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Type 1A diabetes mellitus or T1D is a chronic disease characterized by T cell mediated destruction of the insulin producing β cells in the islets of Langerhans. The classical symptoms include high glucose levels in urine and blood, polyuria, and polydipsia. Complications associated with T1D include blindness, amputations, and end-stage renal disease, and premature death. The non-obese diabetic (NOD) mouse, first described in 1980, is widely used as a model organism for T1D. T1D disease in the NOD mouse shares a number of similarities to human T1D including dependence on genetic and environmental factors. More than 30 disease associated gene regions or loci (termed insulin dependent diabetes, or Idd, loci) have been associated with T1D development in NOD. For some of these Idds, the corresponding region in human has been linked to the development of T1D in human. T1D, both in humans and mice, is recognized as a T cell mediated disease. However, many studies have shown the importance of both the metabolome and the immune system in the pathogenesis of the disease. Appearance of autoantibodies in the serum of patients is the first sign of pathogenesis. However, molecular and cellular events precede the immune attack on the β-cell immunity. It has been shown that patients who developed T1D have an altered metabolome prior to the appearance of autoantibodies. Although much is known about the pathogenesis of T1D, the contribution of the environment/immune factors triggering the disease is still to be revealed.  In the present study both metabolic and immune deviations observed in the NOD mouse was analyzed. Serum metabolome analysis of the NOD mouse revealed striking resemblance to the human metabolic profile, with many metabolites in the TCA cycle significantly different from the non-diabetic control B6 mice. In addition, an increased level of glutamic acid was of the most distinguishing metabolite. A detailed bioinformatics analysis revealed various genes/enzymes to be present in the Idd regions. Compared to B6 mice, many of the genes correlated to the metabolic pathways, showed single nucleotide polymorphism (SNP), which can eventually affect the functionality of the protein. A genetic analysis of the increased glutamic acid revealed several Idd regions to be involved in this phenotype. The regions mapped in the genetic analysis harbor important enzymes and transporters related to glutamic acid. In-vitro islet culture with glutamic acid led to increased beta cell death indicating a toxic role of glutamic acid specifically towards insulin producing beta cells. In the analysis of the immune system, B cells from NOD mice, which are known to express high levels of TACI, were stimulated with APRIL, a TACI ligand. This resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. NOD mice have previously been shown to react vigorously to T-dependent antigens upon immunization. In this study we confirmed this as NOD mice showed an enhanced and prolonged immune response to hen egg lysozyme. Thus, serum IgG levels were significantly increased in the NOD mice and were predominantly of the IgG1 subtype. Immunofluorescence analysis revealed increased number of germinal centers in the NOD mice. Transfer of purified B and T cells from NOD to an immune deficient mouse could reproduce the original phenotype as seen in the NOD mice.     Collectively, this thesis has analyzed the metabolomics and immune deviations observed in the NOD mice.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Chronic HIV-1 infection is characterized by immune activation and dysregulation of immune homeostasis, which impacts on multiple immune cell types. The B-cell compartment, which plays an important role in the producing neutralizing antibodies, is also dysregulated in HIV- 1 infection. In this study we investigated peripheral blood B-cell subset distribution, and changes in expression of cellular activation, inhibition, and apoptosis signaling markers in both untreated chronic HIV-1 infected individuals and healthy uninfected controls. The neuropeptide immune modulator, vasoactive intestinal peptide (VIP) is known to selectively down-regulate activation of CD4+ T-cells in various disease settings including HIV-1, however to our knowledge, no studies have investigated the effect of VIP inhibition on B-cell activation. Materials & Methods: A total of 21 HIV+ve (CD4 count >250 cells/µl), and 19 HIV-ve individuals were recruited from the Emavundleni voluntary testing and counseling clinic in Crossroads, Western Province, South Africa. Whole blood was stained to distinguish B-cell subsets (activated memory (AM: CD21-CD27+), resting memory (RM: CD21+CD27+), mature naïve (MN: CD21+CD27-), or tissue-like memory (TLM: CD21loCD27lo). In addition expression of markers of B-cell activation (CD126, CD86, CD38, CD284, CD287), inhibition (CD72, CD85j, CD300a, CD305, CD307d), and apoptosis signaling (CD95), was assessed ex vivo by flow cytometry (BD FACSCanto II). For determination of functional responsiveness isolated B-cells (RosetteSep, Stemcell Technologies) were cultured for 18h (37°C, 5%CO2) without stimulation or stimulated with TLR ligands (LPS or R848). Stimulation experiments were also performed in the presence or absence of VIP. Results: Chronic HIV-1 infection affected B-cell subset distribution. The percentage (%) of TLM was increased by 59.24%, and %RM was decreased by 22.73% (both p<0.01). Total expression of the VIP receptor VPAC2 was decreased by 47.35% (p=0.0296). Subsets had a mixed phenotype ex vivo; HIV infection upregulated CD38 (by 59.56%, p=0.0004), CD72 (by 60.70%, p=0.0396), CD307d (by 68.63%, p=0.0015) on AM, while RM B-cells had increased expression of TLR4 (by 107.04%, p=0.0057) and TLR7 (by 208.14%, p=0.0199). TLM B cells (i.e. exhausted phenotype) displayed upregulated TLR7 (by 550%, p=0.0128) and CD307d (by 72.40% p=0.045) expression. MN B-cells had increased CD72 expression (by 70.98%, p=0.0026). R848 upregulated CD86 expression by 42.20% on AM (p<0.01), and by 56.06% on RM B-cells (p<0.01), which was significantly downregulated with VIP inhibition (both p<0.05). Similarly, CD95 expression on RM, TLM, and MN B-cells increased by 31.10% (p<0.001), 21.46% (p<0.01), and 39.92% (p<0.01) with R848 stimulation respectively, which was also significantly downregulated with VIP inhibition. Conclusion: These data indicate that B-cells in untreated HIV infection display increased levels of activation, and also the potential for increased susceptibility to apoptosis as evidenced by increased FAS (CD95) expression. VIP significantly down-regulated markers of activation, inhibition, and apoptosis signaling. Dysregulation of B-cells is thus apparent in asymptomatic stable chronic HIV-1 infection, which may impact on both inefficient neutralizing antibody production and hypergammaglobulinemia. The ability of VIP to prevent stimulationassociated marker upregulation may indicate that VIP is a potential therapeutic agent. Its immuno-modulatory properties were demonstrated to limit B-cell hyperactivation, and selectively down-regulate apoptosis and mark it out for further investigation. / AFRIKAANSE OPSOMMING: Inleiding: Immunaktivering en ongekoppelde immuun-homeostase is kenmerke van chroniese MIVinfeksie. Ons het perifere bloed B-sel subgroep-verspreiding, en veranderinge in die uitdrukking van merkers van aktivering, inhibisie, en apoptose in 'n onbehandelde MIV-1 besmettende groep ondersoek (in vergelyk met 'n gesonde onbesmettende kontrole). Die immuun-moduleerder, vasoaktiewe intestinale peptied (VIP) is bekend om aktivasie van geaktiveerde CD4+ T-selle te verminder, maar tot ons kennis, is daar geen studies wat die effek van VIP-inhibisie op B-sel aktivering ondersoek het, in die konteks van MIV-1 infeksie. Materiaal & Metodes: MIV+we individue (CD4-telling >250 selle/µl) , en MIVwe kontroles is gewerf uit die vrywillige toetsing en berading Emavundleni kliniek, Crossroads, Westelike Provinsie, Suid-Afrika. Bsel subgroepe is gedefinieer as geaktiveerde geheue (AM: CD21- CD27+ ), rusende geheue (RM: CD21+ CD27+ ), volwasse naïef (MN: CD21+ CD27- ), of weefsel-agtige geheue (TLM: CD21loCD27lo). Merkers van aktivering (CD126, CD86, CD38, CD284, CD287), inhibisie (CD72, CD85j, CD300a, CD305, CD307d), en apoptose signalering (CD95) is via vloeisitometrie (BD FACSCanto II) op B-selle ex vivo en ook op geïsoleerde B-selle (RosetteSep, Cell Technologies) ondersoek. Vir die bepaling van funksionele responsiwiteit, geïsoleerde B-selle (RosetteSep, StemCell Technologies) was vir 18h (37°C, 5%CO2) gekweek, sonder stimulasie of gestimuleer met TLR ligande (LPS of R848). Stimulasie eksperimente het ook in die teenwoordigheid of afwesigheid van VIP plaasgevind. Resultate: Chroniese MIV-1 infeksie het B-sel subset verspreiding geraak. Die persentasie (%) van TLM is verhoog deur 59,24%, en% RM het met 22.73% afgeneem (beide p <0,01). Totale uitdrukking van die VIP reseptor VPAC2 het met 47,35% afgeneem (p = 0,0296). Subgroepe het 'n gemengde ex vivo fenotipe; MIV-infeksie het CD38 (deur 59,56%, p=0,0004), CD72 (deur 60,70%, p=0,0396), CD307d (deur 68,63%, p=0,0015) op AM verhoog, terwyl RM Bselle het verhoogde uitdrukking van TLR4 (deur 107,04%, p=0,0057) en TLR7 (deur 208,14%, p=0,0199). TLM B-selle (die uitgeputtende fenotiep) het verhoogde TLR7 (deur 550%, p=0,0128) en CD307d (deur 72,40% p=0.045) uitdrukking gewys. MN B-selle het verhoogde uitdrukking van CD72 (deur 70,98%, p = 0,0026). R848 het CD86 uitdrukking op AM deur 42,20%, en op RM deur 56,06% toegeneem (beide p <0,01). Dit het met VIP inhibisie beduidend afgeneem (beide p <0.05). CD95 uitdrukking was soortgelyk verhoog op RM, TLM, en MN B-selle met 31.10% (p <0.001), 21,46% (p <0,01), en 39,92% (p <0,01) met R848 stimulasie. Al drie het beduidend afgeneem met VIP inhibisie. Gevolgtrekking: Hierdie data dui daarop dat B-selle in onbehandelde MIV-infeksie vertoon verhoogde aktiveringsvlakke, en ook die potensiaal vir verhoogde vatbaarheid vir apoptose soos bewys deur verhoogde uitdrukking van FAS (CD95). VIP het beduidend merkers van aktivering, inhibisie, en apoptose af-gereguleer. Wanfunksie van B-selle is dus in asimptomatiese stabiele chroniese MIV-1 infeksie duidelik, wat impak kan hê op beide oneffektiewe neutraliserende teenliggaampie produksie, en hiepergammaglobulinimie. Die vermoë van VIP stimulasie-verwante merker opregulasie te voorkom kan aandui dat VIP 'n potensiële terapeutiese agent is. VIP se immuno-moduleerende eiendomme is gedemonstreer om Bsel hieperaktiveering te beperk, en selektief apoptose afreguleer, en merk dit vir verdere ondersoek.

Cartilaginous fish are the most ancient lineage to possess an adaptive immune system however nothing is known about the processes involved in the development, maintenance or proliferation of B cells in this group. In this thesis studies were undertaken to explore whether transcription factors, cytokines and cell surface markers that are involved in B cell immune responses in mammals are also present in cartilaginous fish. Through sequence database mining forty-one genes involved in B cell immune biology were found; of these twelve were B cell transcription factors. The presence of important B cell transcription factors, such as EBF1, pax5, E2A, Blimp1, PU.1 and Bcl6 suggests that B cells in cartilaginous fish probably undergo a similar developmental pathway as those in mammals. Eight cytokines, eleven cytokine receptors and four B cell surface markers were also identified, including CD40L, BAFF and CD79α that were further studied in this thesis. I cloned CD40L and BAFF from cartilaginous fish, both members of the tumour necrosis factor family and found cartilaginous fish BAFF genes have an extra exon that forms a unique α-helix insertion, and which may impact on receptor binding. The importance of all three molecules for the adaptive immune response was indicated by relatively high expression in shark immune tissue, such as spleen, gut-associated lymphoid tissue, gill and the Leydig organ, and the fact that their expression could be induced by immunostimulants, such as pokeweed mitogen. The finding that CD79α expression significantly correlates with those of immunoglobulin heavy-chains and the co-expression of CD79α and IgM on the B cell surface indicate that as in mammals CD79α in cartilaginous fish is expressed by B cells and associates with surface-bound immunoglobulin to form the active B cell receptor. Thus CD79α may serve as a pan B cell marker in future immunological studies in cartilaginous fish.

published_or_final_version / Medicine / Master / Master of Philosophy

B cells have unique machinery to make up a large pool of antibody repertoire. After V(D)J recombination in early B cell development, the rearranged immunoglobulin genes are further diversified by somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Acitvation induced cytidine deaminase (AID) is a key initiating factor for SHM, GC and CSR. A majority of research data supports the model that AID modifies Ig genes at the DNA level by deaminating cytosines to uracils. The mutagenic activity of AID is largely restricted to Ig genes to avoid genomic instability in general. The specificity cannot be attributed to the primary sequence of the Ig genes since unrelated DNA is mutated by AID in the context of Ig genes. A clue to this problem is that AID function is dependent on transcription. Since not all transcribed genes are mutated by AID, there must be something special about the transcription of Ig genes, and the reasoning has prompted extensive analysis of Ig promoters and enhancers. We addressed this question in chicken B cell line DT40. We identified a 2.4-kilobase regulatory region which is important for AID function both within and outside of Ig locus. This regulatory region contains binding sites for multiple transcription factors. Mutation of these binding sites impairs AID mediated gene conversion. In addition, ablation of NF-κB family member, c-Rel and p50, reduces the AID targeting function of this regulatory region. Since the implicated transcription factors have been reported to associate with histone acetylases, the regulatory region may function by facilitating the access of AID to target DNA. To test this hypothesis, we used the I-SceI endonuclease and dam methylase as probes for chromatin structure. We found that the regulatory region does not increase chromatin accessibility to these probes. In fact, the regulatory region appears to interfere with the cleavage of target DNA by I-SceI. Another possible role of the regulatory region could be direct recruitment of AID to Ig genes. To test this hypothesis, we utilized Dam identification method. Surprisingly, we found that the regulatory region facilitates AID targeting to the Igλ locus. / text

<p>Natural IgM (nIgM) is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. nIgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. However, the cells that maintain high titers of nIgM in the circulation had not yet been identified. Several studies have linked serum nIgM with the presence of fetal-lineage B cells, and others have detected IgM secretion directly by B1a cells in various tissues. Nevertheless, a substantial contribution of undifferentiated B1 cells to nIgM titers is doubtful, as the ability to produce large quantities of antibody (Ab) is a function of the phenotype and morphology of differentiated plasma cells (PCs). No direct evidence exists to support the claim that a B1-cell population directly produces the bulk of circulating nIgM. The source of nIgM thus remained uncertain and unstudied.</p><p>In the first part of this study, I identified the primary source of nIgM. Using enzyme-linked immunosorbent spot (ELISPOT) assay, I determined that the majority of IgM Ab-secreting cells (ASCs) in naïve mice reside in the bone marrow (BM). Flow cytometric analysis of BM cells stained for intracellular IgM revealed that nIgM ASCs express IgM and the PC marker CD138 on their surface, but not the B1a cell marker CD5. By spinning these cells onto slides and staining them, following isolation by fluorescence-activated cell sorting (FACS), I found that they exhibit the typical morphological characteristics of terminally differentiated PCs. Transfer experiments demonstrated that BM nIgM PCs arise from a progenitor in the peritoneal cavity (PerC), but not isolated PerC B1a, B1b, or B2 cells. Immunoglobulin (Ig) gene sequence analysis and examination of B1-8i mice, which carry an Ig knockin that prohibits fetal B-cell development, indicated that nIgM PCs differentiate from fetal-lineage B cells. BrdU uptake experiments showed that the nIgM ASC compartment contains a substantial fraction of long-lived plasma cells (LLPCs). Finally, I demonstrated that nIgM PCs occupy a survival niche distinct from that used by IgG PCs.</p><p>In the second part of this dissertation, I characterized the unique survival niche of nIgM LLPCs, which maintain constitutive high titers of nIgM in the serum. By using genetically deficient or Ab-depleted mice, I found that neither T cells, type 2 innate lymphoid cells, nor mast cells, the three major hematopoietic producers of IL-5, were required for nIgM PC survival in the BM. However, IgM PCs associate strongly with IL-5-expressing BM stromal cells, which support their survival in vitro when stimulated. In vivo neutralization of IL-5 revealed that, like individual survival factors for IgG PCs, IL-5 is not the sole supporter of IgM PCs, but is likely one of several redundant molecules that together ensure uninterrupted signaling. Thus, the long-lived nIgM PC niche is not composed of hematopoietic sources of IL-5, but a stromal cell microenvironment that provides multiple redundant survival signals.</p><p>In the final part of my study, I identified and characterized the precursor of nIgM PCs, which I found in the first project to be resident in the PerC, but not a B1a, B1b, or B2 cell. By transferring PerC cells sorted based on expression of CD19, CD5, and CD11b, I found that only the CD19+CD5+CD11b- population contained cells capable of differentiating into nIgM PCs. Transfer of decreasing numbers of unfractionated PerC cells into Rag1 knockouts revealed an order-of-magnitude drop in the rate of serum IgM reconstitution between stochastically sampled pools of 106 and 3x105 PerC cells, suggesting that the CD19+CD5+CD11b- compartment comprises two cell types, and that interaction between the two necessary for nIgM-PC differentiation. By transferring neonatal liver, I determined that the early hematopoietic environment is required for nIgM PC precursors to develop. Using mice carrying a mutation that disturbs cKit expression, I also found that cKit appears to be required at a critical point near birth for the proper development of nIgM PC precursors.</p><p>The collective results of these studies demonstrate that nIgM is the product of BM-resident PCs, which differentiate from a PerC B cell precursor distinct from B1a cells, and survive long-term in a unique survival niche created by stromal cells. My work creates a new paradigm by which to understand nIgM, B1 cell, and PC biology.</p> / Dissertation

The non-obese diabetic (NOD) mouse is widely used model Type 1 diabetes (T1D), a chronic inflammatory disease characterized by destruction of the insulin producing β cells in the islets of Langerhans by immune cells. The classical symptoms include increased glucose levels in urine and blood, frequent urination and enhanced thirst. The disease has a strong genetic component and is also influenced by the environment. NOD mice develop T1D spontaneously. The disease occurs in two phases; insulitis - the infiltration of immune cells in the islets of Langerhans and overt diabetes caused by the destruction of insulin producing β cells. Several disease associated gene regions or loci [termed insulin dependent diabetes (Idd) loci] have been associated with T1D development. Although, T1D is recognized as a T cell mediated disease in both mouse and man, many studies have shown the importance of B cells in the pathogenesis of the disease. Autoantibodies appear prior to islet infiltration and several molecular and cellular events precede this beta-cell autoimmunity. Although the pathogenesis of T1D is well characterized, less is known about the environmental and immunological factors that trigger the disease. In this thesis, we studied the contribution of B cell anomalies to the skewed immune response observed in the NOD mouse. In our studies covered in the thesis we observed that NOD mice display enhanced IgE in the serum already at one week of age. In addition, upon treatment of pre-diabetic NOD mice with anti-IgE antibodies, diabetes incidence was delayed. We hypothesize that the presence of IgE in the system may be explained due to enhanced class switching. Antibody feedback however, is an essential component of the immune response and can lead to either enhanced or dampened responses. Thus, increased IgE may provide positive feedback that might sustain an immune response. We also aimed to analyze the biological consequence of this feature. In vitro stimulation of B cells by the TACI ligand APRIL resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. In addition, TACI+ cells were observed in NOD germinal centers facilitating increased BAFF uptake and subsequent escape of low affinity antibody producing clones. NOD mice elicited an enhanced and prolonged immune response towards T-dependent antigens such as hen-egg lysozyme (HEL). Serum HEL-specific IgG level was significantly increased and was predominantly of the IgG1 isotype. Immunofluorescence analysis of NOD spleen revealed the presence of spontaneous germinal centers which others have perceived to provide a ready niche for the entry of naïve B cells that encountered novel antigen. Adoptive transfer experiments of purified B and T cells from NOD into NOD.Rag2-/- (NOD-RAG) mice illustrated the importance of B cell intrinsic defects in the reproduction of the original phenotype as observed in NOD.

In these studies we sought to determine the role of Fyn kinase and ADAM10 in B cell biology. A disintegrin and metalloproteinase 10 (ADAM10) is a zinc dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. In particular, ADAM10 has been identified as a key regulator of lymphocyte development. Here we report that ADAM10 is dispensable for early B cell development within the bone marrow. However, deletion of ADAM10 from all peripheral B cells or in post-switch cells leads to severe impairments in humoral responses. When ADAM10 was deleted from all peripheral B cells a decrease in antigen specific IgG production was seen both with respect to serum levels and IgG ASCs, indicating that plasma cell (PC) differentiation is influenced. Cells producing high affinity antigen specific antibodies were particularly affected, consistent with defects in germinal center (GC) reactions. Moreover, changes in lymphoid architecture were also observed. Consistent with these findings, follicular dendritic cell (FDC)-reticula was undetectable following immunization. On the other hand, when ADAM10 was deleted in post-switch B cells, GC formation and lymphoid architecture were not impaired. Despite normal architecture, however, antibody production was still affected, likely due to abnormal gene expression in ADAM10-deficient PCs. Consistent with this hypothesis, PCs isolated from ADAM10Δ/ΔIgG1-cre+/- showed decreased expression of genes that facilitate plasma cell differentiation and function and increased expression of Bcl6, an inhibitor of PC differentiation. Fyn kinase is a member of the Src protein tyrosine kinase. Fyn is widely expressed in many cell types, including lymphocytes. Fyn has been shown to interact with both the B cell and T cell receptor (BCR and TCR, respectively). While Fyn-deletion did not impair the development of immature T cells and B cells, TCR signaling was altered in mature T cells. Our results demonstrate that Fyn-KO mice have significantly low basal levels of IgG1 and IgG2a. Additionally, these mice displayed delayed kinetics in the production of NP-specific IgG1 and IgG2b, and significantly low NP-specific IgG2a after a T-dependent immunization protocol. Defects in antibody production correlated with significantly reduced numbers of GC B cells, TFH cells and splenic PCs. Moreover, Fyn-KO B cells showed decreased production antibody following in vitro activation. Our results thus demonstrate that Fyn-mediated signaling and B cell ADAM10 expression are necessary for optimal humoral responses.

The vitamin D receptor (VDR) is a transcription factor mediating genomic responses to the biologically active form of vitamin D, 1,25(OH)2D3, a key modulator of the immune system. Knowledge on how these polymorphisms modulate the vitamin D endocrine system and confer risk of disease is hindered by the fact that several of the associated allelic variants are located in introns or are synonymous and likely serve as markers within an extended haplotype covering disease-causing alleles. The functional relevance of VDR polymorphisms need to be studied in the context of the haplotype, comparing haplotypes with the process of DNA transcription, protein levels and biological function. These functional studies should be performed using techniques reflecting the in vivo, naturally occurring milieu as close as possible. VDR has several known allelic variants including a FokI restriction fragment length polymorphism in exon II, BsmI and ApaI polymorphisms in the intron VIII, and a synonymous TaqI variant in exon IX. The aim of the current study was the identification of sequence variants in VDR, to define haplotype patterns in the Caucasian population and to understand the functional consequences of single nucleotide polymorphisms (SNPs) and haplotypes. Methods: EBV transformed B-lymphocyte cell lines, from twenty-three individuals, within the Caucasian population were established. Polymorphisms and haplotypes in VDR were identified by genotyping and sequencing. Quantification of the VDR protein level measured with flow cytometry was studied together with the genotype and haplotype data to determine possible influence of genotypes or haplotype and VDR protein levels. Biological function was analysed by the percentage inhibition that each individual experienced in the presence of 1,25(OH)2D3, measured by the Alamar Blue assay and the Trypan blue dye exclusion method. Results: The results showed thatVDR genotypes and haplotypes may not influence VDR protein level although certain genotypes and haplotypes significantly influenced biological function. It was proposed that VDR variants may account for significant influences on cellular responsiveness to 1,25(OH)2D3 as mediated by VDR. Conclusions: The findings of the current study suggest that individual SNPs and haplotypes of VDR influences quality of the repose in the presence of 1,25(OH)2D3, rather than quantity of the VDR levels. This knowledge may permit a rational choice of polymorphisms to use in epidemiology studies or improve our understanding of the significance of VDR genetic polymorphisms on biological function. Keywords: functionality, polymorphism, haplotype, vitamin D receptor, VDR, 1,25(OH)2D3, structure-function analysis, biological responsiveness. / Prof. L. Bornman