Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively.

Is microchimerism a sign of imminent disease recurrence after allogeneic hematopoietic stem cell transplantation? A systematic review of the literature.

Chimerism analysis following hematopoietic stem cell transplantation (HSCT) for leukemia is routinely applied in parallel with quantification of minimal residual disease (MRD) to identify imminent relapse. In the past decades, new methods with a lower limit of detection compared to standard methods have been developed, so-called microchimerism analysis. Microchimerism analysis is fast, simple, applicable across pre-HSCT disease-type and can be applied on peripheral blood allowing frequent testing during follow-up.

Highly sensitive chimerism detection in blood is associated with increased risk of relapse after allogeneic hematopoietic cell transplantation in childhood leukemia.

Analysis of chimerism in blood post-HCT using STR-PCR is routinely applied in parallel with quantification of MRD to predict relapse of leukemia. RQ-PCR chimerism is 10- to 100-fold more sensitive, but clinical studies in children are sparse. We analyzed IMC in blood samples following transplantation for acute lymphoblastic or myeloid leukemia in 56 children.

T-cell chimerism is valuable in predicting early mortality in steroid-resistant acute graft-versus-host disease after myeloablative allogeneic cell transplantation.

The main aim of this study was to evaluate the impact of early T-cell chimerism status on the incidence and clinical course of acute graft-versus-host disease (aGVHD) in allogeneic transplant recipients after myeloablative conditioning. Of 62 patients, 38 (61%) had complete T-cell donor chimerism (DC), and 24 (39%) had mixed T-cell chimerism (MC). The incidence of early death (before day 100) with steroid-resistant aGVHD was significantly higher in patients with complete DC compared to patients with MC (6/38 vs.

Smoking and polymorphisms of genes encoding mannose-binding lectin and surfactant protein-D in patients with rheumatoid arthritis.

To investigate whether polymorphisms in genes coding for mannose-binding lectin (MBL) and surfactant protein-D (SP-D) are associated directly or by interaction with smoking with rheumatoid arthritis (RA), anti-citrullinated peptide antibody (ACPA) positive RA, and erosive RA. MBL2 genotypes, SFTPD genotype at codon 11, and HLA-shared epitope were determined in 456 patients with rheumatoid arthritis and 533 sex- and age-matched controls. Patients were grouped according to the presence of ACPA antibodies and RA-associated bone erosions and sub-stratified according to smoking status as never or ever smokers.

Genotyping of FCN and MBL2 polymorphisms using pyrosequencing.

Pyrosequencing represents one of the most thorough methods used to analyze polymorphisms. One advantage of using pyrosequencing for genotyping is the ability to identify not only single-nucleotide polymorphisms (SNPs) but also tri-allelic variations, insertions and deletions (InDels). In contrast to most other genotyping assays the sequence surrounding the polymorphism provides an internal control making this method highly reliable.

Different immunological phenotypes associated with preserved CD4+ T cell counts in HIV-infected controllers and viremic long term non-progressors.

Background: HIV-infected controllers control viral replication and maintain normal CD4+ T cell counts. Long Term Non-Progressors (LTNP) also maintain normal CD4+ T cell counts, but have on-going viral replication. We hypothesized that different immunological mechanisms are responsible for preserved CD4+ T cell counts in controllers and LTNP.

FoxP3 mRNA splice forms in synovial CD4+ T cells in rheumatoid arthritis and psoriatic arthritis.

Our aim was to elucidate the relative amount of the different splice forms of FoxP3 mRNA in CD4+ T cells in peripheral blood (PB) compared to synovial fluid (SF) in RA and PsA patients. FoxP3 mRNA was measured using a quantitative real-time PCR method. CD4+ T cells were isolated from 17 paired samples of PB and SF from RA and PsA patients, and PB from 10 controls.

Approaches for cytogenetic and molecular analyses of small flow-sorted cell populations from childhood leukemia bone marrow samples.

Discordances between minimal residual disease estimates obtained by different methods are a problem in childhood acute lymphoblastic leukemia. We aimed to optimize methods allowing the biological exploration of such discrepancies, i.e.

Prevalence of t(12;21)[ETV6-RUNX1]-positive cells in healthy neonates.

t(12;21)(p13;q22)[ETV6-RUNX1] is the most common chromosomal translocation in childhood acute lymphoblastic leukemia, and it can often be backtracked to Guthrie cards supporting prenatal initiation and high levels of circulating t(12;21)-positive cells at birth. To explore the prevalence of ETV6-RUNX1-positive cells in healthy neonates, mononuclear cells from 1417 umbilical cord blood samples were isolated within 24 hours from birth and subsequently screened for ETV6-RUNX1 transcripts using a highly sensitive real-time reverse transcription polymerase chain reaction assay. In first-run polymerase chain reaction, 14 samples were positive at levels below 10(-5), of which specific hybridization reflecting the relevant genetic region was positive in 9 cases.

Increased risk of ALL among premature infants is not explained by increased prevalence of pre-leukemic cell clones.

The multi-hit hypothesis for paediatric leukemogenesis states that an initial genetic hit (often occurring prenataly) must be followed by one or more hit(s) before a cell become leukeamic. Studies have demonstrated the presence of pre-leukaemic t(12;21)-positive cells at levels 10(-3) to 10(-4) in 1% of newborns (i.e.

Mannose-binding lectin gene polymorphisms are associated with disease activity and physical disability in untreated, anti-cyclic citrullinated peptide-positive patients with early rheumatoid arthritis.

Objective: To study the association between polymorphisms in the mannose-binding lectin gene (MBL2) and disease activity, physical disability, and joint erosions in patients with newly diagnosed rheumatoid arthritis (RA).

Methods: Patients with early RA (n=158) not previously treated with disease modifying antirheumatic drugs, participating in a treatment trial (CIMESTRA study) were examined at inclusion for MBL2 pooled structural genotypes (O/O, A/O, A/A), regulatory MBL2 promoter polymorphism in position -221 (XX, XY, YY), anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2), disease activity by Disease Activity Score-28 (DAS28 score), physical disability by Health Assessment Questionnaire (HAQ) score, and erosive changes in hands and feet (Sharp-van der Heijde score).

Results: Eight patients were homozygous MBL2 defective (O/O), 101 belonged to an intermediate group, and 49 were MBL2 high producers (YA/YA).

Genetically determined high serum levels of mannose-binding lectin and agalactosyl IgG are associated with ischemic heart disease in rheumatoid arthritis.

Objective: Patients with rheumatoid arthritis (RA) have excess morbidity and mortality due to ischemic heart disease. It has been suggested that high serum levels of mannose-binding lectin (MBL) and agalactosyl IgG (IgG-G0) are associated with increased inflammation in RA. MBL also enhances inflammation-mediated tissue injury during postischemic reperfusion.

Ficolin-2 recognizes DNA and participates in the clearance of dying host cells.

Ficolin-2 is a serum opsonin, which has been shown to be a pattern recognition molecule in the lectin complement activation pathway. Because innate immune mechanisms are involved in maintaining tissue homeostasis we hypothesized that Ficolin-2 also participate in the clearance of dying host cells. We found that Ficolin-2 binds to late apoptotic cells, as well as to apoptotic bodies and necrotic cells, but not to early apoptotic cells.

Increased frequency of mannose-binding lectin insufficiency among children with acute lymphoblastic leukemia.

Epidemiological data indicate that acute lymphoblastic leukemia (ALL) could be induced by interactions between the immune system and early childhood infections. Mannose-binding lectin (MBL) plays a critical role in the immune response in early childhood before specific immune protection develops. We investigated whether there may be an association between childhood ALL and low-producing MBL genotypes.

Mannose-binding lectin variant alleles and HLA-DR4 alleles are associated with giant cell arteritis.

Objective: To determine whether variant alleles of the mannose-binding lectin (MBL) gene causing low serum concentrations of MBL and/or polymorphisms of HLA-DRB1 are associated with increased susceptibility to polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) or particular clinical phenotypes of PMR/GCA.

Methods: MBL and HLA-DRB1 alleles were determined by polymerase chain reaction in 102 Danish patients with PMR (n = 37) or GCA (n = 65). Two hundred fifty and 193 healthy individuals served as controls for MBL and HLA genotyping, respectively.