Linda Larcombe

Functional Gene Polymorphisms in Canadian Aboriginal Populations with High Rates of Tuberculosis

The present study determined whether a pattern of functional single-nucleotide polymorphisms (SNPs) was present that could predispose a Dené cohort to a suboptimal response to Mycobacterium tuberculosis. Compared with a Caucasian cohort, the Dené and Cree were found to maintain a significantly higher frequency of SNPs associated with low expression of vitamin D receptor (VDR), interferon (IFN)-gamma (+874), and tumor necrosis factor-alpha (-308) and high production of monocyte chemoattractant protein (MCP)-1 (-2518) and interleukin (IL)-6 (-174). Given the roles played by IFN-gamma and VDR in facilitating macrophage containment of M. tuberculosis and the opposing role of MCP-1 and IL-6, the observed allelic variation by ethnicity may in part contribute to the high rates of tuberculosis among the Dené.

Vitamin D in a Northern Canadian First Nation Population: Dietary Intake, Serum Concentrations and Functional Gene Polymorphisms

The wide spectrum of vitamin D activity has focused attention on its potential role in the elevated burden of disease in a northern Canadian First Nations (Dené) cohort. Vitamin D insufficiency, and gene polymorphisms in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP) have been implicated in susceptibility to infectious and chronic diseases. The objectives of this study were to determine the contribution of vitamin D from food, and measure the serum concentrations of 25-hydroxyvitamin D3 (25-OHD3) and VDBP in Dené participants. Single nucleotide polymorphisms (SNPs) associated with the dysregulation of the innate immune response were typed and counted. Potential correlations between the SNPs and serum concentrations of 25-OHD3 and VDBP were evaluated. Venous blood was collected in summer and winter over a one-year period and analyzed for 25-OHD3 and VDBP concentrations (N = 46). A questionnaire was administered to determine the amount of dietary vitamin D consumed. Sixty-one percent and 30% of the participants had 25-OHD3 serum concentrations <75 nmol/L in the winter and summer respectively. Mean vitamin D binding protein concentrations were within the normal range in the winter but below normal in the summer. VDBP and VDR gene polymorphisms affect the bioavailability and regulation of 25-OHD3. The Dené had a high frequency of the VDBP D432E-G allele (71%) and the Gc1 genotype (90%), associated with high concentrations of VDBP and a high binding affinity to 25-OHD3. The Dené had a high frequency of VDR Fok1-f allele (82%), which has been associated with a down-regulated Th1 immune response. VDBP and VDR polymorphisms, and low winter 25-OHD3 serum concentrations may be risk factors for infectious diseases and chronic conditions related to the dysregulation of the vitamin D pathway.

Effect of vitamin D supplementation on Mycobacterium tuberculosis-induced innate immune responses in a Canadian Dené First Nations cohort.

Canadian First Nations (FN) population experiences a high burden of tuberculosis. Vitamin D is known to enhance the expression of innate immune effectors, including cathelicidin LL-37, for protection against infections. In this study we performed longitudinal analyses to investigate the impact of vitamin D supplementation on macrophage responses to Mycobacterium tuberculosis (Mtb) lipoprotein (TLR2/1L), in Canadian Dené FN participants compared to Caucasian participants. Serum 25(OH)D and LL-37 levels were evaluated by ELISA. Transcriptional responses and protein expression of TLR2/1L-induced LL-37 and other innate immune cytokines were monitored in monocyte-derived macrophages (MDMs) before and after 8 months of vitamin D supplementation. In this study we showed that serum levels of LL-37 decreased after vitamin D supplementation in both Dené and Caucasian participants. There was no difference in TLR2/1L-induced LL-37 expression in MDMs in the two groups, either pre- or post-vitamin D supplementation. However, vitamin D supplementation markedly enhanced TLR2/1L-induced responses in MDMs e.g. IL-6, IL-12 and IL-23 among Caucasians but not in the Dené participants. In contrast, after vitamin D supplementation TLR2/1L-induced responses e.g. IL-1β, IL-8 and IL-12 were significantly reduced in the Dené MDMs. These results indicate that vitamin D supplementation enhanced TLR2/1L-induced innate immune macrophage responses in the Caucasian but not in the Dené participants. We hypothesize that cytokines may be differentially regulated in Canadian FN compared to Caucasians, in particular those that influence Th-1 and Th-17 responses required for the control of Mtb.

Housing conditions in 2 Canadian First Nations communities

Objectives. Housing conditions were assessed in 2 Canadian First Nations communities. Possible associations with tuberculosis (TB) were explored. Study design. Participatory community-based survey. Methods. Qualitative and quantitative data on housing and health were collected in the northern Denè community at Lac Brochet (LB), which has experienced endemic and epidemic TB, and the southern Ojibwa community at Valley River (VR), which has not. Results. 72 of 135 (53%) houses at LB and 57 of 95 (60%) houses at VR were enrolled. Houses in both communities were small (mean 882 and 970 sq. ft., respectively) compared to the Manitoba average (1,200 sq. ft.). Crowding was evident at LB (mean persons per room [ppr] 1.1) and VR (mean ppr 0.9). The provincial mean ppr is 0.5. However, only 49% of householders at LB and 19% at VR felt “crowded” in their homes. More than two-thirds of houses had absent or non-functional heat recovery ventilation systems. Mould was observed in 44% of LB houses and 19% of VR houses. At LB a significant association was found between the number of permanent residents in the house and the presence of selfreported latent or active TB, either currently or during residence in that house (p=0.001). Conclusions. Houses that were studied in these 2 First Nations communities were predominantly small, crowded and in poor repair. An association was found between the number of persons in a house and self-reported TB. Improved housing conditions in First Nations communities are indicated to promote and sustain health as well as human and Indigenous rights.

Killer Immunoglobulin-Like Receptor (KIR) Centromeric-AA Haplotype Is Associated with Ethnicity and Tuberculosis Disease in a Canadian First Nations Cohort

Killer immunoglobulin-like receptors (KIR) on natural killer (NK) cells interact with other immune cells to monitor the immune system and combat infectious diseases, such as tuberculosis (TB). The balance of activating and inhibiting KIR interactions helps determine the NK cell response. In order to examine the enrichment or depletion of KIRs as well as to explore the association between TB status and inhibitory/stimulatory KIR haplotypes, we performed KIR genotyping on samples from 93 Canadian First Nations (Dene, Cree, and Ojibwa) individuals from Manitoba with active, latent, or no TB infection, and 75 uninfected Caucasian controls. There were significant differences in KIR genes between Caucasians and First Nations samples and also between the First Nations ethnocultural groups (Dene, Cree, and Ojibwa). When analyzing ethnicity and tuberculosis status in the study population, it appears that the KIR profile and centromeric haplotype are more predictive than the presence or absence of individual genes. Specifically, the decreased presence of haplotype B centromeric genes and increased presence of centromeric-AA haplotypes in First Nations may contribute to an inhibitory immune profile, explaining the high rates of TB in this population.

Dietary intake of vitamin D in a northern Canadian Dene´ First Nation community

Background Increased awareness of the wide spectrum of activity of vitamin D has focused interest on its role in the health of Canadas Aboriginal peoples, who bear a high burden of both infectious and chronic disease. Cutaneous vitamin D synthesis is limited at northern latitudes, and the transition from nutrient-dense traditional to nutrient-poor market foods has left many Canadian Aboriginal populations food insecure and nutritionally vulnerable. Objective The study was undertaken to determine the level of dietary vitamin D in a northern Canadian Aboriginal (Dené) community and to determine the primary food sources of vitamin D. Design Cross-sectional study. Methods Dietary vitamin D intakes of 46 adult Dené men and women were assessed using a food frequency questionnaire and compared across age, gender, season and body mass index. The adequacy of dietary vitamin D intake was assessed using the 2007 Adequate Intake (AI) and the 2011 Recommended Dietary Allowance (RDA) values for Dietary Reference Intake (DRI). Results Mean daily vitamin D intake was 271.4 IU in winter and 298.3 IU in summer. Forty percent and 47.8% of participants met the vitamin D 1997 AI values in winter and summer, respectively; this dropped to 11.1 and 13.0% in winter and summer using 2011 RDA values. Supplements, milk, and local fish were positively associated with adequate vitamin D intake. Milk and local fish were the major dietary sources of vitamin D. Conclusions Dietary intake of vitamin D in the study population was low. Only 2 food sources, fluid milk and fish, provided the majority of dietary vitamin D. Addressing low vitamin D intake in this population requires action aimed at food insecurity present in northern Aboriginal populations.

The biochemical signatures of stress: A preliminary analysis of osteocalcin concentrations and macroscopic skeletal changes associated with stress in the 13th - 17th centuries black friars population.

OBJECTIVE As a chemical precursor to the hard tissue changes well-studied in bioarchaeological research, osteocalcin provides a unique opportunity to assess stress via fluctuations in bone metabolism. The main objectives of this research were 1) to successfully extract osteocalcin from the Black Friars skeletal population; 2) to assess the diagenetic change between individual bone samples; and 3) to compare osteocalcin concentrations across sex, age, time period and macroscopic indicators of stress. METHODS Twenty adult individuals were selected from the 13th-17th centuries Black Friars skeletal population with bone samples taken from the clavicle and femur. Total protein was assessed through a MicroBCA analysis with osteocalcin quantified using a Human Quantikine ELISA kit. Diagenetic change was assessed using Fourier transform infrared spectroscopy and the attenuated total reflectance method. RESULTS Osteocalcin concentrations showed no significant differences between sex or age groups; however, between time period the post-medieval individuals showed a significant reduction of osteocalcin in both the clavicle and the femur. There were no significant differences in osteocalcin concentrations between those with and without past stress indicators and only one significant difference among the chronic indicators. The diagenetic results demonstrated a similar degree of crystallinity between all samples. CONCLUSIONS While preliminary in nature, this study was successful in demonstrating the potential use of osteocalcin in future health-related research and how the study of osteocalcin may contribute to a better understanding of how and when stress begins to affect the skeletal tissues.

IFN-γ promoter polymorphisms do not affect QuantiFERON ® TB Gold In-Tube test results in a Canadian population

BACKGROUND Several studies have shown polymorphisms within the interferon-gamma (IFN-γ) promoter influence cytokine expression. The interferon-gamma release assay (IGRA) relies on the ability to produce IFN-γ in response to tuberculosis (TB) specific antigens. This study determined the relationship between the IFN-γ +874 A/T promoter polymorphism and the performance of the QuantiFERON®-TB Gold In-Tube (QFT-GIT) test in an ethnically diverse Canadian population. METHODS A total of 190 participants were categorised into three groups based on history of and exposure to TB: active TB (n = 55), TB exposed (n = 55) and presumably TB unexposed controls (n = 80). All participants underwent QFT-GIT testing, and DNA was extracted from whole blood and probed for polymorphism at position +874 (T/A) of intron 1 of IFN-γ. Statistical relationships between the QFT-GIT results, polymorphisms and demographic data were evaluated. RESULTS IFN-γ +874 genotype frequencies among the entire study population (n = 190) were A/A (45.8%), T/A (39.5%), and T/T (14.7%). Among the three study groups, there was no correlation between QFT-GIT results and the IFN-γ +874 A/T genotype, and no correlation of genotype with IFN-γ production in response to either Mycobacterium tuberculosis antigens or mitogenic stimulation. CONCLUSION Our results indicate that the IFN-γ +874 promoter polymorphism does not influence QFT-GIT performance in this study population.

Vitamin D, serum 25(OH)D, LL-37 and polymorphisms in a Canadian First Nation population with endemic tuberculosis

Background Canadian First Nation populations have experienced endemic and epidemic tuberculosis (TB) for decades. Vitamin D–mediated induction of the host defence peptide LL-37 is known to enhance control of pathogens such as Mycobacterium tuberculosis. Objective Evaluate associations between serum levels of 25-hydroxy vitamin D (25(OH)D) and LL-37, in adult Dene First Nation participants (N = 34) and assess correlations with single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP). Design Venous blood was collected from all participants at baseline (winter and summer) and in conjunction with taking vitamin D supplements (1,000 IU/day) (winter and summer). Samples were analysed using ELISA for concentrations of vitamin D and LL-37, and SNPs in the VDR and VDBP regions were genotyped. Results Circulating levels of 25(OH)D were not altered by vitamin D supplementation, but LL-37 levels were significantly decreased. VDBP and VDR SNPs did not correlate with serum concentrations of 25(OH)D, but LL-37 levels significantly decreased in individuals with VDBP D432E T/G and T/T, and with VDR SNP Bsm1 T/T genotypes. Conclusions Our findings suggest that vitamin D supplementation may not be beneficial as an intervention to boost innate immune resistance to M. tuberculosis in the Dene population.Background Canadian First Nation populations have experienced endemic and epidemic tuberculosis (TB) for decades. Vitamin D-mediated induction of the host defence peptide LL-37 is known to enhance control of pathogens such as Mycobacterium tuberculosis. Objective Evaluate associations between serum levels of 25-hydroxy vitamin D (25(OH)D) and LL-37, in adult Dene First Nation participants (N = 34) and assess correlations with single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP). Design Venous blood was collected from all participants at baseline (winter and summer) and in conjunction with taking vitamin D supplements (1,000 IU/day) (winter and summer). Samples were analysed using ELISA for concentrations of vitamin D and LL-37, and SNPs in the VDR and VDBP regions were genotyped. Results Circulating levels of 25(OH)D were not altered by vitamin D supplementation, but LL-37 levels were significantly decreased. VDBP and VDR SNPs did not correlate with serum concentrations of 25(OH)D, but LL-37 levels significantly decreased in individuals with VDBP D432E T/G and T/T, and with VDR SNP Bsm1 T/T genotypes. Conclusions Our findings suggest that vitamin D supplementation may not be beneficial as an intervention to boost innate immune resistance to M. tuberculosis in the Dene population.

HLA-A, B, DRB1, DQA1, DQB1 alleles and haplotype frequencies in Dene and Cree cohorts in Manitoba, Canada

BACKGROUND First Nations in the Canadian province of Manitoba have disproportionately high rates of epidemic and endemic TB. Gene polymorphisms that modulate HLA Class I and II antigens are among the risk markers for TB, along with other biologic, and social determinants of health. HLA-A, B, DRB1, DQA1, DQB1 were typed in two Manitoba First Nation indigenous groups to identify and compare the frequency of gene polymorphisms that may influence susceptibility or resistance to TB. METHODS Participants who self-identified as either Dene or Cree enrolled into the study from two First Nation communities in Manitoba, Canada. Genomic DNA was extracted from blood samples collected with informed consent from Dene (N=63) and Cree (N=42) First Nation study participants. Participants self-reported having treated active TB, treated latent TB or no TB. HLA Class I and II molecules were typed using sequence-specific oligonucleotide (SSO) probes from commercially available kits. RESULTS The rates of treated active and latent TB were marginally higher among the Dene than the Cree participants (p=0.112). Class I and II HLA loci were in Hardy-Weinberg equilibrium in both the Dene and Cree groups. In this exploratory analysis of TB and HLA allele frequencies in Dene and Cree cohorts HLA-A*03 and HLA-DQB1*05:03 were significantly associated with TB. CONCLUSIONS The high incidence of TB in both Dene and Cree populations in Canada requires both biomedical and socioeconomic prevention and control measures. Among the former, an understanding of HLA diversity among First Nations groups may aid the development of new effective vaccine and therapeutic modalities that depend on the interaction between small molecules and specific HLA epitopes.