Abstract Expression of leucine-rich α2 glycoprotein (LRG), a member of the leucine-rich repeat family of proteins, was recently shown to be up-regulated during neutrophil differentiation. Its precise role in granulopoiesis, however, remains unknown. In this paper, we show that the transcription factors PU.1 and C/EBPε that regulate the expression of multiple myeloid-specific genes also bind to the LRG promoter. We also demonstrate that LRG localizes to the same cytoplasmic compartment as myeloperoxidase and that G-CSF treatment of the 32Dcl3 myeloid cell line induces nuclear translocation of LRG. Stable transfection of LRG into 32Dcl3 cells resulted in accelerated, G-CSF-mediated neutrophil differentiation and induction of CD11b expression. In contrast, constitutive expression of LRG in 32Dwt18 cells, expressing a chimeric erythropoietin (Epo)/G-CSFR consisting of the EpoR extracellular domain fused to the G-CSFR transmembrane and cytoplasmic domains, failed to induce accelerated neutrophil differentiation and CD11b expression in response to Epo stimulation. LRG-mediated accelerated differentiation and CD11b expression were found to correlate with an increased level of phospho-Stat3 but not with PU.1 or p27kip1 levels. Hence, similar to other genes involved in neutrophil differentiation, the expression of LRG also appears to be regulated by PU.1 and C/EBPε. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and expression of CD11b via nonredundant G-CSFR signals.
High-dose cyclophosphamide (Cy) is a common component of conditioning regimens that patients receive prior to hematopoietic cell transplant (HCT). Due to concerns for increased risk of toxicity, obese patients at Virginia Commonwealth University Health System (VCUHS) receive doses of Cy based on an adjusted body weight (ABW; ABW = [(total body weight [TBW] – ideal body weight [IBW]) x 0.4] + IBW) whereas non-obese patients receive doses based on TBW. However, these practices are inconsistent with the American Society of Clinical Oncology (ASCO) 2012 guidelines, which recommend that obese patients receive full-dose chemotherapy. Interestingly, these guidelines excluded patients with hematological malignancies undergoing HCT from their literature review. Using a retrospective medical record review, we examined whether there is a difference in effectiveness and toxicity between obese and non-obese patients who receive high-dose Cy prior to HCT. The primary objective of this study was to determine if there is a difference in toxicity between obese versus non-obese patients during the first 60 days following transplant. Toxicity was measured as a composite endpoint of overall toxicity, which was comprised of any grade 3 or 4 non-hematological toxicity, the incidence of pulmonary, hepatic, renal, or cardiac toxicity, and each component individually. Secondary objectives were to compare the differences in effectiveness between obese and non-obese patients assessed by relapse at day +100, relapse at one year, death at one year, chimerisms at days +30, +60, and +90, and the incidence of acute graft versus host disease (aGVHD). We also sought to evaluate the difference in time to engraftment and rates of infection between groups. Chi-squared test and Fisher's exact test were used to assess the primary and secondary endpoints and two-sample t-tests were used to assess time to engraftment. Sixty-one patients met the inclusion criteria. Of these, 28 were considered to be obese while 33 were considered to be non-obese. Mean BMI was 24.7 ± 2.5 in non-obese patients and 32.5 ± 4.3 in obese patients. The mean Cy dose in non-obese and obese patients was 56.80 ± 10.4 mg/kg and 48.55 ± 3.3 mg/kg of TBW, respectively. The rate of overall toxicity was greater in the obese patients compared to non-obese patients (82% vs 52%, OR 4.3 [95% CI 1.3-14.1]), which was driven by a significantly greater incidence of renal dysfunction (79% vs 48%, OR 3.9 [95% CI 1.3-12.1]). There were no differences in rates of grade 3 or 4 toxicity, hepatic dysfunction, or any measure of effectiveness between groups. Although obese patients are at increased risk of toxicity, they appear to achieve the same effectiveness as non-obese patients. Since the increased risk of toxicity is due to increases in grade 2 renal toxicity, there is no evidence to support changing the dosing practices at VCUHS.
Mercury has been identified as a risk factor for cardiovascular disease among humans. Through diet, mainly fish consumption, humans are exposed to methylmercury, the biomethylated organic form of environmental mercury. As the endothelium is an important player in homeostasis of the cardiovascular system, here, the authors tested their hypothesis that methylmercury activates the lipid signaling enzyme phospholipase A(2) (PLA(2)) in vascular endothelial cells (ECs), causing upstream regulation of cytotoxicity. To test this hypothesis, the authors used bovine pulmonary artery ECs (BPAECs) cultured in monolayers, following labeling of their membrane phospholipids with [(3)H]arachidonic acid (AA). The cells were exposed to methylmercury chloride (MMC) and then the release of free AA (index of PLA(2) activity) and lactate dehydrogenase (LDH; index of cytotoxicity) were determined by liquid scintillation counting and spectrophotometry, respectively. MMC significantly activated PLA(2) in a dose-dependent (5 to 15 microM) and time-dependent (0 to 60 min) fashion. Sulfhydryl (thiol-protective) agents, calcium chelators, antioxidants, and PLA(2)-specific inhibitors attenuated the MMC-induced PLA(2) activation, suggesting the role of thiols, reactive oxygen species (ROS), and calcium in the activation of PLA(2) in BPAECs. MMC also induced the loss of thiols and increase of lipid peroxidation in BPAECs. MMC induced cytotoxicity in BPAECs as observed by the altered cell morphology and LDH leak, which was significantly attenuated by PLA(2) inhibitors. This study established that PLA(2) activation through thiols, calcium, and oxidative stress was associated with the cytotoxicity of MMC in BPAECs, drawing attention to the involvement of PLA(2) signaling in the methylmercury-induced vascular endothelial dysfunctions.
Abstract TGF-β plays an important role in the genesis and progression of pulmonary fibrosis. We sought to determine the role of mononuclear phagocytes in the activation of TGF-β and found that freshly isolated peripheral blood monocytes spontaneously released TGF-β. Stimulating these monocytes with GM-CSF or LPS, but not MCSF, augmented the activation of TGF-β. In human monocytes, the free thiol compounds DTT and NAC decreased the activity of TGF-β, without affecting TGF-β mRNA transcription. Both NAC and DTT lessened the biological activity of recombinant active TGF-β in a cell-free system. We found that NAC and DTT reduced dimeric active TGF-β from a 25 kDa protein to 12.5 kDa inactive monomer. This conversion was reversed using the oxidizing agent diamide. Diamide also restored biological activity to NAC or DTT-treated TGF-β. Reduction of TGF-β to monomers could competitively inhibit active dimeric TGF-β and block intracellular signaling events. Our observations suggest that modulation of the oxidative state of TGF-β may be a novel therapeutic approach for patients with pulmonary fibrosis.
Activation of both positive and "negative" or anti-proliferative signals has emerged as a common paradigm for regulation of cell growth through cell surface receptors that regulate immune responses. SHP-1 and -2 and the novel 5'-inositol phosphatase SHIP have recently been shown to function as growth inhibitory molecules in immune receptor signaling. In the current study, we have identified distinct regions in the granulocyte colony-stimulating factor receptor (G-CSFR) distal to the conserved box 2 motif necessary for mitogenesis, which exert positive and negative influences on growth signaling in Ba/F3 pro-B lymphoid cells. The region spanning amino acids 682 to 715 mediates activation of phosphatidylinositol 3'(PI3)-kinase. Activation of PI3-kinase leads to inhibition of apoptosis, promotion of cell survival, and enhanced proliferative responses to G-CSF. We show that the region of 98 amino acids in the distal tail of the class I G-CSFR down-modulates proliferative signaling, not only in myeloid cell lines, as previously reported, but also in Ba/F3 cells. This same region recruits SHIP to the signaling cascade through a mechanism involving Shc, with the formation of Shc/SHIP complexes. Our data suggest a model in which PI3-kinase and SHIP coordinately regulate growth signaling through the G-CSFR.
Top risk factors • Age between the onset of menarche and prior to menopause, early age at menarche, and short menstrual cycle length are cited as the top risk factors for endometriosis.
Women are at high risk of dying from unrecognized cardiovascular disease. Many differences in cardiovascular disease between men and women appear to be mediated by vascular smooth muscle cells (SMC). Because estrogen reduces the proliferation of SMC, we hypothesized that activation of estrogen receptor-alpha (ERalpha) by agonists or by growth factors altered SMC function. To determine the effect of growth factors, estrogen, and ERalpha expression on SMC differentiation, human aortic SMC were cultured in serum-free conditions for 10 days. SMC from men had lower spontaneous expression of ERalpha and higher levels of the differentiation markers calponin and smooth muscle alpha-actin than SMC from women. When SMC containing low expression of ERalpha were transduced with a lentivirus containing ERalpha, activation of the receptor by ligands or growth factors reduced differentiation markers. Conversely, inhibiting ERalpha expression by small interfering RNA (siRNA) in cells expressing high levels of ERalpha enhanced the expression of differentiation markers. ERalpha expression and activation reduced the phosphorylation of Smad2, a signaling molecule important in differentiation of SMC and initiated cell death through cleavage of caspase-3. We conclude that ERalpha activation switched SMC to a dedifferentiated phenotype and may contribute to plaque instability.
