The evolutionary origins of Southeast Asian Ovalocytosis.

Southeast Asian Ovalocytosis (SAO) is a common red blood cell disorder that is maintained as a balanced polymorphism in human populations. In individuals heterozygous for the SAO-causing mutation there are minimal detrimental effects and well-documented protection from severe malaria caused by Plasmodium vivax and Plasmodium falciparum; however, the SAO-causing mutation is fully lethal in utero when homozygous. The present-day high frequency of SAO in Island Southeast Asia indicates the trait is maintained by strong heterozygote advantage. Our study elucidates the evolutionary origin of SAO by characterizing DNA sequence variation in a 9.5 kilobase region surrounding the causal mutation in the SLC4A1 gene. We find substantial haplotype diversity among SAO chromosomes and estimate the age of the trait to be approximately 10,005 years (95% CI: 4930-23,200 years). This date is far older than any other human malaria-resistance trait examined previously in Southeast Asia, and considerably pre-dates the widespread adoption of agriculture associated with the spread of speakers of Austronesian languages some 4000 years ago. Using a genealogy-based method we find no evidence of historical positive selection acting on SAO (s=0.0, 95% CI: 0.0-0.03), in sharp contrast to the strong present-day selection coefficient (e.g., 0.09) estimated from the frequency of this recessively lethal trait. This discrepancy may be due to a recent increase in malaria-driven selection pressure following the spread of agriculture, with SAO targeted as a standing variant by positive selection in malarial populations.

Geographical barriers and climate influence demographic history in narrowleaf cottonwoods.

Studies of genetic variation can clarify the role of geography and spatio-temporal variation of climate in shaping demography, particularly in temperate zone tree species with large latitudinal ranges. Here, we examined genetic variation in narrowleaf cottonwood, Populus angustifolia, a dominant riparian tree. Using multi-locus surveys of polymorphism in 363 individuals across the species' 1800 km latitudinal range, we found that, first, P. angustifolia has stronger neutral genetic structure than many forest trees (simple sequence repeat (SSR) FST=0.21), with major genetic groups corresponding to large apparent geographical barriers to gene flow. Second, using SSRs and putatively neutral sequenced loci, coalescent simulations indicated that populations diverged before the last glacial maximum (LGM), suggesting the presence of population structure before the LGM. Third, the LGM and subsequent warming appear to have had different influences on each of these distinct populations, with effective population size reduction in the southern extent of the range but major expansion in the north. These results are consistent with the hypothesis that climate and geographic barriers have jointly affected the demographic history of P. angustifolia, and point the importance of both factors as being instrumental in shaping genetic variation and structure in widespread forest trees.

Genetic exchange in 2La inversion heterokaryotypes of Anopheles gambiae.

In the malaria vector Anopheles gambiae, alternative arrangements of chromosome 2 (2La and 2L+(a)) vary in relative frequency along clines of aridity, suggesting the action of natural selection on targets within the inversion. Our long term goal of detecting such targets depends in part on the level of genetic exchange between arrangements. Accordingly, we estimated recombination rates on 2L from the backcross progeny of 2La/+(a) heterokaryotypes and as a control, from 2L+(a) homokaryotypes. In homokaryotypes, the recombination rate was uniform at ~2.0 centimorgans per megabase (cM/Mb). In heterokaryotypes, recombination within the rearranged region was reduced to < 0.5 cM/Mb, with slightly higher but nevertheless reduced levels (< 1.0 cM/Mb) flanking the rearrangement. Yet, gene exchange was recorded between nearly all markers, including those very near the distal inversion breakpoint. These results suggest that reduced recombination is a necessary but not sufficient mechanism for genetic isolation between alternative arrangements, and that the targets of natural selection can be identified against the different chromosomal backgrounds.

Contrasting modes of natural selection acting on pigmentation genes in the Drosophila dunni subgroup.

Genes that encode for divergent adaptive traits may have genealogies that contrast with those from loci that are not functionally involved in differentiation. Here, we examine DNA sequence variation among the species of the eastern Caribbean Drosophila dunni subgroup at two loci, yellow and dopa decaboxylase (Ddc), which both play integral roles in pigmentation patterning of adult Drosophila. Phylogenetic analyses of these loci produce gene genealogies with topologies that mirror those described for other nuclear genes: the six morphologically distinct species within the subgroup are divided into only three lineages, with one lineage containing four species that share extensive ancestral polymorphism. At the Ddc locus these major lineages are delineated only by silent site variation. We observe a significantly higher rate of synonymous site divergence than non-synonymous divergence, consistent with strong purifying selection acting on the locus. In contrast, the yellow locus exhibits patterns of amino acid divergence and nucleotide diversity that are consistent with recent diversifying selection acting in two different lineages. This selection appears to be targeting amino acid variants in the signal sequence of the Yellow protein, a region which is tightly constrained among members of the larger D. cardini radiation. This result highlights not only the potential importance of yellow in the evolution of divergent pigmentation patterns among members of the D. dunni subgroup, but also hints that variation in signal peptide sequences may play a role in phenotypic diversification.

Recent radiation of endemic Caribbean Drosophila of the dunni subgroup inferred from multilocus DNA sequence variation.

Studies of island endemism provide a unique opportunity to elucidate fundamental mechanisms of speciation. Here we examine intra- and interspecific DNA sequence variation at four unlinked genetic loci among populations of the Drosophila dunni subgroup to provide a detailed genealogical portrait of the process of speciation among these island endemic species. Our data indicate two major rounds of diversification that have shaped the D. dunni subgroup. The first occurred 1.6-2.6 million years ago and separated three major lineages, one in Puerto Rico and the Virgin Islands, a second in the northern Lesser Antilles and Barbados, and a third in St. Vincent and Grenada. A second round of diversification occurred in the last 96,000 years in the northern Lesser Antilles and Barbados. The four distinct species that resulted from this recent round of diversification maintain relatively high amounts of genetic variation, similar to that of a closely related mainland species, and share extensive ancestral polymorphism. These data suggest a minimal role for population bottlenecks linked to founder events in the history of the D. dunni subgroup. Further, the recent divergence of these island populations highlights the extremely rapid development of reproductive isolation and distinct patterns of abdominal pigmentation that has occurred in these species.

Drosophila melanogaster and D. simulans rescue strains produce fit offspring, despite divergent centromere-specific histone alleles.

The interaction between rapidly evolving centromere sequences and conserved kinetochore machinery appears to be mediated by centromere-binding proteins. A recent theory proposes that the independent evolution of centromere-binding proteins in isolated populations may be a universal cause of speciation among eukaryotes. In Drosophila the centromere-specific histone, Cid (centromere identifier), shows extensive sequence divergence between D. melanogaster and the D. simulans clade, indicating that centromere machinery incompatibilities may indeed be involved in reproductive isolation and speciation. However, it is presently unclear whether the adaptive evolution of Cid was a cause of the divergence between these species, or merely a product of postspeciation adaptation in the separate lineages. Furthermore, the extent to which divergent centromere identifier proteins provide a barrier to reproduction remains unknown. Interestingly, a small number of rescue lines from both D. melanogaster and D. simulans can restore hybrid fitness. Through comparisons of cid sequence between nonrescue and rescue strains, we show that cid is not involved in restoring hybrid viability or female fertility. Further, we demonstrate that divergent cid alleles are not sufficient to cause inviability or female sterility in hybrid crosses. Our data do not dispute the rapid divergence of cid or the coevolution of centromeric components in Drosophila; however, they do suggest that cid underwent adaptive evolution after D. melanogaster and D. simulans diverged and, consequently, is not a speciation gene.

All in the family: evolutionary and functional relationships among death receptors.

Over the last decade, significant progress has been made towards identifying the signaling pathways within mammalian cells that lead to apoptosis mediated by death receptors. The simultaneous expression of more than one death receptor in many, if not all, cell types suggests that functional innovation has driven the divergence of these receptors and their cognate ligands. To better understand the physiological divergence of the death receptors, a phylogenetic analysis of vertebrate death receptors was conducted based upon amino-acid sequences encoding the death domain regions of currently known and newly identified members of the family. Evidence is presented to indicate an ancient radiation of death receptors that predates the emergence of vertebrates, as well as ongoing divergence of additional receptors both within several receptor lineages as well as modern taxonomic lineages. We speculate that divergence among death receptors has led to their functional specialization. For instance, some receptors appear to be primarily involved in mediating the immune response, while others play critical roles during development and tissue differentiation. The following represents an evolutionary approach towards an understanding of the complex relationship among death receptors and their proposed physiological functions in vertebrate species.

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup.

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.

Ovalbumin aerosols induce airway hyperreactivity in naïve DO11.10 T cell receptor transgenic mice without pulmonary eosinophilia or OVA-specific antibody.

The pathobiology of allergic asthma is being studied using murine models, most of which use systemic priming followed by pulmonary challenges with the immunizing antigen. In general, mice develop eosinophilic pulmonary inflammation, increased antigen-specific immunoglobulins, and airway hyperreactivity (AHR), all of which are dependent on antigen-specific T cell activation. To establish a model of allergic asthma, which did not require systemic priming, we exposed DO11.10 T cell receptor transgenic mice, which have an expanded repertoire of ovalbumin (OVA), peptide-specific T cells, to limited aerosols of OVA protein. DO11.10 +/- mice developed AHR in the absence of increases in total serum IgE, OVA-specific IgG, or eosinophilia. The AHR was accompanied by pulmonary recruitment of antigen-specific T cells with decreased expression of CD62L and CD45RB and increased expression of CD69, a phenotype indicative of T cell activation. Our results support recent hypotheses that T cells mediate AHR directly.

Ig heavy chain complex-linked genes influence the immune response in a murine cryptococcal infection.

A murine pulmonary infection with Cryptococcus neoformans (Cne) has been used to determine mechanisms regulating effective T cell-mediated immunity in the lungs. In BALB/c and C.B-17 mice, following intratracheal deposition of Cne, the fungus initially grows rapidly and is then progressively cleared from the lungs. Cne clearance in C.B-17 mice requires CD4 and CD8 T cells, IFN-gamma, and NO. Clearance in congenic BALB/c mice proceeds more slowly than in C.B-17 mice, even though the only genetic difference between these strains is at the Ig H chain-containing region of chromosome 12. Examination of the pulmonary immune response in the two strains revealed that both cleared lung Cne by T cell-dependent mechanisms and generated equivalent levels of NO. Furthermore, both strains recruited equal numbers of macrophages, lymphocytes, and neutrophils to the lungs, although BALB/c mice recruited higher numbers of eosinophils. Notably, leukocytes isolated from BALB/c lungs during infection secreted lower levels of IFN-gamma and higher levels of the Th2 cytokines IL-4 and IL-5 as compared with lung leukocytes from C.B-17 mice. Furthermore, serum levels of IgM, IgG1, IgG2a, and IgG3 anti-Cne Abs generated during infection were significantly greater in BALB/c mice than C.B-17 mice. These data suggest that although both BALB/c and C.B-17 mice clear pulmonary cryptococcosis through T cell-mediated mechanisms, Ig H chain-linked genes in BALB/c mice are associated with a decreased effectiveness of the host response, which we suggest might influence the balance in Th1/Th2 T cell subset development or increase anti-Cne Abs, or both.

The mechanism of activation of NK-cell IFN-gamma production by ligation of CD28.

We have investigated the mechanism by which anti-CD28 antibodies activates IFN-gamma production by murine NK cells. These studies reveal that engagement of CD28 alone by this antibody is a poor activator of this cytokine response. Effective stimulation requires simultaneous ligation of the receptor for Fc (FcgammaRIII, CD16) which on its own is also a poor inducer of murine NK cells. The mechanism by which immobilized anti-CD28 increases IFN-gamma mRNA abundance involves both upregulation of transcription as well as induction of mRNA stabilization. However, the elevation of transcription is not as evident as that induced by IL-12 which, in contrast, does not induce message stabilization. Thus ligation of CD28 in the presence of IL-12 results in a synergistic increase in production of the cytokine. Using this assay we have also determined that immobilized anti-CD28 cannot induce resting NK cells to produce IFN-gamma. In contrast, the same cells can be induced by BCL1-C11 tumor cells that express high amounts of the CD28 ligand, B7-2. These studies provide important insights into the ability of cells bearing counter-receptor for CD28 to activate NK cell-cytokine production in vivo.

Dissociation of airway hyperresponsiveness from immunoglobulin E and airway eosinophilia in a murine model of allergic asthma.

Nonspecific airway hyperresponsiveness (AHR) is a hallmark of human asthma. Both airway eosinophilia and high serum levels of total and antigen-specific immunoglobulin E (IgE) are associated with AHR. It is unclear, however, whether either eosinophilia or increased IgE levels contribute directly to, or predict, the development of AHR. Investigations conducted with various murine models of asthma and different mouse strains have resulted in conflicting evidence about the roles that IgE and airway eosinophilia play in the manifestation of AHR. We show that systemic priming with ovalbumin (OVA) in alum, followed by a single day of OVA aerosol challenge, is sufficient to induce AHR, as measured by increased pulmonary resistance in response to intravenously delivered methacholine in BALB/c, but not C57BL/6 or B6D2F1, mice. This was observed despite the fact that OVA-challenged BALB/c mice had less airway eosinophilia and smaller increases in total IgE than either C57BL/6 or B6D2F1 mice, and had less pulmonary inflammation and OVA-specific IgE than B6D2F1 mice. We conclude that airway eosinophilia, pulmonary inflammation, and high serum levels of total or OVA-specific IgE are all insufficient to induce AHR in C57BL/6 and B6D2F1 mice, whereas BALB/c mice demonstrate AHR in the absence of airway eosinophilia. These data confirm that the development of AHR is genetically determined, not only in naive mice, but also in actively immunized ones, and cannot be predicted by levels of airway eosinophilia, pulmonary inflammation, total IgE, or antigen-specific IgE.

Immune dysregulation as a cause for allergic asthma.

Allergic asthma is being increasingly understood as a disease caused by Th2-mediated immune responses to inhaled allergens. Most individuals fail to respond to allergens with a Th2 response, and thus, allergic asthma can be considered the result of an abnormally regulated or dysregulated immune response. The prevalence of asthma has risen precipitously in urbanized cultures, as contrasted with third world countries. This observation underlies the heightened efforts in the past few years of basic and applied research efforts to gain a better understanding of both normal and dysregulated immunity to antigens introduced via the airways. This review focuses on recent human studies into the immune dysregulation that results in the asthma phenotype, but also cites selected relevant papers from research with experimental animals.

The role of NK cells during in vivo antigen-specific antibody responses.

Investigations into the role of NK cells in regulating Ab responses have yielded variable results, some suggesting that NK cells can down-regulate Ag-specific Ig production and others proposing an enhancing effect. These apparently inconsistent findings may stem partially from the specificity of reagents used in purifying cell populations and/or the nature of the in vitro systems used to study these events. We chose to investigate the ability of either resting or poly(I:C)-activated NK cells to alter an in vivo Ab response in mice given a T-independent (TNP-LPS) or T-dependent (TNP-keyhole limpet hemocyanin (KLH)) Ag. By using a more specific Ab, anti-NK-1.1, to deplete NK cells, we were able to clearly show that resting, endogenous NK cells do not affect either type of response, as measured by serum Ag-specific Ig levels quantitated by isotype-specific ELISA. In contrast, activation of NK cells by poly(I:C) increased Ag-specific IgC2a as well as IgG1 levels. Interestingly, only the effect on IgG2a production is reversible by depletion of NK cells.

Regulation of IFN-gamma mRNA production in murine natural killer cells.

We have previously reported that large, presumably in vivo activated, B cells stimulate murine natural killer (NK) cells to secrete increased levels of IFN-gamma. In order to further understand the mechanism of IFN-gamma induction, we compared the regulation of IFN-gamma mRNA production after stimulation of NK cells with either B lymphocytes or phorbol myristate acetate (PMA)+ionomycin. Here we show that stimulation of NK cells by either stimuli results increase in IFN-gamma mRNA, albeit with different kinetics. Although the induction requires new RNA synthesis, we could not detect increased transcription of the IFN-gamma gene after stimulation. Measurement of the rate of mRNA degradation after IFN-gamma mRNA has accumulated demonstrates that this mRNA is more stable than IFN-gamma mRNA from unstimulated NK cells. Together, these results suggest that the increase in IFN-gamma mRNA and protein in NK cells, stimulated by B cells or PMA+ionomycin, results from stabilization of pre-existing IFN-gamma message. Our results also suggest that induction of the factor which stabilizes the mRNA, although as yet unknown, requires new RNA synthesis.

Interactions between B lymphocytes and NK cells.

The ability of natural killer (NK) cells to secrete lymphokines confers upon them the potential to regulate cell types via mechanisms other than direct cytotoxicity. During the past few years increasing evidence has been accumulating to show that NK and B cells can interact productively. First, NK cells cocultured with B cells can induce them to initiate polyclonal Ig secretion. This help is mediated by a soluble factor (or factors) that appears to be different from any known cytokine. Second, preactivated B lymphocytes can induce NK cells to produce greater amounts of IFN-gamma via an interaction that requires direct cell contact. Third, in contrast to previous suggestions, NK cells do not have the ability to kill primary B lymphocytes regardless of their stage of differentiation. Evaluation of the in vivo relevance of these interactions revealed that activated NK cells can increase the IgG2a response to a specific protein antigen. Without activation, NK cells neither enhance nor inhibit B cell responses to antigens. The deviation of the isotype distribution may allow increased NK cell specificity for certain pathogens by enhancing antibody-dependent cytotoxicity.

Mechanism of lipopolysaccharide-mediated transcriptional enhancement of the mu gene.

LPS induces both B cell proliferation and differentiation to Ig secretion. By treating stimulated cells for a brief period with staurosporine, and inhibitor of protein kinase C, it is possible to allow continued proliferation but partially inhibit differentiation. Analysis of the molecular basis for the decrease in IgM production shows that the increased transcription of the Ig-H chain gene induced by LPS is abrogated by staurosporine treatment whereas alteration of 3' end processing is not affected. These experiments indicate that LPS continues to mediate its effect on some of the more distal differentiative events through protein kinase C even after initial cell activation.

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.

The influence of lipopolysaccharide content on the apparent B cell stimulating activity of anti-mu preparations.

Anti-mu preparations differ greatly in their ability to stimulate mouse B cells to incorporate tritiated thymidine (TdR). We have found that these differences may be due in part to different levels of lipopolysaccharide (LPS) content. In this report we show that LPS concentrations as low as 0.025 ng/ml stimulate the proliferation of T-depleted (C57BL/6 X DBA/2)F1 (B6D2F1) spleen cells, provided that 5 X 10(-5) M 2-mercaptoethanol is also present. Each of six commercial anti-mu preparations tested for LPS content contained more than this amount. We describe a technique that uses polymyxin B-agarose to remove nanogram quantities of LPS from anti-mu preparations. In B6D2F1 B cells, LPS-depleted anti-mu preparations induced much more uniform tritiated thymidine incorporation than did non-depleted preparations; but there was little difference between the two preparations when tested on B cells from C3H/HeJ (LPS hyporesponsive) mice.