Genetic variation at twentythree microsatellite loci in sixteen human populations

Artículo científico -- Universidad de Costa Rica, Instituto de Investigaciones en Salud. 1999We have analysed genetic variation at 23 microsatellite loci in a global sample of 16 ethnically and geographically diverse human populations. On the basis of their ancestral heritage and geographic locations, the studied populations can be divided into five major groups, viz. African, Caucasian, Asian Mongoloid, American Indian and Pacific Islander. With respect to the distribution of alleles at the 23 loci, large variability exists among the examined populations. However, with the exception of the American Indians and the Pacific Islanders, populations within a continental group show a greater degree of similarity. Phylogenetic analyses based on allele frequencies at the examined loci show that the first split of the present-day human populations had occurred between the Africans and all of the non-African populations, lending support to an African origin of modern human populations. Gene diversity analyses show that the coefficient of gene diversity estimated from the 23 loci is, in general, larger for populations that have remained isolated and probably of smaller effective sizes, such as the American Indians and the Pacific Islanders. These analyses also demonstrate that the component of total gene diversity, which is attributed to variation between groups of populations, is significantly larger than that among populations within each group. The empirical data presented in this work and their analyses reaffirm that evolutionary histories and the extent of genetic variation among human populations can be studied using microsatellite loci.Universidad de Costa Rica. Instituto de Investigaciones en SaludUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA

Genetic Variation at 9 Autosomal Microsatellite Loci in Asian and Pacific Populations

Genetic variation at 9 autosomal microsatellite loci (CFS1R, TH01, PLA2A, F13A1, CYP19, LPL, D20S481, D20S473, and D20S604) has been characterized in 16 Asian and Oceanic populations, mostly from mainland and insular Southeast Asia. The neighbor-joining tree and the principal coordinates analysis of the genetic relationships of these populations show a clear separation of Papua New Guinea Highlanders and, to a lesser extent, Malayan aborigines (Orang Asli or Semai) from the rest of the populations. Although the number of markers used in this study appears to be inadequate for clarifying the patterns of genetic relationships among the studied populations, in the principal coordinates analysis a geographic trend is observed in the mainland and insular Southeast Asian populations. Furthermore, in an attempt to contrast the extent of variation between autosomal and Y-chromosome-specific microsatellite loci and to reveal potential differences in the patterns of male and female migrations, we have also compared genetic variation at these 9 autosomal loci with variation observed at 5 Y-chromosome-specific microsatellites in a common set of 14 Asian populations

Recipient natural killer cells alter the course of rejection of allogeneic heart grafts in rats.

Rejection of solid organ grafts is regarded to be dependent on T cell responses. Nonetheless, numerous studies have focused on the contribution of NK cells in this process, resulting in contradictory theories. While some conclude that there is no participation of NK cells, others found an inflammatory or regulative role of NK cells. However, the experimental settings are rarely comparable with regard to challenged species, strain combinations or the nature of the graft. Thus, clear definition of NK cell contribution might be impeded by these circumstances. In this study we performed heterotopic heart transplantation (HTx) in rats, choosing one donor-recipient-combination leading to a fast and a second leading to a prolonged course of graft rejection. We intervened in the rejection process, by depletion of recipient NK cells on the one hand and by injection of activated NK cells syngeneic to the recipients on the other. The fast course of rejection could not be influenced by any of the NK cell manipulative treatments. However, the more prolonged course of rejection was highly susceptible to depletion of NK cells, resulting in significant acceleration of rejection, while injection of NK cells induced acceptance of the grafts. We suggest that, depending on the specific setting, NK cells can attenuate the first trigger of immune response, which allows establishing the regulatory activity leading to tolerance of the graft

The c.503T>C Polymorphism in the Human <i>KLRB1</i> Gene Alters Ligand Binding and Inhibitory Potential of CD161 Molecules

<div><p>Studying genetic diversity of immunologically relevant molecules can improve our knowledge on their functional spectrum in normal immune responses and may also uncover a possible role of different variants in diseases. We characterized the c.503T>C polymorphism in the human <i>KLRB1</i> gene (Killer cell lectin-like receptor, subfamily B, member 1) coding for the cell surface receptor CD161. CD161 is expressed by subsets of CD4⁺ and CD8⁺ T cells and the great majority of CD56⁺ natural killer (NK) cells, acting as inhibitory receptor in the latter population. Genotyping a cohort of 118 healthy individuals revealed 40% TT homozygotes, 46% TC heterozygotes, and 14% carriers of CC. There was no difference in the frequency of CD161 expressing CD4⁺ and CD8⁺ T cells between the different genotypes. However, the frequency of CD161⁺ NK cells was significantly decreased in CC carriers as compared to TT homozygotes. c.503T>C causes an amino acid exchange (p.Ile168Thr) in an extracellular loop of the CD161 receptor, which is regarded to be involved in binding of its ligand Lectin-like transcript 1 (LLT1). Binding studies using soluble LLT1-Fc on 293 transfectants over-expressing CD161 receptors from TT or CC carriers suggested diminished binding to the CC variant. Furthermore, triggering of CD161 either by LLT1 or anti-CD161 antibodies inhibited NK cell activation less effectively in cells from CC individuals than cells from TT carriers. These data suggest that the c.503T>C polymorphism is associated with structural alterations of the CD161 receptor. The regulation of NK cell homeostasis and activation apparently differs between carriers of the CC and TT variant of CD161.</p></div

Characterization of 293 transfectants. 293 cells were “mock”-transfected with the empty pIRES2-AcGFP1 vector or vectors containing CD161 from a homozygous TT or CC individual.

<p>Analyses were performed after selection of stable transfectants. (A) Flow cytometry analysis of 293-mock, 293-CD161/TT, and 293-CD161/CC cells after staining with the PE-conjugated anti-CD161 mAb B199.2. Grey histograms represent staining with an isotype control mAb. The numbers represent mean fluorescence intensity obtained by anti-CD161 staining. (B) Detection of CD161 by Western-blotting. 293 transfectants were lysed, and protein lysates were separated by 9% SDS-PAGE. After transferring to PVDF membranes, samples were probed with the anti-CD161 mAb (B199.2). Binding of B199.2 was visualized by HRP-conjugated goat anti-mouse-IgG and ECL reagents. Protein loading was controlled by staining with a mAb against α-Actin. Representative results of at least two independent experiments are shown. Lysates of PBMC activated for five days with IL-12 were used as a further control to estimate the molecular weight of CD161 molecules.</p

Inhibition of NKp46-induced NK cell activation by antibody-mediated triggering of CD161.

<p>PBMC were isolated and stimulated for three hours with the anti-NKp46 mAb 9E2 combined with the anti-CD161 mAb 191B8 or an isotype matched control antibody. The antibodies had been coupled to Fc-receptors on P815 cells. Activation of NK cells was assessed by monitoring CD107a expression on gated CD3^-CD56⁺ cells. (A) Representative dot-plots showing weaker down-regulation of NK cell activation by anti-CD161 triggering in NK cells from a CC carrier compared to cells from a TT individual. (B) Frequency of activated NK cells after stimulation with anti-NKp46 plus isotype control and anti-NKp46 plus anti-CD161. PBMC from nine individuals with CD161 TT and nine carriers of CC were studied. (C) Diminished capacity of CD161 from CC carriers to inhibit NKp46-induced NK cell activation. % Inhibition was calculated as follows: 100 - [(%CD107a⁺ NK cells after stimulation with NKp46-CD161 / % CD107a⁺ NK cells after stimulation with NKp46-isotype) x 100]; *p<0.05 (Unpaired t-test).</p

Inhibition of NK cell activation by LLT1-mediated triggering of CD161.

<p>PBMC were isolated and co-cultivated for three hours with 293-mock or 293-LLT1 transfectants. Activation of NK cells was assessed by monitoring CD107a expression on gated CD3^-CD56⁺ and CD3^-CD56⁺CD161⁺/CD161^- cells. (A) Abrogation of inhibition by antibody-mediated blocking of CD161/LLT1 interactions. Co-cultivation of PBMC with 293 transfectants was performed in the presence of the anti-CD161 mAb 191B8 or an isotype-matched control antibody. (B) Frequency of activated NK cells after cultivation with 293-mock or 293-LLT1 cells. PBMC from nine individuals with CD161 TT and four carriers of CC were studied. (C) Diminished capacity of CD161 from CC carriers to inhibit NK cell activation. % Inhibition was calculated as follows: 100 - [(%CD107a⁺ NK cells in 293-LLT1 co-cultures / %CD107a⁺ NK cells in 293-mock co-cultures) x 100];*p<0.05 (Mann-Whitney). (D) LLT1 induced CD161 down-regulation on IL-12 activated NK cells. PBMC were co-cultured for three hours with either mock transfected 293 cells or cells expressing LLT1. CD161 expression was monitored on gated CD3^-CD56⁺ NK cells. Depicted histograms show CD161 expression in untreated NK cells (open) and after treatment with 293-LLT1 transfectants (filled histograms). The broken vertical line indicates the peak of CD161 observed in LLT1-treated NK cells from a TT individual. The graph summarizes data obtained in experiments using cells from five TT and three CC individuals (mean ± SEM).</p

Assessment of lymphocyte subsets in individuals carrying different CD161 genotypes.

<p>PBMC were isolated and stained with the antibody combinations CD161/CD3/CD4 or CD161/CD3/CD56. Analyses were performed in gated viable lymphocytes as defined by forward- and side-scatter characteristics. (A) CD161 expression patterns on CD3^- NK cells. Representative dot-plots obtained after staining of cells from one TT, TC, and CC individual are shown. Boxes indicate the CD56^brightCD161⁺CD3^- NK cell subset and the frequency. The numbers indicate % positive cells in each quadrant. (B) Decreased frequency of CD3^-CD56⁺ NK cells in CC carriers. PBMC from 27 TT, 51 TC, and 16 carriers of CC were analyzed. Results are expressed as % NK cells among gated lymphocytes; the mean value calculated for each cohort is indicated by the horizontal line; *p< 0.05 (ANOVA). (C) Ratios of CD56^dim to CD56^bright NK cells. The frequencies of CD56^dimCD161⁺CD3^- and CD56^brightCD161⁺CD3^- NK cells were determined in gated lymphocytes from 22 TT, 46 TC, and 15 carriers of CC. The horizontal broken line is an arbitrary cut-off value. (D) Level of CD161 expression. Data are expressed as mean fluorescence intensity/MFI on gated CD3^-CD56⁺ NK cells. (E) Proportion of NK cells co-expressing CD161. Data are expressed as % CD161⁺ cells in gated CD3^-CD56⁺ NK cells. (F) CD161 expression patterns on CD3⁺ T cells. The numbers indicate % positive cells in each quadrant. (G) Proportion of CD4⁺ T cells co-expressing CD161. (H) Proportion of CD8⁺ T cells co-expressing CD161^dim or CD161^bright.</p