Expansion of highly activated invariant natural killer T cells with altered phenotype in acute dengue infection.

Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)-γ and interleukin (IL)-4 ex-vivo enzyme-linked immunospot (ELISPOT) assays following stimulation with alpha-galactosyl-ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4(+) subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus-specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl-6 (P = 0·0003) and both Bcl-6 and inducible T cell co-stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4(+) iNKT cells, with reduced expression of CD161 markers.

Training and practice in bronchoscopy a national survey in Italy.

BACKGROUND AND AIM: Bronchoscopy is performed in a variety of different settings in Italy. The surveys conducted so far have highlighted the heterogeneity of the procedures and the frequent inability to adhere to the guidelines. The aim of this survey was to analyse procedures, training, and opinions of Italian respiratory physicians performing interventional bronchology in the clinical practice. METHODS: The study was conducted retrospectively on 300 pulmonologists. From January to June 2008, these were invited to participate in an email survey to be sent out monthly to each participant for four consecutive months. RESULTS: Two hundred and one respiratory physicians took part in the study, most of whom (83.5%) work in either Pulmonology or Interventional Pulmonology Units. The year before the survey, 21.2% of the participants had performed fewer than 100 examinations, 42.3% 100 to 300, and 36.6% more than 300 bronchoscopies; 53.9% were familiar with the international guidelines on the topic. Among the responders, 34.1% had received less than 6 months training, 55.3% considered further training in rigid bronchoscopy, laser procedures and thoracoscopy, invaluable for their professional activity. Adequate training for transbronchial needle aspirates, was reported by 49.6% of respondents. CONCLUSIONS: Our data show that interventional bronchoscopy procedures are regularly performed according to current recommendations by over half of the Italian Pulmonologists participating in our survey. The need for more comprehensive basic education and training was put forward by the majority of physicians.

The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.

Degradation of T cell receptor (TCR)-CD3-zeta complexes after antigenic stimulation.

T cell activation by specific antigen results in a rapid and long-lasting downregulation of triggered T cell receptors (TCRs). In this work, we investigated the fate of downregulated TCR- CD3-zeta complexes. T cells stimulated by peptide-pulsed antigen-presenting cells (APCs) undergo an antigen dose-dependent decrease of the total cellular content of TCR-beta, CD3-epsilon, and zeta chains, as detected by FACS(R) analysis on fixed and permeabilized T-APC conjugates and by Western blot analysis on cell lysates. The time course of CD3-zeta chain consumption overlaps with that of TCR downregulation, indicating that internalized TCR-CD3 complexes are promptly degraded. Inhibitors of lysosomal function (bafilomycin A1, folimycin) markedly reduced zeta chain degradation, leading to the accumulation of zeta chain in large Lamp1(+) vesicles. These results indicate that in T cell-APC conjugates, triggered TCRs are rapidly removed from the cell surface and are degraded in the lysosomal compartment.

Quantitative contribution of CD4 and CD8 to T cell antigen receptor serial triggering.

CD4 and CD8 are thought to function as coreceptors by binding to the cognate major histocompatibility complex (MHC) molecules recognized by the T cell antigen receptor (TCR) and initiating the signal transduction cascade. We report that during T cell-antigen-presenting cell interaction, triggered TCRs and coreceptors are downregulated and degraded with identical kinetics. This coordinated disappearance takes place whenever the TCR is triggered, even when the coreceptor does not engage the cognate MHC molecule and is the consequence of binding of the coreceptor-associated Lck to ZAP-70. The interaction of coreceptor and cognate MHC molecules is dispensable when T cells are stimulated by optimal ligands, but becomes crucial when suboptimal ligands are used. In the latter case the coreceptor increases the efficiency of TCR triggering without changing the activation threshold or the quality of the T cell response.

Maturation, activation, and protection of dendritic cells induced by double-stranded RNA.

The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses.

Essential and partially overlapping role of CD3gamma and CD3delta for development of alphabeta and gammadelta T lymphocytes.

CD3gamma and CD3delta are two highly related components of the T cell receptor (TCR)-CD3 complex which is essential for the assembly and signal transduction of the T cell receptor on mature T cells. In gene knockout mice deficient in either CD3delta or CD3gamma, early thymic development mediated by pre-TCR was either undisturbed or severely blocked, respectively, and small numbers of TCR-alphabeta+ T cells were detected in the periphery of both mice. gammadelta T cell development was either normal in CD3delta-/- mice or partially blocked in CD3gamma-/- mice. To examine the collective role of CD3gamma and CD3delta in the assembly and function of pre-TCR and in the development of gammadelta T cells, we generated a mouse strain with a disruption in both CD3gamma and CD3delta genes (CD3gammadelta-/-). In contrast to mice deficient in either CD3gamma or CD3delta chains, early thymic development mediated by pre-TCR is completely blocked, and TCR-alphabeta+ or TCR-gammadelta+ T cells were absent in the CD3gammadelta-/- mice. Taken together, these studies demonstrated that CD3gamma and CD3delta play an essential, yet partially overlapping, role in the development of both alphabeta and gammadelta T cell lineages.

Selective inhibition of Ii-dependent antigen presentation by Helicobacter pylori toxin VacA.

A major virulence factor in the stomach chronic infection by Helicobacter pylori is a protein toxin (VacA), which alters cell membrane trafficking of late endosomal/prelysosomal compartments. Its role in the chronic infection established by H. pylori is unknown. To test the possibility that VacA alters antigen processing taking place in prelysosomal compartments, we have used the well-established model of antigen processing and presentation consisting of tetanus toxoid-specific human (CD4(+)) T cells stimulated by autologous antigen-pulsed Epstein-Barr virus-transformed B cells. We found that VacA interferes with proteolytic processing of tetanus toxin and toxoid and specifically inhibits the Ii-dependent pathway of antigen presentation mediated by newly synthesized major histocompatibility complex (MHC) class II, while leaving unaffected the presentation pathway dependent on recycling MHC class II. The results presented here suggest that VacA may contribute to the persistence of H. pylori by interfering with protective immunity and that this toxin is a new useful tool in the study of the different pathways of antigen presentation.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Regression of diabetic complications by islet transplantation in the rat.

AIMS/HYPOTHESIS: Type 1 diabetes is a chronic disease leading to complications such as peripheral neuropathies, nephropathy and cardiovascular disease. Pancreatic islet transplantation is being extensively investigated for blood glucose control in animals and in human type 1 diabetic patients, but the question of whether it can reverse long-term diabetic complications has not been fully explored. We investigated the effects of islet transplantation on diabetic complications in a rat model of streptozotocin-induced diabetes. METHODS: Three groups of rats were used: healthy controls, diabetic and diabetic rats transplanted with microencapsulated islets at 2 months after diabetes induction, when neuropathy was detectable by a decrease in tail nerve conduction velocity (NCV) and impaired nociceptive thresholds. Blood glucose levels and body weight were measured weekly. The variables considered were: thermal (hot plate test) and mechanical sensitivity (Randal-Selitto paw withdrawal test), NCV and Na+, K+-ATPase activity in the sciatic nerve. At the end of the experiments hearts were removed for morphometric determination and myocyte number, and kidneys removed for histological examination. RESULTS: Islet transplantation in diabetic rats induced normoglycaemia in a few days, accompanied by a rapid rise in body weight and amelioration of impaired nociceptive thresholds, as well as normalisation of NCV and Na(+), K(+)-ATPase, which were both about 25% below normal in diabetic rats. Myocyte loss was reduced (-34%) by islet transplantation and the observed mild kidney damage of diabetic rats was prevented. CONCLUSIONS/INTERPRETATION: Besides controlling glycaemia, transplantation of microencapsulated pancreatic islets induced almost complete regression of neuropathy and prevented cardiovascular alterations.

Incidence of complications in bronchoscopy. Multicentre prospective study of 20,986 bronchoscopies.

AIM: To evaluate the frequency of complications in bronchoscopy from data compiled between 1/2/2002 to 1/2/2003. MATERIALS AND METHODS: Nineteen Italian centres of thoracic endoscopy participated in the study, for a total of 20,986 bronchoscopies (FBS), including 10,658 explorative bronchoscopies (EB) (50.79%), 5,520 bronchial biopsies (BB) (26.30%), 1,660 transbronchial biopsies (TBB) (7.91%), 1,127 broncho-alveolar lavages (BAL) (5.37%), 930 transbronchial needle-aspirates (TBNA) (4.43%), 1.091 therapeutic bronchoscopies (TB), comprising ND-YAG Laser, argon-plasma, electrocautery knife, stent insertion (5.20%). 82.4% of the procedures involved the use of a flexible bronchoscope, 16.3% were carried out using a rigid bronchoscope and 1.3% using the mixed technique. RESULTS: The total number of complications recorded was 227 (1.08% of the cases examined), including 20 (0.09%) during local anesthesia and pre-medication, 195 (0.92%) during the endoscopic procedures and 12 (0.05%) in the two hours following FBS. The total number of deaths was 4 (0.02%), due to cardiac arrest, pulmonary edema, delayed respiratory failure and shock in pre-medication, respectively. 68.28% of the complications were treated medically, 25.99% by means of endoscopy and 5.72% with surgery. The healing percentage was 98.2%. CONCLUSIONS: This study has shown that bronchoscopy is a safe method with low incidence of mortality and complications. The preparation, experience and continuous training of the operators of the medical and nursing team seem to play a fundamental role in reducing the incidence of complications at least in certain procedures such as BB and TBB.

Harnessing NKT cells for therapeutic applications.

Activation of NKT cells leads to the maturation of dendritic cells and efficiently assists priming of antigen-specific immune responses. The lack of polymorphism of CDld molecules and the evolutionary conservation of NKT cell responses highlight the important role of these cells in bridging innate and adaptive immune responses and advocate the value of harnessing this system in clinical settings. Compounds capable of fine tuning NKT cell activation should be actively exploited as potent adjuvants in vaccination strategies or as immunomodulators of autoimmune diseases.

Chest wall involvement by lung cancer: computed tomographic detection and results of operation.

The aim of this prospective study was to evaluate: (1) the role of computed tomographic scanning in predicting chest wall invasion by peripheral lung cancer and (2) the results of operation according to the depth of chest wall involvement and other potential indicators of long-term survival. One hundred twelve patients with non-small cell lung cancer adjacent to the pleural surface who underwent computed tomographic scanning and subsequent thoracotomy were entered into this study. Tumor invasion was confined to the visceral pleura in 53 patients, to the parietal pleura in 18 patients, and to intercostal muscles in 25 patients; invasion extended beyond this layer in 16 patients. The computed tomographic criteria for chest wall invasion were (1) obliteration of the extrapleural fat plane, (2) the length of the tumor-pleura contact, (3) the ratio between the tumor-pleura contact and the tumor diameter, (4) the angle of the tumor with the pleura, (5) a mass involving the chest wall, and (6) rib destruction. The computed tomographic criteria 1 and 3 were significantly related to pathologic findings. Sensitivity was 85% for criterion 1 and 83% for criterion 3, specificity being 87% and 80%, respectively. Long-term survival of patients with T3 disease critically depended on the lymph node state and completeness of resection. The adenocarcinoma cell type and the T4 category were unfavorable prognostic factors. The depth of chest wall invasion did not affect survival, except for extensive rib and soft tissue infiltration. En bloc resection yielded better results than discontinuous resection.

[Allergic bronchial asthma: an evaluation of immunofluorescence technics on bronchial biopsies]. / Asma bronchiale allergico: valutazione delle tecniche di immunofluorescenza su biopsia bronchiale.

Although the immunofluorescence technique has mostly been applied to renal diseases, it may be of use in the examination of lung tissue. Immunofluorescence tests on bronchial bioptic samples can show: 1) presence of IgE, IgA, IgM, IgG and complement factors; 2) their location on the bronchial mucosa; 3) their relationships with epithelioid cells, mast cells and plasma cells. Patients with atopic asthma were examined. Bioptic samples were taken from the main bronchi and treated by immunofluorescence. Cellular positivity was found for IgE, IgA, IgM and complement components.

[Transthoracic needle aspiration in infectious pathology]. / L'agoaspirazione transtoracica nella patologia infettiva.

A study was conducted on 20 patients with isolated or multiple patches of doubtful significance in the framework of neoplastic or infectious pathologies or in the presence of probably infectious diffuse or localised pulmonary infiltrations in immunodepressed patients or those in whom broad spectrum antibiotic treatment had failed. Percutaneous needle aspiration and sterile brushing was performed for the purpose of bacteriological examination in all patients. The result showed the clear superiority of needle aspiration (75% positive) over sterile brushing (25%). The first method is therefore recommended for use in the diagnosis of so-called "difficult" lung pathologies.

[Percutaneous needle aspiration biopsy in the diagnosis of pulmonary neoplasms]. / L'agobiopsia percutanea aspirativa nella diagnosi delle neoplasie polmonari.

The diagnostic efficacy of percutaneous needle biopsy (PNB) was assessed in 220 lung tumour patients. The results obtained prove this diagnostic technique to be extremely sensitive even though personal experience suggests that slight complications (pneumothorax, homophthoe) may occur.

[Isolated endobronchial metastases]. / Metastasi endobronchiali isolate.

Endobronchial metastases account for about 5% of autoptic findings in patients with multiple secondary locations. Five cases are reported of patients with malignant neoplasms in various organs with metastases to the trachea and the large bronchi without involvement of the pulmonary parenchyma and therefore with negative radiographic and CT scan findings.

[Echography in the study of thoracic pathology. I--Indications and limitations]. / L'ecografia nello studio della patologia toracica. Nota I--Indicazioni e limiti.

The role of echography in the study of the thorax is evaluated: after reporting the technical limits due to the peculiar anatomy of this region, personal experience is presented. This method extremely precise to define the solid or liquid nature of tightly adherent to the chest wall lesions, but it is non specific to assess their benign or malignant behaviour. Ultrasounds have their on limits in drawing the extension of such lesions; these limits have been overcome by CT and MR. Finally the usefulness of the method in studying the diaphragm and its pathology is briefly described.

[Antibacterial and antifungal activity of isoflurane and common anesthetic gases]. / Attività antibatterica ed antifungina dell'isofluorano e dei comuni vapori anestetici.

An in vitro analysis was conducted to investigate the hypothetical antibacterial and antimycotic activity of the common anesthetic gases (halothane, enflurane, isoflurane, methoxyflurane) in view of the clinical absence of bronchopulmonary pathology after inhalation narcosis despite the many risk factors involved. For this purpose scalar dilutions of the four gases were prepared on cultures of Klebsiella pneumoniae and Candida albicans and the antibacterial action of the gases was tested in vitro. Even with the weaker concentrations used, halothane and methoxyflurane totally inhibited both microorganisms. Enflurane had less effect on Klebsiella p. and almost none on Candida. Isoflurane, a new halogen ether anesthetic was found to have an excellent inhibitory effect. In conclusion it is hypothesised that the anesthetic gases considered might have an in vivo antibacterial activity considering the experimental results obtained in vitro.