There has been an increase in rates of chromosomal abnormalities in newborns as a result of reproductive aging. For the past decades, a lot of effort has been placed on identifying pregnancies at risk of genetic defects. Conventional prenatal genetic diagnosis is achieved by invasive procedures that have been associated with an increased risk of pregnancy loss. This has led the researchers to explore the use of non-/minimally invasive techniques for prenatal diagnosis. Trophoblasts are known to be shed from regressing chorionic villi into the lower uterine pole of pregnant women during the first trimester. These cells are trapped within cervical mucus, which can be retrieved with a cytobrush. By using human leukocyte antigen G (HLA-G) and cytokeratin-7 (CK7) as trophoblast markers, this study aims to investigate the possibility of isolating individual fetal trophoblast from transcervical samples for genetic diagnosis. 195 healthy pregnant women requesting for legal termination of pregnancy (TOP) were recruited in this study. Transcervical cells were collected from them with the use of a cytobrush before TOP. HLA-G+ or CK7+ cells were then isolated by a combination of mucolytic action, fluorescent immunohistochemistry, and micromanipulation. The origin of these cells was subsequently investigated by either fluorescent in situ hybridization (FISH) or allelic profiling by quantitative fluorescent polymerase chain reaction (QF-PCR) based on chromosome 16, chromosome X, amelogenin gene and sex determining region Y (SRY) gene. This study first demonstrated the presence of fetal cells in transcervical samples based on the detection of chromosome Y signal by ordinary PCR. Cells expressing HLA-G and CK7 were also identified among transcervical cells. Immunopositive cells were isolated by micromanipulation under fluorescent microscopy. One isolated cell expressing CK7 was shown to inherit paternal allele at a locus on chromosome 16, suggesting the possible fetal origin of this cell. However, this study was still hampered by a number of technical factors. Further optimization of the protocol is required before transcervical trophoblasts can be retrieved in a reliable manner. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Les molécules du complexe majeur d'histocompatibilité de classe II (CMH II) sont exprimées exclusivement à la surface des cellules présentatrices d'antigènes et servent à stimuler les cellules CD4+ initiant une réponse immunitaire. Le chargement peptidique sur HLA-DR se produit dans les endosomes tardifs et les lysosomes sous l'action de HLA-DM. Cette molécule de classe II non-classique enlève les fragments peptidiques de la chaîne invariante (Ii) restés associés aux molécules de classe II (CLIP) et édite leur répertoire d'antigènes présentés. En utilisant une forme mutante de HLA-DM (HLA-DMy) qui s'accumule à la surface plasmique, nous avons observé que HLA-DMy augmente les chargements de peptides exogènes et aussi la réponse des cellules T en comparaison avec HLA-DM sauvage. Il a été démontré que des molécules chimiques, comme le n-propanol, pouvait avoir le même effet que HLA-DM en remplaçant les peptides associés aux molécules de classe II de la surface cellulaire. De plus, HLA-DMy et le n-propanol ont présenté un effet additif sur la présentation de peptides exogènes. Certaines protéines de la voie endocytique, comme HLA-DR, HLA-DM, HLA-DO et Ii sont ciblés aux compartiments multivésiculaires (MVB) et peuvent être ciblées aux exosomes. Suite à une fusion entre les MVB et la membrane plasmique, les exosomes sont relâchés dans le milieu extracellulaire. Nous avons déterminé que le motif tyrosine de HLA-DMβ et son interaction avec HLA-DR n'affectaient pas le ciblage aux exosomes, sauf la molécule HLA-DO. Cette étude nous a permis de démontrer que HLA-DMy augmente la quantité de peptides exogènes chargés sur les CPA et que HLA-DM et HLA-DMy sont incorporés dans les exosomes. / Major histocompatibility class II (MHC II) molecules are expressed exclusively on the surface of antigen presenting cells and serve to stimulate CD4+ cells to initiate an immune response. Peptide loading on MHC II molecules occurs in late endosomal and lysosomal compartments by the catalytic action of HLA-DM. This non-classical class II molecule removes class-II-associated invariant-chain peptide (CLIP) from class II molecules and edits their repertoire of antigenic peptides loaded. Using a mutant form of HLA-DM (HLA-DMy) that is targeted to the plasma membrane, we observed in the case of HLA-DMy, there is an increase of the loading of exogenous peptides and also significantly increased T cell response in comparison with HLA-DM wild-type. It was found that some chemical molecules, like n-propanol, could mimic the effect of HLA-DM by removing peptides from cell surface class II molecules. Interestingly, HLA-DMy and n-propanol seem to have an additive effect on the exogenous peptide loading. Some proteins of the endosomal pathway, like HLA-DR, HLA-DM, HLA-DO and Ii are targeted to microvesicules-containing compartments called MVB and they can be introduced into exosomes. Following the fusion between MVB and plasma membrane, exosomes are released in extracellular environment. We have determined that tyrosine motif of HLA-DMβ and interaction with HLA-DR does not affect HLA-DM targeting to exosomes, except for the HLA-DO molecule. In conclusion, we showed that HLA-DMy increases the quantity and the quality of exogenous peptides loading on APC and HLA-DM and HLA-DMy are incorporated to exosomes.

Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein, plays an essential role in immune tolerance and is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). To examine the pattern of HLA-G expression post-transplantation we determined that HLA-G can be up-regulated in smooth muscle cells (SMCs) following exposure to everolimus. We also determined that HLA-G at 500 and 1000 ng/ml reduces SMC proliferation. In further studies, treatment with HLA-G inhibited TNFα-stimulated neutrophil adhesion to endothelial cells (ECs) at all concentrations tested (0.1-1 ng/ml), suggesting a role in inflammation. The expression of HLA-G is influenced by a polymorphism in the HLA-G gene. We sought to determine if the 14bp insertion/deletion polymorphism can predict the development of CAV. There was no association between this polymorphism and CAV; however, this study had a small number of patients; therefore further investigations are needed to confirm these findings.

No description available.

Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein, plays an essential role in immune tolerance and is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). To examine the pattern of HLA-G expression post-transplantation we determined that HLA-G can be up-regulated in smooth muscle cells (SMCs) following exposure to everolimus. We also determined that HLA-G at 500 and 1000 ng/ml reduces SMC proliferation. In further studies, treatment with HLA-G inhibited TNFα-stimulated neutrophil adhesion to endothelial cells (ECs) at all concentrations tested (0.1-1 ng/ml), suggesting a role in inflammation. The expression of HLA-G is influenced by a polymorphism in the HLA-G gene. We sought to determine if the 14bp insertion/deletion polymorphism can predict the development of CAV. There was no association between this polymorphism and CAV; however, this study had a small number of patients; therefore further investigations are needed to confirm these findings.

Deficiency of the cytochrome P-450 steroid 21-hydroxylase (21-OHase) which causes Congenital Adrenal Hyperplasia (CAH) is a monogenic autosomal recessive disorder which is linked to HLA. There are two 21-OHase genes in man, A and B, and they are mapped to the HLA class III region ~ 3 kb 3' to the complement genes C4A and C4B, respectively. Two genes encoding 21-OHase were isolated, characterized and sequenced. Both 21-OHase genes are ~ 3.3 kb in length and are split into 10 exons by nine introns. Comparison of the two genes showed that although they are highly conserved, there are three deleterious mutations in the 21-OHase A gene which cause frameshifts and introduce in phase premature termination codons. Thus the 21-OHase A gene is a pseudogene. Comparison of the 21-OHase B gene to the other cytochrome P-450 sequences revealed that although the cysteine-429 was conserved in 21-OHase, there is very little homology with other cytochrome P-450, indicating it belongs to a separate family of genes within the superfamily. Clear evidence of polymorphism in 21-OHase is apparent on comparison with other 21-OHase B sequences. There is a size polymorphism of 494 and 495 amino acids. The differing severities of 21-OHase deficiency in CAH may be due to allelic variants of the 21-OHase B gene, since in most cases the defect is not due to gene deletion (Rumsby et al., 1986). A 21-OHase B gene from a patient with CAH was characterized and sequenced. There were 13 nucleotide alterations in his single 21-OHase B gene, one of which at codon 269 caused a serine to change to a threonine residue. The G → C transversion in the 21-OHase B gene from the patient at codon 269 introduced a new NcoI restriction site into the gene. This restriction fragment length polymorphism (RFLP) was used to study other patients with CAH and normal individuals. The NcoI RFLP was found not to be confined to the 21-OHase B gene but was also present in some 21-OHase A genes. It is likely therefore that the mutation occurred in the pseudogene first and then transferred to some 21-OHase B genes.

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

HLA-G is a protein normally expressed during pregnancy, protecting the fetus from the maternal immune system. Previous studies have shown an association between HLA-G expression post-transplantation and lower incidences of organ rejection. To further examine this beneficial role, we conducted a prospective study following a cohort of heart transplant recipients for one year and measuring their plasma HLA-G levels at various time points. Preliminary analyses failed to reveal an association between HLA-G and various parameters of rejection and vasculopathy. However, we decided to examine the in vitro effects of HLA-G in a smooth muscle cell (SMC) migration assay and whether HLA-G can be modulated pharmacologically. We made the novel observations that purified HLA-G was capable of inhibiting migration of SMCs, a key event in the development of cardiac allograft vasculopathy. IL-10, an anti-inflammatory cytokine, was capable of upregulating HLA-G in a Jeg-3 cell line. The modulation of HLA-G may represent a strategy to protect again vasculopathy, which is a leading cause of morbidity and mortality in heart transplant recipients.

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

HLA-G is a protein normally expressed during pregnancy, protecting the fetus from the maternal immune system. Previous studies have shown an association between HLA-G expression post-transplantation and lower incidences of organ rejection. To further examine this beneficial role, we conducted a prospective study following a cohort of heart transplant recipients for one year and measuring their plasma HLA-G levels at various time points. Preliminary analyses failed to reveal an association between HLA-G and various parameters of rejection and vasculopathy. However, we decided to examine the in vitro effects of HLA-G in a smooth muscle cell (SMC) migration assay and whether HLA-G can be modulated pharmacologically. We made the novel observations that purified HLA-G was capable of inhibiting migration of SMCs, a key event in the development of cardiac allograft vasculopathy. IL-10, an anti-inflammatory cytokine, was capable of upregulating HLA-G in a Jeg-3 cell line. The modulation of HLA-G may represent a strategy to protect again vasculopathy, which is a leading cause of morbidity and mortality in heart transplant recipients.