EEG), were normal. Administration of oxcarbazepine was started but at 8 months was stopped after a normal EEG during nocturnal sleep was obtained. After 15 months, the patient has not presented any more episodes. CONCLUSIONS: The paroxysmal character of the disorder together with normal interictal periods, the normality of the neuroimages, and the speedy recovery achieved after the administration of phenytoin support the notion of an epileptic origin. We believe that we are dealing with a bilateral anterior opercular syndrome due to a non-convulsive epileptic state, compatible with the presentation of benign rolandic epilepsy.

[Complexity index and analysis of digestive endoscopies at a university hospital]. / Indice de complejidad y análisis de los costes de las endoscopias digestivas en un hospital universitario.

BACKGROUND: In Spain neither the cost of digestive endoscopies nor the complexity index (CI) have been objectively estimated. Consequently, the profitability of an endoscopy unit cannot be evaluated. OBJECTIVES: 1: To classify endoscopies according to the time taken to accomplish them in order to estimate the CI and determine the relative value unit (RVU). 2: To evaluate the cost of each examination in order to determine the profitability of endoscopy as a diagnostic and therapeutic tool. 3: To measure unnecessary delays in examinations and to determine whether there are differences in the time an examination takes according to the endoscopist's experience and the patient's age or sex. MATERIAL AND METHODS: The cost per hour of an endoscopy room was estimated by dividing the cost per year of employees, equipment, maintenance and the hospital's general costs between the hours of work per year of the endoscopy team. The time taken to perform endoscopies was estimated and the result was used to calculate the RVI, the CI and the cost of 500 consecutive endoscopies. RESULTS: The annual cost amounted to 349,617.69 E. Staffing costs represented 65.5%, of which 56.3% were direct costs. Gastroscopy was taken as the RVU (cost = 27.52 E). Anoscopy was the simplest procedure (RVU = 0.61, cost = 15.08 E) and colonoscopy plus polypectomy was the most complex procedure (RVU = 4.41, cost = 74.28 E). The greater the experience of the member of staff performing the procedure, the less time it took (p < 0.01). CONCLUSION: CI was lowest for anoscopy (URV = 0.61) and highest for total colonoscopy plus polypectomy (URV = 4.41). Cost and exploration time depended on the endoscopist's experience. Staff represented the highest percentage of cost.

A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis.

In normal epidermis, beta1 integrin expression is confined to the basal layer, whereas in hyperproliferative epidermis, integrins are also expressed in the suprabasal layers. Transgenic mice in which integrins are expressed suprabasally via the involucrin promoter have a sporadic psoriatic phenotype; however, the mechanism by which integrins contribute to the pathogenesis of psoriasis is unknown. We observed activation of mitogen-activated protein kinase (MAPK) in basal and suprabasal keratinocytes of human and transgenic mouse psoriatic lesions and healing mouse skin wounds, correlating in each case with suprabasal integrin expression. Phenotypically normal human and transgenic mouse epidermis did not contain activated MAPK. Transgene-positive keratinocytes produced more IL-1alpha than controls did, and keratinocyte MAPK could be activated by ligation of suprabasal integrins or treatment with IL-1alpha. Constitutive activation of MAPK increased the growth rate of human keratinocytes and delayed the onset of terminal differentiation, recreating many of the histological features of psoriatic epidermis. We propose that activation of MAPK by integrins, either directly or through increased IL-1alpha production, is responsible for epidermal hyperproliferation in psoriasis and wound healing, and that the sporadic phenotype of the transgenic mice may reflect the complex mechanisms by which IL-1 release and responsiveness are controlled in skin.

Analysis of cultured keratinocytes from a transgenic mouse model of psoriasis: effects of suprabasal integrin expression on keratinocyte adhesion, proliferation and terminal differentiation.

Many important transgenic mouse models of benign and neoplastic skin diseases have been generated through the use of promoters that target transgene expression to the different epidermal layers. However, more mechanistic studies of the specific effects of the transgenes on keratinocytes have been hampered by difficulties in culturing keratinocytes from adult mouse epidermis and by the low differentiation potential of many established mouse keratinocyte lines. We have used the Rheinwald & Green technique to cultivate primary adult keratinocytes and to generate keratinocyte lines from transgenic mice which have a sporadic psoriatic phenotype due to expression of human integrin subunits under the control of the involucrin promoter. We show that the transgenes are induced when keratinocytes are placed in suspension and that the transgenic integrins are capable of clustering in focal adhesions and mediating cell adhesion and spreading. We also show that suprabasal integrin expression has no direct effect on proliferation of cells in the underlying basal layer, ruling this out as a possible explanation for the epidermal hyperproliferation observed in the transgenic mice.

Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.

Integrin expression is normally confined to the basal layer of the epidermis, but when epidermal homeostasis is perturbed, the receptors are also expressed by suprabasal, differentiating keratinocytes. We have used the involucrin promoter to express functional human integrin subunits alpha 2, alpha 5, and beta 1 in the suprabasal epidermal layers of transgenic mice. In mice expressing alpha 5 or beta 1 alone or alpha 2 beta 1 or alpha 5 beta 1 heterodimers, there were hair and whisker abnormalities and a failure of eyelid fusion. In addition, mice expressing beta 1 alone or in combination with alpha 2 or alpha 5 exhibited epidermal hyper-proliferation, perturbed keratinocyte differentiation, and skin inflammation, all of which are features of a common human skin disease, psoriasis.

Alpha/beta inversion of the Euplotes and Oxytricha tubulins.

Immunoblotting studies, using a polyclonal antibody specific for the alpha-tubulin of ciliates and an anti-beta-tubulin monoclonal antibody, demonstrated that the tubulins of the ciliates Euplotes and Oxytricha show alpha/beta inversion although less accentuated than that observed in Paramecium and Tetrahymena. Results suggest that (a) the alpha/beta inversion may be a common characteristic within the Phylum Ciliophora; and (b) the electrophoretic behaviour of the alpha-tubulin may be useful for establishing evolutionary relatedness between the ciliates.