Unexpectedly late expression of intracellular CD3epsilon and TCR gammadelta proteins during adult thymus development.

During adult thymus development immature CD4(-)CD8(-) [double-negative (DN)] precursor cells pass through four phenotypically distinct stages defined by expression of CD44 and CD25: CD44(hi)CD25(-) (DN1), CD44(hi)CD25(+) (DN2), CD44(lo)CD25(+) (DN3) and CD44(lo)CD25(-) (DN4). Although it is well established that the TCR beta, gamma and delta genes are rearranged and expressed in association with the CD3 components in DN thymocytes, the precise timing of expression of the TCR and CD3 proteins has not been determined. In this report we have utilized a sensitive intracellular (ic) staining technique to analyze the expression of ic CD3epsilon, TCR beta and TCR gammadelta proteins in immature DN subsets. As expected from previous studies of TCR beta rearrangement and mRNA expression, icTCR beta(+) cells were first detected in the DN3 subset and their proportion increased thereafter. Surprisingly, however, both icCD3epsilon(+) and icTCR gammadelta(+) cells were detected at later stages of development than was predicted by molecular studies. In particular icCD3epsilon protein expression coincided with the transition from the DN2 to DN3 stage of development, whereas icTCR gammadelta protein expression was only detected in a minor subset of DN4 cells. The implications of these findings for alphabeta lineage divergence will be discussed.

Immunity to intracellular bacteria

Immunity to intracellular bacteria including Mycobacterium tuberculosis. Mycobacterium leprae, and Listeria monocytogenes depends on specific T cells. Evidence to be described suggests that CD4 (alpha/beta)T cells which interact with each other and with macrophages contribute to acquired resistence against as well as pathogenesis of intracellular bacterial infections.

Ligand binding transmits conformational changes across the membrane-spanning region to the intracellular side of the 5-HT3 serotonin receptor.

Conformational changes of channel activation: Five enhanced green fluorescent protein (EGFP) molecules (green cylinders) were integrated into the intracellular part of the homopentameric ionotropic 5-HT3 receptor. This allowed the detection of extracellular binding of fluorescent ligands (?) to EGFP by FRET, and also enabled the quantification of agonist-induced conformational changes in the intracellular region of the receptor by homo-FRET between EGFPs. The approach opens novel ways for probing receptor activation and functional screening of therapeutic compounds.

Systemic effects of epidural Methylprednisolone injection on glucose tolerance in diabetic patients.

ABSTRACT: BACKGROUND: Several studies have shown that in diabetic patients, the glycemic profile was disturbed after intra-articular injection of corticosteroids. Little is known about the impact of epidural injection in such patients. The goal of this study was double, at first comparing the glycaemic profile in diabetic patients after a unique injection of 80 mg of acetate methylprednisolone either intra-articular or epidural and secondly to compare the amount of systemic diffusion of the drug after both procedures. METHODS: Seventeen patients were included. Glycemic changes were compared in 9 diabetic patients following intra-articular (4 patients) and epidural injections (5 patients). Epidural injections were performed using the sacral route under fluoroscopic control in patients with lumbar spinal stenosis. Diabetes control had to stable for more than 10 days and the renal function to be preserved. Blood glucose was monitored using a validated continuous measuring device (GMS, Medtronic) the day before and for two days following the injection. Results were expressed in the form of daily glycemic profiles and as by mean, peak and minimal values +/ SD. The urinary excretion of methylprednisolone after the 2 routes of injection was analyzed in 8 patients (4 in each group). Urine samples were cropped one hour before the injections, then 4 times during the first day and 3 times a week for 2 weeks. The measurements included the free and conjugated fraction RESULTS: The glycaemic profile remains unchanged with no significant changes in the group of the 5 diabetic patients receiving epidural injections. On the other end, the average peak and mean values were enhanced up to 3 mmol/l above baseline two days after the infiltration in the groups of the 4 diabetic patients infiltrated intra-articular. The mean urinary excretion of the steroid was about ten times higher in the intra-articular versus epidural group: 7000 ng/ml versus 700 ng/ml. Looking at each individual there were marked differences especially after intra-articular injections. CONCLUSION: This is the first study to show that a single epidural steroid injection of 80 mg depot methylprednisolone had no effect on the glycemic control in diabetic patients. The absence of glycemic control changes correlated well with the very low urinary excretion of the drug after epidural injection. Trial registration NCT01420497.

Intracellular bacteria and adverse pregnancy outcomes.

Clin Microbiol Infect 2011; 17: 1312-1322 ABSTRACT: This review considers the role of intracellular bacteria in adverse pregnancy outcomes, such as miscarriage, stillbirths, and preterm labour. The cause of miscarriage, stillbirth and preterm labour often remains unexplained. Intracellular bacteria that grow either poorly or not at all on media used routinely to detect human pathogens could be the aetiological agents of these obstetric conditions. For example, Listeria monocytogenes and Coxiella burnetti are intracellular bacteria that have a predilection for the fetomaternal unit and may induce fatal disease in the mother and/or fetus. Both are important foodborne or zoonotic pathogens in pregnancy. Preventive measures, diagnostic tools and treatment will be reviewed. Moreover, we will also address the importance in adverse pregnancy outcomes of other intracellular bacteria, including Brucella abortus and various members of the order Chlamydiales. Indeed, there is growing evidence that Chlamydia trachomatis, Chlamydia abortus and Chlamydia pneumoniae infections may also result in adverse pregnancy outcomes in humans and/or animals. Moreover, newly discovered Chlamydia-like organisms have recently emerged as new pathogens of both animals and humans. For example, Waddlia chondrophila, a Chlamydia-related bacterium isolated from aborted bovine fetuses, has also been implicated in human miscarriages. Future research should help us to better understand the pathophysiology of adverse pregnancy outcomes caused by intracellular bacteria and to determine the precise mode of transmission of newly identified bacteria, such as Waddlia and Parachlamydia. These emerging pathogens may represent the tip of the iceberg of a large number of as yet unknown intracellular pathogenic agents.

Mechanisms of formation and function of eosinophil lipid bodies: inducible intracellular sites involved in arachidonic acid metabolism

Lipid bodies, inducible lipid-rich cytoplasmic inclusions, are characteristically abundant in cells associated with inflammation, including eosinophils. Here we reviewed the formation and function of lipid bodies in human eosinophils. We now have evidence that the formation of lipid bodies is not attributable to adverse mechanisms, but is centrally mediated by specific signal transduction pathways. Arachidonic acid and other cis fatty acids by an NSAID-inhibitable process, diglycerides, and PAF by a 5-lipoxygenase dependent pathway are potent stimulators of lipid body induction. Lipid body formation develops rapidly by processes that involve PKC, PLC, and de novo mRNA and protein synthesis. These structures clearly serve as repositoires of arachidonyl-phospholipids and are more than inert depots. Specific enzymes, including cytosolic phospholipase A2, MAP kinases, lipoxygenases and cyclooxygenases, associate with lipid bodies. Lipid bodies appear to be dynamic, organelle-like structures involved in intracellular pathways of lipid mobilization and metabolism. Indeed, increases in lipid body numbers correlated with enhanced production of both lipoxygenase- and cyclooxygenase-derived eicosanoids. We hypothesize that lipid bodies are distinct inducible sites for generating eicosanoids as paracrine mediators with varied activities in inflammation. The capacity of lipid body formation to be specifically and rapidly induced in leukocytes enhances eicosanoid mediator formation, and conversely pharmacologic inhibition of lipid body induction represents a potential novel and specific target for anti-inflammatory therapy.

Cellular pathology of hilar neurons in Ammon's horn sclerosis.

In addition to functionally affected neuronal signaling pathways, altered axonal, dendritic, and synaptic morphology may contribute to hippocampal hyperexcitability in chronic mesial temporal lobe epilepsies (MTLE). The sclerotic hippocampus in Ammon's horn sclerosis (AHS)-associated MTLE, which shows segmental neuronal cell loss, axonal reorganization, and astrogliosis, would appear particularly susceptible to such changes. To characterize the cellular hippocampal pathology in MTLE, we have analyzed hilar neurons in surgical hippocampus specimens from patients with MTLE. Anatomically well-preserved hippocampal specimens from patients with AHS (n = 44) and from patients with focal temporal lesions (non-AHS; n = 20) were studied using confocal laser scanning microscopy (CFLSM) and electron microscopy (EM). Hippocampal samples from three tumor patients without chronic epilepsies and autopsy samples were used as controls. Using intracellular Lucifer Yellow injection and CFLSM, spiny pyramidal, multipolar, and mossy cells as well as non-spiny multipolar neurons have been identified as major hilar cell types in controls and lesion-associated MTLE specimens. In contrast, none of the hilar neurons from AHS specimens displayed a morphology reminiscent of mossy cells. In AHS, a major portion of the pyramidal and multipolar neurons showed extensive dendritic ramification and periodic nodular swellings of dendritic shafts. EM analysis confirmed the altered cellular morphology, with an accumulation of cytoskeletal filaments and increased numbers of mitochondria as the most prominent findings. To characterize cytoskeletal alterations in hilar neurons further, immunohistochemical reactions for neurofilament proteins (NFP), microtubule-associated proteins, and tau were performed. This analysis specifically identified large and atypical hilar neurons with an accumulation of low weight NFP. Our data demonstrate striking structural alterations in hilar neurons of patients with AHS compared with controls and non-sclerotic MTLE specimens. Such changes may develop during cellular reorganization in the epileptogenic hippocampus and are likely to contribute to the pathogenesis or maintenance of temporal lobe epilepsy.

Glucocorticoids induce retinal toxicity through mechanisms mainly associated with paraptosis.

PURPOSE: Corticosteroids have recorded beneficial clinical effects and are widely used in medicine. In ophthalmology, besides their treatment benefits, side effects, including ocular toxicity have been observed especially when intraocular delivery is used. The mechanism of these toxic events remains, however, poorly understood. In our present study, we investigated the mechanisms and potential pathways of corticosteroid-induced retinal cell death. METHODS: Rats were sacrificed 24 h and 8 days after an intravitreous injection of 1 microl (40 microg) of Kenacort Retard. The eyes were processed for ultra structure analysis and detection of activated caspase-3, cytochrome-C, apoptosis-inducing factor (AIF), LEI-L-Dnase II, terminal transferase dUTP nick end labeling (TUNEL), and microtubule-associated protein 1-light chain 3 (MAP-LC3). In vitro, rat retinal pigment epithelial cells (RPE), retinal Müller glial cells (RMG) and human ARPE-19 cells were treated with triamcinolone acetonide (TA) or other glucocorticoids. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) assay and cell counts. Nuclei staining, TUNEL assay, annexin-V binding, activated caspase-3 and lactate dehydrogenase (LDH) production characterized cell death. Localization of cytochrome-C, AIF, LEI-and L-Dnase II, and staining with MAP-LC3 or monodansylcadaverine were also carried out. Finally, ARPE-19 cells transfected with AIP-1/Alix were exposed to TA. RESULTS: In vitro incubation of retinal cell in the presence of corticosteroids induced a specific and dose-dependent reduction of cell viability. These toxic events were not associated with the anti-inflammatory activity of these compounds but depended on the hydro solubility of their formulation. Before cell death, extensive cytoplasmic vacuolization was observed in the retinal pigment epithelial (RPE) cells in vivo and in vitro. The cells however, did not show known caspase-dependent or caspase-independent apoptotic reactions. These intracellular vacuoles were negative for MAP-LC3 but some stained positive for monodansylcadaverine. Furthermore, over expression of AIP-1/Alix inhibited RPE cell death. CONCLUSIONS: These observations suggest that corticosteroid-induced retinal cell death may be carried out mainly through a paraptosis pathway.

Drug uptake in a rodent sarcoma model after intravenous injection or isolated lung perfusion of free/liposomal doxorubicin.

The distribution of free and liposomal doxorubicin (Liporubicin) administered by intravenous injection (IV) or isolated lung perfusion (ILP) was compared in normal and tumor tissues of sarcoma bearing rodent lungs. A single sarcomatous tumor was generated in the left lung of 35 Fischer rats, followed 10 days later by left-sided ILP (n=20) or IV drug administration (n=12), using 100 microg and 400 microg free or liposomal doxorubicin, respectively. The tumor and lung tissue drug concentration was measured by HPLC. Free doxorubicin administered by ILP resulted in a three-fold (100 microg) and 10-fold (400 microg) increase of the drug concentration in the tumor and normal lung tissue compared to IV administration. In contrast, ILP with Liporubicin resulted in a similar drug uptake in the tumor and lung tissue compared to IV injection. For both drug formulations and dosages, ILP resulted in a higher tumor to lung tissue drug ratio but also in a higher spatial heterogeneity of drug distribution within the lung compared to IV administration. ILP resulted in a higher tumor to lung tissue drug ratio and in a more heterogeneous drug distribution within the lung compared to IV drug administration.

Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes.

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (i) the hyperglycemic response to i.p. glucagon injection; (ii) the in vivo measurement of glucose turnover rate; and (iii) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [14C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [14C]glucose, we demonstrated that this pathway was sensitive to low temperature (12 degreesC). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.

Organometallic cages as vehicles for intracellular release of photosensitizers.

Water-soluble metalla-cages were used to deliver hydrophobic porphin molecules to cancer cells. After internalization, the photosensitizer was photoactivated, significantly increasing the cytotoxicity in cells. During the transport, the photosensitizer remains nonreactive to light, offering a new strategy to tackle overall photosensitization, a limitation often encountered in photodynamic therapy.

Discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Evaluation of tumor vascularisation in two rat sarcoma models for studying isolated lung perfusion : Injection route determines the origin of tumour vessels

Résumé Introduction: La perfusion isolée cytostatique du poumon est une technique attractive qui permet l'administration des doses élevées d'un agent cytostatique tout en épargnant dans la mesure du possible la circulation systémique. Cependant, la perfusion de l'artère pulmonaire risque d'épargner le territoire pulmonaire vascularisé par l'intermédiaire des artères bronchiques, ce qui pourrait diminuer l'efficacité de ce traitement au cas où la lésion ciblée est vascularisée par les artères bronchiques. Ce travail est destiné au développement d'un modèle tumoral au niveau des poumons de rongeur (rat) porteur d'un sarcome pulmonaire afin de déterminer si la voie d'injection des cellules tumorales (intraveineuse, versus intratumorale) influencera la vascularisation des tumeurs (provenant du système artères pulmonaires ou artères bronchiques). Méthod: Des tumeurs de sarcomes pulmonaires ont été générées par injection d'une suspension cellulaire de sarcome, soit par injection intraveineuse, soit directement dans le parenchyme pulmonaire par thoracotomie. Ensuite, une perfusion isolée du poumon porteur de la tumeur à l'aide de l'encre a été effectuée, soit par l'artère pulmonaire, soit par le système des artères bronchiques. La distribution de l'encre dans les vaisseaux tumoraux ainsi que dans les vaisseaux non tumoraux du poumon adjacent a été investiguée à l'aide d'une analyse histologique des poumons perfusés. Résultat: L'administration intraveineuse et intratumorale de la suspension de cellules tumorales résulte en des tumeurs similaires sur le plan histologique. Néanmoins, l'injection intra-parenchymateuse démontre des tumeurs plus homogènes et avec un développement plus prédictible, était associée à une survie plus longue qu'après injection intraveineuse. Les analyses histologiques après perfusion isolée à l'aide de l'encre démontre que les tumeurs résultant de l'injection intraveineuse ont développé une vascularisation se basant sur le système d'artères pulmonaires tandis que les tumeurs émergeant après injection intraparenchymateuse ont développé une vascularisation provenant du système des artères bronchiques. Conclusion: Ce travail démontre pour la première fois l'importance du mode de génération de tumeurs pulmonaires en ce qui concerne leur future vascularisation, ce qui pourrait avoir un impact sur leur traitement par perfusion isolée du poumon. Abstract Isolated cytostatic lung perfusion (ILP) is an attractive technique allowing delivery of a high-dose of cytostatic agents to the lungs while limiting systemic toxicity. In developing a rat model of ILP, we have analysed the effect of the route of tumour cell injection on the source of tumour vessels. Pulmonary sarcomas were estab¬lished by injecting a sarcoma cell suspension either by the intravenous (i.v.) route or directly into the lung paren¬chyma. Ink perfusion through either pulmonary artery (PA) or bronchial arteries (BA) was performed and the characteristics of the tumour deposits defined. i.v. and direct injection methods induced pulmonary sarcoma nodules, with similar histological features. The intraparenchymal injection of tumour cells resulted in more reli¬able and reproducible tumour growth and was associat¬ed with a longer survival of the animals. i.v. injected tumours developed a PA-derived vascular tree whereas directly injected tumours developed a BA-derived vasculature.