Cellular and subcellular localization of the small G protein RhoA in the human and rat embryonic and adult kidney

Rho proteins, a subgroup of the Ras GTPase superfamily, control many cellular processes and morphogenetic events by acting as signaling molecules in the transduction pathways of various receptors. Among the "Rho-dependent" receptors are the extracellular matrix- and growth factor-binding sites; these are particularly involved in the modulation of renal development since they control the epithelial-mesenchymal interactions that drive kidney organogenesis. The present study has addressed the immunohistochemical localization of RhoA in developing and adult kidneys of rats and humans because: a) Rho proteins are known to have a morphogenetic role, b) data in the literature on expression of Rho GTPases during mammalian histogenesis and organogenesis are scarce, and c) their involvement in the transduction pathways of receptors is implicated in kidney development. In particular, RhoA peptide was found to be localized in the mesonephric duct and vesicles in both rats and humans; metanephric anlagen were mainly stained in ampullar-derived cells. Periglomerular tubules of fetal and adult kidneys as well as collecting ducts of adult kidneys showed intense staining. Therefore, the present study provides new information on the distribution patterns of RhoA during early stages of mammalian kidney development suggesting that this signaling molecule may take part in epithelial-mesenchymal induction processes that control kidney organogenesis. RhoA expression in adult structures may be linked with renewal of renal epithelial cells and the maintenance of their morphology and polarity.     

Hyperbaric oxygenation mitigates focal cerebral injury and reduces striatal dopamine release in a rat model of transient middle cerebral artery occlusion

The usefulness of the administration of hyperbaric oxygen (HBO) in the treatment of acute focal cerebral ischemia remains debatable. A significant association exists between focal cerebral injury and an excessive release of extracellular dopamine (DA). In vivo microdialysis was used in the present study to examine the effect of HBO on DA release in the striatum during ischemia and reperfusion in rats. The histological changes occurring were also evaluated. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery (MCA) using a surgically placed intraluminal filament. Control rats (n=8) were subjected to 1 h of ischemia, whilst the study rats (n=8) were in addition treated with HBO (2.8 atmospheres of absolute pressure 100% O₂) during ischemia. Both groups were returned to breathing room air at normal pressure during reperfusion. Microdialysis samples were continuously collected at 15 min intervals at 2 μl·min^–1. The [mean (SE)] increase in release of striatal DA attained significance after 30 min of occlusion of MCA [170 (24)%], and continued to increase [268 (26)% at 45 min] reaching a peak level at 60 min [672 (59)%] before returning to the baseline level during the late reperfusion phase. There was no significant change in the level of DA in HBO treated rats during the period of ischemia. A significant reduction in edema and neuronal shrinkage were observed by histological examination in HBO treated rats when compared to the control rats. The results showed that HBO, when administered during ischemia, offered significant neuroprotection in our experimental model of transient focal cerebral ischemia in the rat. The mechanism seems to imply, at least in part, a reduced level of DA. Electronic Publication     

Interactions of human phagocytes with moulds Fusarium spp. and Verticillium nigrescens possessing different pathogenicity.

Fusarium spp. are emerging as important causes of invasive fungal infections. They tend to have decreased susceptibility to antifungal agents, making host defences very important. The ability of human phagocytes to cause damage to hyphae of Fusarium solani, F. oxysporum and Verticillium nigrescens, a mould with very low pathogenicity, was assessed using the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]2H-tetrazolium-5-carboxanilide (XTT) metabolic assay. The oxidative burst, evidenced as superoxide anion (O2-) production, of phagocytes in response to hyphae was also investigated, as well as phagocytosis of conidia by monocyte (MNC)-derived macrophages (MDM). Hyphal damage by polymorphonuclear leukocytes (PMNL) and MNC showed a linear trend increasing with effector cell:target cell (E:T) ratio. Although no significant differences were observed for PMNL, MNC-induced damage to F. solani hyphae was lower than that seen with F. oxysporum hyphae at an E:T ratio of 20:1 and with V. nigrescens hyphae at ratios of 1:1, 5:1 and 20:1 (P < 0.05). In contrast, levels of O2- production by phagocytes in response to F. oxysporum were lower than those induced in response to the other fungi (P < 0.01). The average number of V. nigrescens conidia ingested by MDM was higher than that of conidia of the other fungi (P < 0.01). Phagocytes respond to the test fungi differentially, with F. solani being the least susceptible to damage by MNC. This may correlate with the observation that, compared to the other fungi studied, it causes a relatively high incidence of infections in neutropenic patients.     

Association of interleukin 1 gene family polymorphisms with duodenal ulcer disease

Cytokine genes taking part in the immunological response to Helicobacter pylori infection are good candidates to study for genetic predisposition to duodenal ulcer disease (DU). Among cytokines, interleukin (IL)-1beta and its natural specific inhibitor, the interleukin-1 receptor antagonist, are cytokines that play a key role in regulating gastric acid secretion and modulating the immune response in the gastrointestinal mucosa. We aimed to investigate whether polymorphisms in the IL-1B and IL-1RN genes are involved in the susceptibility to duodenal ulcer. DNA from 131 unrelated Spanish Caucasian patients with DU and 105 ethnically matched healthy controls was typed for the IL-1B-511, IL-1B-31, and IL-1B + 3954 gene polymorphisms, and the VNTR polymorphism in intron 2 of the IL-1RN gene by polymerase chain reaction (PCR)-based methods and TaqMan assays. H. pylori status and non-steroidal anti-inflammatory drugs (NSAIDs) use was determined in all patients and controls. Logistic regression analysis identified H. pylori infection (OR: 9.74; 95%CI = 3.53-26.89) and NSAIDs use (OR: 8.82; 95%CI = 3.51-22.17) as independent risk factors for DU. In addition, the simultaneous carriage of IL-1RN*2, IL-1B-511*C, IL-1B-31*T and IL-1B + 3954*C alleles was a genetic risk factor for DU in patients with H. pylori infection (OR: 3.22; 95%CI = 1.09-9.47). No significant differences in IL-1RN and IL-1B genotypes were found when patients were categorized according to gender, age of onset, smoking habit, NSAIDs use, type of complication and positive family history. Our results provide further evidence that host genetic factors play a key role in the pathogenesis of duodenal ulcer.     

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.     

Encuesta nacional sobre valoración de la actividad de los médicos internos residentes en los servicios de urgencias españoles

Introduction

The care activity of internal resident doctors is common to practically all medical and surgical training programs; however, there are no national data available regarding the situation of this activity from the perspective of the resident. The present study has sought to collect the opinion of the resident internal physicians on the health care performance and the teaching character that links them to their corresponding emergency departments, in the following areas: overall training value, appreciation of the workload, characteristics of the morning rotation, supervision or tutoring, etc.

B-1 cells are pivotal for in vivo inflammatory giant cell formation

The mechanisms that govern giant cell (GC) formation in inflammatory, neoplastic and physiologic conditions are far from being understood. Here, we demonstrate that B-1 cells are essential for foreign-body GC formation in the mouse. GCs were analysed on the surface of glass cover slips implanted into the subcutaneous tissue of the animals. It was demonstrated that GCs are almost absent on cover slips implanted into the subcutaneous tissue of BALB/c or CBA/N X-linked immunodeficient mice. As these animals do not have B-1 cells in the peritoneal cavity, they were reconstituted with B-1 cells obtained from cultures of adherent mouse peritoneal cells. Results showed that in B-1-reconstituted animals, the number of GCs on the implant surface surpassed the values obtained with preparations from wild animals. In animals selectively irradiated (pleural and peritoneal cavities) to deplete these cavities of B-1 cells, GCs were also not formed. Enriched suspensions of B-1 cells grown in culture were labelled with [(3)H]-tymidine and injected into the peritoneal cavity of naive mice before implantation of glass cover slips. After 4 days, about 17% of mononuclear cells had their nuclei labelled, and almost 70% of GCs had one or more of their nuclei labelled when analysed by histoautoradiographic technique. A few GCs expressed an immunoglobulin M when analysed by immunostaining and confocal microscopy. Overall, these data demonstrate that B-1 cells are pivotal in the mechanisms of foreign-body GC formation in the mouse.     

Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.