Pas de résumé en français / Malaria is a devastating global health problem that affected 219 million people and caused 660,000 deaths in 2010. Inaccurate estimation of the level of infection may have adverse clinical and therapeutic implications for patients, and for epidemiological endpoint measurements. The level of infection, expressed as the parasite density (PD), is classically defined as the number of asexual parasites relative to a microliter of blood. Microscopy of Giemsa-stained thick blood smears (TBSs) is the gold standard for parasite enumeration. Parasites are counted in a predetermined number of high-power fields (HPFs) or against a fixed number of leukocytes. PD estimation methods usually involve threshold values; either the number of leukocytes counted or the number of HPFs read. Most of these methods assume that (1) the distribution of the thickness of the TBS, and hence the distribution of parasites and leukocytes within the TBS, is homogeneous; and that (2) parasites and leukocytes are evenly distributed in TBSs, and thus can be modeled through a Poisson-distribution. The violation of these assumptions commonly results in overdispersion. Firstly, we studied the statistical properties (mean error, coefficient of variation, false negative rates) of PD estimators of commonly used threshold-based counting techniques and assessed the influence of the thresholds on the cost-effectiveness of these methods. Secondly, we constituted and published the first dataset on parasite and leukocyte counts per HPF. Two sources of overdispersion in data were investigated: latent heterogeneity and spatial dependence. We accounted for unobserved heterogeneity in data by considering more flexible models that allow for overdispersion. Of particular interest were the negative binomial model (NB) and mixture models. The dependent structure in data was modeled with hidden Markov models (HMMs). We found evidence that assumptions (1) and (2) are inconsistent with parasite and leukocyte distributions. The NB-HMM is the closest model to the unknown distribution that generates the data. Finally, we devised a reduced reading procedure of the PD that aims to a better operational optimization and a practical assessing of the heterogeneity in the distribution of parasites and leukocytes in TBSs. A patent application process has been launched and a prototype development of the counter is in process.

Background: Leukocytes in peripheral blood (PB) are prognostic biomarkers in head and neck squamous cell carcinoma cancer patients (HNSCC-CPs), but differences between HNSCC-CPs and healthy adults (HAs) are insufficiently described. Methods: 10-color flow cytometry (FCM) was used for in-depth immunophenotyping of PB samples of 963 HAs and 101 therapy-naïve HNSCC-CPs. Absolute (AbsCC) and relative cell counts (RelCC) of leukocyte subsets were determined. A training cohort (TC) of 43 HNSCC-CPs and 43 HAs, propensity score (PS)-matched according to age, sex, alcohol, and smoking, was used to develop a score consecutively approved in a validation cohort (VC). Results: Differences in AbsCC were detected in leukocyte subsets (p < 0.001), but had low power in discriminating HNSCC-CPs and HAs. Consequently, RelCC of nine leukocyte subsets in the TC were used to calculate 36 ratios; receiver operating characteristic (ROC) curves defined optimum cut-off values. Binary classified data were combined in a score based on four ratios: monocytes-to-granulocytes (MGR), classical monocytes-to-monocytes (clMMR), monocytes-to-lymphocytes (MLR), and monocytes-to-T-lymphocytes (MTLR); ≥3 points accurately discriminate HNSCC-CPs and HAs in the PS-matched TC (p = 2.97 × 10−17), the VC (p = 4.404 × 10−178), and both combined (p = 7.74 × 10−199). Conclusions: RelCC of leukocyte subsets in PB of HNSCC-CPs differ significantly from those of HAs. A score based on MGR, clMMR, MLR, and MTLR allows for accurate discrimination.

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[pt] Os analisadores hematológicos são sistemas de medição que permitem identificar elementos sanguíneos, incluindo a contagem diferencial dos cinco tipos de leucócitos encontrados no sangue periférico: neutrófilos, linfócitos, monócitos, eosinófilos e basófilos. Devido à inexistência de materiais de referência certificados ou métodos de referência para a análise diferencial de leucócitos, tem sido habitual avaliar o desempenho de novos analisadores comparando-os com sistemas tradicionalmente disponíveis e bem consolidados. Em 2015, foi lançada uma nova tecnologia portátil e de baixo custo, o modelo DxH500 (Beckman Coulter), baseada na perda axial de luz usando LED azul como emissor, combinada à medição de impedância. O presente trabalho compara o desempenho do DxH500 com um modelo bastante utilizado desde seu lançamento em 2001 para exames laboratoriais em larga escala, o modelo LH750 (Beckman Coulter), baseado na combinação dos princípios de impedância, condutividade e dispersão de luz (VCS). No estudo foram examinadas 310 amostras pareadas. A análise comparativa entre os resultados fornecidos por cada dispositivo indicou uma boa correlação para a caracterização de neutrófilos, linfócitos, monócitos e eosinófilos. Apesar da reduzida correlação observada para a contagem de basófilos, esse resultado não apresenta relevância clínica já que os valores obtidos para as amostras avaliadas foram muito reduzidos, inferiores aos limites de referência. Embora a análise comparativa realizada tenha apontado para um desempenho equivalente utilizando-se tecnologias com princípios diversos, a adequada avaliação dos dispositivos de análise hematológica requer o desenvolvimento de materiais de referência certificados, uma demanda fundamental para a garantia da confiabilidade na quantificação diferencial de leucócitos. / [en] Hematology analyzers are measurement systems that allow the identification of blood elements, including differential counting of the five types of leukocytes found in peripheral blood: neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Because of the lack of certified reference materials or reference methods for differential leukocyte analysis, it has been customary to evaluate the performance of new analyzers by comparing them with traditionally available and well-established systems. In 2015, a new low-cost, portable technology was introduced, the Beckman Coulter DxH500 model, based on the axial loss of light using blue LED as a combined transmitter for impedance measurement. The present work compares the performance of the DxH500 with a widely used model since its launch in 2001 for large-scale laboratory tests, the Beckman Coulter model LH750, based on the combination of impedance, conductivity and scatter (VCS). In the study, we examined 310 paired samples. The comparative analysis between differential leukocyte evaluation results provided by each device indicated a good correlation for the characterization of neutrophils, lymphocytes, monocytes, and eosinophils. Despite the low correlation observed for basophil counts, this result is not clinically relevant since the values obtained for the evaluated samples were very low, lower than the reference limits. Although the comparative analysis carried out pointed to an equivalent performance using technologies with different principles, the adequate evaluation of hematological analysis devices requires the development of certified reference materials, fundamental demand for the reliability of the differential quantification of leukocytes.

Le système de différenciation entre le « soi » et le « non-soi » des vertébrés permet la détection et le rejet de pathogènes et de cellules allogéniques. Il requiert la surveillance de petits peptides présentés à la surface cellulaire par les molécules du complexe majeur d’histocompatibilité de classe I (CMH I). Les molécules du CMH I sont des hétérodimères composés par une chaîne lourde encodée par des gènes du CMH et une chaîne légère encodée par le gène β2-microglobuline. L’ensemble des peptides est appelé l’immunopeptidome du CMH I. Nous avons utilisé des approches en biologie de systèmes pour définir la composition et l’origine cellulaire de l’immunopeptidome du CMH I présenté par des cellules B lymphoblastoïdes dérivés de deux pairs de fratries avec un CMH I identique. Nous avons découvert que l’immunopeptidome du CMH I est spécifique à l’individu et au type cellulaire, qu’il dérive préférentiellement de transcrits abondants, est enrichi en transcrits possédant d’éléments de reconnaissance par les petits ARNs, mais qu’il ne montre aucun biais ni vers les régions génétiques invariables ni vers les régions polymorphiques. Nous avons également développé une nouvelle méthode qui combine la spectrométrie de masse, le séquençage de nouvelle génération et la bioinformatique pour l’identification à grand échelle de peptides du CMH I, dont ceux résultants de polymorphismes nucléotidiques simples non-synonymes (PNS-ns), appelés antigènes mineurs d’histocompatibilité (AMHs), qui sont les cibles de réponses allo-immunitaires. La comparaison de l’origine génomique de l’immunopeptidome de soeurs avec un CMH I identique a révélé que 0,5% des PNS-ns étaient représentés dans l’immunopeptidome et que 0,3% des peptides du CMH I seraient immunogéniques envers une des deux soeurs. En résumé, nous avons découvert des nouveaux facteurs qui modèlent l’immunopeptidome du CMH I et nous présentons une nouvelle stratégie pour l’indentification de ces peptides, laquelle pourrait accélérer énormément le développement d’immunothérapies ciblant les AMHs. / The self/nonself discrimination system of vertebrates allows detection and rejection of pathogens and allogeneic cells. It requires the surveillance of short peptides presented by major histocompatibility class I (MHC I) molecules on the cell surface. MHC I molecules are heterodimers that consist of a heavy chain produced by MHC genes and a light chain encoded by the β2-microglobulin gene. The peptides presented by MHC I molecules are collectively referred to as the MHC I immunopeptidome. We employed systems biology approaches to define the composition and cellular origin of the self MHC I immunopeptidome presented by B lymphoblastoid cells derived from two pairs of MHC-identical siblings. We found that the MHC I immunopeptidome is subject- and cell-specific, derives preferentially from abundant transcripts, is enriched in transcripts bearing microRNA response elements and shows no bias toward invariant vs. polymorphic genomic sequences. We also developed a novel personalized approach combining mass-spectrometry, next-generation sequencing and bioinformatics for high-throughput identification of MHC I peptides including those caused by nonsynonymous single nucleotide polymorphisms (ns-SNPs), termed minor histocompatibility antigens (MiHAs), which are the targets of allo-immune responses. Comparison of the genomic landscape of the immunopeptidome of MHC-identical siblings revealed that 0.5% of ns-SNPs were represented in the immunopeptidome and that 0.3% of the MHC I-peptide repertoire would be immunogenic for one of the siblings. We discovered new factors that shape the self MHC I immunopeptidome and present a novel strategy for the identification of MHC I-associated peptides that could greatly accelerate the development of MiHA-targeted immunotherapy.

La présentation d'antigène par les molécules d'histocompatibilité majeure de classe I (CMHI) permet au système immunitaire adaptatif de détecter et éliminer les agents pathogènes intracellulaires et des cellules anormales. La surveillance immunitaire est effectuée par les lymphocytes T CD8 qui interagissent avec le répertoire de peptides associés au CMHI présentés à la surface de toutes cellules nucléées. Les principaux gènes humains de CMHI, HLA-A et HLA-B, sont très polymorphes et par conséquent montrent des différences dans la présentation des antigènes. Nous avons étudié les différences qualitatives et quantitatives dans l'expression et la liaison peptidique de plusieurs allotypes HLA. Utilisant la technique de cytométrie de flux quantitative nous avons établi une hiérarchie d'expression pour les quatre HLA-A, B allotypes enquête. Nos résultats sont compatibles avec une corrélation inverse entre l'expression allotypique et la diversité des peptides bien que d'autres études soient nécessaires pour consolider cette hypothèse. Les origines mondiales du répertoire de peptides associés au CMHI restent une question centrale à la fois fondamentalement et dans la recherche de cibles immunothérapeutiques. Utilisant des techniques protéogénomiques, nous avons identifié et analysé 25,172 peptides CMHI isolées à partir des lymphocytes B de 18 personnes qui exprime collectivement 27 allotypes HLA-A,B. Alors que 58% des gènes ont été la source de 1-64 peptides CMHI par gène, 42% des gènes ne sont pas représentés dans l'immunopeptidome. Dans l'ensemble, l’immunopeptidome présenté par 27 allotypes HLA-A,B ne couvrent que 17% des séquences exomiques exprimées dans les cellules des sujets. Nous avons identifié plusieurs caractéristiques des transcrits et des protéines qui améliorent la production des peptides CMHI. Avec ces données, nous avons construit un modèle de régression logistique qui prédit avec une grande précision si un gène de notre ensemble de données ou à partir d'ensembles de données indépendants génèrerait des peptides CMHI. Nos résultats montrent la sélection préférentielle des peptides CMHI à partir d'un répertoire limité de produits de gènes avec des caractéristiques distinctes. L'idée que le système immunitaire peut surveiller des peptides CMHI couvrant seulement une fraction du génome codant des protéines a des implications profondes dans l'auto-immunité et l'immunologie du cancer. / Antigen presentation by major histocompatibility complex class I (MHCI) molecules allows the adaptive immune system to detect and eliminate intracellular pathogens or abnormal cells. Immune surveillance is executed by CD8 T cells that monitor the repertoire of MHCI-associated peptides (MAPs) presented at the surface of all nucleated cells. The primary human MHCI genes, HLA-A and HLA-B, are highly polymorphic and consequentially demonstrate differences in antigen presentation. We investigated qualitative and quantitative differences in expression and peptide binding. Using quantitative flow cytometry we establish clear hierarchy of expression for the four HLA-A,B allotypes investigated. Our results are consistent with an inverse correlation between expression and peptide diversity although further work is necessary to solidify this hypothesis. The global origins of the MAP repertoire remains a central question both fundamentally and in the search for immunotherapeutic targets. Using proteogenomics, we identified and analyzed 25,172 MAPs isolated from B lymphocytes of 18 individuals who collectively expressed 27 HLA-A,B allotypes. While 58% of genes were the source of 1-64 MAPs per gene, 42% of genes were not represented in the immunopeptidome. Overall, we estimate the immunopeptidome presented by 27 HLA-A,B allotypes covered only 17% of exomic sequences expressed in subjects’ cells. We identified several features of transcripts and proteins that enhance MAP production. From these data we built a logistic regression model that predicts with high accuracy whether a gene from our dataset or from independent datasets would generate MAPs. Our results show preferential selection of MAPs from a limited repertoire of gene products with distinct features. The notion that the immune system can monitor MAPs covering only a fraction of the protein coding genome has profound implications in autoimmunity and cancer immunology.

OBJECTIVES: The integrin α4β7 is the gut-homing receptor for lymphocytes. It also is an important co-receptor for human immunodeficiency virus (HIV) via glycoprotein (gp)120 binding. Depletion of gut cluster of differentiation (CD)4 T cells is linked to chronic inflammation in patients with HIV; however, measuring CD4 cells in the gut is invasive and not routine. As such, establishing a peripheral marker for CD4 depletion of the gut is needed. We hypothesized that α4β7 CD4 T cells are depleted in the peripheral blood of treatment-naïve patients with HIV compared with healthy controls. METHODS: The study groups were treatment-naïve patients with HIV and uninfected controls. Subjects were included if they were 18 years or older with no history of opportunistic infections, active tuberculosis, or cancer. We collected peripheral blood and examined on whole blood using flow cytometry for the following cell surface markers: CD4, CD45RO, chemokine receptor type 5, C-X-C chemokine receptor type 4 (CXCR4), and the integrin β7. We collected demographic information, including age, sex, and ethnicity, as well as viral load (VL) and CD4 count. Two-samplettests and Fisher exact tests were used to compare the differences between the two groups. Spearman correlation coefficients were calculated between CD4 count and log10-VL and percentage of CD4+/CD45RO+/β7+and log10-VL in patients. RESULTS: Twenty-two subjects were enrolled in the study (12 patients with HIV and 10 controls). There were no differences in age or sex between the two groups. There were more Hispanics and fewer Asians in the group comprising patients with HIV compared with the control group (7 vs 2 and 0 vs 4,P= 0.05, respectively). Patients infected with HIV had significantly lower frequencies of CD4+/CD45RO+/β7+cells (median 12%, range 5-18 compared with uninfected controls: median 20%, range 11-26,P= 0.0007). There was a statistically significant difference in the percentage of CD4+/CD45RO+/C-X-C chemokine receptor type 4+cells between patients (72%, range 60%-91%) compared with controls (79%, range 72%-94%,P= 0.04). The percentage of CD4+/CD45RO+/chemokine receptor type 5+did not differ between the group of patients with HIV and the control groups (22%, range 11%-57% vs 27%, range 14%-31%;P= 0.8, respectively). There was no correlation between percentage of CD4+/CD45RO+/β+cells and log10-VL as measured by the Spearman correlation coefficient (r= 0.05,P= 0.88) in patients infected with HIV. CONCLUSIONS: Memory CD4 β7+cells are reduced significantly in the peripheral blood of untreated patients infected with HIV, which could be used as a noninvasive indicator of intestinal CD4 T cell loss and recovery. Further studies are needed to examine whether depletion of these CD4+/CD45RO+/β7+cells in the peripheral blood parallels depletion in the gut of treatment-naïve patients with HIV and whether levels return to control levels after treatment.

Inflammatory bowel diseases, (IBD), are a group of chronic disorders of the gastro-intestinal tract, and include Crohn’s disease (CD) and Ulcerative Colitis (UC). The pathogenesis is not known, but involves at least in part a loss of tolerance towards the commensal colonic microbiota. In this thesis, we show in animal models of CD and UC that the colonic mucosal blood flow increased compared to healthy animals. This blood flow increase is due to an up regulation of endothelial nitric oxide synthase (NOS). Further, we show in the UC model that the thickness of the firmly adherent colonic mucus layer increased compared to healthy animals. This increase is due to an up regulation of inducible NOS in the epithelium. Both the blood flow and mucus thickness increase appear to be protective mechanisms.  We demonstrate that the firmly adherent colonic mucus layer acts as a partial barrier towards luminal bacteria. In the UC model, this barrier is destroyed, causing increased bacterial translocation. The adhesion molecule P-selectin was up regulated in the UC model, leading to increased interactions between leukocytes and the endothelium, but also increased interactions between platelets and the endothelium. This indicates that not only leukocytes, but also platelets are involved in colonic inflammation. The addition of the probiotic bacterial strain Lactobacillus reuteri prevented disease by normalizing P-selectin levels and endothelial interactions with leukocytes and platelets. Lactobacillus reuteri also decreased bacterial translocation over the epithelium. In summary, this thesis highlights the importance of colonic barrier functions, and investigates the role of the microbiota in experimental IBD.

Malaria is a devastating global health problem that affected 219 million people and caused 660,000 deaths in 2010. Inaccurate estimation of the level of infection may have adverse clinical and therapeutic implications for patients, and for epidemiological endpoint measurements. The level of infection, expressed as the parasite density (PD), is classically defined as the number of asexual parasites relative to a microliter of blood. Microscopy of Giemsa-stained thick blood smears (TBSs) is the gold standard for parasite enumeration. Parasites are counted in a predetermined number of high-power fields (HPFs) or against a fixed number of leukocytes. PD estimation methods usually involve threshold values; either the number of leukocytes counted or the number of HPFs read. Most of these methods assume that (1) the distribution of the thickness of the TBS, and hence the distribution of parasites and leukocytes within the TBS, is homogeneous; and that (2) parasites and leukocytes are evenly distributed in TBSs, and thus can be modeled through a Poisson-distribution. The violation of these assumptions commonly results in overdispersion. Firstly, we studied the statistical properties (mean error, coefficient of variation, false negative rates) of PD estimators of commonly used threshold-based counting techniques and assessed the influence of the thresholds on the cost-effectiveness of these methods. Secondly, we constituted and published the first dataset on parasite and leukocyte counts per HPF. Two sources of overdispersion in data were investigated: latent heterogeneity and spatial dependence. We accounted for unobserved heterogeneity in data by considering more flexible models that allow for overdispersion. Of particular interest were the negative binomial model (NB) and mixture models. The dependent structure in data was modeled with hidden Markov models (HMMs). We found evidence that assumptions (1) and (2) are inconsistent with parasite and leukocyte distributions. The NB-HMM is the closest model to the unknown distribution that generates the data. Finally, we devised a reduced reading procedure of the PD that aims to a better operational optimization and a practical assessing of the heterogeneity in the distribution of parasites and leukocytes in TBSs. A patent application process has been launched and a prototype development of the counter is in process.