Critical Transition in Tissue Homeostasis Accompanies Murine Lung Senescence

BACKGROUND: Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The susceptibility to pulmonary insults is attributed to "low pulmonary reserve", ostensibly reflecting a combination of age-related musculoskeletal, immunologic and intrinsic pulmonary dysfunction. METHODS/PRINCIPAL FINDINGS: Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age. Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4 (toll receptor 4) and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace enlargement during late life stages. CONCLUSION/SIGNIFICANCE: Our findings establish that a tissue-specific aging program is evident during a presenescent interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These signature events, during middle age, indicate that early stages of the aging immune system may have important correlates in the maintenance of tissue morphology. We further show that time-course analyses of aging models, when informed by structural surveys, can reveal nonintuitive signatures of organ-specific aging pathology

Fas-Mediated Apoptosis Regulates the Composition of Peripheral αβ T Cell Repertoire by Constitutively Purging Out Double Negative T Cells

BACKGROUND: The Fas pathway is a major regulator of T cell homeostasis, however, the T cell population that is controlled by the Fas pathway in vivo is poorly defined. Although CD4 and CD8 single positive (SP) T cells are the two major T cell subsets in the periphery of wild type mice, the repertoire of mice bearing loss-of-function mutation in either Fas (lpr mice) or Fas ligand (gld mice) is predominated by CD4(-)CD8(-) double negative alphabeta T cells that also express B220 and generally referred to as B220+DN T cells. Despite extensive analysis, the basis of B220+DN T cell lymphoproliferation remains poorly understood. In this study we re-examined the issue of why T cell lymphoproliferation caused by gld mutation is predominated by B220+DN T cells. METHODOLOGY AND PRINCIPAL FINDINGS: We combined the following approaches to study this question: Gene transcript profiling, BrdU labeling, and apoptosis assays. Our results show that B220+DN T cells are proliferating and dying at exceptionally high rates than SP T cells in the steady state. The high proliferation rate is restricted to B220+DN T cells found in the gut epithelium whereas the high apoptosis rate occurred both in the gut epithelium and periphery. However, only in the periphery, apoptosis of B220+DN T cell is Fas-dependent. When the Fas pathway is genetically impaired, apoptosis of peripheral B220+DN T cells was reduced to a baseline level similar to that of SP T cells. Under these conditions of normalized apoptosis, B220+DN T cells progressively accumulate in the periphery, eventually resulting in B220+DN T cell lymphoproliferation. CONCLUSIONS/SIGNIFICANCE: The Fas pathway plays a critical role in regulating the tissue distribution of DN T cells through targeting and elimination of DN T cells from the periphery in the steady state. The results provide new insight into pathogenesis of DN T cell lymphoproliferation

Comparison of phospholipase D activity in vasopressin- and phorbol ester-stimulated fibroblasts

AbstractPhospholipase D (PLD) activation by vasopressin (VP) was compared to activation by TPA in REF52 cells prelabeled with [3H]glycerol and [14C]myristic acid. Upon VP-treatment, the formation of [3H] and [14c]phosphatidic acid (PA) and phosphatidylethanol (PEt) was accompanied by the loss of radioactivity from PC and PI. However, upon TPA-treatment, radioactivity was lost from PC only. No significant changes of phosphatidylethanolamine and phosphatidylserine were detected in the same samples. The inclusion of 5μM staurosporine for 10 min diminished the production of [3H]PEt and [14C]PEt by 27% and 53% in VP-treated cells, and by 100% and 75% in TPA-treated cells, respectively. Adding 1 mM EGTA to chelate extracellular Ca2+ inhibited [3H]PEt by approximately 31% and [14C]PEt by 17% after VP-stimulation. In contrast, EGTA had no effeet on TPA-stimulation. The data suggest that REF52 cells contain dual PLD activities. The first is stimulated only by VP, requires Ca2+ and hydrolyzes PI. The second is stimulated by both TPA and VP, activated by protein kinase C and hydrolyzes PC

Gender Counts: Defining the Core Genetic Profile of Polycythemia Vera.

Abstract Intra-abdominal venous thrombosis and exuberant extramedullary hematopoiesis leading to painful splenomegaly and hepatomegaly are two of the most serious consequences of polycythemia vera. Although polycythemia vera is slightly more common in men, both of these complications appear to be more common in women, in whom the disease also presents at an earlier age. We previously reported that polycythemia vera peripheral blood (pb) CD34+ cells could be distinguished from their immunophenotypically similar normal counterparts by gene expression profiling. We, therefore, hypothesized that phenotypic differences in disease behavior between men and women with polycythemia vera were a consequence of gender-based genotypic differences and have employed gene expression profiling to examine this issue. For this purpose, cRNA was prepared from the total RNA of pb CD34+ cells of eleven polycythemia vera patients and six normal controls purified by immunomagnetic bead chromatography. The cRNA was hybridized to an Affymetrix HU133 high-density oligonucleotide microarray chip representing 22,000 genes. Approximately 30–45 % of chip genes were recorded as present in the RNA samples. GC-RMA (Robust Multiarray Analysis) was used for normalization, to adjust for probe effects and for signal estimation. A parametric empirical Bayes statistical modeling method was used for differential gene expression analysis between the patients and controls. A posterior probability of &gt;0.5 was taken to indicate significant differential gene expression. As previously reported, all polycythemia vera patients could be unequivocally distinguished from the controls, indicating that gene expression profiling can be employed as diagnostic test for polycythemia vera. With respect to gender, comparing male patients with male controls, 1106 genes were differentially expressed in the patient group; comparing female patients with female controls, 461 genes were differentially expressed in the patient group. Using unsupervised hierarchical clustering, the control patients segregated as a single group while the polycythemia vera patients segregated into two groups based on exuberant extramedullary hematopoiesis but this segregation was not gender-based despite the marked differences in gender-specific gene expression. Importantly, however, after eliminating gender-specific genes, only 93 genes were concordantly expressed in the male and female patients (59 up regulated and 34 down regulated). This small group appears to represent core genes in polycythemia vera whose behavior may be modified by gender-specific genes but the specific up regulation or repression of which is essential for disease expression. These data indicate that gender-specific effects on gene expression must be addressed in order to determine the basic genetic signature of a hematologic malignancy and they also define a core genetic profile in polycythemia vera.</jats:p

Prognosis Is Genetically Fixed In Polycythemia Vera and Definable by Gene Expression Profiling

Abstract Abstract 1987 Polycythemia vera (PV) is a chronic, clonal, stem cell disorder whose natural history is usually indolent but an undefined fraction of patients have a clinical course characterized by extramedullary hematopoiesis, myelofibrosis, massive splenomegaly, bone marrow failure, and a propensity to develop spontaneous acute leukemia or early death. Recently, a prognostic scoring system was developed to determine the survival of such patients after the onset of bone marrow failure (Blood, 2008; 111:3383-7). However, at that point, for most such patients, only palliative therapy is available. We have been conducting a gene expression profiling (GEP) study in PV peripheral blood (pb) CD34+ cells using oligonucleotide microarray technology, confirmed by QPCR, and now report that in patients who develop an aggressive form of PV, the associated genetic abnormalities are time independent, providing an opportunity to identify patients at risk before bone marrow failure ensues. Our observations were obtained in a cross-sectional study of 19 PV patients (8 men and 11 women) from whom pb CD34+ cells were harvested for GEP in comparison to immunophenotypically similar G-CSF mobilized pb CD34+cells from normal individuals (3 men and 3 women). The median age (55 years; range 33–71), disease duration (11.5 years; range 1–33) and JAK2 V617F allele burden (92%: range 50–100) of the men at the time of study were not significantly different from the women (median age 51 years; range 42–70); disease duration, (8 years; range 1–13) and JAK2 V617F allele burden (100%; range 49–100);16 patients were homozygous for the mutation. Gender-specific differences in PV clinical features (Haematologica 2010; 95:1090-7) were mirrored by gender-specific differences in GEP between the men and women patients, allowing us to identify a core set of 89 genes (31 down regulated and 58 up regulated) shared in common. Using this core gene set and unsupervised hierarchical clustering, the 19 PV patients could be segregated into a small homogeneous group (6 patients; 3 men and 3 women) and a larger heterogeneous group(5 men and 8 women), which did not differ with respect to median age (64.5 vs 67 years); median disease duration (10.5; range 5–20, vs 7 years; range 1–33); JAK2 V617F allele burden (100% vs 88%), or leukocyte and platelet counts, but did differ with respect to their clinical characteristics with the smaller group having a lower median hemoglobin level (10.7 gm% vs 13.1 gm%; p= 0.01), a greater number of thrombotic events (p = 0.017), greater spleen size, (p= 0.01), a need for splenectomy (p= 0.017), a need for chemotherapy (p =0.009), and a shorter median survival time (17 years vs 25 years; log rank p value 0.02). Since the median disease duration of the two groups did not differ, the underlying genetic abnormalities must be time independent. Analysis of the core gene set revealed a “stromal gene signature” consisting of 16 matricellular and collagen genes, which, when subjected to unsupervised hierarchical clustering, not only led to the same group distinctions but resolved the genetic heterogeneity of the larger group. Remarkably, the stromal genes were down regulated in the more aggressive group and up regulated in the more indolent one. To determine the overall genetic differences between these two clinically distinct groups, we used Gene Set Enrichment Analysis to identify the top 50 up or down regulated genes in each. Among the notable genes down regulated in the indolent group were BAALC, DACH1, CD44, DDX17 and VIM, and in the aggressive group, FOXO1, IRF8, ABCD1, HOXA5, SOX4 and CRIM1. In the indolent group, LSM1, FADS2, CDKN1A, E2F1, MAZ and FHL2 were up regulated. FHL2 was also the only top 50 up regulated gene shared in common with the aggressive group, and one of the top two most up regulated genes in the patient population as a whole, LSM1 being the other, emphasizing the importance of these genes in disease pathogenesis. Equally important, in the aggressive group, there was an up regulated 17 gene signature involving DNA replication unique to it alone (p = 0.032). These data solidify the contention that despite the commonality of JAK2 V617F, PV is not a monolithic illness, that prognosis is independent of disease duration, provide insight into the pathogenesis of PV and offer the possibility of genetically identifying those patients most at risk of disease-associated morbidity and mortality when they can still benefit from therapeutic intervention. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Molecular cloning and characterization of LKv1, a novel voltage-gated potassium channel in leech

We have cloned a novel voltage-gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage-gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I(K). Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage-gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker-related K channels in jellyfish. Thus, this analysis indicates that this group of voltage-gated K channels contains several evolutionarily divergent lineages