B6.g7 mice reconstituted with BDC2·5 non-obese diabetic (BDC2·5NOD) stem cells do not develop autoimmune diabetes.

In BDC2·5 non-obese diabetic (BDC2·5NOD) mice, a spontaneous model of type 1 diabetes, CD4(+) T cells express a transgene-encoded T cell receptor (TCR) with reactivity against a pancreatic antigen, chromogranin. This leads to massive infiltration and destruction of the pancreatic islets and subsequent diabetes. When we reconstituted lethally irradiated, lymphocyte-deficient B6.g7 (I-A(g7+)) Rag(-/-) mice with BDC2·5NOD haematopoietic stem and progenitor cells (HSPC; ckit(+)Lin(-)Sca-1(hi)), the recipients exhibited hyperglycaemia and succumbed to diabetes. Surprisingly, lymphocyte-sufficient B6.g7 mice reconstituted with BDC2·5NOD HSPCs were protected from diabetes. In this study, we investigated the factors responsible for attenuation of diabetes in the B6.g7 recipients. Analysis of chimerism in the B6.g7 recipients showed that, although B cells and myeloid cells were 98% donor-derived, the CD4(+) T cell compartment contained ∼50% host-derived cells. These host-derived CD4(+) T cells were enriched for conventional regulatory T cells (Tregs ) (CD25(+) forkhead box protein 3 (FoxP3)(+)] and also for host- derived CD4(+)CD25(-)FoxP3(-) T cells that express markers of suppressive function, CD73, FR4 and CD39. Although negative selection did not eliminate donor-derived CD4(+) T cells in the B6.g7 recipients, these cells were functionally suppressed. Thus, host-derived CD4(+) T cells that emerge in mice following myeloablation exhibit a regulatory phenoytpe and probably attenuate autoimmune diabetes. These cells may provide new therapeutic strategies to suppress autoimmunity.

Enhancement of vaccine-specific cellular immunity in infants by passively acquired maternal antibody.

The known protective effects of passively acquired maternal antibody on the resistance of newborns to infections have prompted widespread interest in maternal vaccination. However, a range of animal model and human studies indicate potential inhibitory effects of maternal antibody on vaccine-specific humoral responses in infants. In the present study we have examined the relationship between maternally acquired TT-specific IgG present before DTaP vaccination and subsequent TT-specific T-cell memory responses at 12 and 18 months, in a cohort of 118 infants. We demonstrate a strong positive association between TT-specific cellular immunity as evidenced by increased IL-4, IL-5 and IL-13 responses, and maternal TT-specific IgG.

Association between antenatal cytokine production and the development of atopy and asthma at age 6 years.

BACKGROUND: Various lines of evidence suggest that antenatal factors are important in determining susceptibility to atopy and asthma. One possible mechanism is cytokines, production of which in the placenta is high throughout gestation and which protect placental integrity via control of local immunological homoeostasis. We investigated antenatal cytokine concentrations in a prospective birth cohort, intensively monitored for atopy and asthma outcomes at age 6 years. METHODS: Cryopreserved cord-blood serum samples from 407 children were assayed for interleukins 4, 5, 6, 10, 12, and 13, interferon gamma, and tumour necrosis factor alpha (TNFalpha). Associations between family, antenatal, and perinatal factors, cord-blood cytokine concentrations, and atopy or asthma outcomes were analysed by logistic regression. Causal effects of cytokines on outcomes were estimated by propensity scores based on family, antenatal, and perinatal factors. FINDINGS: Detectable cord-blood concentrations of interleukin 4 and interferon gamma were each associated with lower risk of physician-diagnosed asthma (adjusted odds ratios 0.60 [95% CI 0.37-0.99] and 0.60 [0.37-0.97] respectively), current asthma (0.59 [0.33-1.00] and 0.39 [0.22-0.71]), and current wheeze (0.55 [0.32-0.93] and 0.52 [0.31-0.90]) and atopy (sensitisation to some inhalant allergens) outcomes at 6 years. High concentrations of TNFalpha were associated with lower risk of atopy but not with asthma risk. These associations were broadly unaltered by propensity-score adjustment. Maternal smoking was associated with higher risk of both wheeze at 6 years and lower concentrations of interleukin 4 and interferon gamma in cord blood. INTERPRETATION: The mechanism underlying attenuated T-helper-1/T-helper-2 cytokine production in high-risk children also apparently operates in control of cytokine production in the fetoplacental unit. The finding that this mechanism is dysregulated by maternal smoking suggests it is a target for antenatal environmental factors relevant to asthma aetiology.

Reciprocal patterns of allergen-induced GATA-3 expression in peripheral blood mononuclear cells from atopics vs. non-atopics.

BACKGROUND: T helper (Th)2 cytokines are considered to play a central role in the induction and expression of allergic disease. However, the relative importance of individual cytokines is unclear, and overall disease pathogenesis appears to involve the coordinate activities of a range of Th2 cytokines acting in sequence or in parallel. The present study examines an alternative approach to the study of cytokine gene function in atopy, focusing instead upon T cell transcription factors (TFs) which play a role in the regulation of multiple cytokine genes. OBJECTIVE: To investigate the allergen-induced expression of the TF GATA-3 and c-Maf in peripheral blood mononuclear cells (PBMCs) and in cytokine-driven Th polarization. METHODS: PBMC from house dust mite (HDM)-atopic and non-atopics were stimulated in vitro with allergen or anti-CD3/IL-2. TF expression was analysed by semiquantitative RT-PCR and major findings were validated by real-time PCR. Cell separations were performed to analyse the contribution of CD45RO+ cells. CD4+ cord blood cells were Th1 or Th2 polarized in vitro by exogenous cytokines and TF expression analysed by Northern blot and real-time PCR. Results We demonstrate for the first time that during differentiation of CD4+ CD45RA+ naïve human T cells towards Th2 commitment, and during allergen-specific reactivation of peripheral CD4+ CD45RO+ Th2 memory cells in established atopics, expression of the Th2-associated TF GATA-3 is rapidly up-regulated, whereas T cells from non-atopics display equally rapid GATA-3 down-regulation under identical conditions of allergen stimulation. CONCLUSION: These findings identify Th2-associated TFs as key determinants of the atopic phenotype, suggesting their unique potential as therapeutic targets for disease control.

"Bystander" amplification of PBMC cytokine responses to seasonal allergen in polysensitized atopic children.

BACKGROUND: Atopic children show increased expression and production of the Th2-associated cytokines IL-4, IL-5, IL-13, and IL-9 from PBMCs after stimulation with allergen, but it has previously not been clearly determined whether the Th2-cytokine production is restricted to the inhalant allergen the child is sensitized to, and whether perennial or seasonal allergens induce different cytokine responses. Our purpose was to determine whether in vitro Th2 cytokine production is specific to the sensitizing allergen, and to compare the cytokine responses to a perennial and a seasonal allergen in monosensitized and polysensitized children. METHODS: Using semiquantitative RT-PCR, we analyzed the expression of the cytokines IL-4, IL-5, IL-13, IL-9, IL-10, and IFN-gamma after stimulation of PBMCs with house-dust-mite (HDM) or ryegrass allergen. The cells were sampled from groups of 6-year-old children sensitized to either HDM (n=20) or ryegrass (n=24), or to both allergens (n=20), as well as from a nonatopic group (n=20). RESULTS: After stimulation with HDM allergen, PBMCs from children sensitized only to HDM expressed increased mRNA levels of the Th2 cytokines, but not of IL-10 and IFN-gamma, whereas ryegrass stimulation did not result in increased cytokine expression. PBMCs from children sensitized to HDM and ryegrass expressed increased Th2 cytokines after stimulation with either of the two allergens. In contrast, PBMCs from children sensitized only to ryegrass did not express increased levels after stimulation with either of the allergens. CONCLUSIONS: The expression of Th2 cytokines after in vitro stimulation of PBMCs from atopic children is specific to the sensitizing allergen, indicating that atopic status per se does not affect the type of T-cell response. In addition, T cells specific to seasonal allergens circulate in the blood out of season only if the child is concomitantly sensitized to a perennial allergen.

Heterogeneity in diphtheria-tetanus-acellular pertussis vaccine-specific cellular immunity during infancy: relationship to variations in the kinetics of postnatal maturation of systemic th1 function.

Cellular immunity to vaccines is highly variable during infancy. This study addressed the hypothesis that these responses are governed by the pace of maturational changes in adaptive immune competence, in particular, cellular functions that underlie the postnatal transition from Th2 to Th1 "bias." Tetanus-specific cytokine responses were tracked in peripheral blood mononuclear cells collected from infants at months 2, 4, 6, 12, and 18. These were compared with polyclonal responses. Results show that the Th2 component of the vaccine response develops rapidly and remains stable, unlike interferon (IFN)-gamma production, which also is initiated early but commonly declines after the final priming dose at 6 months. However, between 12 and 18 months, the IFN-gamma component of the vaccine-specific response has a spontaneous resurgence that coincides with a parallel increase in overall IFN-gamma production capacity. The Th2 component of vaccine-specific responses was more prominent in children with atopic family history.

Regulation of cytokine production in T-cell responses to inhalant allergen:GATA-3 expression distinguishes between Th1- and Th2-polarized immunity.

BACKGROUND: The precise nature of allergen-specific cytokine responses in atopics versus non-atopics, in particular the 'Th1 polarity' of responses in non-atopics, remains controversial. This is due in part to the relative insensitivity of cytokine detection systems, and associated variations in kinetics of cytokine production and catabolism in in vitro culture systems. As an alternative to cytokine measurement, this study focuses on expression of the transcription factor GATA-3 for analysis of allergen-specific Th cell responses. METHODS: Cord blood mononuclear cells were Th1- or Th2-polarized by culture in IL-12- or IL-4-employing established methods; PBMC from house dust mite (HDM)-sensitive atopics and controls were stimulated overnight with HDM; cytokine production was measured by ELISA and GATA-3 mRNA expression by PCR. RESULTS: Cytokine-driven Th2 polarization of naive T cells is associated with marked upregulation of GATA-3 expression, whereas a reciprocal expression pattern accompanies differentiation towards the Th1 cytokine phenotype. In T cells from HDM skin prick test-positive (HDM-SPT+/HDM-IgE+) volunteers, overnight stimulation results in marked upregulation of GATA-3 expression, compared to an equally marked downregulation of expression in T cells from SPT-/IgE- subjects. In subjects who are HDM-SPT+ but IgE-, GATA-3 expression levels remained relatively stable during culture with HDM. CONCLUSIONS: Upregulation of GATA-3 expression in PBMC is a hallmark of the early phase of Th2 recall responses to specific allergen in atopics. The reciprocal expression pattern observed in HDM-specific recall responses of non-atopics provides independent confirmation of the presence of underlying Th1-like immunity in these subjects. The parallel findings in neonatal T cells suggest that the same approach may be utilized for monitoring the progress of allergen-specific Th1/Th2 memory development during early childhood, and hence in assessment of risk for future allergic disease.

Primary sensitization to inhalant allergens.

The neonatal T-cell system is capable of responding to allergens at birth, indicating the occurrence of prenatal sensitization, and the cytokine profile of these responses is skewed towards the Th-2 type. This response is further modified by postnatal exposure to different types of allergens. In relation to inhalant allergen (employed by HDM) the low level fetal Th-2 responses in non-atopics appear to be down-regulated rapidly after birth, parallel to an increase in allergen-specific IFN-gamma production. In contrast, atopics appear to consolidate their initial Th-2 responses, and around the age of 6 exhibit a cytokine response profile similar to the adult pattern. A pre-existing deficiency in IFN-gamma production may be one of the key factors determining the postnatal persistence of Th-2 responses in atopics.

Antigen-specific responses to diphtheria-tetanus-acellular pertussis vaccine in human infants are initially Th2 polarized.

Immune responses to exogenous antigens in infant experimental animals display various degrees of Th2 polarization. Preliminary evidence from small human studies suggest a similar age-dependent response pattern to vaccines, but detailed investigations on vaccine immunity during infancy have not yet been undertaken. We report below the results of a comprehensive prospective study on responses to the tetanus component of the diphtheria, tetanus, acellular pertussis (DTaP) vaccine in a cohort of 55 healthy children, employing peripheral blood mononuclear cells (PBMC) collected at the 2-, 4-, and 6-month vaccinations and at 12 months. Antigen-specific production of interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10, IL-13, and gamma interferon (IFN-gamma) was determined at each sample point, in parallel with polyclonal (phytohemagglutinin PHA-induced) cytokine responses. Our results indicate early and persistent Th2 responses to the vaccine, in contrast to a more delayed and transient pattern of IFN-gamma production. This initial disparity between the Th1 and Th2 components of the vaccine response was mirrored by patterns of polyclonally induced cytokine production, suggesting that the delayed maturation of the Th1 component of the vaccine response during infancy is secondary to developmental processes occurring within the overall Th cell system.

Development of immunologic memory against tetanus toxoid and pertactin antigens from the diphtheria-tetanus-pertussis vaccine in atopic versus nonatopic children.

BACKGROUND: Recent findings suggest that a hallmark of the atopic phenotype is reduced capacity to respond to vaccine antigens, as well as to environmental allergens, during infancy. This deficiency, which is most marked for the cytokine IFN-gamma, appears transient but can result in a long-lasting imbalance within T helper cell (T(H)) memory responses to allergens. Indirect evidence suggests that parallel effects may occur within immunologic memory responses against vaccine antigens in atopic children. OBJECTIVE: Our purpose was to compare vaccine antigen-specific T(H) memory responses in atopic and nonatopic children. METHODS: We analyzed specific serum IgG and cytokine responses to pertactin and tetanus antigens as well as to mitogen (PHA) and house dust mite (HDM) allergen in 25 HDM-sensitized atopic and 25 nonatopic 6-year-old children who were vaccinated and boosted with diphtheria-tetanus-pertussis (DTP) vaccine. RESULTS: PBMCs from the atopic subjects produced higher levels of T(H)1 and T(H)2 cytokines to HDM allergen and PHA. Vaccine antibody titers were normal in the atopic subjects; vaccine-specific T(H)2 responses were rarely detectable, yet T(H)1 (IFN-gamma) responses, in particular against tetanus, were frequent and higher in the atopic subjects (121.5 [SE 64.3] vs 8.0 [3.5] pg/mL culture fluid, P =.04). Corresponding pertactin responses were comparable in both groups. CONCLUSIONS: At the completion of the full primer-booster DTP vaccination regimen, levels of vaccine-specific immunity in atopic 6-year-old children are at least equivalent to their nonatopic counterparts, indicating that the transient atopy-associated deficiency in T(H)1 function in childhood can be successfully overcome by appropriate vaccination and boosting regimens.

The role of allergy in the development of asthma.

Recent studies have shown that initial sensitization to airborne environmental allergens occurs typically in early childhood, but subsequent progression to persistent atopic asthma, which may not manifest for several years, is restricted to only a subset of atopics. The key to establishing the link between atopy and asthma lies in the development of persistent inflammation in the airway wall, resulting in structural and functional changes in local tissues which are responsible for the symptoms of the disease. This review summarizes recent findings on the nature of the cellular and molecular mechanisms underlying this process, and addresses the issue of why the intensity and duration of these tissue-damaging responses in the airway wall apparently exceeds the critical threshold required for development of persistent asthma in only a minority of allergy sufferers.

Allergen-induced cytokine secretion in relation to atopic symptoms and immunoglobulin E and immunoglobulin G subclass antibody responses.

There are few studies on allergen-induced cytokine production in allergic children, and little is known of antigen-specific cytokine regulation of human immunoglobulin (Ig) G subclass antibody responses. An association with T-helper 1 (Th1)-like immunity and complement-activating antibodies remains to be demonstrated in humans. We have previously observed that atopic symptoms are associated with high levels of IgG subclass, especially IgG4, antibodies to birch and beta-lactoglobulin. The differences were seen early in life for the food allergen and increased with age for the inhaled allergen. The aim of this study was to investigate the association between atopic symptoms, birch allergen-, and beta-lactoglobulin-induced cytokine production in peripheral blood mononuclear cells (PBMC), and serum IgE and IgG subclass antibody responses to these allergens in children in order to further clarify the role of Th1- and Th2-like immunity in responses to various antigens. PBMC from 55 eight-year old children, who had been followed prospectively from birth, were stimulated with birch- and beta-lactoglobulin. Production of interleukin (IL)-5, IL-6, IL-10, IL-13 and interferon (IFN)-gamma was analysed by ELISA and expression of IL-4 and IL-9 mRNA by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). IgG subclass antibody levels to birch- and beta-lactoglobulin in serum were determined by ELISA, and IgE antibodies by Magic-Lite and CAP-RAST, respectively. Birch-induced expression of IL-4, but not of the other cytokines, was associated with IgE antibodies to birch. Furthermore, the IL-4 expression and IL-6 production correlated with serum IgG4 antibody levels to this allergen, and IFN-gamma secretion with IgG1 antibody responses. There were no correlations between beta-lactoglobulin-stimulated cytokine production and IgG subclass antibody levels to that allergen, except for a negative association between beta-lactoglobulin-stimulated IL-4 expression and IgG1 antibodies. Atopic children tended to have high levels of birch and beta-lactoglobulin-induced IL-5, IL-6 and IL-10 secretion. Birch-induced IL-4 expression may be the major factor in determining IgE antibody formation to that allergen, while allergen-induced IL-5, IL-6 and IL-10 secretion in PBMC is associated with atopic symptoms. Th1-like immunity to inhaled allergens could be associated with production of the opsonizing and complement-activating IgG1 antibody subclass, and Th2-like immunity with IgG4 antibody responses.

Regulation of T-helper cell responses to inhalant allergen during early childhood.

BACKGROUND: Recent evidence suggests that preschool children manifest patterns of allergen-specific skin prick test (SPT) reactivity and in vitro T-cell cytokine production which are similar to that of either atopic or nonatopic adults. However, published studies on this age group involve small sample sizes and a restricted number of cytokines, usually in response to polyclonal stimuli. OBJECTIVE: To elucidate the relationship between in vivo and in vitro immune responses to a major inhalant allergen house dust mite (HDM) in preschoolers. METHODS: Peripheral blood mononuclear cells (PBMCs) from matched groups of HDM-SPT+ and SPT- 6-year-olds (n = 30 and 29, respectively) tested for PBMC responses to HDM, and cytokine production measured at both the protein and mRNA levels. Immunoglobulin (Ig) E and IgG subclass antibody titres were determined in serum. Interrelationships between in vitro and in vivo HDM responses were examined via multivariate analyses. RESULTS: SPT reactivity to HDM was associated with in vitro production by putative T cells of interleukin (IL) -4, IL-5, IL-9, IL-10, IL-13 and low level IFNgamma, and with production in vivo of IgE and (all) IgG subclass antibodies; HDM responses in the SPT- group were restricted mainly to IL-10 and IFNgamma and very low levels of IL-4; IL-6 production from non-T-cell sources was common. The cytokine most associated with positive SPT responses was IL-9; SPT weal diameter correlated positively with IL-4, IL-5 and IL-13 and negatively with IL-10. CONCLUSION: Detailed analysis of cytokine responses in this very young age group have the potential to uncover subtle relationships between in vivo and in vitro allergen reactivity which may be less clear in adults, in whom T-cell response patterns are modified via chronic stimulation. The present findings which suggest potentially important roles for IL-9 and IL-10 in the early phase of allergic disease, may be one such example.

Microbial stimulation as an aetiologic factor in atopic disease.

Epidemiologic evidence from various sources suggests that exposure to microbial stimuli during early childhood can influence the induction and expression of atopic diseases, particularly in the respiratory tract. Moreover, these effects may have long-lasting consequences in relation to expression of the atopic phenotype in adulthood. This review discusses key aspects of this evidence in relation to the underlying mechanisms which regulate T-helper (Th)-cell function; in particular, the generation of Th-memory cells responsive to inhalant allergens.

Development of allergen-specific T-cell memory in atopic and normal children.

BACKGROUND: In the past 20-30 years, there has been an increase in prevalence of allergic respiratory diseases, particularly amongst children. This study is a prospective analysis of the postnatal maturation of T-helper cell (Th) responses to aeroallergens in atopic and non-atopic infants. METHODS: We measured mononuclear-cell proliferative and cytokine responses to specific allergens and tetanus toxoid in blood samples from atopic and non-atopic infants every 6 months from birth to 2 years of age. Cytokine analyses of responses to housedust-mite allergen used ELISA and reverse-transcriptase PCR. We also measured responses to Fel d1 (cat allergen) and tetanus toxoid. FINDINGS: Samples from 18 atopic and 13 non-atopic infants showed low-level Th2-skewed allergen-specific responses at birth, with little accompanying specific interferon-gamma production. Neonatal Th2 responses were lower in the atopic group than in the non-atopic group; the differences were significant for interleukin-4 (mRNA: beta-actin ratio 0.48 [SE 0.15] vs 0.15 [0.06], p=0.049), interleukin-6 (4750 [48] vs 1352 [51] pg/mL culture fluid, p=0.003), interleukin-10 (1162 [228] vs 485 [89], p=0.015), and interleukin-13 (7.1 [0.9] vs 0.9 [0.3], p=0.008). There was rapid suppression of Th2 responses during the first year of life in non-atopic children, but there was consolidation of responses in atopic children, associated with defective neonatal interferon-gamma production. INTERPRETATION: The continuation of fetal allergen-specific Th2 responses during infancy is a defining feature of the inductive phase of atopic disease, and is associated with decreased capacity for production of the Th1 cytokine interferon y by atopic neonates. These findings provide a plausible mechanism for persistence of the fetal Th2 responses during early childhood in atopic individuals and subsequent expression of disease.

Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the Th2 cytokine profile.

The expression of Th2-skewed immunity against soluble protein Ags present in the normal environment is recognized as the primary cause of allergic inflammation in atopics. In contrast, nonallergic normal individuals display low level Th1-skewed immunity against the same Ags ("allergens"), which is perceived as conferring protection against Th2-dependent allergic sensitization. The type of T cell memory that develops against these Ags is currently believed to be the result of complex interactions between environmental and genetic susceptibility factors, which occur postnatally when the naive immune system directly confronts the outside environment. The results of the present study challenge this general concept. We demonstrate here for the first time that Th2-skewed responses to common environmental allergens, comprising IL-4, IL-5, IL-6, IL-9, and IL-13, are present in virtually all newborn infants and are dominated by high level production of IL-10. Moreover, these responses are demonstrable within 24 h of culture initiation, arguing against a significant contribution from covert in vitro T cell priming and/or differentiation. These findings imply that the key etiologic factor in atopic disease may not be the initial acquisition of allergen-specific Th2-skewed immunity per se, but instead may be the efficiency of immune deviation mechanisms, which in normal (nonatopic) individuals redirect these fetal immune responses toward the Th1 cytokine phenotype.

Reciprocal age-related patterns of allergen-specific T-cell immunity in normal vs. atopic infants.

By adulthood there is almost universal immunological memory to aeroallergens, and the presence of allergic disease appears to be related to the nature of the underlying T-helper (Th) cell cytokine responses. The hypothesis of this study is that adult patterns of allergen specific Th-cell memory (Th-2 polarized in atopics vs. Th1 in non-atopics) can be determined in early infancy. Mononuclear cell cytokine responses to house-dust mite were measured at 6-monthly intervals from birth to 2 years of age, using ELISA (IL-10, IL-13, IFN-gamma) and sqRT/PCR (IL-4, IL-5, IL-9, IFN-gamma) in normal infants (n = 14) with no family history or allergic symptoms, and infants with a family history and definite atopy by 2 years (n = 16). Both normals and atopics showed low-level Th2 skewed allergen-specific responses at birth with little accompanying IFN-gamma. The Th2 responses to house-dust mite were higher in normal newborns, who then show a rapid downregulation of these responses in the first year of life. Atopic infants instead show a consolidation of their neonatal patterns of Th2 polarized allergen specific immunity. Earlier studies indicate that neonates at high risk of atopy display diminished capacity for production of the Th1 cytokine IFN-gamma. The present study suggests for the first time that neonates who subsequently develop atopy also initially have reduced capacity to mount Th2 responses. However, in contrast to non-atopics who selectively downregulate their fetal Th2 polarized allergen-specific responses, atopic children display age-associated upregulation of Th2 immunity.

The complex mutation pattern of a microsatellite.

DQCAR is a (CA)n microsatellite located in the HLA class II region and tightly linked to HLA-DQB1. Previous studies showed a strikingly low level of size variation in DQCAR alleles within an extensive subfamily of HLA-DQ subtypes (DQ1). DQCAR alleles in non-DQ1 subtypes showed a higher degree of size polymorphism. In this study sequence analysis demonstrates that DQ1-associated DQCAR alleles have a single C-->A nucleotide substitution interrupting the CA repeat array. Frequent CA-->GA mutations are also observed in DQ1-associated microsatellites with identical allele sizes. In contrast, DQCAR alleles associated with non-DQ1 haplotypes display a perfect CA repeat sequence and the variation in allele size is attributable only to differences in the number of CA repeats. Our results imply that several mutational mechanisms are involved in the generation of allelic diversity within the same microsatellite locus. The possibility of different mutation rates in the same locus should to be taken into account when using these markers in evolutionary and disease studies.