Citations

Background— Preclinical diastolic dysfunction (PDD) has been broadly defined as subjects with left ventricular diastolic dysfunction, without the diagnosis of congestive heart failure (HF) and with normal systolic function. Our objective was to determine the risk factors associated with the progression from PDD (stage B) HF to symptomatic (stage C) HF. Methods and Results— Using the resources of the Rochester Epidemiology Project, all residents of Olmsted County, MN, who underwent echocardiography between January 1, 2004, and December 31, 2005, and had grade 2–4 diastolic dysfunction and ejection fraction ≥50% were identified. Patients with a diagnosis of HF before or within 30 days of the echocardiogram were excluded. Patients were also excluded if they had a diagnosis of atrial fibrillation or severe mitral or aortic valve regurgitation at the time of the echocardiogram. A total of 388 patients met the inclusion criteria. The mean age of the cohort was 67±12 years, with a female (57%) predominance. Prevalence of renal insufficiency (estimated glomerular filtration rate <60 mL/min per 1.73 m 2 ) was 34%. The 3-year cumulative probabilities of development of (stage C) HF, development of atrial fibrillation, cardiac hospitalization, and mortality were 11.6%, 14.5%, 17.7%, and 10.1% respectively. In multivariable Cox proportional hazard regression analysis, we determined that age, renal dysfunction, and right ventricular systolic pressure were independently associated with the development of HF. Conclusions— This population-based study demonstrated that in PDD (stage B) HF, there was a moderate degree of progression to symptomatic (stage C) HF over 3 years, and renal dysfunction was associated with this progression independent of age, sex, hypertension, coronary disease, and ejection fraction.

ABSTRACT To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.

Abstract Maintaining results of successful induction therapy is an important goal in multiple myeloma. Here, members of the International Myeloma Working Group review the relevant data. Thalidomide maintenance therapy after autologous stem cell transplantation improved the quality of response and increased progression-free survival (PFS) significantly in all 6 studies and overall survival (OS) in 3 of them. In elderly patients, 2 trials showed a significant prolongation of PFS, but no improvement in OS. A meta-analysis revealed a significant risk reduction for PFS/event-free survival and death. The role of thalidomide maintenance after melphalan, prednisone, and thalidomide is not well established. Two trials with lenalidomide maintenance treatment after autologous stem cell transplantation and one study after conventional melphalan, prednisone, and lenalidomide induction therapy showed a significant risk reduction for PFS and an increase in OS in one of the transplant trials. Maintenance therapy with single-agent bortezomib or in combination with thalidomide or prednisone has been studied. One trial revealed a significantly increased OS with a bortezomib-based induction and bortezomib maintenance therapy compared with conventional induction and thalidomide maintenance treatment. Maintenance treatment can be associated with significant side effects, and none of the drugs evaluated is approved for maintenance therapy. Treatment decisions for individual patients must balance potential benefits and risks carefully, as a widely agreed-on standard is not established.

The targeting of lysosomal transmembrane (TM) proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of mucolipin‐1 (Mcoln1), the TM protein defective in the autosomal recessive disease, mucolipidosis type IV, was studied by overexpressing full‐length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53‐amino acid C‐terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ∼35‐kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that the co‐expression of full‐length Mcoln1 with kinase‐inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident TM proteins from the Golgi to the lysosomes.

Single cycle reporter viruses that preserve the majority of the HIV-1 genome, long terminal repeat-promoted transcription and Rev-dependent structural protein expression are useful for investigating the viral life cycle. Reporter viruses that encode the viral proteins in cis in this way have been lacking for feline immunodeficiency virus (FIV), where the field has used genetically minimized transfer vectors with viral proteins supplied in trans. Here we report construction and use of a panel of single cycle FIV reporter viruses that express fluorescent protein markers. The viruses can be produced to high titer using human cell transfection and can transduce diverse target cells. To illustrate utility, we tested versions that are (+) and (-) for OrfA, an FIV accessory protein required for replication in primary lymphocytes and previously implicated in down-regulation of the primary FIV entry receptor CD134. We observed CD134 down-regulation after infection with or without OrfA, and equivalent virion production as well. These results suggest a role for FIV proteins besides Env or OrfA in CD134 down-regulation.

AbstractTo identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

AbstractBackgroundA fundamental obstacle to using retroviral‐mediated gene transfer (GT) to treat human diseases is the relatively low transduction levels that have been achieved in clinically relevant human cells. We previously showed that performing GT in utero overcomes this obstacle and results in significant levels of transduction within multiple fetal organs, with different tissues exhibiting optimal transduction at different developmental stages. We undertook the present study aiming to elucidate the mechanism for this age‐dependent transduction, testing the two factors that we hypothesized could be responsible: (i) the proliferative status of the tissue at the time of GT and (ii) the expression level of the amphotropic PiT‐2 receptor.MethodsImmunofluorescence was performed on tissues from sheep of varying developmental stages to assess the proliferative status of the predominant cells within each organ as a function of age. After developing an enzyme‐linked immunosorbent assay (ELISA) and a quantitative reverse transcription chain reaction (qRT‐PCR) assay, we then quantified PiT‐2 expression at the protein and mRNA levels, respectively.ResultsThe results obtained indicate that the proliferative status of organs at the time of fetal GT is not the major determinant governing transduction efficiency. By contrast, our ELISA and qRT‐PCR analyses demonstrated that PiT‐2 mRNA and protein levels vary with gestational age, correlating with the observed differences in transduction efficiency.ConclusionsThe findings of the present study explain the age‐related differences that we previously observed in transduction efficiency after in utero GT. They also suggest it may be possible to achieve relatively selective GT to specific tissues by performing in utero GT when levels of PiT‐2 are maximal in the desired target organ. Copyright © 2012 John Wiley & Sons, Ltd.