John J. Murphy

European development of clofarabine as treatment for older patients with acute myeloid leukemia considered unsuitable for intensive chemotherapy.

PURPOSE Treatment options for older patients with acute myeloid leukemia (AML) who are not considered suitable for intensive chemotherapy are limited. We assessed the second-generation purine nucleoside analog, clofarabine, in two similar phase II studies in this group of patients. PATIENTS AND METHODS Two consecutive studies, UWCM-001 and BIOV-121, recruited untreated older patients with AML to receive up to four or six 5-day courses of clofarabine. Patients in UWCM-001 were either older than 70 years or 60 to 69 years of age with poor performance status (WHO > 2) or with cardiac comorbidity. Patients in BIOV-121 were >or= 65 years of age and deemed unsuitable for intensive chemotherapy. RESULTS A total of 106 patients were treated in the two monotherapy studies. Median age was 71 years (range, 60 to 84 years), 30% had adverse-risk cytogenetics, and 36% had a WHO performance score >or= 2. Forty-eight percent had a complete response (32% complete remission, 16% complete remission with incomplete peripheral blood count recovery), and 18% died within 30 days. Interestingly, response and overall survival were not inferior in the adverse cytogenetic risk group. The safety profile of clofarabine in these elderly patients with AML who were unsuitable for intensive chemotherapy was manageable and typical of a cytotoxic agent in patients with acute leukemia. Patients had similar prognostic characteristics to matched patients treated with low-dose cytarabine in the United Kingdom AML14 trial, but had significantly superior response and overall survival. CONCLUSION Clofarabine is active and generally well tolerated in this patient group. It is worthy of further evaluation in comparative trials and might be of particular use in patients with adverse cytogenetics.

Regulation of human metabolism by hypoxia- inducible factor

The hypoxia-inducible factor (HIF) family of transcription factors directs a coordinated cellular response to hypoxia that includes the transcriptional regulation of a number of metabolic enzymes. Chuvash polycythemia (CP) is an autosomal recessive human disorder in which the regulatory degradation of HIF is impaired, resulting in elevated levels of HIF at normal oxygen tensions. Apart from the polycythemia, CP patients have marked abnormalities of cardiopulmonary function. No studies of integrated metabolic function have been reported. Here we describe the response of these patients to a series of metabolic stresses: exercise of a large muscle mass on a cycle ergometer, exercise of a small muscle mass (calf muscle) which allowed noninvasive in vivo assessments of muscle metabolism using 31P magnetic resonance spectroscopy, and a standard meal tolerance test. During exercise, CP patients had early and marked phosphocreatine depletion and acidosis in skeletal muscle, greater accumulation of lactate in blood, and reduced maximum exercise capacities. Muscle biopsy specimens from CP patients showed elevated levels of transcript for pyruvate dehydrogenase kinase, phosphofructokinase, and muscle pyruvate kinase. In cell culture, a range of experimental manipulations have been used to study the effects of HIF on cellular metabolism. However, these approaches provide no potential to investigate integrated responses at the level of the whole organism. Although CP is relatively subtle disorder, our study now reveals a striking regulatory role for HIF on metabolism during exercise in humans. These findings have significant implications for the development of therapeutic approaches targeting the HIF pathway.

The RNA binding protein Zfp36l1 is required for normal vascularisation and post-transcriptionally regulates VEGF expression

The Zfp36l1 gene encodes a zinc finger‐containing mRNA binding protein implicated in the posttranscriptional control of gene expression. Mouse embryos homozygous for a targeted mutation in the Zfp36l1 locus died mid‐gestation and exhibited extraembryonic and intraembryonic vascular abnormalities and heart defects. In the developing placenta, there was a failure of the extraembryonic mesoderm to invaginate the trophoblast layer. The phenotype was associated with an elevated expression of vascular endothelial growth factor (VEGF)‐A in the embryos and in embryonic fibroblasts cultured under conditions of both normoxia and hypoxia. VEGF‐A overproduction by embryonic fibroblasts was not a consequence of changes in Vegf‐a mRNA stability; instead, we observed enhanced association with polyribosomes, suggesting Zfp36l1 influences translational regulation. These data implicate Zfp36l1as a negative regulator of Vegf‐a gene activity during development. Developmental Dynamics 235:3144–3155, 2006.

TIS11 Family Proteins and Their Roles in Posttranscriptional Gene Regulation

Posttranscriptional regulation of gene expression of mRNAs containing adenine-uridine rich elements (AREs) in their 3′ untranslated regions is mediated by a number of different proteins that interact with these elements to either stabilise or destabilise them. The present review concerns the TPA-inducible sequence 11 (TIS11) protein family, a small family of proteins, that appears to interact with ARE-containing mRNAs and promote their degradation. This family of proteins has been extensively studied in the past decade. Studies have focussed on determining their biochemical functions, identifying their target mRNAs, and determining their roles in cell functions and diseases.

Anti -β2GPI -antibody -induced endothelial cell gene expression profiling reveals induction of novel pro-inflammatory genes potentially involved in primary antiphospholipid syndrome

Objective: To determine the effects of primary antiphospholipid syndrome (PAPS)-derived anti-β2GPI antibodies on gene expression in human umbilical vein endothelial cells (HUVEC) by gene profiling using microarrays. Methods: Anti-β2GPI antibodies purified from sera of patients with PAPS or control IgG isolated from normal subjects were incubated with HUVEC for 4 h before isolation of RNA and processing for hybridisation to Affymetrix Human Genome U133A-2.0 arrays. Data were analysed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. For selected genes microarray data were confirmed by real-time PCR analysis or at the protein level by ELISA. Results: A total of 101 genes were found to be upregulated and 14 genes were downregulated twofold or more in response to anti-β2GPI antibodies. A number of novel genes not previously associated with APS were induced, including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1, and growth factors CSF2, CSF3 IL-6, IL1β and FGF18. The majority of downregulated genes were transcription factors/signalling molecules including ID2. Quantitative real-time RT-PCR analysis confirmed the microarray results for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C). Conclusions: This study reveals a complex gene expression response in HUVEC to anti-β2GPI antibodies with multiple chemokines, pro-inflammatory cytokines, pro-thrombotic and pro-adhesive genes regulated by these antibodies in vitro. Some of these newly identified anti-β2GPI antibody-regulated genes could contribute to the vasculopathy associated with this disease.

A B cell specific immediate early human gene is located on chromosome band 1q31 and encodes an α helical basic phosphoprotein

We have determined the cDNA sequence of a human B cell specific, immediate early gene, designated 1R20, which is inducible in response to several B cell activation signals. The cDNA sequence predicts a 196 amino acid open reading frame comprising numerous highly basic residues and the predicted structure contains several potential alpha helical domains together with eight consensus protein phosphorylation sites. The 1R20 gene has been localised by fluorescence in situ hybridisation to chromosome band 1q31, a region known to be implicated in the pathogenesis of haemopoietic malignancies.

A novel immunoglobulin superfamily receptor (19A) related to CD2 is expressed on activated lymphocytes and promotes homotypic B-cell adhesion

A novel lymphocyte-specific immunoglobulin superfamily protein (19A) has been cloned. The predicted 335-amino-acid sequence of 19A represents a Type 1 membrane protein with homology with the CD2 family of receptors. A molecular model of the two predicted extracellular immunoglobulin-like domains of 19A has been generated using the crystal structure of CD2 as a template. In isolated lymphocytes, expression of 19A is induced by various activation stimuli, and enforced expression of the 19A gene promotes homotypic cell adhesion in a B-cell-line model. Collectively these data imply that the 19A protein plays a role in regulation of lymphocyte adhesion.

Screening of a HUVEC cDNA library with transplant‐associated coronary artery disease sera identifies RPL7 as a candidate autoantigen associated with this disease

A HUVEC cDNA library was screened with sera from two patients who had developed transplant‐associated coronary artery disease (TxCAD) following cardiac transplantation. A total of six positive clones were isolated from a primary screen of 40 000 genes. Subsequent DNA sequence analysis identified these to be lysyl tRNA synthetase, ribosomal protein L7, ribosomal protein L9, β transducin and TANK. Another gene whose product could not be identified showed homology to a human cDNA clone (DKFZp566M063) derived from fetal kidney. Full‐length constructs of selected genes were expressed as his‐tag recombinant fusion proteins and used to screen a wider patient base by ELISA to determine prevalence and association with TxCAD. Of these ribosomal protein L7 showed the highest prevalence (55·6%) with TxCAD sera compared to 10% non‐CAD.

Importance of dose of type II collagen in suppression of collagen-induced arthritis by nasal tolerance

OBJECTIVE To determine the influence of the dose of collagen given nasally on the induction of specific mucosal tolerance in collagen-induced arthritis. METHODS The severity of clinical arthritis induced in DBA/1 mice was studied after the nasal administration (before disease induction) of 1 of 4 doses (across a 2-log range) of bovine type II collagen (CII). Parameters of immunity included lymphocyte proliferation and cytokine production in vitro in response to antigen stimulation, and the production of anticollagen IgG antibody subclasses. RESULTS The 3 highest doses (20, 80, and 320 microg) ameliorated disease severity, whereas the lowest dose (5 microg) aggravated disease. These findings correlated well with antigen-specific T cell proliferation and cytokine and antibody production. T cell proliferation was suppressed by the higher doses of CII, whereas the low dose enhanced T cell proliferation, indicating it primed the T cells. Suppression of T cell proliferation could be overcome by the addition of exogenous interleukin-2 (IL-2) to these cultures. Decreased T cell proliferation was associated with suppression of both Th1 (interferon-gamma [IFNgamma]) and Th2 (IL-4) cytokines and all the subclasses of anticollagen IgG in mice receiving 20, 80, or 320 microg of collagen. Overall, the highest dose of collagen (320 microg) was less effective at suppressing the immune response and disease than the 20-microg or 80-microg doses. There was an increased production of antibodies of all IgG isotypes, and of the Th1-associated cytokines IFNgamma and IL-2, in animals that had received the lowest dose of 5 microg collagen nasally. CONCLUSION Nasal administration of antigens is effective in inducing tolerance and reducing disease severity, but the effects are dose dependent. Low doses can prime the immune system and aggravate disease; high doses may not suppress disease. Suppression of the immune response, which correlates with suppression of disease, is not obviously associated with a type I to type II T cell switch, but rather with an overall suppression of both forms of T cell response, with a potential role for anergy of T cells in this process.

Phorbol ester induction of early response gene expression in lymphocytic leukemia and normal human B-cells

The spectrum of early response genes induced following phorbol ester (phorbol 12-myristate 13-acetate-PMA) stimulation of B-chronic lymphocytic leukemia cells was compared with that in normal tonsillar B-cells by using a panel of 20 gene probes. Of these, 2 (fos-B and Fra-1) were not expressed in either cell type; 11 displayed a comparable pattern and magnitude of induction in both the cell types, and one anonymous gene (5L3) that was inducible in leukemic cells was not detectably expressed in normal B-cells. Four further anonymous cDNAs (1R21, 10A, 1R19 and 3L11) detected transcripts that were constitutively expressed in normal B-cells with a concomitant reduction in inducibility compared with leukemic B-cells, whilst jun-B and jun-D, which were both inducible in normal B-cells, were constitutively expressed and only marginally PMA-inducible in leukemic B-cells. These data demonstrate clear differences in the PMA-activated early response gene regulatory pathways between normal and lymphocytic leukemia B-cells, which may reflect perturbations in signal transduction pathways that manifest in the differentiation arrest characteristic of these malignant B-cells.