Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.

The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes.

Rapid development of myeloproliferative neoplasm in mice with mutation and haploinsufficient for .

gain-of-function mutation is the most common mutation found in patients with juvenile myelomonocytic leukemia and DNMT3A loss occurs in over 20% of acute myeloid leukemia patients. We studied the combined effect of both gain-of-function mutation (D61Y) and haploinsufficiency on mouse hematopoiesis, the presence of which has been described in both juvenile myelomonocytic leukemia and acute myeloid leukemia patients. Double mutant mice rapidly become moribund relative to any of the other genotypes, which is associated with enlargement of the spleen and an increase in white blood cell counts.

Pharmacologic inhibition of PI3K p110δ in mutant Shp2E76K-expressing mice.

Juvenile myelomonocytic leukemia is a childhood malignancy that lacks effective chemotherapies and thus has poor patient outcomes. PI3K p110δ has been found to promote hyperproliferation of cells expressing mutant Shp2. In this study, we tested the efficacy of a PI3Kδ inhibitor in mice expressing the Shp2 gain-of-function mutation, E76K.

Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease.

Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4CD146CCR5 T cell population is Th17 prone and increased by ICOS stimulation.

Defects in the Fanconi Anemia Pathway in Head and Neck Cancer Cells Stimulate Tumor Cell Invasion through DNA-PK and Rac1 Signaling.

Purpose: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies.

Innate immune induction and influenza protection elicited by a response-selective agonist of human C5a.

The anaphylatoxin C5a is an especially potent mediator of both local and systemic inflammation. However, C5a also plays an essential role in mucosal host defense against bacterial, viral, and fungal infection. We have developed a response-selective agonist of human C5a, termed EP67, which retains the immunoenhancing activity of C5a at the expense of its inflammatory, anaphylagenic properties.

Age-associated changes in miRNA expression profiles in thymopoiesis.

During adult life, the thymus involutes and thymic output of mature T cells drastically declines. The molecular events underlying this process are not well understood. Here, we present evidence of the importance of miRNAs in regulating T cell differentiation in the aged.

A novel approach to thymic rejuvenation in the aged.

With advancing age, the mammalian thymus undergoes involution, a progressive loss of architectural integrity and lymphoid cellularity that results in reduced T lymphopoiesis. Thymic involution also is associated with extreme malnutrition and states of immune deficiency, such as active HIV infection, after chemotherapy, or during pregnancy. Immune recovery appears to require restoration of normal thymopoiesis.

IL-7 gene therapy in aging restores early thymopoiesis without reversing involution.

Thymic involution begins early in life and continues throughout adulthood, resulting in a decreased population of naive T cells in the periphery and a reduced ability to fight off newly encountered infectious diseases. We have previously shown that the first step of thymopoiesis is specifically blocked in aging. This block at the DN1 to DN2 transition and the subsequent loss of thymic output in old age mirrors the changes seen in IL-7-deficient mice, and it is hypothesized that decreased intrathymic IL-7 is involved in age-related thymic involution.

A CD45 minigene restores regulated isoform expression and immune function in CD45-deficient mice: therapeutic implications for human CD45-null severe combined immunodeficiency.

Transgenic mice have been generated that carry a CD45 minigene under control of the human leukocyte function-associated antigen (LFA-1, CD11a) promoter. CD45-null mice carrying the transgene exhibit the lymphocyte lineage-specific isoform expression patterns of wild-type mice. Furthermore, these mice have normal thymocyte development and peripheral T-cell numbers.

The role of intron sequences in high level expression from CD45 cDNA constructs.

Consistent expression from CD45 cDNA constructs has proven difficult to achieve. Through the use of new CD45 cDNA constructs and reporter genes, the role 5', 3', and intron sequences play in CD45 expression was determined. The CD45 polyadenylation signal sequence was fully functional in a beta-galactosidase reporter construct.

Expression of CD45 isoforms lacking exons 7, 8 and 10.

The CD45 exon usage pattern of various CD8+ and CD4+ T cell lines was studied. By using the reverse transcription-polymerase chain reaction (RT PCR) and Southern analysis with exon specific or exon junction probes, we showed that all of the cytotoxic T cell lines and the majority of the helper T cells expressed the 789 isoform as a major splice variant. Expression of the splice product lacking exons 4-7 (isoform 89) was not as ubiquitous.

Murine mast cells and monocytes express distinctive sets of CD45 isoforms.

CD45 isoform expression patterns were examined in various mast and monocyte cell populations. The reverse transcription-polymerase chain reaction (RT/PCR) and Southern analysis showed these myeloid cells express characteristic sets of CD45 isoforms. Mast cells produce mRNA for two splice variants, one containing exons 5, 7 and 8 of the alternatively expressed exons (therefore lacking exons 4 and 6) and another containing variable exons 7 and 8.

Common regulatory elements control symbiotic and microaerobic induction of nifA in Rhizobium meliloti.

We have previously demonstrated that the nifA promoter (nifAp) of Rhizobium meliloti is inducible under microaerobic conditions in the absence of alfalfa. Here we show that microaerobic activation of nifAp involves both cis- and trans-acting regulatory controls identical to those used symbiotically. The start site for nifA mRNA synthesis was found to be the same during symbiosis and microaerobiosis, and a deletion analysis of nifAp demonstrated that DNA between positions -62 and -45 is essential for induction.

The nifA gene of Rhizobium meliloti is oxygen regulated.

Experiments using plasmid-borne gene fusions and direct RNA measurements have revealed that expression from the nifA gene is induced in Rhizobium meliloti when the external oxygen concentration is reduced to microaerobic levels. Induction occurs in the absence of alfalfa and in the presence of fixed nitrogen and does not require ntrC. The production of functional nifA gene product (NifA) can be demonstrated by its ability to activate the nitrogenase promoter P1.

Analysis of transfer of tumor-inducing plasmids from Agrobacterium tumefaciens to Petunia protoplasts.

Petunia protoplasts were infected with the virulent Agrobacterium tumefaciens strain A348 or the avirulent strain A136 (lacking a Ti plasmid). The infection process was stopped at various time intervals up to 24 h after inoculation, and the DNA from the plant cells was isolated. Southern blot analysis indicated that the DNA isolated from infected Petunia cells was not detectably contaminated by bacterial DNA from lysed Agrobacterium cells.