Emergence of translocation t(9;11)-positive leukemia during treatment of childhood acute lymphoblastic leukemia

Therapy-related acute myeloid leukemia (t-AML) characterized by the t(9;11)(p22;q23) translocation is one of the most frequent secondary malignancies. The timing of the initiation of translocation and of development of the malignant t(9;11) clone during chemotherapy is presently unknown. In the present study, we backtracked bone marrow samples from three children during treatment for acute lymphoblastic leukemia (ALL). Two patients developed a t(9;11)-positive t-AML 19 and 30 months after therapy start, whereas the third patient, diagnosed with a rare t(9;11)-positive ALL, suffered from an ALL relapse 23 months after initial diagnosis. The genomic MLL-MLLT3 (MLL-AF9) fusion site was amplified by a multiplex, nested long-range PCR and used as a clonal marker for quantification of the MLL-MLLT3-positive cells during chemotherapy. The t(9;11)-positive clone was detectable 13 and 18 months after therapy start in both t-AML cases, which was 6-12 months before clinical diagnosis of the secondary malignancy. In the t(9;11)-positive ALL patient, the identical leukemic clone reoccurred during maintenance therapy after a short molecular remission, 8 months before clinically overt ALL relapse. The time course and characteristics of the genomic breakpoints in the present t-AML cases support the hypothesis of translocation formation as a result of defective breakage repair after topoisomerase II cleavage.     

The MDR1 polymorphisms at exons 21 and 26 predict steroid weaning in pediatric heart transplant patients

Various polymorphisms of the MDR1 gene that encodes for P-glycoprotein (P-gp), a transmembrane pump, have been identified. A silent mutation C3435T in exon 26 and a G2677T mutation in exon 21 have been correlated with P-gp expression and function in humans. The objectives of this study were (a) to determine whether the MDR1 exon 21 and exon 26 polymorphisms were related to steroid weaning in a pediatric heart transplant (HTx) population, and (b) to determine whether an association exist between the MDR1 exon 21 and exon 26 polymorphisms in these patients. Sixty-nine pediatric HTx patients were studied. MDR1 genotyping was determined by polymerase chain reaction amplification, sequencing the DNA, and sequence evaluation using Polyphred software (University of Washington) to identify genotypes. The steroid dose at 1 year post-transplantation was recorded. For steroid weaning at one year post-HTx for MDR1 C3435T, 12 of 18 (67%) patients in the CC genotype were still on prednisone, whereas only 18 of 47 (38%) of the CT/TT group were still receiving prednisone (p = 0.04). Similar results were observed for the MDR1 G2677T genotyping and steroid weaning. Forty-three of 46 patients (93.5%) who have MDR1 C3435T allele also have a mutant G2677T allele (p < 0.001). We conclude that (a) a significantly larger number of MDR1 3435 CC HTx patients remain on steroids at 1 year after transplantation, and (b) the MDR1 C3435T genotype is associated with the G2677 genotype in pediatric HTx patients.     

Antibodies to the polymeric immunoglobulin receptor with different binding and trafficking patterns

The polymeric immunoglobulin receptor (pIgR) has been proposed as a therapeutic target, but its potential depends on the efficiency of uptake and trafficking of the receptor ligand. Mouse monoclonal antibodies (Mabs) directed against pIgR, selected for strong binding to secretory component (SC) and secretory IgA (sIgA), were tested in a transcytosis assay in 16HBEo--cells (human bronchial epithelial cell line) transfected with human pIgR. Intracellular trafficking was followed by confocal microscopy. Mabs fell into two classes. For two Mabs, transcytosis from basolateral to apical surface is rapid, unidirectional, and little Mab is retained in the cell. For three Mabs, basolateral to apical transcytosis occurs to a significantly lesser extent, reverse transcytosis is permitted, and some of the Mab is retained in the perinuclear region even after 24 h. When tested for their ability to recognize and immunoprecipitate pIgR with systematic truncations and deletions of the five immunoglobulin (Ig)-like domains, all Mabs bound to the fifth Ig-like domain, but three of them also bound to the C-terminal region of pIgR near the plasma membrane. Different binding sites probably account for the different trafficking of these Mabs and may predict differential therapeutic utility.     

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

Summary We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt ⁺ and mt ^– vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt ⁺ gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.     

Neurological manifestations of human parvovirus B19 infection

Since its discovery, human parvovirus B19 has been linked with a broad spectrum of clinical syndromes. An aetiological role for the virus has been confirmed in erythema infectiosum, transient aplastic crisis, persistent infection manifesting as pure red cell aplasia in immunocompromised persons, non-immune hydrops fetalis and arthritis. Less commonly recognised, but receiving increasing attention recently, are the neurological manifestations, a variety of which have been described in patients with either clinically diagnosed or laboratory confirmed B19 infection. The purpose of this review is to summarise present knowledge of B19, its known and potential pathogenic mechanisms and its association with human diseases, particularly those with neurological manifestations. The outcome of the review supports an aetiological role of the virus in neurological disease. However, the pathogenesis remains unknown and elucidating this is a priority.     

A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice

Studies in gene-targeted mice have demonstrated that factor B of the alternative complement pathway plays an important role in several disease models, but an exogenous inhibitor of factor B has not previously been available. We have developed an inhibitory monoclonal antibody directed against a critical epitope on mouse factor B and have tested it in a model of antiphospholipid (aPL) antibody (Ab)-induced fetal loss. Gene-targeted factor B-deficient mice (fB-/-) were injected with a fusion protein comprised of the second and third short consensus repeat (SCR) domains of mouse factor B linked to a mouse IgG1 Fc domain. Hybridomas were made from splenocytes of the immunized mouse. One mAb, designated 1379, produced an IgG1 antibody that inhibited alternative pathway activation in vitro and in vivo by preventing formation of the C3bBb complex. Strikingly, this mAb inhibited alternative pathway activation in serum from mice, rats, humans, monkeys, pigs and horses. Fab fragments made from this mAb also inhibited alternative pathway activation. Epitope mapping demonstrated that this antibody binds to factor B within the third SCR domain. When mAb 1379 was administered to mice that also received human IgG containing antiphospholipid antibodies, it provided significant protection from antiphospholipid antibody-induced complement activation and fetal loss. Thus, this mAb to factor B has broad species reactivity and effectively inhibits alternative pathway activation. The mAb protects mice in an in vivo model of antiphospholipid antibody syndrome, demonstrating the therapeutic potential for the inhibition of factor B in this disease.     

Mechanisms of MHC class I-restricted antigen processing and cross-presentation

Summary: In this review, we discuss recent data from our laboratory that address two aspects of major histocompatibility complex (MHC) class I‐restricted antigen processing. First, we consider the nature of the peptide‐loading complex, which is the assembly of proteins in the endoplasmic reticulum (ER) into which newly synthesized MHC class I‐β₂ microglobulin (β₂m) heterodimers are incorporated, and the mechanisms involved in MHC class I assembly and peptide loading that are facilitated by the peptide‐loading complex. Second, we discuss mechanisms of cross‐presentation, the phenomenon whereby extracellular and luminal protein antigens can be processed by antigen‐presenting cells, particularly dendritic cells, and presented by MHC class I molecules to CD8⁺ T cells. The focus of the discussion is mainly on the human MHC class I system.     

Dental abnormalities in individuals with pathogenic germline variation in DICER1

Pathogenic germline variation in the microRNA processing gene DICER1 gives rise to an autosomal dominant, tumor‐predisposition disorder. Conditional deletion of Dicer1 in murine dental epithelium shows that it controls tooth patterning, size, number, and shape. The human dental phenotype of people with germline pathogenic variation in DICER1 is unknown. DICER1‐carriers (n = 57) and family controls (n = 55) were evaluated at the NIH Clinical Center dental clinic as part of a comprehensive medical evaluation. Digital panoramic radiographs, bite‐wing radiographs, and oral photographs were collected. A single observer, blind to DICER1 status, reviewed the dental records and determined the presence or absence of 11 dental characteristics as described in the clinic notes, radiographs, or oral photographs. Subjective phenotypes were reviewed on radiographs by two examiners (blind to DICER1 status) for the presence or absence of the dental characteristics to reduce inconsistencies. By simple association, bulbous crown, periodontitis, and taurodontism were all significant (p < .05). Logistic regression with chi‐square maximum likelihood estimates showed that bulbous crown and periodontitis remained significant. Recognition of these phenotypes may aid identification of individuals and families at risk for DICER1‐associated neoplasms. These findings may also guide dental care for individuals with germline DICER1 pathogenic variation.     

Immunolocalization of Alk8 during replacement tooth development in zebrafish

The novel type I transforming growth factor-beta (TGF-beta) family member receptor Alk8 was previously identified in a degenerate RT-PCR screen for zebrafish type I and II TGF-beta family member receptors. Functional analyses revealed that Alk8 acts through Bmp signaling pathways in early embryonic dorsoventral patterning, in neural crest cell specification, and in patterning and differentiation of neural crest cell-derived pharyngeal arch cartilages. In addition, Alk8 forms active signaling complexes with TGF-beta1 and the TGF-beta RII receptor, suggesting that Alk8 mediates cross talk between Bmp and TGF-beta subfamily members. In this study, immunohistochemical analysis was performed on zebrafish aged 2 days postfertilization to 1 year, revealing immunolocalization of Alk8 to tissues of the tooth-bearing ceratobranchial 5 (cb5) arch including dental epithelial and mesenchymal tooth tissues of developing primary and replacement teeth, mucous-producing crypt epithelium, keratinized bite plate, and developing taste buds. These results suggest roles for Alk8 in patterning tooth-bearing pharyngeal epithelium, in the initiation of tooth development, in odontoblast and ameloblast differentiation, and in osteoblast maturation. The ability for zebrafish to continuously form teeth throughout their lives allows for the comparison of Alk8 expression in both primary and replacement tooth development, revealing identical Alk8 expression profiles. This study advances our current understanding of the functions of Alk8, particularly with respect to primary and replacement tooth formation, reveals additional roles for Alk8 in dental epithelial patterning and in odontoblast, ameloblast and osteoblast differentiation, and demonstrates the utility of the zebrafish as a model for primary and replacement tooth development.