L’importance de la coopération de TRAF1 et LSP1 en aval du récepteur 4-1BB(CD137) pour la survie des lymphocytes

4-1BB (CD137) est un membre de la superfamille TNFR qui est impliqué dans la transmission des signaux de survie aux lymphocytes. TRAF1 est une protéine adaptatrice qui est recrutée par 4-1BB et autres TNFRs et est caractérisée par une expression très restreinte aux lymphocytes, cellules dendritiques et certaines cellules épithéliales. TRAF1 est nécessaire pour l’expansion et la survie des cellules T mémoire en présence d'agonistes anti-4-1BB in vivo. De plus, TRAF1 est requise en aval de 4-1BB pour activer (phosphoryler) la MAP kinase Erk impliquée dans la régulation de la molécule pro-apoptotique Bim. Suite à l’activation du récepteur 4-1BB, TRAF1 et ERK sont impliqués dans la phosphorylation de Bim et la modulation de son expression. L’activation et la régulation de TRAF1 et Bim ont un rôle important dans la survie des cellules T CD8 mémoires. Dans cette étude, nous avons utilisé une approche protéomique afin de pouvoir identifier de nouveaux partenaires de liaison de TRAF1. Utilisant cette stratégie, nous avons identifié que LSP1 (Leukocyte Specific Protein 1) est recruté dans le complexe de signalisation 4-1BB de manière TRAF1 dépendante. Une caractérisation plus poussée de l’interaction entre TRAF1 et LSP1 a montré que LSP1 lie la région unique N-terminal de TRAF1 de façon indépendante de la région conservée C-terminal. À l’instar des cellules T déficientes en TRAF1, les cellules T déficientes en LSP1 ne sont pas capables d’activer ERK en aval de 4-1BB et par conséquent ne peuvent pas réguler Bim. Ainsi, TRAF1 et LSP1 coopèrent en aval de 4-1BB dans le but d’activer ERK et réguler en aval les niveaux de Bim dans les cellules T CD8. Selon la littérature, le récepteur 4-1BB n’est pas exprimé à la surface des cellules B murines, mais le récepteur 4-1BB favorise la prolifération et la survie des cellules B humaines. Cependant, il est important d'étudier l'expression du récepteur 4-1BB dans les cellules B murines afin de disposer d'un modèle murin et de prédire la réponse clinique à la manipulation de 4-1BB. En utilisant différentes stimulations de cellules B murines primaires, nous avons identifié que le récepteur 4-1BB est exprimé à la surface des cellules B de souris suite à une stimulation avec le LPS (Lipopolysaccharides). Une caractérisation plus poussée a montré que le récepteur 4-1BB est induit dans les cellules B murines d'une manière dépendante de TLR4 (Toll Like Receptor 4). Collectivement, notre travail a démontré que la stimulation avec le LPS induit l’expression du récepteur 4-1BB à la surface des cellules B murines, menant ainsi à l'induction de TRAF1. De plus, TRAF1 et LSP1 coopèrent en aval de 4-1BB pour activer la signalisation de la Map kinase ERK dans les cellules B murines de manière similaire aux cellules T. Les cellules B déficientes en TRAF1 et les cellules B déficientes en LSP1 ne sont pas en mesure d'activer la voie ERK en aval de 4-1BB et montrent un niveau d’expression du récepteur significativement diminué comparé aux cellules B d’une souris WT. Ainsi, TRAF1 et LSP1 sont nécessaires pour une expression maximale du récepteur 4-1BB à la surface cellulaire de cellules B murines et coopèrent en aval de 4-1BB afin d'activer la cascade ERK dans les cellules B murines.

Expression of the HPV16E7 oncoprotein by thymic epithelium is accompanied by disrupted T cell maturation and a failure of the thymus to involute with age

Transgenic mice expressing the E7 protein of HPV16 from the keratin 14 promoter demonstrate increasing thymic hypertrophy with age. This hypertrophy is associated with increased absolute numbers of all thymocyte types, and with increased cortical and medullary cellularity. In the thymic medulla, increased compartmentalization of the major thymic stromal cell types and expansion of thymic epithelial cell population is observed. Neither an increased rate of immature thymocyte division nor a decreased rate of immature thymocyte death was able to account for the observed hypertrophy. Thymocytes with reduced levels of expression of CD4 and/or CD8 were more abundant in transgenic (tg) mice and became increasingly more so with age. These thymic SP and DP populations with reduced levels of CD4 and/or CD8 markers had a lower rate of apoptosis in the tg than in the non-tg mice. The rate of export of mature thymocytes to peripheral lymphoid organs was less in tg animals relative to the pool of available mature cells, particularly for the increasingly abundant CD4lo population. We therefore suggest that mature thymocytes that would normally die in the thymus gradually accumulated in E7 transgenic animals, perhaps as a consequence of exposure to a hypertrophied E7-expressing thymic epithelium or to factors secreted by this expanded thymic stromal cell population. The K14E7 transgenic mouse thus provides a unique model to study effects of the thymic epithelial cell compartment on thymus development and involution.

Host B cells produce IL-10 following TBI and attenuate acute GVHD after allogeneic bone marrow transplantation

Host antigen-presenting cells (APCs) are known to be critical for the induction of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but the relative contribution of specific APC subsets remains unclear. We have studied the role of host B cells in GVHD by using B-cell-deficient mu MT mice as BMT recipients in a model of CD4-dependent GVHD to major histocompatlibility complex antigens. We demonstrate that acute GVHD is initially augmented in mu MT recipients relative to wild-type recipients (mortality: 85% vs 44%, P < .01), and this is the result of an increase in donor T-cell proliferation, expansion, and inflammatory cytokine production early after BMT. Recipient B cells were depleted 28-fold at the time of BMT by total body irradiation (TBI) administered 24 hours earlier, and we demonstrate that TBI rapidly induces sustained interleukin-110 (IL-10) generation from B cells but not dendritic cells (DCs) or other cellular populations within the spleen. Finally, recipient mice in which B cells are unable to produce IL-10 due to homologous gene deletion develop more severe acute GVHD than recipient mice in which B cells are wild type. Thus, the induction of IL-10 in host B cells during conditioning attenuates experimental acute GVHD.

Structural and cellular characterization of Idiopathic Pulmonary Fibrosis

La Fibrosi Polmonare Idiopatica (IPF) è una malattia polmonare cronica, irreversibile la cui eziologia risulta essere ignota, caratterizzata da un processo fibrotico progressivo che inizia nel tratto respiratorio inferiore. Le persone affette da IPF presentano età media compresa tra 55 e 77 anni. L’incidenza annuale di IPF è stata recentemente stimata tra 14 e 42,7 casi per 100.000 persone e tale dato risulta essere in aumento. IPF fa parte delle malattie Polmonari Idiopatiche Interstiziali (IIP) che comprendono patologie con quadri istologici e clinici differenti. Le affezioni su cui si concentrerà questo studio sono: UIP (Usual Interstitial Pneumonia) caratterizzata da fibrosi interstiziale e dalla presenza di foci fibrotici connessi alla pleura e corrispondente al quadro anatomopatologico della maggior parte dei casi di IPF; NSIP (Non Specific Interstitial Pneumonia) simile alla UIP ma con maggiore uniformità temporale e spaziale delle manifestazioni; Sarcoidosi, malattia granulomatosa ad eziologia ignota. Attualmente la gravità della IPF, che implica una mortalità del 50% dei pazienti a 5 anni dall’esordio, e la scarsa efficacia farmacologica nel rallentarne la progressione vedono il trapianto polmonare come unica possibilità di sopravvivenza nelle forme più severe. Al momento non è chiaro il meccanismo patogenetico di insorgenza e progressione della IPF anche se sono stati individuati alcuni fattori scatenanti quali fumo di sigaretta, infezioni respiratorie e inquinanti atmosferici; tuttavia nessuno di tali elementi può da solo determinare un così esteso e progressivo rimodellamento del parenchima polmonare. Numerose sono le evidenze di come il substrato genetico, le alterazioni del rapporto morte/proliferazione cellulare e le citochine svolgano un ruolo nella genesi e nella progressione della malattia, ma non sono ancora chiari i fenomeni biologico-cellulari che la sostengono e, quindi, quali siano i punti di attacco per poter incidere terapeuticamente nel modificare l’evoluzione della IPF. Poiché il nostro laboratorio ha partecipato alla scoperta dell’esistenza di cellule staminali nel polmone umano normale, uno degli obiettivi finali di questo progetto si basa sull’ipotesi che un’alterazione del compartimento staminale svolga un ruolo cruciale nella eziopatogenesi di IPF. Per questo in precedenti esperienze abbiamo cercato di identificare nella IPF cellule che esprimessero antigeni associati a staminalità quali c-kit, CD34 e CD133. Questo lavoro di tesi si è proposto di condurre un’indagine morfometrica ed immunoistochimica su biopsie polmonari provenienti da 9 pazienti affetti da UIP, 3 da NSIP e 5 da Sarcoidosi al fine di valutare le alterazioni strutturali principali imputabili alle patologie. Preparati istologici di 8 polmoni di controllo sono stati usati come confronto. Come atteso, è stato osservato nelle tre patologie esaminate (UIP, NSIP e Sarcoidosi) un significativo incremento nella sostituzione del parenchima polmonare con tessuto fibrotico ed un ispessimento dei setti alveolari rispetto ai campioni di controllo. L’analisi dei diversi pattern di fibrosi presenti fa emergere come vi sia una netta differenza tra le patologie con una maggiore presenza di fibrosi di tipo riparativo e quindi altamente cellulata nei casi di UIP, e NSIP mentre nelle Sarcoidosi il pattern maggiormente rappresentato è risultato essere quello della fibrosi replacement o sostitutiva. La quantificazione delle strutture vascolari è stata effettuata tenendo separate le aree di polmone alveolare rispetto a quelle occupate da focolai sostitutivi di danno (componente fibrotica). Nei campioni patologici analizzati era presente un significativo riarrangiamento di capillari, arteriole e venule rispetto al polmone di controllo, fenomeno principalmente riscontrato nel parenchima fibrotico. Tali modifiche erano maggiormente presenti nei casi di NSIP da noi analizzati. Inoltre le arteriole subivano una diminuzione di calibro ed un aumento dello spessore in special modo nei polmoni ottenuti da pazienti affetti da Sarcoidosi. Rispetto ai controlli, nella UIP e nella Sarcoidosi i vasi linfatici risultavano inalterati nell’area alveolare mentre aumentavano nelle aree di estesa fibrosi; quadro differente si osservava nella NSIP dove le strutture linfatiche aumentavano in entrambe le componenti strutturali. Mediante indagini immunoistochimiche è stata documentata la presenza e distribuzione dei miofibroblasti, positivi per actina muscolare liscia e vimentina, che rappresentano un importante componente del danno tissutale nella IPF. La quantificazione di questo particolare fenotipo è attualmente in corso. Abbiamo inoltre analizzato tramite immunoistochimica la componente immunitaria presente nei campioni polmonari attraverso la documentazione dei linfociti T totali che esprimono CD3, andando poi a identificare la sottopopolazione di T citotossici esprimenti la glicoproteina CD8. La popolazione linfocitaria CD3pos risultava notevolmente aumentata nelle tre patologie analizzate soprattutto nei casi di UIP e Sarcoidosi sebbene l`analisi della loro distribuzione tra i vari distretti tissutali risultasse differente. Risultati simili si sono ottenuti per l`analisi dei linfociti CD8pos. La componente monocito-macrofagica è stata invece identificata tramite la glicoproteina CD68 che ha messo in evidenza una maggiore presenza di cellule positive nella Sarcoidosi e nella UIP rispetto ai casi di NSIP. I dati preliminari di questo studio indicano che il rimodellamento strutturale emo-linfatico e cellulare infiammatorio nella UIP si differenziano rispetto alle altre malattie interstiziali del polmone, avanzando l’ipotesi che il microambiente vascolare ed immunitario giochino un ruolo importante nella patogenesi della malattia

Étude fonctionnelle de lymphocytes T extrathymiques produits sous l'effet de l'oncostatin M

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Étude fonctionnelle de lymphocytes T extrathymiques produits sous l'effet de l'oncostatin M

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

ROLE OF STAT1 AND CXCL10 IN THE TUMOUR IMMUNE MICROENVIRONMENT OF HIGH-GRADE SEROUS OVARIAN CANCER

High-grade serous ovarian cancer (HGSC) is the most prevalent epithelial ovarian cancer characterized by late detection, metastasis and resistance to chemotherapy. Previous studies on the tumour immune microenvironment in HGSC identified STAT1 and CXCL10 as the most differentially expressed genes between treatment naïve chemotherapy resistant and sensitive tumours. Interferon-induced STAT1 is a transcription factor, which induces many genes including tumour suppressor genes and those involved in recruitment of immune cells to the tumour immune microenvironment (TME), including CXCL10. CXCL10 is a chemokine that recruits tumour infiltrating lymphocytes (TILs) and exhibits angiostatic function. The current study was performed to determine the effects of differential STAT1 and CXCL10 expression on HGSC disease progression and TME. STAT1 expression and intratumoural CD8+ T cells were evaluated as prognostic and predictive biomarkers via immunohistochemistry on 734 HGSC tumours accrued from the Terry Fox Research Institute-Canadian Ovarian Experimental Unified Resource. The combined effect of STAT1 expression and CD8+ TIL density was confirmed as prognostic and predictive companion biomarkers in the second independent biomarker validation study. Significant positive correlation between STAT1 expression and intratumoral CD8+ TIL density was observed. The effects of enforced CXCL10 expression on HGSC tumour growth, vasculature and immune tumour microenvironment were studied in the ID8 mouse ovarian cancer cell engraftment in immunocompetent C57BL/6 mice. Significant decrease in tumour progression in mice injected with ID8 CXCL10 overexpressing cells compared to mice injected with ID8 vector control cells was observed. Multiplexed cytokine analysis of ascites showed differential expression of IL-6, VEGF and CXCL9 between the two groups. Endothelial cell marker staining showed differences in tumour vasculature between the two groups. Immune transcriptomic profiling identified distinct expression profiles in genes associated with cytokines, chemokines, interferons, T cell function and apoptosis between the two groups. These findings provide evidence that STAT1 is an independent biomarker and in combination with CD8+ TIL density could be applied as novel immune-based biomarkers in HGSC. These results provide the basis for future studies aimed at understanding mechanisms underlying differential tumour STAT1 and CXCL10 expression and its role in pre-existing tumour immunologic diversity, thus potentially contributing to biomarker guided immune modulatory therapies.

Anomalies immunitaires chez les enfants exposés au VIH mais non infectés.

La thérapie antirétrovirale prévient la transmission mère-enfant du VIH dans plus de 98% des cas lorsqu’administrée pendant la grossesse, le travail et au nouveau-né. L’accessibilité à la thérapie antirétrovirale dans près de 70% des 1,5 millions cas de grossesses VIH+ dans le monde mène à la naissance de plus d’un million d’enfants exposés non infectés chaque année. Le nombre d’enfants exposés non infectés est à la hausse ainsi que les préoccupations concernant leur santé. En effet, plusieurs groupes ont signalé une augmentation de la morbidité et de la mortalité chez les enfants exposés non infectés. L’analyse des données rétrospectives de 705 enfants exposés non infectés de la cohorte mère-enfant du CMIS a révélé qu’à 2 mois d’âge, les enfants nés de mères ayant une charge virale supérieure à 1,000 copies d’ARN / ml avaient une fréquence de lymphocytes B significativement plus élevés par rapport aux enfants exposés non infectés nés de mères ayant une charge virale indétectable. L’objectif de cette étude est de caractériser ces anomalies. Les lymphocytes, provenant du sang de cordon ombilical et de sang veineux obtenu à 6 et 12 mois d’âge, ont été phénotypés par cytométrie en flux à l’aide des marqueurs CD3 / CD10 / CD14 / CD16 / CD19 / CD20 / CD21 / CD27 / IgM pour les lymphocytes B et CD4 / CD8 / CD3 / CCR7 / CD45RA pour les lymphocytes T. De plus, afin d’étudier les capacités fonctionnelles des lymphocytes B CD19+, la réponse antigène-spécifique au vaccin antitétanique a été mesurée par marquage avec des tétramères fluorescents de fragment C du toxoïde tétanique. Nos travaux ont mis en évidence des différences statistiquement significatives entre les enfants exposés non-infectés (ENI) nés de mères avec une charge virale détectable comparativement à ceux nés de mères avec une charge virale indétectable. À la naissance, les enfants ENI nés de mères avec une charge virale détectable avaient significativement moins de lymphocytes B totaux, plus de lymphocytes B mémoires classiques, activés, plasmablastes et lymphocytes T CD8+ mémoires centrales. À 6 mois, ils avaient significativement plus de lymphocytes B naïfs et significativement moins de lymphocytes T CD8+ effecteurs mémoires. À 12 mois d’âge, ils avaient significativement plus de lymphocytes B et T CD8+ totaux; significativement moins de lymphocytes T CD4+ totaux et leurs lymphocytes T affichaient un profil significativement plus activé (plus de cellules mémoires). L’analyse de la réponse antigène-spécifique a révélé une fréquence plus élevé de lymphocytes B mémoires IgM+ suggérant que les enfants nés de mères avec une virémie détectable ont plus de mal à établir une mémoire immunitaire efficace face au vaccin antitétanique. Nos données suggèrent qu’il y a exposition durant le premier trimestre de grossesse à la virémie maternelle et que cette exposition impacte le système immunitaire en développement du fœtus. Les mécanismes sous-jacents causant ces anomalies doivent encore être élucidés et l’épuisement du compartiment T à la naissance et à 6 mois reste à être investigué. Dans un pays industrialisé où l’accès aux soins est facilité, ces anomalies ont des conséquences modérées mais dans des pays à faible et moyen revenu, les conséquences peuvent être beaucoup plus tragiques voir fatales.

Anomalies immunitaires chez les enfants exposés au VIH mais non infectés

La thérapie antirétrovirale prévient la transmission mère-enfant du VIH dans plus de 98% des cas lorsqu’administrée pendant la grossesse, le travail et au nouveau-né. L’accessibilité à la thérapie antirétrovirale dans près de 70% des 1,5 millions cas de grossesses VIH+ dans le monde mène à la naissance de plus d’un million d’enfants exposés non infectés chaque année. Le nombre d’enfants exposés non infectés est à la hausse ainsi que les préoccupations concernant leur santé. En effet, plusieurs groupes ont signalé une augmentation de la morbidité et de la mortalité chez les enfants exposés non infectés. L’analyse des données rétrospectives de 705 enfants exposés non infectés de la cohorte mère-enfant du CMIS a révélé qu’à 2 mois d’âge, les enfants nés de mères ayant une charge virale supérieure à 1,000 copies d’ARN / ml avaient une fréquence de lymphocytes B significativement plus élevés par rapport aux enfants exposés non infectés nés de mères ayant une charge virale indétectable. L’objectif de cette étude est de caractériser ces anomalies. Les lymphocytes, provenant du sang de cordon ombilical et de sang veineux obtenu à 6 et 12 mois d’âge, ont été phénotypés par cytométrie en flux à l’aide des marqueurs CD3 / CD10 / CD14 / CD16 / CD19 / CD20 / CD21 / CD27 / IgM pour les lymphocytes B et CD4 / CD8 / CD3 / CCR7 / CD45RA pour les lymphocytes T. De plus, afin d’étudier les capacités fonctionnelles des lymphocytes B CD19+, la réponse antigène-spécifique au vaccin antitétanique a été mesurée par marquage avec des tétramères fluorescents de fragment C du toxoïde tétanique. Nos travaux ont mis en évidence des différences statistiquement significatives entre les enfants exposés non-infectés (ENI) nés de mères avec une charge virale détectable comparativement à ceux nés de mères avec une charge virale indétectable. À la naissance, les enfants ENI nés de mères avec une charge virale détectable avaient significativement moins de lymphocytes B totaux, plus de lymphocytes B mémoires classiques, activés, plasmablastes et lymphocytes T CD8+ mémoires centrales. À 6 mois, ils avaient significativement plus de lymphocytes B naïfs et significativement moins de lymphocytes T CD8+ effecteurs mémoires. À 12 mois d’âge, ils avaient significativement plus de lymphocytes B et T CD8+ totaux; significativement moins de lymphocytes T CD4+ totaux et leurs lymphocytes T affichaient un profil significativement plus activé (plus de cellules mémoires). L’analyse de la réponse antigène-spécifique a révélé une fréquence plus élevé de lymphocytes B mémoires IgM+ suggérant que les enfants nés de mères avec une virémie détectable ont plus de mal à établir une mémoire immunitaire efficace face au vaccin antitétanique. Nos données suggèrent qu’il y a exposition durant le premier trimestre de grossesse à la virémie maternelle et que cette exposition impacte le système immunitaire en développement du fœtus. Les mécanismes sous-jacents causant ces anomalies doivent encore être élucidés et l’épuisement du compartiment T à la naissance et à 6 mois reste à être investigué. Dans un pays industrialisé où l’accès aux soins est facilité, ces anomalies ont des conséquences modérées mais dans des pays à faible et moyen revenu, les conséquences peuvent être beaucoup plus tragiques voir fatales.

Characterization of the immune response induced by a commercially available inactivated bluetongue virus serotype 1 vaccine in sheep

The protective immune response generated by a commercial monovalent inactivated vaccine against bluetongue virus serotype 1 (BTV1) was studied. Five sheep were vaccinated, boost-vaccinated, and then challenged against BTV1 ALG/2006. RT-PCR did not detect viremia at any time during the experiment. Except a temperature increase observed after the initial and boost vaccinations, no clinical signs or lesions were observed. A specific and protective antibody response checked by ELISA was induced after vaccination and boost vaccination. This specific antibody response was associated with a significant increase in B lymphocytes confirmed by flow cytometry, while significant increases were not observed in T lymphocyte subpopulations (CD4(+), CD8(+), and WC1(+)), CD25(+) regulatory cells, or CD14(+) monocytes. After challenge with BTV1, the antibody response was much higher than during the boost vaccination period, and it was associated with a significant increase in B lymphocytes, CD14(+) monocytes, CD25(+) regulatory cells, and CD8(+) cytotoxic T lymphocytes.

A USANS/SANS study of the accessibility of pores in the barnett shale to methane and water

Shale is an increasingly important source of natural gas in the United States. The gas is held in fine pores that need to be accessed by horizontal drilling and hydrofracturing techniques. Understanding the nature of the pores may provide clues to making gas extraction more efficient. We have investigated two Mississippian Barnett Shale samples, combining small-angle neutron scattering (SANS) and ultrasmall-angle neutron scattering (USANS) to determine the pore size distribution of the shale over the size range 10 nm to 10 μm. By adding deuterated methane (CD4) and, separately, deuterated water (D2O) to the shale, we have identified the fraction of pores that are accessible to these compounds over this size range. The total pore size distribution is essentially identical for the two samples. At pore sizes >250 nm, >85% of the pores in both samples are accessible to both CD4 and D2O. However, differences in accessibility to CD4 are observed in the smaller pore sizes (∼25 nm). In one sample, CD4 penetrated the smallest pores as effectively as it did the larger ones. In the other sample, less than 70% of the smallest pores (<25 nm) were accessible to CD4, but they were still largely penetrable by water, suggesting that small-scale heterogeneities in methane accessibility occur in the shale samples even though the total porosity does not differ. An additional study investigating the dependence of scattered intensity with pressure of CD4 allows for an accurate estimation of the pressure at which the scattered intensity is at a minimum. This study provides information about the composition of the material immediately surrounding the pores. Most of the accessible (open) pores in the 25 nm size range can be associated with either mineral matter or high reflectance organic material. However, a complementary scanning electron microscopy investigation shows that most of the pores in these shale samples are contained in the organic components. The neutron scattering results indicate that the pores are not equally proportioned in the different constituents within the shale. There is some indication from the SANS results that the composition of the pore-containing material varies with pore size; the pore size distribution associated with mineral matter is different from that associated with organic phases.

Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to Uropathogenic Escherichia coli

Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC) being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10) in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

Airway Inflammation and Bronchial Hyperresponsiveness in Elite Cross-Country Skiers and in Patients with Newly Diagnosed Asthma: A Bronchial Biopsy Study

The objective of these studies was to evaluate possible airway inflammation and remodeling at the bronchial level in cross-country skiers without a prior diagnosis of asthma, and relate the findings to patients with mild chronic asthma and patients with newly diagnosed asthma. We also studied the association of airway inflammatory changes and bronchial hyperresponsivess (BHR), and treatment effects in cross-country skiers and in patients with newly diagnosed asthma. Bronchial biopsies were obtained from the subjects by flexible bronchoscopy, and the inflammatory cells (eosinophils, mast cells, T-lymphocytes, macrophages, and neutrophils) were identified by immunohistochemistry. Tenascin (Tn) immunoreactivity in the bronchial basement membrane (BM) was identified by immunofluorescence staining. Lung function was measured with spirometry, and BHR was assessed by methacholine (skiers) or histamine (asthmatics) challenges. Skiers with BHR and asthma-like symptoms were recruited to a drug-intervention study. Skiers were given treatment (22 weeks) with placebo or budesonide (400 µg bid). Patients with newly diagnosed asthma were given treatment for 16 weeks with placebo, salmeterol (SLM) (50 µg bid), fluticasone propionate (FP) (250 µg bid), or disodium cromoglicate (DSCG) (5 mg qid). Bronchial biopsies were obtained at baseline and at the end of the treatment period. In the skiers a distinct airway inflammation was evident. In their bronchial biopsy specimens, T-lymphocyte, macrophage, and eosinophil counts were, respectively greater by 43-fold (P<0.001), 26-fold (P<0.001, and 2-fold (P<0.001) in skiers, and by 70-fold (p>0.001), 63-fold (P<0.001), and 8-fold (P<0.001) in asthmatic subjects than in controls. In skiers, neutrophil counts were more than 2-fold greater than in asthmatic subjects (P<0.05). Tn expression was higher in skiers than in controls and lower in skiers than in mild asthmatics. No significant changes were seen between skiers with or without BHR in the inflammatory cell counts or Tn expression. Treatment with inhaled budesonide did not attenuate asthma-like symptoms, the inflammatory cell infiltration, or BM Tn expression in the skiers. In newly diagnosed asthmatic patients, SLM, FP, and DSCG reduced asthma symptoms, and need for rescue medication (P<0.04). BHR was reduced by doubling doses 2.78, 5.22, and 1.35 respectively (all P<0.05). SLM and placebo had no effect on cell counts or Tn expression. FP and DSCG reduced eosinophil counts in the bronchial biopsy specimens (P<0.02 and <0.048, respectively). No significant change in tenascin expression appeared in any treatment group. Regarding to atopy, no significant differences existed in the inflammatory cell counts in the bronchial mucosa of subjects with newly diagnosed asthma or in elite cross country skiers. Tn expression in the BM was significantly higher in atopic asthma than in those with nonatopic asthma. Airway inflammation occurred in elite cross-country skiers with and without respiratory symptoms or BHR. Their inflammatory cell pattern differed from that in asthma. Infiltration with eosinophils, macrophages, and mast cells was milder, but lymphocyte counts did not differ from counts in asthmatic airways. Neutrophilic infiltration was more extensive in skiers than in asthmatics. Remodeling took place in the skiers’ airways, as reflected by increased expression of BM tenascin These inflammatory changes and Tn expression may be caused by prolonged exposure of the lower airways to inadequately humidified cold air. In skiers inflammatory changes and remodeling were not reversed with anti-inflammatory treatment. In contrast, in patients with newly diagnosed asthma, anti-inflammatory treatment did attenuate eosinophilic inflammation in the bronchial mucosa. In skiers, anti-inflammatory treatment did not attenuate BHR as it did in asthmatic patients. The BHR in skiers was attenuated spontaneously during placebo treatment, with no difference from budesonide treatment. Lower training intensity during the treatment period may explain this spontaneous decrease in BHR. The origin of BHR probably differs in skiers and in asthmatics. No significant association between BHR and inflammatory cell counts or between BHR and Tn expression was evident in cross-country skiers or asthmatic subjects. Airway remodeling differed between atopic and nonatopic asthma. As opposed to nonatopic asthma, Tn expression was higher in atopic asthma and is related to inflammatory cell densities.

Tumour islet Foxp3+ T-cell infiltration predicts poor outcome in nonsmall cell lung cancer

The impact of host immunity on outcome in nonsmall cell lung cancer (NSCLC) is controversial. We examined the relationship between lymphoid infiltration patterns in NSCLC and prognosis. Tumour- and stroma-infiltrating CD3+, CD8+ and forkhead box P3 (Foxp3)+ T-lymphocytes were identified using immunohistochemistry and a novel image analysis algorithm to assess total, cytotoxic and regulatory T-lymphocyte counts, respectively, in 196 NSCLC cases. The median cell count was selected as a cut-point to define patient subgroups and the ratio of the corresponding tumour islet:stroma (TI/S) counts was determined. There was a positive association between overall survival and increased CD8+ TI/S ratio (hazard ratio (HR) for death 0.44, p<0.001) but an inverse relationship between Foxp3+ TI/S ratio and overall survival (HR 4.86, p<0.001). Patients with high CD8+ islet (HR 0.48, p<0.001) and Foxp3+ stromal (HR 0.23, p<0.001) counts had better survival, whereas high CD3+ and CD8+ stromal counts and high Foxp3+ islet infiltration conferred a worse survival (HR 1.55, 2.19 and 3.14, respectively). By multivariate analysis, a high CD8+ TI/S ratio conferred an improved survival (HR 0.48, p=0.002) but a high Foxp3+ TI/S ratio was associated with worse survival (HR 3.91, p<0.001). Microlocalisation of infiltrating T-lymphocytes is a powerful predictor of outcome in resected NSCLC.

Designed Cyclic Permutants of HIV-1 gp120: Implications for Envelope Trimer Structure and Immunogen Design

Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120-gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. We have therefore designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cycl, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with similar to 20-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface.