Methylation at global LINE-1 repeats in human blood are affected by gender but not by age or natural hormone cycles

Previously, we reported on inter-individual and gender specific variations of LINE-1 methylation in healthy individuals. In this study, we investigated whether this variability could be influenced by age or sex hormones in humans. To this end, we studied LINE-1 methylation in vivo in blood-derived DNA from individuals aged 18 to 64 years and from young healthy females at various hormone levels during the menstrual cycle. Our results show that no significant association with age was observed. However, the previously reported increase of LINE-1 methylation in males was reconfirmed. In females, although no correlation between LINE-1 or Alu methylation and hormone levels was observed, a significant stable individual specific level of methylation was noted. In vitro results largely confirmed these findings, as neither estrogen nor dihydrotestosterone affected LINE-1 or Alu methylation in Hek293T, HUVEC, or MDA-kb2 cell lines. In contrast, a decrease in methylation was observed in estrogen-treated T47-Kbluc cell lines strongly expressing estrogen receptor. The very low expression of estrogen receptor in blood cells could explain the observed insensitivity of methylation at LINE-1 to natural hormonal variations in females. In conclusion, neither natural cycle of hormones nor age has a detectable effect on the LINE-1 methylation in peripheral blood cells, while gender remains an important factor.     

Age-Related Changes in the Global DNA Methylation Profile of Leukocytes Are Linked to Nutrition but Are Not Associated with the MTHFR C677T Genotype or to Functional Capacities

Global DNA methylation of peripheral blood leukocytes has been recently proposed as a potential biomarker for disease risk. However, the amplitude of the changes in DNA methylation associated with normal aging and the impacts of environmental changes on this variation are still unclear. In this context, we evaluated the association of global DNA methylation with nutritional habits, tobacco smoking, body mass index (BMI), clinical laboratory parameters, polymorphism C677T MTHFR, functional cognition and the daily practice of physical activity in a cancer-free older population. Leukocyte global DNA methylation from 126 older individuals was quantified using a high-throughput ELISA-based method. Global DNA hypomethylation was observed in older individuals when compared to a younger population (p = 0.0469), confirming changes in DNA methylation in the aging process. Furthermore, the methylation profile of elders was correlated with the daily ingestion of carbohydrates (p = 0.0494), lipids (p = 0.0494), vitamin B6 (p = 0.0421), magnesium (p = 0.0302), and also to the serum levels of total protein (p = 0.0004), alpha 2 globulin (p = 0.0013) and albumin (p = 0.0015). No statistically significant difference was observed when global DNA methylation were stratified according to C677T MTHFR genotypes (p = 0.7200), BMI (p = 0.1170), smoking habit (p = 0.4382), physical activity in daily life (p = 0.8492), scored cognitive function (p = 0.7229) or depression state (p = 0.8301). Our data indicate that age-related variations in the global DNA methylation profile of leukocytes might be modulated by the daily intake of carbohydrates, lipids, vitamin B6, and magnesium and be associated with serum protein levels, however it is independent of C677T MTHFR genotype and not correlated with BMI, smoking habit, cognitive function or the routine physical activities.     

Proliferation and Propagation of Human Melanocytes in Vitro Are Affected by Donor Age and Anatomical Site

We report the effects of two factors, donor age and anatomical site, on the proliferation and melanization of human melanocytes (MC) derived from (1) neonatal foreskins, (2) adult foreskins, or (3) adult breast or arm skin. Two different growth media have been used for this purpose. Medium I supports the long-term proliferation of neonatal MC, and medium II supports the growth of both neonatal and adult MC. We found that neonatal foreskin MC proliferated equally well in both media for more than 12 passages. However, adult MC behaved differently in the two growth media. In very early passages (passages 1-5), all three types of MC proliferated well and reached comparable final cell densities in medium I, but adult foreskin MC proliferated at a higher rate than neonatal or adult non-foreskin MC in medium II. The non-foreskin adult MC had the least proliferative rate in medium II. Unlike neonatal MC, both adult foreskin and non-foreskin MC underwent a drastic reduction in their proliferative rate after only a few (9-10) passages in culture. While the three types of MC differed in their proliferative rates, they responded similarly to melanogenic stimulation by cholera toxin (CT) and isobutyl methylxanthine (IBMX). These results demonstrate that the proliferation of human MC in standard culture media is affected by the donor age and the anatomical site from which these cells are derived. We conclude that human MC are heterogeneous, and caution must be used in extrapolating the results of studies on MC derived from different anatomical sites or age groups.     

Breadth of Neutralizing Antibody Response to Human Immunodeficiency Virus Type 1 Is Affected by Factors Early in Infection but Does Not Influence Disease Progression

The determinants of a broad neutralizing antibody (NAb) response and its effect on human immunodeficiency virus type 1 (HIV-1) disease progression are not well defined, partly because most prior studies of a broad NAb response were cross-sectional. We examined correlates of NAb response breadth among 70 HIV-infected, antiretroviral-naïve Kenyan women from a longitudinal seroincident cohort. NAb response breadth was measured 5 years after infection against five subtype A viruses and one subtype B virus. Greater NAb response breadth was associated with a higher viral load set point and greater HIV-1 env diversity early in infection. However, greater NAb response breadth was not associated with a delayed time to a CD4⁺ T-cell count of <200, antiretroviral therapy, or death. Thus, a broad NAb response results from a high level of antigenic stimulation early in infection, which likely accounts for prior observations that greater NAb response breadth is associated with a higher viral load later in infection.Some human immunodeficiency virus (HIV)-infected individuals develop broad neutralizing antibody (NAb) responses, but the factors that lead to NAb response breadth remain elusive. Several cross-sectional studies have found that individuals with greater NAb response breadth have higher contemporaneous viral loads, suggesting that the presence of a greater amount of viral antigen may promote a greater NAb response breadth (9, 10, 25, 30, 32). However, because viral load and NAb response breadth were measured at the same time after HIV type 1 (HIV-1) acquisition in prior studies, it is difficult to discern cause and effect. There is also evidence that NAbs adapt in response to the evolving HIV-1 population throughout infection (11, 29, 35), which may contribute to a greater overall response breadth. Together, these studies support a model in which a greater NAb response breadth is driven by a higher level of antigenic stimulation, in terms of both the absolute level of virus and viral diversity. Confirmation of this model requires an assessment of the temporal relationship of viral load, HIV-1 diversity, and NAb response breadth.In addition to uncertainty regarding the determinants of NAb response breadth, the consequences of a broad NAb response for HIV-1 disease progression remains controversial. Broad NAb responses have been found in long-term nonprogressors (LTNPs) in some studies, suggesting that NAbs may contribute to control of infection in these individuals (6-8, 22, 27, 37). Other studies have found no evidence for NAb control in LTNPs (1, 2, 14, 18), including studies in which NAb response breadth was lower in LTNPs (10) or elite controllers (15, 25) than in viremic individuals. A detailed analysis of NAb response breadth versus clinical outcome has not yet been conducted, particularly for individuals with typical HIV-1 disease progression.To investigate the determinants and consequences of NAb response breadth in HIV-1 infection, we examined NAb responses in women in a seroincident cohort in Mombasa, Kenya, that began in 1993 (19-21). For each woman, the time of infection was defined by both HIV-1 serology and RNA testing (17). Women who had a banked plasma sample ∼5 years after the estimated time of HIV-1 infection were included in this study. This time period was chosen to maximize the chances for the NAb response to broaden while generally testing prior to the beginning of clinical immunodeficiency. We only included samples prior to the initiation of antiretroviral therapy (ART), which in this cohort began in March 2004, according to the WHO and Kenyan National guidelines. Plasma samples meeting these criteria were identified from 70 women and came from a median of 5.0 (range, 4.5 to 6.8) years postinfection (ypi). This subset of women was representative of the entire cohort in terms of their behavioral, clinical, and demographic characteristics (data not shown).HIV-1 subtype A accounts for most of the infections in this cohort (28), including 72% of the 53 women in this study for whom env subtype information was available (Fig. ​(Fig.1).1). Therefore, to test neutralization of viruses relevant to women in this population, we measured NAb response breadth against a panel of five recently transmitted subtype A viruses from other individuals in this cohort, which represented a spectrum of neutralization sensitivities (4). We also included one commonly studied, easy-to-neutralize subtype B virus (SF162) for comparison to other studies. The TZM-bl neutralization assay, using pseudoviruses prepared with these six envelope variants and TZM-bl indicator cells, was performed as described previously (4, 36). The median inhibitory concentration (IC₅₀) was defined as the reciprocal dilution of plasma that resulted in 50% inhibition. Figure ​Figure11 shows the IC₅₀ for each plasma-virus pair, averaged across three independent experiments that included duplicate testing of each pair.Open in a separate windowFIG. 1.Summary of the IC₅₀s and NAb response breadth scores of 70 plasma samples. The first column indicates the subject identifier of each plasma sample, and the next three columns indicate the env V1 to V5 subtype (available for 53/70 women), the set point viral load (available for 64 women), and the viral load at ∼5 ypi, when the NAb response breadth was measured. Data not available are indicated by a period. Each subsequent column shows the results with one panel virus (indicated at the top of the column). Results are the average of three experiments in which each plasma-virus pair was tested in duplicate. In the case of Q769 and Q259, two closely related viruses from the same individual were used in one (Q769.h5, Q259.d217) and two (Q769.b9, Q259.d226) of the three experiments. The IC₅₀ for each plasma-virus pair is the reciprocal dilution of plasma that led to a 50% reduction in infectivity, averaged across the three experiments. IC₅₀s are shown in gray scale to represent increasing neutralization sensitivity, with white for values of <100, light gray for values of >101 and <1,000, and dark gray for values of >1,001. Plasma-virus pairs in which 50% neutralization was not detected at the highest plasma dilution (1:50) are indicated by a pair of dashes. The NAb response breadth score for each plasma sample was calculated as follows. For each experiment, the median IC₅₀ for each virus (across all 70 plasma samples) was determined. Plasma samples were assigned a score of 1 for every virus against which their IC₅₀ was greater than the median IC₅₀, and the score was summed across all six viruses. The NAb response breadth scores that are shown here (and which were used for analysis) were calculated by taking the average response breadth score across the three independent experiments; they were not calculated from the average IC₅₀s shown.In general, we found that the viruses that had been easily neutralized in prior screening with pooled plasma, {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1 and Q168b23 (4), were the most readily neutralized by individual plasma samples from women in this study (Fig. ​(Fig.1).1). Of the 70 plasma samples tested, 68 (97%) showed detectable neutralization activity (IC₅₀, >50) against {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1 and 60 (86%) showed activity against Q168b23. Most (76%) of the plasma samples also neutralized variant Q842d16 at detectable levels, although generally with lower IC₅₀s. By contrast, only about half of the plasma samples neutralized envelope variants Q769b9 and {"type":"entrez-protein","attrs":{"text":"Q259d2","term_id":"122195760","term_text":"Q259D2"}}Q259d2.26 (51% and 46%, respectively). Almost all (93%) of the plasma samples neutralized SF162.Given the different neutralization sensitivities of these viruses, we quantified the NAb responses in these individuals by using a previously described NAb response breadth score that takes into consideration the neutralization sensitivity of each virus (5). Briefly, the NAb response breadth score represents the number of viruses (out of six) that a given plasma sample neutralized at an IC₅₀ that was higher than the median IC₅₀ for that virus (across all 70 plasma samples). The response breadth score was calculated independently for each of three experiments, and the average scores are listed in Fig. ​Fig.1.1. Among all of the individuals, the median response breadth score was 2 and the response breadth scores ranged from 0 to 5.3. A potential limitation of this approach is that response breadth was calculated by using a relatively small number of viruses. However, we found that NAb response breadth measured against this 6-virus panel was highly correlated with the NAb response breadth measured against an expanded 17-virus panel (including these 6 viruses plus an additional 11 viruses representing subtypes A, C, D, A/D, and B; J. Overbaugh et al., unpublished data), for a subset of 29 women whose plasma samples were tested against the expanded panel (Spearman''s rho = 0.62, P < 0.001). Furthermore, the NAb response breadth score measured against this six-virus panel was highly correlated with NAb potency (Spearman''s rho = 0.81, P < 0.001), a measure we have used in prior studies that takes into consideration the magnitude of the IC₅₀ for each plasma-virus pair (5). These findings suggest that the NAb response breadth score measured against the six-virus panel is representative of the overall NAb response breadth.We investigated whether NAb response breadth was associated with the contemporaneous plasma viral load, which was measured at the same time as NAb response breadth (4.5 to 6.8 ypi). Viral loads ranged from 1.7 to 6.7 log₁₀ copies/ml among all of the individuals, with a median of 4.7 log₁₀ copies/ml. As shown in Fig. ​Fig.2a,2a, individuals with higher viral loads had greater NAb response breadth (Spearman''s rho = 0.31, P = 0.009), consistent with prior studies (9, 10, 30, 32). A similar relationship was observed between viral load set point and NAb potency, a measure that takes into account the magnitude of neutralization (data not shown). There was no association between NAb response breadth and CD4⁺ T-cell count (Spearman''s rho = −0.15, P = 0.2) among the 64 women with contemporaneous CD4⁺ T-cell counts available.Open in a separate windowFIG. 2.Associations between NAb response breadth and viral load. In each plot, the NAb response breadth score is indicated on the y axis and the contemporaneous (∼5 ypi) viral load (a) or viral load set point (b) is indicated on the x axis. Each point represents one individual. The results of Spearman correlation analysis are shown above the plots.To further assess whether the viral load may drive NAb response breadth, we examined the relationship between the viral load set point and NAb response breadth. For each individual, the viral load set point was defined as the first available viral load measurement 4 to 24 months after infection (16), and this ranged from 2.1 to 6.2 log₁₀ copies/ml (median, 4.6 log₁₀ copies/ml) among the 64 individuals for whom this measurement was available. As shown in Fig. ​Fig.2b,2b, individuals with higher viral load set points had greater NAb response breadth at ∼5 ypi (Spearman''s rho = 0.35, P = 0.005). The viral load set point was also highly correlated with the viral load measured at ∼5 ypi (Spearman''s rho = 0.42, P = 0.001). Therefore, we investigated whether the relationship between NAb response breadth and the contemporaneous (∼5 ypi) viral load could be explained by the viral load set point. In multivariate linear regression analysis, NAb response breadth was significantly associated with the viral load set point (coefficient of variation = 0.55, P = 0.02) but not with the contemporaneous viral load (coefficient of variation = 0.25, P = 0.3). Thus, the relationship between the contemporaneous viral load and NAb response breadth appeared to be driven by the viral load set point, with each 1-log increase in the viral load set point associated with an increase in the response breadth score of 0.55.Given this association between the viral load set point and NAb response breadth, we wondered whether another factor in early infection—HIV-1 sequence diversity—might influence the development of NAb response breadth. Proviral HIV-1 sequences were available from 26 individuals and had been sampled a median of 87 (range, 17 to 299) days postinfection. For each individual, gag and env V1 to V5 diversity was calculated from a median of seven single-copy sequences per gene as described previously (26). Across all 26 individuals, the median env diversity was 0.28% (range, 0 to 4.0%) and the median gag diversity was 0.19% (range, 0 to 1.28%). Individuals with greater env diversity early in infection had greater NAb response breadth at ∼5 ypi (Spearman''s rho = 0.51, P = 0.008). However, there was no association between early gag diversity and NAb response breadth (Spearman''s rho = 0.10, P = 0.6). Although both early env diversity and the viral load set point were associated with NAb response breadth, there was no association between these factors among the women in this study (Spearman''s rho = 0.21, P = 0.3). However, in a larger study of 156 women in this cohort, women with greater early env heterogeneity (as measured by heteroduplex mobility assay) had higher viral load set points (31). Further work is needed to clarify whether early env diversity and the viral load set point are independent determinants of NAb response breadth or whether early env diversity may drive both the viral load and NAb response breadth.Because the viral load set point and early env diversity have also been shown to be associated with HIV-1 disease progression in this cohort (17, 31), we explored the relationship of NAb response breadth, the viral load set point, and disease progression. We performed Cox proportional hazard analysis by using a composite survival outcome of time to the first occurrence of a CD4⁺ T-cell count of <200, ART initiation, or death. Among all 70 women, 45 reached this composite outcome over a median of 6.8 years of follow-up after HIV-1 infection (range, 1.2 to 14.2 years). In univariate analysis, a greater NAb response breadth was associated with an increased risk of HIV-1 disease progression (Table ​(Table1,1, hazard ratio [HR], 1.27 per unit increase in breadth, P = 0.03). However, this association was attenuated, and no longer statistically significant, in a multivariate analysis adjusting for the viral load set point (HR = 1.06, P = 0.6). In this multivariate model, a higher viral load set point was associated with a greater risk of HIV-1 disease progression (HR = 2.02, P = 0.003), as expected. In a second multivariate analysis considering only those outcome events that occurred after NAb response measurement (n = 25 events among 50 women), there was an association between NAb response breadth and HIV-1 disease outcomes (HR = 1.39, P = 0.03) but again this did not persist after adjustment for the viral load (HR = 1.17, P = 0.4). Thus, we found no evidence that NAb response breadth affected HIV-1 disease progression independently of the viral load set point.

TABLE 1.

Association between NAb response breadth and risk of HIV-1 disease progression^a

Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human Sera,and Their Number Is Influenced By Age,Gender, and Disease

The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer’s and Parkinson’s diseases.     

Sterol Uptake in Saccharomyces cerevisiae Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.     

Raised Thyrotrophin-Releasing Hormone, Pyroglutamylamino Peptidase, and Proline Endopeptidase Are Present in the Spinal Cord of Wobbler Mice but Not in Human Motor Neurone Disease

The Wobbler mouse (wr) is a mutant that exhibits loss of anterior horn cells in the spinal cord and brainstem and subsequent muscle wasting, particularly of the forelimbs and neck. The wr mice, 2-3 months of age, were found to have increased levels of immunoreactive-thyrotrophin-releasing hormone (ir-TRH) in the spinal cord and pons and medulla, but not in other CNS areas. This increase was observed in dorsal and ventral cord and at cervical, thoracic, and lumbar levels and was confirmed by HPLC to be authentic TRH. The levels of immunoreactive-somatostatin, -neurotensin, and -substance P were not raised in the CNS of wr mice. The activities of two peptidases capable of degrading TRH, pyroglutamylaminopeptidase (PGAP, EC 3.4.11.8) and proline endopeptidase (PEP, EC 3.4.21.26), and the level of 5-hydroxyindoleacetic acid were also raised in the spinal cord of 2-3-month-old wr mice although the activities of alanine aminopeptidase and lactate dehydrogenase and the level of 5-hydroxytryptamine were not. Increased spinal cord levels of ir-TRH and PGAP and PEP activities were not observed in the 1-month-old wr mice. In addition, a pilot study using spinal cord obtained at autopsy from three patients with motor neurone disease and 12 control subjects indicated no increase in spinal cord ir-TRH, PGAP, or PEP in human motor neurone disease.     

Intronic Parent-of-Origin Dependent Differential Methylation at the Actn1 Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression

Parent-of-origin differential DNA methylation has been associated with regulation of the preferential expression of paternal or maternal alleles of imprinted genes. Based on this association, recent studies have searched for parent-of-origin dependent differentially methylated regions in order to identify new imprinted genes in their vicinity. In a previous genome-wide analysis of mouse brain DNA methylation, we found a novel differentially methylated region in a CpG island located in the last intron of the alpha 1 Actinin (Actn1) gene. In this region, preferential methylation of the maternal allele was observed; however, there were no reports of imprinted expression of Actn1. Therefore, we have tested if differential methylation of this region is common to other tissues and species and affects the expression of Actn1. We have found that Actn1 differential methylation occurs in diverse mouse tissues. Moreover, it is also present in other murine rodents (rat), but not in the orthologous human region. In contrast, we have found no indication of an imprinted effect on gene expression of Actn1 in mice: expression is always biallelic regardless of sex, tissue type, developmental stage or isoform. Therefore, we have identified a novel parent-of-origin dependent differentially methylated region that has no apparent association with imprinted expression of the closest genes. Our findings sound a cautionary note to genome-wide searches on the use of differentially methylated regions for the identification of imprinted genes and suggest that parent-of-origin dependent differential methylation might be conserved for functions other that the control of imprinted expression.     

Campylobacter Colonization and Proliferation in the Broiler Chicken upon Natural Field Challenge Is Not Affected by the Bird Growth Rate or Breed

The zoonotic association between Campylobacter bacteria in poultry and humans has been characterized by decades of research which has attempted to elucidate the epidemiology of this complex relationship and to reduce carriage within poultry. While much work has focused on the mechanisms facilitating its success in contaminating chicken flocks (and other animal hosts), it remains difficult to consistently exclude Campylobacter under field conditions. Within the United Kingdom poultry industry, various bird genotypes with widely varying growth rates are available to meet market needs and consumer preferences. However, little is known about whether any differences in Campylobacter carriage exist across this modern broiler range. The aim of this study was to establish if a relationship exists between growth rate or breed and cecal Campylobacter concentration after natural commercial flock Campylobacter challenge. In one investigation, four pure line genotypes of various growth rates were grown together, while in the second, eight different commercial broiler genotypes were grown individually. In both studies, the Campylobacter concentration was measured in the ceca at 42 days of age, revealing no significant difference in cecal load between birds of different genotypes both in mixed- and single-genotype pens. This is important from a public health perspective and suggests that other underlying reasons beyond genotype are likely to control and affect Campylobacter colonization within chickens. Further studies to gain a better understanding of colonization dynamics and subsequent proliferation are needed, as are novel approaches to reduce the burden in poultry.     

Stability and Change in In-Group Mate Preferences among Young People in Ethiopia Are Predicted by Food Security and Gender Attitudes,but Not by Expected Pathogen Exposures

There is broad anthropological interest in understanding how people define “insiders” and “outsiders” and how this shapes their attitudes and behaviors toward others. As such, a suite of hypotheses has been proposed to account for the varying degrees of in-group preference between individuals and societies. We test three hypotheses related to material insecurity, pathogen stress, and views of gender equality among cross-sectional (n = 1896) and longitudinal (n = 1002) samples of young people in Ethiopia (aged 13–17 years at baseline) to explore stability and change in their preferences for coethnic spouses. We show that food insecurity is associated with a greater likelihood of intolerant mate preferences. We also find that young people who hold more gender equitable attitudes tended to hold more tolerant mate preferences. Finally, we find no support for the hypothesis that expected pathogen exposure is associated with intolerant mate preferences. Our results most strongly support a material insecurity hypothesis of in-group bias, which assumes that uncertainty over meeting basic needs leads people to favor those in their in-group. As such, our findings join a small but growing group of studies that highlight the importance of material insecurity for understanding tolerance, xenophobia, in-group bias, and favoritism.