Sami B. Kanaan

Maternal Microchimerism Predicts Increased Infection but Decreased Disease due to Plasmodium falciparum During Early Childhood

Background A mothers infection with placental malaria (PM) can affect her childs susceptibility to malaria, although the mechanism remains unclear. The fetus acquires a small amount of maternal cells and DNA known as maternal microchimerism (MMc), and we hypothesized that PM increases MMc and that MMc alters risk of Plasmodium falciparum malaria during infancy. Methods In a nested cohort from Muheza, Tanzania, we evaluated the presence and level of cord blood MMc in offspring of women with and without PM. A maternal-specific polymorphism was identified for each maternal-infant pair, and MMc was assayed by quantitative polymerase chain reaction. The ability of MMc to predict malaria outcomes during early childhood was evaluated in longitudinal models. Results Inflammatory PM increased the detection rate of MMc among offspring of primigravidae and secundigravidae, and both noninflammatory and inflammatory PM increased the level of MMc. Detectable MMc predicted increased risk of positive blood smear but, interestingly, decreased risk of symptomatic malaria and malaria hospitalization. Conclusions The acquisition of MMc may result in increased malaria infection but protection from malaria disease. Future studies should be directed at the cellular component of MMc, with attention to its ability to directly or indirectly coordinate anti-malarial immune responses in the offspring.

Maternal microchimerism is prevalent in cord blood in memory T cells and other cell subsets, and persists post-transplant

ABSTRACT Among reported advantages of umbilical cord blood (CB) in transplantation is lower leukemia relapse probability. Underlying cellular mechanisms of graft-vs.-leukemia (GVL) are thought to include a prominent role for T cells. Cells of the CBs mother, maternal microchimerism (MMc), were recently strongly, but indirectly, implicated in this GVL benefit. We assayed MMc directly and hypothesized benefit accrues from CB maternal T cells. MMc was quantified in 51 CBs and, within memory T, naïve T, B, NK cells, and monocytes in 27 CBs. Polymorphism-specific quantitative-PCR assays targeted maternal genotypes non-shared with CBs. Overall MMc was common and often at substantial levels. It was present in 52.9% of CB and in 33.3–55.6% of tested subsets. Remarkably, MMc quantities were greater in memory T cells than other subsets (p < 0.001). Expressed as genome equivalents (gEq) per 105 total gEq tested (gEq/105), memory T cell MMc averaged 850.2 gEq/105, while other subset mean quantities were 13.8–30.1 gEq/105. After adjustment for proportionality in CB, MMc remained 6–17 times greater in memory T, and 3–9 times greater in naïve T, vs. non-T-cell subsets. Further, CB-origin MMc was detected in vivo in a patient up to 6 mo post-transplantation, including among T cells. Overall, results revealed levels and phenotypes of CB MMc with potential relevance to CB transplantation and, more broadly, to offspring health.

Evaluation of X Chromosome Inactivation with Respect to HLA Genetic Susceptibility in Rheumatoid Arthritis and Systemic Sclerosis

Background Autoimmune diseases, including rheumatoid arthritis (RA) and systemic sclerosis (SSc) are characterized by a strong genetic susceptibility from the Human Leucocyte Antigen (HLA) locus. Additionally, disorders of epigenetic processes, in particular non-random X chromosome inactivation (XCI), have been reported in many female-predominant autoimmune diseases. Here we test the hypothesis that women with RA or SSc who are strongly genetically predisposed are less susceptible to XCI bias. Methods Using methylation sensitive genotyping of the androgen receptor (AR) gene, XCI profiles were performed in peripheral blood mononuclear cells from 161 women with RA, 96 women with SSc and 100 healthy women. HLA-DRB1 and DQB1 were genotyped. Presence of specific autoantibodies was documented for patients. XCI skewing was defined as having a ratio ≥ 80:20 of cells inactivating the same X chromosome. Results 110 women with RA, 68 women with SSc, and 69 controls were informative for the AR polymorphism. Among them 40.9% of RA patients and 36.8% of SSc patients had skewed XCI compared to 17.4% of healthy women (P = 0.002 and 0.018, respectively). Presence of RA-susceptibility alleles coding for the “shared epitope” correlated with higher skewing among RA patients (P = 0.002) and such correlation was not observed in other women, healthy or with SSc. Presence of SSc-susceptibility alleles did not correlate with XCI patterns among SSc patients. Conclusion Data demonstrate XCI skewing in both RA and SSc compared to healthy women. Unexpectedly, skewed XCI occurs more often in women with RA carrying the shared epitope, which usually reflects severe disease. This reinforces the view that loss of mosaicism in peripheral blood may be a consequence of chronic autoimmunity.

HLA-DRB1, DQA1, and DQB1 in Juvenile-Onset Systemic Sclerosis.

Systemic sclerosis (SSc) is a rare disease that is particularly uncommon in children. Specific HLA alleles have been associated with SSc in adults. This study was undertaken to investigate HLA class II alleles in juvenile‐onset SSc.

Brief Report: HLA–DRB1, DQA1, and DQB1 in Juvenile-Onset Systemic Sclerosis

Systemic sclerosis (SSc) is a rare disease that is particularly uncommon in children. Specific HLA alleles have been associated with SSc in adults. This study was undertaken to investigate HLA class II alleles in juvenile‐onset SSc.

A6.40 Copy number increase of TLR7 and TLR8 genes in men with rheumatoid arthritis

Background and objectives Men are less often affected by Rheumatoid arthritis (RA) than women. In mice studies, it was shown that a duplication in Toll-like receptor 7 (Tlr7) gene is sufficient to potentiate autoimmunity in males. We therefore propose to investigate whether an increase in copy number (CN) of TLR7, and its neighbouring paralog TLR8 on X chromosome, could contribute to the pathogenesis of RA in men. Materials and methods In a pilot analysis, we had first tested our hypothesis by real-time quantitative PCR assay to assess CN of TLR7 gene and TLR8 compared to Beta-Globin gene (HBB), using the relative standard curve calculation method, on DNA from PBMCs of 49 men with RA and 42 healthy controls. TLR7/8 copy numbers significantly increased in PBMCs from men with RA when compared to healthy men. Nevertheless, because men with RA were significantly older than healthy donors, the increased TLR7/8 CN could be age-dependent. In the current study, we have tested whether TLR7/8 CN varies with age on a large number of controls from birth to 82 years old (160 men and 180 women). We also tested an additional autosomal RPP30 gene to further validate our results. DNA samples from peripheral blood of 49 men with RA versus 160 healthy men and 44 women with RA versus 180 healthy women were tested for TLR7/8 CN compared to mean CN values of HBB and RPP30. Similarly, DNA samples from PBMCs of 53 men with RA versus 42 healthy men and 40 women with RA versus 41 healthy women were analysed. Results TLR7 or TLR8 CN does not vary with age in healthy individuals (women or men). Similar findings were observed in patients with RA. Nevertheless, we confirmed that men with RA had an increased TLR7 and TLR8 CN in peripheral blood compared to healthy men (p < 0.0001 and p = 0.002 respectively). A rather opposite trend was observed in women with RA. Similar results were observed in PBMCs. Conclusions We have presented evidence that copy number of X-linked TLR7 and TLR8 genes increases in men with RA. This increase is not only disease dependent but also sex-dependent. We are currently validating our results by digital droplet polymerase chain reaction to confirm this genetic heterogeneity between men and women with RA.

1.65 Copy number variation of TLR7 and TLR8 genes is age and sex biased: which role in autoimmunity?

Background and Objectives Women, having two X chromosomes, are more predisposed than men to autoimmune diseases. The X chromosome contains many genes linked to immunity which may contribute to this gender bias. In a mouse model, a duplication of the innate immunity X-linked toll like receptor 7 (Tlr7) gene has been shown to potentiate autoimmunity in males. We then proposed to investigate whether TLR7 gene and its neighboring paralog TLR8 could have variations in their copy numbers, contributing to the pathogenesis of rheumatoid arthritis (RA) in men. Methods A real-time quantitative PCR protocol was developed to assess copy number variation (CNV) of TLR7 and TLR8 gene, using sensitive and optimised ΔΔCt and standard curve methods, in DNA from peripheral blood mononuclear cells of 60 patients with RA (including 49 men) and 64 healthy controls (including 42 men). Among them, 31 men with RA and 18 healthy men were further screened for TLR7/8 CNV in 4 subpopulations: B cells, T cells, granulocytes and the depleted fraction of the former 3. Results TLR7/8 copy numbers significantly increased with age in PBMCs from all men (P < 0.0001, Spearman’s rank correlation test), whether they had RA or not. The increase had mean amplitude of 20%, spanning from the age of 20 until 80, according to the linear-regression-curve’s best fit. This age-dependent and disease-independent CNV increase was also observed in all cell subsets. Interestingly, such increase was not observed in women, healthy or with RA, but rather an opposite trend. Conclusion and Perspectives For the first time we showed an increased CNV in TLR7 and TLR8 genes which is age and sex-mediated. Several hypotheses could explain such phenomenon. For example, somatically acquired duplications can affect some cells over time and result in an increase with age in men. In parallel, X chromosome monosomy, previously described in aging women could account for the opposite trend in those. Another explanation for men can be due to the presence of feminine microchimerism, arising from feto-maternal or twin sister exchange during in utero life, as previously described. Such cells would carry 2 X chromosomes and contribute to the increased pool of X-linked genes among XY host cells. Investigating these hypotheses would provide better understanding of age-associated X-linked genetic modifications and the role of the X chromosome in gender differences in health and disease. Abstract topicOther

Fetal microchimerism by mode of delivery: a prospective cohort study

To compare fetal microchimerism (FMc) in pregnancies with uncomplicated vaginal delivery (VD) versus caesarean delivery (CD).

Persistence of the losing cord blood unit following double cord blood transplantation: finding the unseen.

To the editor: Double cord blood (CB) transplantation (dCBT) is an accepted treatment of patients with hematologic malignancies.[1][1],[2][2] In the vast majority of dCBT recipients, 1 unit emerges as the sole source of long-term hematopoiesis.[3][3] As measured by standard clinical testing for

New BRAF (v raf murine sarcoma viral oncogene homologue B1) mutation in rheumatoid arthritis

Rheumatoid arthritis (RA)–associated autoantibodies include those directed at the kinase site of BRAF (v‐Raf murine sarcoma viral oncogene homolog B1), a serine–threonine kinase involved in the MAPK signaling pathway. To understand anti‐BRAF immunization, we sought to identify BRAF mutations in the peripheral blood lymphocytes (PBLs) of patients with RA.