Dissertations / Theses on the topic 'Leukocyte populations'

The immune system plays a dual role in cancer progression, either preventing or promoting tumor progression and the fine regulation of the complex interaction between immune system and tumor determines patient outcome. It has been demonstrated that also in brain tumors a combination of signals and soluble factors secreted by tumor, immune and stromal cells are able to potentiate tumor progression. Therefore, the interest towards the characterization of tumor microenvironment in these tumors is growing and the presence of tumor infiltrating immune cells of myeloid origin has already been reported, but a clear phenotypic and functional characterization in human brain tumors still lacking. For this reason, in this research project we performed a deep analysis of both circulating and tumor-infiltrating leukocytes present in meningioma (MNG) and glioblastoma (GBM) patients in order to dissect their properties, with the ultimate goal of finding new immunological therapeutic strategies. We thus performed an extensive immunophenotyping of peripheral blood and fresh tumor tissue at surgery by multiparametric flow cytometry in 34 patients affected by MNG (WHO grade I-II) and in 76 patients affected by GBM (WHO grade IV glioma), along with immunosuppressive activity of sorted cells of myeloid origin. In the peripheral blood, we observed a number of significant alterations in myeloid cell subsets indicating a specific monocyte subsets as the main cell subset actively recruited to the tumor. Moreover, four subsets of myeloid-derived suppressor cells (MDSCs) are detectable in the blood and in the tumor tissue of patients affected by MNG and GBM. Three of these subsets are significantly expanded in the blood of patients, whereas two of them are significantly expanded in the tumor tissue. In addition, we assayed ARG-1 (arginase 1) levels and activity in plasma samples from patients affected by MNG and GBM, and observed both a significantly increased level and a boost of its functional activity, compared to the control group. At the tumor site, we observed a large leukocyte infiltrate, predominantly constituted by CD33+ myeloid cells, largely composed of macrophages endowed with suppressive activity and significantly expanded in both types of tumor. Based on the expression of different markers, in GBM patients, we were able to discriminate bone marrow-derived (BMDM) macrophages from resident microglia (MG). These populations showed a different suppressive activity, since BMDMs displayed a higher immunosuppressive activity compared to MG cells that showed low or no suppressive immune regulatory ability. Taken together the results of this study shed light on the complex interaction between immune system and the main tumors of the brain.
Il sistema immunitario svolge un duplice ruolo nella progressione del cancro, è in grado sia di prevenire che di promuovere la progressione tumorale e la regolazione della complessa interazione tra sistema immunitario ed il tumore è in grado di determinare la prognosi del paziente. Inoltre, è stato dimostrato che, anche nei tumori cerebrali, una combinazione di segnali e di fattori solubili secreti dal tumore, dalle cellule immunitarie e dalle cellule stromali, è in grado di potenziare la progressione tumorale. Pertanto, in questi tumori sta crescendo l'interesse verso la caratterizzazione del microambiente tumorale ed è già stata dimostrata la presenza di cellule immunitarie di origine mieloide infiltranti il tumore, ma una chiara caratterizzazione fenotipica e funzionale di queste popolazioni non è ancora stata documentata. Pertanto, in questo progetto di ricerca abbiamo eseguito un'analisi approfondita sia dei leucociti circolanti che dei leucociti infiltranti il tumore nei pazienti affetti da meningioma (MNG) e glioblastoma (GBM), al fine di studiarne le caratteristiche, con l'obiettivo finale di trovare nuove strategie terapeutiche. Abbiamo pertanto eseguito un’accurata immunofenotipizzazione del sangue periferico e del tessuto tumorale, analizzato subito dopo la resezione chirurgica, mediante citofluorimetria a flusso multi-parametrica in 34 pazienti con MNG (grado I-II OMS) e in 76 pazienti con GBM (glioma di grado IV OMS). Abbiamo inoltre testato l’attività immunosoppressiva delle popolazioni di origine mieloide isolate da biopsia. Nel sangue periferico abbiamo osservato delle alterazioni significative nelle sottopopolazioni di cellule di origine mieloide, rivelando che un particolare sottogruppo di monociti viene attivamente reclutato al sito tumorale. Inoltre, quattro sottopopolazioni di cellule soppressorie di derivazione mieloide (MDSC) sono rilevabili nel sangue e nel tessuto tumorale dei pazienti affetti da MNG e GBM. Tre di queste popolazioni sono significativamente espanse nel sangue dei pazienti, mentre due di esse sono significativamente espanse nel tessuto tumorale. In questo studio, abbiamo analizzato anche i livelli plasmatici di arginasi 1 (ARG-1) e la sua attività funzionale in campioni di plasma di pazienti con MNG o GBM ed abbiamo osservato sia un aumento significativo della sua concentrazione plasmatica che della sua attività funzionale, rispetto al gruppo di controllo. Analizzando il tessuto tumorale, abbiamo osservato la presenza di un importante infiltrato leucocitario, costituito prevalentemente da cellule mieloidi CD33+ ed in particolare da macrofagi dotati di attività soppressiva e la cui percentuale è notevolmente elevata in entrambi i tipi di tumore. Nei pazienti con GBM, sulla base dell'espressione di diversi marcatori, abbiamo potuto discriminare i macrofagi derivati dal midollo osseo (BMDM) dalla microglia (MG). Queste popolazioni macrofagiche hanno dimostrato avere una diversa attività soppressoria, infatti, i BMDM risultano essere più immunosoppressivi rispetto alla MG che invece ha una bassa o irrilevante capacità soppressoria. I risultati di questo studio sottolineano quindi l’esistenza di una complessa interazione tra sistema immunitario ed i principali tumori cerebrali.

Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123dim and CD11c– CD123high) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2–1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class II molecules, CD33 and low levels of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc=γRIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell priming
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123dim and CD11c– CD123high) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2–1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class II molecules, CD33 and low levels of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc=γRIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell priming.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Il microbiota intestinale (insieme dei microrganismi che si trovano all’interno dell’apparato gastroenterico) svolge diverse funzioni e, tra queste, di particolare interesse è il suo rapporto con il sistema immunitario. Infatti, diversi studi hanno evidenziato la presenza di disbiosi non solo in corso di patologie gastroenteriche, ma anche autoimmuni ed infettive. Gli studi in medicina veterinaria sull’argomento sono ancora pochi e proprio per questo motivo, all’interno di questo progetto, è stato scelto di indagare la possibile relazione tra il microbiota intestinale e due particolari patologie infettive (la peritonite infettiva felina -FIP- e la leishmaniosi canina) la cui patogenesi è fortemente influenzata dal tipo di risposta immunitaria sviluppata dall’ospite. Gli scopi di questo progetto sono quindi stati: la valutazione del microbiota intestinale in gatti affetti o meno da FIP (studio I). Dal momento che diagnosi in vivo di FIP risulta spesso difficoltosa, è stato valutato il potenziale, come biomarker di FIP, della paraoxonasi-1, una proteina di fase acuta negativa fortemente influenzata da importanti stati ossidativi (studi II e III). Per lo stesso motivo è stata valutata la correlazione tra le performance diagnostiche di istopatologia, immunoistochimica e RT-PCR su differenti organi (studio IV). Infine, è stata indagata la composizione del microbiota intestinale in cani infetti o meno da Leishmania spp., correlando i risultati ottenuti con le differenti popolazioni leucocitarie valutate mediante citofluorimetria (studi V e VI). I risultati ottenuti da questo progetto hanno fornito delle indicazioni preliminari sulla composizione del microbiota intestinale in gatti affetti da FIP o positivi per Coronavirus, che necessitano però un approfondimento su un gruppo di studio più ampio (studio I). È stato possibile determinare gli intervalli di riferimento della paraoxonasi-1 nel gatto ed evidenziare le sue buone performance come marker diagnostico in corso di FIP (studi II e III). Nonostante l’immunoistochimica rimanga il gold standard per la diagnosi di FIP, l’associazione con RT-PCR potrebbe ridurre gli errori diagnostici, vista la buona correlazione tra le due metodiche (studio IV). Infine, la valutazione della composizione del microbiota e delle popolazioni leucocitarie in cani affetti da leishmaniosi ha messo in luce delle differenze significative sia rispetto ai cani sani, che agli esposti asintomatici. Questi risultati sono incoraggianti e possono fungere da punto di partenza per ulteriori indagini (studi V e VI).
The gut microbiota (consortium of all the microorganisms that inhabit the gastrointestinal tract) plays different roles in the host. Among these, its relationship with the immune system has been of great interest in the last few years. Indeed, several studies highlight the presence of dysbiosis not only in gastrointestinal diseases, but also during autoimmune or infectious diseases. Literature about this topic is scarce in veterinary medicine. Thus, in this project, the possible relationship between gut microbiota and two specific diseases (feline infectious peritonis -FIP- and canine leishmaniasis) was investigated. These diseases were chosen due to the pivotal role of the immune response in their pathogenesis. The aims of this projects were: the evaluation of gut microbiota of cats with and without FIP (study I). Since in vivo diagnosis of FIP is quite challenging, the potential role of paroxonase-1 (a negative acute phase protein strongly influenced by oxidation) as a biomarker of FIP was investigated (studies II-III). For the same reason, the diagnostic agreement among histopathology, immunohistochemistry and RT-PCR on different organs was evaluated (study IV). Finally, the gut microbiota composition in dogs infected or not by Leishmania spp. was investigated. The results were correlated with the leukocyte populations studied by flow cytometry (studies V-VI). Results obtained in this project provided preliminary data about gut microbiota composition in cats affected by FIP or only Coronavirus positive. This achievement needs to be further investigated on a bigger sample size (study I). Paraoxonase-1 reference interval and its good performance as a diagnostic biomarker of FIP were determined (studies II-III). Despite the immunohistochemistry is still the gold standard for FIP diagnosis, the good diagnostic agreement obtained in the study suggested that a possible association with RT-PCR could minimize diagnostic errors (study IV). Finally, the gut microbiota composition and leukocyte populations of leishmaniotic dogs highlighted some significant differences compared with both healthy and exposed asymptomatic dogs. These promising results could be a starting point for further researches (studies V-VI).

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Tuberculosis (TB) disease results in approximately 2 million deaths annually and is the leading cause of death due to a single infectious agent. Previous studies have indicated that host genetics play an important role in the development of TB. This together with pathogen and environmental factors intensifies the complexity of this disease. The Major Histocompatibility Complex (MHC) and Leukocyte Receptor Complex (LRC) comprise several genes which are known to be important modulators of the host immune response. The human leukocyte antigen (HLA) class-I genes of the MHC are involved in the presentation of pathogenic antigens on the surfaces of infected cells, while the killer cell immunoglobulin-like receptors (KIRs) of the LRC are involved in the recognition of self and non-self cells. Natural Killer (NK) cells through their KIRs are thus able to kill non-self cells through recognition of the class-I molecules expressed. Additionally, HLAs and KIRs are extremely polymorphic and differ markedly across populations of different ethnicities. Here we studied these genes and their polymorphisms in the South African Coloured (SAC) population to determine their involvement in susceptibility to TB, susceptibility to disease caused by specific Mycobacterium tuberculosis subtypes, and understanding their ancestral contribution to the SAC with regards to the development of TB. We showed that the KIR3DS1 gene and KIR genotypes with five or more activating KIRs, and the presence of 3DS1, protected against the development of active TB in the SAC population. Several HLA class-I alleles were identified as susceptibility factors for TB disease. With regards to genes of the MHC and LRC, several loci were found to alter susceptibility to TB in the SAC population, including MDC1, BTNL2, HLA-DOA, HLA-DOB, C6orf10, TAP2, LILRA5, NCR1, NLRP7 and the intergenic regions between HLA-C/WASF5P and LAIR1/TTYH1. We showed that the Beijing strain occurred more frequently in individuals with multiple disease episodes, with the HLA-B27 allele lowering the odds of having an additional episode. Associations were identified for specific HLA types and disease caused by the Beijing, Latin America-Mediterranean (LAM), Low-Copy Clade (LCC), and Quebec strains. HLA types were associated with disease caused by strains from the Euro-American or East Asian lineages, and the frequencies of these alleles in their sympatric human populations identified potential co-evolutionary events between host and pathogen. Finally, we showed that the SAC population is the most diverse SA population with regards to HLA alleles and KIR genotypes, as would be expected given the admixture of the SAC. Based on the HLA allele class-I profiles across SA populations, we noted that the Ag85BESAT- 6, Ag85B-TB10.4 and Mtb72f vaccines currently undergoing clinical trials would have low efficacy across most SA populations. We showed that the MHC and LRC regions in SAC healthy controls are predominantly of European ancestry, and that SAC TB cases are more closely related to Khoisan and black SA population groups. Our work highlights the importance of investigating both host and pathogen genetics when studying TB disease development and that understanding the genetic ancestral contributions to the SAC population can contribute to the identification of true and novel TB causing variants.
AFRIKAANSE OPSOMMING: Tuberkulose (TB) is jaarliks verantwoordelik vir ongeveer 2 miljoen sterftes en is die hoofoorsaak van dood as gevolg van „n aansteeklike siekte. Vorige navorsingstudies het aangedui dat die genetiese samestelling van die gasheer „n beduidende rol speel in die ontwikkeling van TB. Die kompleksiteit van hierdie siekte word vererger deur die betrokkenheid van die gasheer genoom sowel as bakteriële en omgewings faktore. Die Major Histocompatibility Complex (MHC) en Leukocyte Receptor Complex (LRC) bestaan uit verskeie gene wat die gasheer immuunrespons verstel. Die human leukocyte antigen (HLA) klas I gene van die MHC is betrokke by die aanbieding van patogeniese antigene op die oppervlak van geïnfekteerde selle, terwyl die killer cell immunoglobulin-like receptors (KIRs), geleë in die LRC, betrokke is by die herkenning van eie en vreemde selle. NK selle, deur middel van hul KIRs, kan dus vreemde selle uitwis aangesien hulle die uitgedrukte klas I molekules kan herken. Beide HLA en KIRs is hoogs polimorfies en verskil beduidend tussen etniese groepe. In hierdie studie is die bogenoemde gene en hul polimorfismes in die Suid Afrikaanse Kleurling bevolking (SAC) ondersoek om vas te stel tot watter mate dit genetiese vatbaarheid vir TB, asook vatbaarheid vir TB wat deur spesifieke Mycobacterium tuberculosis subtipes veroorsaak word, beïnvloed. Daar is ook gepoog om te verstaan hoe die voorouerlike bydrae van hierdie gene die SAC met betrekking tot TB vatbaarheid affekteer. Die resultate van die studie het aangedui dat die KIR3DS1 geen en KIR genotipes met vyf of meer aktiewe KIRs en die teenwoordigheid van 3DS1, die SAC bevolking beskerm teen die ontwikkeling van aktiewe TB. Verskeie HLA klas I allele is geïdentifiseer as vatbaarheidsfaktore vir TB. Talle lokusse van die MHC en LRC gene is ook as vatbaarheidsfaktore vir TB in die SAC bevolking geïdentifiseer, insluitende MDC1, BTNL2, HLA-DOA, HLA-DOB, C6orf10, TAP2, LILRA5, NCR1, NLRP7 en die intergeniese areas tussen HLA-C/WASF5P en LAIR1/TTYH1. Die studie het aangedui dat die Beijing stam meer voorkom in individue wat verskeie kere TB gehad het en dat die HLA-B27 alleel die kanse om „n verdere episode te hê, verlaag het. Assosiasies is geïdentifiseer tussen spesifieke HLA tipes en siekte veroorsaak deur die Beijing, LAM, LCC, en Quebec TB stamme. HLA tipes was geassosieer met siekte veroorsaak deur TB stamme van Euro-Amerikaanse en Oos-Asiëse afkoms. Die frekwensies van hierdie allele, in hul ooreenstemmende mensbevolkings, dui op „n potensïele koevolusionêre gebeurtenis tussen die gasheer en patogeen. Die studie het ook vasgestel dat die SAC populasie die mees diverse SA bevolking is met betrekking tot die HLA allele en KIR genotipes, soos verwag sou word gegewe die gemengde genetiese herkoms van die SAC. Gebaseer op die HLA allele klas I profiel van verskillende SA bevolkings merk ons op dat die Ag85B-ESAT-6, Ag85B-TB10.4 en Mtb72f vaksiene, wat huidiglik kliniese toetsing ondergaan, nie so effektief in die meeste SA bevolkings sal wees nie. Die studie het ook bewys dat die MHC en LRC streke in gesonde SAC kontroles, grootliks afkomstig was van „n Europese nalatenskap en dat die SAC TB gevalle meer verwant is aan die Khoisan en swart SA bevolkings. Hierdie studie beklemtoon die noodsaaklikheid om beide gasheer en patogeen genetika te bestudeer wanneer die ontwikkeling van TB ondersoek word en dat die verstaan van die genetiese voorouerlike bydrae van die SAC bevolking kan bydra tot die identifisering van ware en nuwe TB-veroorsakende variante.

An individual's Human Leukocyte Antigen (HLA) type is an essential immunogenetic parameter, influencing susceptibility to a variety of autoimmune and infectious diseases, to certain types of cancer and the likelihood of adverse drug reactions. I present and evaluate two models for the accurate statistical determination of HLA types for single-population and multi-population studies, based on SNP genotypes. Importantly, SNP genotypes are already available for many studies, so that the application of the statistical methods presented here does not incur any extra cost besides computing time. HLA*IMP:01 is based on a parallelized and modified version of LDMhc (Leslie et al., 2008), enabling the processing of large reference panels and improving call rates. In a homogeneous single-population imputation scenario on a mainly British dataset, it achieves accuracies (posterior predictive values) and call rates >=88% at all classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DRB1) at 4-digit HLA type resolution. HLA*IMP:02 is specifically designed to deal with multi-population heterogeneous reference panels and based on a new algorithm to construct haplotype graph models that takes into account haplotype estimate uncertainty, allows for missing data and enables the inclusion of prior knowledge on linkage disequilibrium. It works as well as HLA*IMP:01 on homogeneous panels and substantially outperforms it in more heterogeneous scenarios. In a cross-European validation experiment, even without setting a call threshold, HLA*IMP:02 achieves an average accuracy of 96% at 4-digit resolution (>=91% for all loci, which is achieved at HLA-DRB1). HLA*IMP:02 can accurately predict structural variation (DRB paralogs), can (to an extent) detect errors in the reference panel and is highly tolerant of missing data. I demonstrate that a good match between imputation and reference panels in terms of principal components and reference panel size are essential determinants of high imputation accuracy under HLA*IMP:02.

Nineteen yearling Quarter Horses were utilized in a randomized, complete block design to evaluate systemic cytokine gene expression and circulating leukocyte population in young horses following an intra-articular lipopolysaccharide (LPS) challenge. Horses were administered an injection of 0.25 ng (n = 7) or 0.50 ng (n = 6) of LPS or lactated Ringer?s solution (n = 6; control). Blood was collected via jugular catheter at pre-injection h 0 and at 2, 6, 12, and 24 h following aseptic injection of the left radiocarpal joint. Aseptic arthrocentesis was performed at the same times to sample synovial fluid for a companion study. Total RNA was isolated from leukocytes using a commercially available kit and real-time PCR was used to determine relative gene expression of the cytokines; interleukin (IL)-1beta (beta), IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-alpha). Determination of total leukocyte subpopulations and differentials was performed by Texas Veterinary Medical Diagnostic Laboratory. Data were analyzed using the PROC MIX procedure of SAS. Gene expression of all cytokines analyzed was unaffected by treatment. However, changes over time were observed in some cytokines. Interleukin-1? was increased above baseline at 6, 12, and 24 h (P = 0.04), IL-6 was decreased slightly at 6 and 12 h and then increased at 24 h (P = 0.002), and TNF-alpha was increased at 6 and 12 h (P = 0.01). Only IL-8 exceeded a 2-fold change in expression (P = 0.01), peaking at 12 h and indicating greater responsiveness to arthrocentesis than was observed in the other cytokines. No treatment effects on the leukocyte population were observed; however, total circulating leukocytes increased over time (P = 0.04), peaking at 6 h post-injection. Similarly, an increase over time was observed in monocytes (P = 0.002) and in platelets (P = 0.01) at 24 h post-injection. The results indicate that regardless of treatment, a mild immune response was elicited, likely due to repeated arthrocentesis. Future experiments should consider the effects of arthrocentesis and potential systemic inflammatory response, even in control animals, when administering intra-articular LPS to young horses.

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and γδ T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and γδ T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and γδ T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

South Africa is at present encountering one of the worst Tuberculosis (TB) epidemics in the world, accentuating the need for intervention to eradicate TB. Various studies have established that certain population groups are at risk for increased susceptibility to infection with Mycobacterium tuberculosis (M. tuberculosis). This predominantly occurs in populations, like the native African population groups, who were not exposed to TB until the disease arrived in their country with European settlers, colonialists and missionaries. These population groups consequently lack the natural resistance to infection, which other populations developed through years of exposure to the pathogen. Several susceptibility-associated genetic polymorphisms have been proposed to explain differential susceptibility to TB. HLA class II molecules play a pivotal role in the activation of the host immune response against M. tuberculosis. Consequently numerous HLA class II genes have been found to be associated with TB. Among the most commonly observed associations is that of HLA-DR2 with TB, which has been observed in various population groups. Although this association has been observed to transcend ethnic barriers, inter-population variation has also been established regarding HLA-TB associations. In this study, the possible association of HLA class II polymorphisms, specifically of the HLA-DRB1, DQB1, DRB3, DRB4 and DRB5 loci, with TB susceptibility was investigated in the Venda population of South Africa. This was achieved by conducting both a case-control and family-based association study. The results obtained in this study established a unique association between HLA-DRB1*1302, DQ7 and TB susceptibility. A marginally significant association was also observed with DRB1*1301 and DQ6d and possible TB resistance. The above-mentioned results, which were observed in the case-control group, could not be replicated in the family-based study. It was therefore concluded from the results obtained in this study that employing both a case-control and family-based analysis when undertaking an association study is the most beneficial option.
Prof. Liza Bornman

HLA (Human Leukocyte Antigen) molecules provide a framework for T-cell recognition of antigenic peptides and thus play an important role in the immune system and defence against pathogens. HLA molecules are encoded for by genes on the short arm of chromosome six in the human genome, a region of over 4000 kilo bases (kb) known as the human major histocompatibility complex (MHC) or HLA complex. According to structural and functional characteristics, the genes of this region have been classified into three families, namely classes ƒ¹, ƒ¹ƒ¹ and ƒ¹ƒ¹ƒ¹. The HLA genes are highly polymorphic and because of this characteristic, it increases the functional range of recognition of different antigens and contributes to immunological specificity. Previous studies on population groups such as Indians and Cambodians, have identified an association between HLA polymorphisms and susceptibility to TB. A large number of different population groups with diverse gene pools reside in South Africa, of which the Cape Coloured population is one. This anthropologically distinct population group¡¦s diverse gene pool originated from founder individuals of the colonizing population, which came from various nations and cultural backgrounds, including Europe, Africa (such as Khoi, San Xhosa, Sotho and East African populations), Madagascar and the Far East. Unusual MHC allele frequencies and haplotypes have been identified in the Cape Coloured population. The Cape Coloureds are highly susceptible to TB and reside in the Western Cape, which has a TB incidence rate that is higher than that of any other province in South Africa. The recently admixed Coloured population is a valuable candidate population for the identification of genes/mutations underlying complex diseases. This study focussed on polymorphisms of class ƒ¹ƒ¹ genes, specifically HLA-DRB, and their possible contribution to disease susceptibility in populations of South Africa. Two questions have been formulated: 1) What knowledge can we gain from the current literature on HLA variants in the different population groups of South Africa and disease susceptibility? 2) Is there an association between alleles of the most polymorphic class ƒ¹ƒ¹ MHC gene, HLA-DRB, and susceptibility or resistance to TB in the Cape Coloured population? These two primary questions have been addressed by: 1) Reviewing the literature concerning HLA alleles in diverse population groups in South Africa and their contribution to disease susceptibility. Chapter 2 addresses this objective and is written in the format of a review article to facilitate its future publication, 2) Conducting a TB case-control study, typing HLA-DRB in Cape Coloured individuals residing in the Western Cape to investigate the possible association between TB susceptibility or resistance and specific DRB alleles. The HLA-DRB1, DRB3 and DRB4 typing by means of PCR-SSP (polymerase chain reaction ¡V sequence specific primers) was done on DNA isolated from 106 TB patients and 107 controls from the Cape Coloured population. The results obtained for this experimental investigation is presented (also in publication format) in Chapter 3. Summary of main findings: 1) The literature overview of publications describing HLA related disease association in the different population groups in South Africa (Chapter 2), revealed that unique alleles contributing to susceptibility of various diseases have been identified in some South African populations. The large number of ethnic groups in South Africa and unique populations such as the Cape Coloureds provide a genetic resource, which has the potential to be utilized for candidate gene hunting. 2) A weak association between susceptibility for TB and HLA-DRB1*0301-0302 (DR3) and HLA-DRB3*0101-0301 (DR52) exists in the Cape Coloured population (Chapter 3). Since the South African population consists of a large number of different population groups with diverse gene pools, a unique opportunity to study disease predispositions among specific population groups with diverse genetic make-up is presented. With these different populations, often residing in a common environment, the interplay of environmental and genetic factors in disease development could be studied. For example, HLA typing of these populations could clarify the extent to which inter-population HLA variation contributes towards disease susceptibility. The identification of certain HLA-DRB alleles potentially contributing to TB susceptibility, could lead to an understanding of the differential factors involved in disease susceptibility and in turn lead to the understanding of the fundamental mechanisms involved. This could result in therapeutic approaches and treatments that will be advantageous to the population concerned.
Prof. L. Bornman

The human leukocyte antigen (HLA) is the most polymorphic region in the human genome and accounts for more than 10% of human diversity. This region plays an important role in matching donors and recipients for transplantation. The South African Bone Marrow Registry (SABMR) does not reflect the demographics of the South African population. The large number of polymorphisms resulting from HLA diversity in the Black South African population and their limited representation in the SABMR reduce the chances of finding adequate matches between donors and recipients in this group. Umbilical cord blood is an alternative to bone marrow for the treatment of fatal diseases. Less strict HLA matching is required due to the naive nature of the T cells in cord blood. A public umbilical cord blood bank is a necessity in trying to cater for the diverse population in South Africa. However, the ethnic diversity of the South African population poses a great challenge in constituting a public umbilical cord blood bank that is representative of the entire population. The Roche designed next generation sequencing (NGS) high resolution (HR) HLA typing kit enables sequencing of additional HLA exons and could improve the degree of matching between individuals to ultimately decrease adverse reactions. An extensive study of the literature was performed to establish the demographics, linguistics, and HLA diversity of the South African population to determine how a public cord blood bank should be constituted. In addition, HLA genotyping was performed by 454 NGS on 20 samples that had previously been HLA typed by conventional methods. The 454 NGS technique made use of a Roche designed medium and high resolution HLA typing kit to genotype the samples. It was possible to assign accurate genotypes to 95.5% of the loci of interest for the total number of 20 samples using the MR kit, compared with 98.5% using the HR kit. In conclusion, the present study indicates the extreme HLA diversity in the South African population, and therefore, recommends constituting the first public umbilical cord blood bank in Gauteng on the basis of race or major ethnic groupings. A minimum number of 10 000 cord blood units is needed to initiate the bank. Furthermore, the 454 NGS platform together with the HR HLA typing kit display potential as an alternative method to be used in a public cord blood bank, as well as routine clinical and diagnostic laboratories, to ultimately improve HLA matching between donors and recipients.
Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Immunology
unrestricted