Specific T-cell receptor beta-rearrangements of gluten-triggered CD8+ T-cells are enriched in celiac disease patients' duodenal mucosa.

Celiac disease (CeD) is an autoimmune disorder affecting the small intestine with gluten as disease trigger. Infections including Influenza A, increase the CeD risk. While gluten-specific CD4+ T-cells, recognizing HLA-DQ2/DQ8 presented gluten-peptides, initiate and sustain the celiac immune response, CD8+ α/ß intraepithelial T-cells elicit mucosal damage. Here, we subjected TCRs from a cohort of 56 CeD patients and 22 controls to an analysis employing 749 published CeD-related TCRß-rearrangements derived from gluten-specific CD4+ T-cells and gluten-triggered peripheral blood CD8+ T-cells. We show, that in addition to TCRs from gluten-specific CD4+ T-cells, TCRs of gluten-triggered CD8+ T-cells are significantly enriched in CeD duodenal tissue samples. TCRß-rearrangements of gluten-triggered CD8+ T-cells were even more expanded in patients than TCRs from gluten-specific CD4+ T-cells (p < 0.0002) and highest in refractory CeD. Sequence alignments with TCR-antigen databases suggest that a subgroup of these most likely indirectly gluten-triggered TCRs recognize microbial, viral, and autoantigens.

Evidence for a role of RUNX1 as recombinase cofactor for TCRß rearrangements and pathological deletions in ETV6-RUNX1 ALL.

T-cell receptor gene beta (TCRß) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRß sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRß rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRß gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRß gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRß rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.

Donor CD4 T Cell Diversity Determines Virus Reactivation in Patients After HLA-Matched Allogeneic Stem Cell Transplantation.

Delayed reconstitution of the T cell compartment in recipients of allogeneic stem cell grafts is associated with an increase of reactivation of latent viruses. Thereby, the transplanted T cell repertoire appears to be one of the factors that affect T cell reconstitution. Therefore, we studied the T cell receptor beta (TCRß) gene rearrangements of flow cytometry-sorted CD4(+) and CD8(+) T cells from the peripheral blood of 23 allogeneic donors before G-CSF administration and on the day of apheresis. For this purpose, TCRß rearrangements were amplified by multiplex PCR followed by high-throughput amplicon sequencing. Overall, CD4(+) T cells displayed a significantly higher TCRß diversity compared to CD8(+) T cells irrespective of G-CSF administration. In line, no significant impact of G-CSF treatment on the TCR Vß repertoire usage was found. However, correlation of the donor T cell repertoire with clinical outcomes of the recipient revealed that a higher CD4(+) TCRß diversity after G-CSF treatment is associated with lower reactivation of cytomegalovirus and Epstein-Barr virus. By contrast, no protecting correlation was observed for CD8(+) T cells. In essence, our deep TCRß analysis identifies the importance of the CD4(+) T cell compartment for the control of latent viruses after allogeneic stem cell transplantation.

Genetic variation in oxytocin rs2740210 and early adversity associated with postpartum depression and breastfeeding duration.

Mothers vary in duration of breastfeeding. These individual differences are related to a variety of demographic and individual maternal factors including maternal hormones, mood and early experiences. However, little is known about the role of genetic factors. We studied single-nucleotide polymorphisms (SNPs) in the OXT peptide gene (rs2740210; rs4813627) and the OXT receptor gene (OXTR rs237885) in two samples of mothers from the Maternal adversity, Vulnerability and Neurodevelopment study (MAVAN), a multicenter (Hamilton and Montreal, Canada) study following mothers and their children from pregnancy until 7 years of age. Data from the Hamilton site was the primary sample (n = 201) and data from Montreal was the replication sample (n = 151). Breastfeeding duration, maternal mood (measured by the CES-D scale) and early life adversity (measured by the CTQ scale) were established during 12 months postpartum. In our primary sample, polymorphisms in OXT rs2740210, but not the other SNPs, interacted with early life adversity to predict variation in breastfeeding duration (overall F8,125 = 2.361, P = 0.021; interaction effect b = -8.12, t = -2.3, P = 0.023) and depression (overall F8,118 = 5.751, P ≤ 0.001; interaction effect b = 6.06, t = 3.13, P = 0.002). A moderated mediation model showed that higher levels of depression mediated the inverse relation of high levels of early life adversity to breastfeeding duration, but only in women possessing the CC genotype [effect a' = -3.3401, 95% confidence interval (CI) = -7.9466 to -0.0015] of the OXT SNP and not in women with the AA/AC genotype (a' = -1.2942, ns). The latter findings (moderated mediation model) were replicated in our Montreal sample (a' = -0.277, 95% CI = -0.7987 to -0.0348 for CC; a' = -0.1820, ns for AA/AC).

Dopamine receptors D1 and D2 are related to observed maternal behavior.

The dopamine pathway and especially the dopamine receptors 1 and 2 (DRD1 and DRD2) are implicated in the regulation of mothering in rats. Evidence for this in humans is lacking. Here, we show that genetic variation in both DRD1 and DRD2 genes in a sample of 187 Caucasian mothers predicts variation in distinct maternal behaviors during a 30-min mother-infant interaction at 6 months postpartum. Two DRD1 single-nucleotide polymorphisms (SNPs rs265981 and rs686) significantly associated with maternal orienting away from the infant (P = 0.002 and P = 0.003, respectively), as did DRD1 haplotypes (P = 0.03). Two DRD2 SNPs (rs1799732 and rs6277) significantly associated with maternal infant-directed vocalizing (P = 0.001 and P = 0.04, respectively), as did DRD2 haplotypes (P = 0.01). We present evidence for heterosis in DRD1 where heterozygote mothers orient away from their infants significantly less than either homozygote group. Our findings provide important evidence that genetic variation in receptors critical for mothering in non-human species also affect human maternal behaviors. The findings also highlight the importance of exploring multiple dimensions of the complex human mothering phenotype.

Serotonin transporter allelic variation in mothers predicts maternal sensitivity, behavior and attitudes toward 6-month-old infants.

Maternal behavior in the new mother is a multidimensional set of responses to infant cues that are influenced by the mother's early life experiences. In this study, we wanted to test if mothers' early life experiences and mothers' genotype have interactive effects on maternal behaviors and attitudes, something which has not been previously explored. In a sample of 204 mothers, we assessed maternal genotype at the serotonin transporter-linked polymorphic region (5-HTTLPR) and an adjacent upstream polymorphism (rs25531), together giving rise to three alleles: short (S), L(G) and L(A). Controlling for maternal age and parity, we showed that this genotype can predict differences in maternal sensitivity at 6 months postpartum: mothers with an S (or the functionally similar L(G)) allele were more sensitive than mothers who lacked the allele during a 30-min recorded mother-infant interaction (F (4,140) = 3.43; P = 0.01). Furthermore, we found highly significant gene-environment interactions in association with maternal behavior, such that mothers with no S or L(G) alleles oriented away more frequently from their babies if they also reported more negative early care quality (F (5,138) = 3.28; P = 0.008). Finally, we found significant gene-environment associations with maternal attitudes; mothers with the S allele and with greater early care quality scored higher on ratings of their perceived attachment to their baby (F (5,125) = 3.27; P = 0.008). The regression results show significant interactions between the reported quality of care mothers received from their own parents and genotype on both their frequency of orienting away from the infant during the interaction (F(5, 138) = 3.28; P = 0.008, Fig. 1a) and their perceived attachment feelings to the infant (F(5, 125) = 3.27; P = 0.008, Fig. 1b); however the direction of the effects for these two outcome measures were different from one another. With increasing care quality, mothers with the L(A)L(A) genotype (no S or L(G) allele) oriented away less frequently, while S or L(G) allele carriers showed no significant change. In contrast, with increasing early care quality. L(A)L(A) (no S or L(G) allele) mothers scored lower on perceived attachment to their infants, whereas S or L(G) allele carrying mothers scored higher. [corrected].

Detection of novel skeletogenesis target genes by comprehensive analysis of a Runx2(-/-) mouse model.

Runx2 is an essential factor for skeletogenesis and heterozygous loss causes cleidocranial dysplasia in humans and a corresponding phenotype in the mouse. Homozygous Runx2-deficient mice lack hypertrophic cartilage and bone. We compared the expression profiles of E14.5 wildtype and Runx2(-/-) murine embryonal humeri to identify new transcripts potentially involved in cartilage and bone development. Seventy-one differentially expressed genes were identified by two independent oligonucleotide-microarray hybridizations and quantitative RT-PCR experiments. Gene Ontology analysis demonstrated an enrichment of the differentially regulated genes in annotations to terms such as extracellular, skeletal development, and ossification. In situ hybridization on E15.5 limb sections was performed for all 71 differentially regulated genes. For 54 genes conclusive in situ hybridization results were obtained and all of them showed skeletal expression. Co-expression with Runx2 was demonstrated for 44 genes. While 41 of the 71 differentially expressed genes have a known role in bone and cartilage, we identified 21 known genes that have not yet been implicated in skeletal development and 9 entirely new transcripts. Expression in the developing skeleton was demonstrated for 21 of these genes.

A single amphioxus and sea urchin runt-gene suggests that runt-gene duplications occurred in early chordate evolution.

Runt-homologous molecules are characterized by their DNA binding runt-domain which is highly conserved within bilaterians. The three mammalian runt-genes are master regulators in cartilage/bone formation and hematopoiesis. Historically these features evolved in Craniota and might have been promoted by runt-gene duplication events. The purpose of this study was therefore to investigate how many runt-genes exist in the stem species of chordates, by analyzing the number of runt-genes in what is likely to be the closest living relative of Craniota-amphioxus. To acquire further insight into the possible role of runt-genes in early chordate evolution we have determined the number of runt-genes in sea urchins and have analyzed the runt-expression pattern in this species. Our findings demonstrate the presence of a single runt-gene in amphioxus and sea urchin, which makes it highly likely that the stem species of chordates harbored only a single runt-gene. This suggests that runt-gene duplications occurred later in chordate phylogeny, and are possibly also associated with the evolution of features such as hematopoiesis, cartilage and bone development. In sea urchin embryos runt-expression involves cells of endodermal, mesodermal and ectodermal origin. This complex pattern of expression might reflect the multiple roles played by runt-genes in mammals. A strong runt-signal in the gastrointestinal tract of the sea urchin is in line with runt-expression in the intestine of nematodes and in the murine gastrointestinal tract, and seems to be one of the phylogenetically ancient runt-expression domains.

Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by a subtractive hybridization approach.

In this study we have analyzed bacterial lipopolysaccharide (LPS) induced genes in hemocytes of the Lepidopteran species Galleria mellonella using subtractive hybridization, followed by suppressive PCR. We have found genes that show homologies to molecules, such as gloverin, peptidoglycan recognition proteins and transferrin known to be involved in immunomodulation after bacterial infection in other species. In addition, a few molecules previously not described in the innate immune reactions were detected, such as a RNA binding molecule and tyrosine hydroxylase. Furthermore, the full-length cDNA of a LPS-induced molecule with six toxin-2-like domains is described to be a promising candidate to further elucidate the relationship between toxin- and defensin-like domains in arthropod host defense.

Evolution of classic Hodgkin lymphoma in correlation to changes in the lymphoid organ structure of vertebrates.

In order to integrate evolutionary concepts into lymphoma research we mapped features of classic Hodgkin lymphoma (a disease which has been recently described to be derived from germinal center B-cells) onto a phylogenetic tree of vertebrates. Secondly, we matched the phylogenetic occurrence of classic Hodgkin lymphoma to the changes in the lymphoid organ structure during vertebrate evolution. According to our analysis, classic Hodgkin lymphoma evolved exclusively at the developmental stage of mammals. Interestingly the appearance of Hodgkin lymphoma is correlated to the evolution of germinal centers in mammals. This lends some credit to the hypothesis that genes specific to the germinal center reaction are involved in the pathogenesis of Hodgkin lymphoma. However, as evolution did not stop at the developmental stage of the mammalian stem-species, to a certain extent species with specific differences of classic Hodgkin lymphoma can be expected. One such difference is that classic Hodgkin lymphoma occurs with a significantly higher frequency in humans than in all other mammals. This could be partially due to Epstein-Barr virus (EBV) infection in approximately 40%-50% of Hodgkin disease cases, that is associated with an expression of the EBV-encoded oncogen LMP-1. In conclusion we propose that the mapping of lymphoma related characteristics onto a phylogenetic tree is a valuable new tool in lymphoma research.

Analysis of BCL-6 mutations in classic Hodgkin disease of the B- and T-cell type.

BCL-6 is essential for germinal center formation and thus for affinity maturation of immunoglobulin (Ig) genes by somatic mutations. The 5'-noncoding region of the BCL-6 gene is even a target for the mutation machinery. Translocations of the BCL-6 gene to heterologous promoters and mutations of its 5'-noncoding regulatory region were reported to be potential mechanisms for deregulating BCL-6 expression and for playing a role in the genesis of non-Hodgkin lymphoma. In line with this hypothesis is the observation that B-cell lymphoma with somatic mutations, such as diffuse large B-cell lymphoma and follicular lymphoma, also carry BCL-6 mutations, some of which are recurrently detectable. Classic Hodgkin disease (cHD) is also derived from B cells with high loads of somatic mutations and thus a further candidate for BCL-6 mutations. To determine the presence and potential role of BCL-6 mutations in cHD, the 5'-noncoding BCL-6 proportion of single Hodgkin and Reed-Sternberg (HRS) cells from 6 cases of cHD and 6 cases of HD-derived cell lines was analyzed. All B-cell-derived HD cases and cell lines harbored BCL-6 mutations. In contrast, both T-cell-derived HD cases and cell lines were devoid of BCL-6 mutations. With only one exception, there were no lymphoma-specific recurrent BCL-6 mutations detected, and BCL-6 protein was absent from the HRS cells of most cases. In conclusion, (1) somatic BCL-6 mutations are restricted to cHD cases of B-cell origin, and (2) the BCL-6 mutations represent mostly irrelevant somatic base substitutions without consequences for BCL-6 protein expression and the pathogenesis of cHD.

Detection of clonal T-cell receptor gamma-chain gene rearrangements in Reed-Sternberg cells of classic Hodgkin disease.

Recent molecular single-cell studies have shown that in approximately 95% of cases, Reed-Sternberg cells of classic Hodgkin disease (HD) are derived from B cells of germinal center origin. Attempts to determine the cellular nature of the remaining cases have so far failed. To clarify whether they are derived from T cells, this study examined 791 single CD30(+) Hodgkin and Reed-Sternberg (HRS) cells from 13 T-cell marker-positive cases and from 6 cases with null-cell phenotype for rearranged T-cell receptor-gamma (TCR-gamma) genes by single copy polymerase chain reaction. Monoclonally rearranged TCR-gamma genes were detectable in 2 of the 13 classic HD cases with T-cell marker-positive HRS cells, with none detectable in the null-cell cases. Eight of the T-cell marker-positive cases and all 6 null-cell cases were also studied for rearrangements of immunoglobulin genes. Six of the 8 T-cell marker-positive cases harbored clonal immunoglobulin gene rearrangements. The 2 cases without rearranged immunoglobulin genes were those that contained clonal TCR-gamma rearrangements and lacked expression of the B-cell-specific activator protein. From these findings we conclude that cases of classic HD with T-cell-derived HRS cells definitely exist, although their overall incidence at 1% to 2% is very low. Even within the T-cell marker-positive cases only a minority (15%) were derived from T cells. The majority (85%) originated from B cells, indicating that the T-cell antigens expressed by HRS cells are, in contrast to those expressed in non-Hodgkin lymphoma, not lineage specific.

Effects of a school for pregnant students on the incidence of low-birthweight deliveries.

To evaluate effects of an alternative public school for pregnant teenagers in New Haven, CT, medical and school records were reviewed for a 1-year birth cohort of 230 adolescent mothers. Nearly three-quarters of all school-aged primiparas who were enrolled in the city's public schools when they became pregnant attended the alternative school. Because of summer vacation, however, students who conceived in January through April began attending later in pregnancy than did those who conceived in May through December; these mothers were significantly more likely to deliver a preterm, low-birthweight infant. No such seasonal effects were found for other teenagers in the city who were not enrolled in public school at conception. Positive birth outcomes for early program attenders are similar to those reported for a nurse-home-visitation program. The results suggest that school programs have considerable potential to be an effective service delivery model for providing prenatal intervention to adolescents.

Parent-focused intervention: diffusion effects on siblings.

This study examined whether intervention provided to parents of firstborn children produced delayed benefits for later-born children. We studied younger siblings of children in the Yale Child Welfare Project, a family support program previously shown to result in better school adjustment for the firstborns. Information was obtained from the siblings' teachers and school records for 3 academic years. As was true for the older children, intervention group siblings had better school attendance than did control group siblings, were less likely to need supportive or remedial services, and were more likely to be making normal school progress. The results suggest that changes in the caregiving environment resulting from early family support lead to benefits for all the family's children. Parent-focused programs thus appear to provide a particularly efficient strategy for intervention efforts.

Adolescent mothers and repeated childbearing: effects of a school-based intervention program.

This study examined the effectiveness of a public school program for pregnant teenagers in preventing rapid repeated childbearing. Students permitted to attend longer than seven weeks postpartum were much more likely to avoid having another child in the next five years than were students required to return to their regular schools. The results indicate the potential of school-based programs to improve life outcomes for adolescent mothers and their children.

PIP: If an adolescent mother bears a second child soon after her first, her chances of becoming educationally and economically self-sufficient are severely limited. Whereas prenatal services to pregnant adolescents are designed to optimize the obstetrical outcomes, research has indicated that adolescent mothers involved in postnatal programs show a substantial decrease in subsequent pregnancy rates, a decrease which may continue for years beyond actual program involvement. In a study at the Polly T. McCabe Center, an alternate public school for pregnant students in New Haven, Connecticut, students who were allowed to remain at least 7 weeks postnatally were almost 3 times less likely to have another baby within the next 2 years than students who were not allowed to remain so long. The 102 adolescent mothers in this study were part of a cohort of residents of New Haven who delivered a live first-born infant on or between March 1, 1979 and February 29, 1980, were less than 19 years old at delivery, were not high school graduates when they became pregnant, were from low-income families, were Black, were regular attendees at the McCabe Center, and either returned to school or graduated after delivery. The duration of the students' assignment to McCabe depended on the timing of their pregnancy. They were referred to McCabe when their pregnancy became apparent or when they notified their current school that they were pregnant. They typically remained at McCabe during the academic quarter in which their baby was delivered and then returned to their home school. However, students delivering during the third quarter were allowed to complete the year at McCabe. These rules had the effect of random assignment to postpartum services. Information on the deliver of a second child was available for 99 mothers. Medical records were collected for the period from the first prenatal checkup until the first baby was 6-years-old. Postnatal time at McCabe was determined to be short if it was or=7 weeks and long if it was 7 weeks. 19 or 52 (36%) of students with a short postnatal period at the school delivered a second child within 2 years compared with 6 of 50 (12%) with a long period (p=.005). At 5 years, 35 of 50 (70%) of the short time group had additional children vs. 22 of 49 (45%) of the long time group (p=.015). After recoding 4 mothers who miscarried and 6 who had involuntary sterilization, it was found that almost half of the presumably fertile mothers who were at McCabe for a long postnatal time avoided subsequent childbearing for 5 years vs. only about 25% of the fertile short-time mothers. The 2 groups were similar in incidence of abortion, in receiving a postpartum check-up and effective means of contraception, in living arrangements, and in child care help received. These findings indicate that the effectiveness of school programs for pregnant adolescents may be greatly enhanced and lead to a longterm improvement in the young mothers' chances for a self-sufficient life if they are extended into the postpartum period, which is an especially optimum time for intervention.

Effects of an intervention program for pregnant adolescents: educational outcomes at two years postpartum.

Examined postpartum effects of a school-based intervention program for pregnant adolescents. Interviews were conducted with 102 innercity black, low-income, school-aged mothers who had attended the program, and their academic and medical records were reviewed. For teenagers who had been poor students prior to becoming pregnant, a strong linear effect was found for duration of program attendance: with sufficient time in the program, poorer students became indistinguishable from better students in educational success. Most of the better students were educationally successful at 2 years postpartum, independent of their length of time in the program. For all students, longer durations of postnatal intervention were predictive of lower likelihood of subsequent childbearing. Numerous academic, medical, social, and demographic variables were ruled out as possible confounding factors that might have produced the positive educational outcomes for poorer students. The results suggest that adolescents who appear to have minimal academic promise prior to their pregnancy are nevertheless very responsive to school-based intervention.

Imitation, recall, and imitativeness in children with low intelligence of organic and familial etiology.

Groups of noninstitutionalized organic and familial mentally retarded and borderline mentally retarded children at two CA levels were given tasks designed to assess imitation. In contrast to findings from studies of average IQ children, organic low IQ children showed as much imitativeness at the older as at the younger age levels. Consistent with expectations, etiology, independent of IQ, was found to be significant. Familial low IQ children showed more absolute imitation and recall, whereas organic children were more imitative and responsive to the irrelevant behaviors modeled. Findings are discussed in terms of the developmental approach to imitation.