Jason M. Elinoff

A Generic Approach to Pathological Lung Segmentation

In this study, we propose a novel pathological lung segmentation method that takes into account neighbor prior constraints and a novel pathology recognition system. Our proposed framework has two stages; during stage one, we adapted the fuzzy connectedness (FC) image segmentation algorithm to perform initial lung parenchyma extraction. In parallel, we estimate the lung volume using rib-cage information without explicitly delineating lungs. This rudimentary, but intelligent lung volume estimation system allows comparison of volume differences between rib cage and FC based lung volume measurements. Significant volume difference indicates the presence of pathology, which invokes the second stage of the proposed framework for the refinement of segmented lung. In stage two, texture-based features are utilized to detect abnormal imaging patterns (consolidations, ground glass, interstitial thickening, tree-inbud, honeycombing, nodules, and micro-nodules) that might have been missed during the first stage of the algorithm. This refinement stage is further completed by a novel neighboring anatomy-guided segmentation approach to include abnormalities with weak textures, and pleura regions. We evaluated the accuracy and efficiency of the proposed method on more than 400 CT scans with the presence of a wide spectrum of abnormalities. To our best of knowledge, this is the first study to evaluate all abnormal imaging patterns in a single segmentation framework. The quantitative results show that our pathological lung segmentation method improves on current standards because of its high sensitivity and specificity and may have considerable potential to enhance the performance of routine clinical tasks.

Accelerated Metabolism of Voriconazole and Its Partial Reversal by Cimetidine

ABSTRACT We report a case of accelerated metabolism of voriconazole during therapy for invasive pulmonary aspergillosis, resulting in subtherapeutic levels. Target voriconazole levels were restored with high dosages of voriconazole (up to 40 mg/kg of body weight/day) and the addition of cimetidine as a cytochrome P450 enzyme inhibitor.

G Protein-coupled Receptor 40 (GPR40) and Peroxisome Proliferator-activated Receptor γ (PPARγ): AN INTEGRATED TWO-RECEPTOR SIGNALING PATHWAY*

Background: PPARγ ligands are used to treat type 2 diabetes mellitus, but signaling by these drugs is incompletely understood. Results: Rosiglitazone activation of GPR40 markedly enhanced PPARγ-dependent transcription through downstream effects on p38 MAPK, PGC1α, and EP300. Conclusion: GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway. Significance: Future drug development should consider the effects of prospective ligands at both receptors. Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPARγ signaling remains incomplete. In addition to PPARγ, these drugs also activate G protein-coupled receptor 40 (GPR40), a Gαq-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPARγ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPARγ-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPARγ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-Gαq mutant likewise blocked activation of both p38 MAPK and PPARγ reporters. Importantly, RGZ induction of PPARγ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPARγ signal transduction was dependent on p38 MAPK activation and induction of PPARγ co-activator-1 (PGC1α). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1α in PAECs. Knockdown of PGC1α, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPARγ-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPARγ target gene transcription. Thus, GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development.

A pilot study of the effect of spironolactone therapy on exercise capacity and endothelial dysfunction in pulmonary arterial hypertension: study protocol for a randomized controlled trial

BackgroundPulmonary arterial hypertension is a rare disorder associated with poor survival. Endothelial dysfunction plays a central role in the pathogenesis and progression of pulmonary arterial hypertension. Inflammation appears to drive this dysfunctional endothelial phenotype, propagating cycles of injury and repair in genetically susceptible patients with idiopathic and disease-associated pulmonary arterial hypertension. Therapy targeting pulmonary vascular inflammation to interrupt cycles of injury and repair and thereby delay or prevent right ventricular failure and death has not been tested. Spironolactone, a mineralocorticoid and androgen receptor antagonist, has been shown to improve endothelial function and reduce inflammation. Current management of patients with pulmonary arterial hypertension and symptoms of right heart failure includes use of mineralocorticoid receptor antagonists for their diuretic and natriuretic effects. We hypothesize that initiating spironolactone therapy at an earlier stage of disease in patients with pulmonary arterial hypertension could provide additional benefits through anti-inflammatory effects and improvements in pulmonary vascular function.Methods/DesignSeventy patients with pulmonary arterial hypertension without clinical evidence of right ventricular failure will be enrolled in a randomized, double-blinded, placebo-controlled trial to investigate the effect of early treatment with spironolactone on exercise capacity, clinical worsening and vascular inflammation in vivo. Our primary endpoint is change in placebo-corrected 6-minute walk distance at 24 weeks and the incidence of clinical worsening in the spironolactone group compared to placebo. At a two-sided alpha level of 0.05, we will have at least 84% power to detect an effect size (group mean difference divided by standard deviation) of 0.9 for the difference in the change of 6-minute walk distance from baseline between the two groups. Secondary endpoints include the effect of spironolactone on the change in placebo-corrected maximal oxygen consumption; plasma markers of vascular inflammation and peripheral blood mononuclear cell gene expression profiles; sympathetic nervous system activation, renin-angiotensin-aldosterone system activation and sex hormone metabolism; and right ventricular structure and function using echocardiography and novel high-resolution magnetic resonance imaging-based techniques. Safety and tolerability of spironolactone will be assessed with periodic monitoring for hyperkalemia and renal insufficiency as well as the incidence of drug discontinuation for untoward effects.Trial registrationClinicalTrials.gov: NCT01712620

Late multiple organ surge in interferon-regulated target genes characterizes staphylococcal enterotoxin B lethality.

Background Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB) challenge was investigated in six tissues. Results The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC), spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN)/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN)-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature. Conclusion Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes.

Recombinant Human Factor VIIa for Alveolar Hemorrhage Following Allogeneic Stem Cell Transplantation

The mortality rate of alveolar hemorrhage (AH) after allogeneic hematopoietic stem cell transplantation is greater than 60% with supportive care and high-dose steroid therapy. We performed a retrospective cohort analysis to assess the benefits and risks of recombinant human factor VIIa (rFVIIa) as a therapeutic adjunct for AH. Between 2005 and 2012, 57 episodes of AH occurred in 37 patients. Fourteen episodes (in 14 patients) were treated with steroids alone, and 43 episodes (in 23 patients) were treated with steroids and rFVIIa. The median steroid dose was 1.9 mg/kg/d (interquartile range [IQR], 0.8 to 3.5 mg/kg/d; methylprednisolone equivalents) and did not differ statistically between the 2 groups. The median rFVIIa dose was 41 μg/kg (IQR, 39 to 62 μg/kg), and a median of 3 doses (IQR, 2 to 17) was administered per episode. Concurrent infection was diagnosed in 65% of the episodes. Patients had moderately severe hypoxia (median PaO2/FiO2, 193 [IQR, 141 to 262]); 72% required mechanical ventilation, and 42% survived to extubation. The addition of rFVIIa did not alter time to resolution of AH (P = .50), duration of mechanical ventilation (P = .89), duration of oxygen supplementation (P = .55), or hospital mortality (P = .27). Four possible thrombotic events (9% of 43 episodes) occurred with rFVIIa. rFVIIa in combination with corticosteroids did not confer clear clinical advantages compared with corticosteroids alone. In patients with AH following hematopoietic stem cell transplantation, clinical factors (ie, worsening infection, multiple organ failure, or recrudescence of primary disease) may be more important than the benefit of enhanced hemostasis from rFVIIa.

Raf/ERK drives the proliferative and invasive phenotype of BMPR2-silenced pulmonary artery endothelial cells

A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.

Mineralocorticoid Receptor (MR) trans-Activation of Inflammatory AP-1 Signaling: DEPENDENCE ON DNA SEQUENCE, MR CONFORMATION, AND AP-1 FAMILY MEMBER EXPRESSION.

Glucocorticoids are commonly used to treat inflammatory disorders. The glucocorticoid receptor (GR) can tether to inflammatory transcription factor complexes, such as NFκB and AP-1, and trans-repress the transcription of cytokines, chemokines, and adhesion molecules. In contrast, aldosterone and the mineralocorticoid receptor (MR) primarily promote cardiovascular inflammation by incompletely understood mechanisms. Although MR has been shown to weakly repress NFκB, its role in modulating AP-1 has not been established. Here, the effects of GR and MR on NFκB and AP-1 signaling were directly compared using a variety of ligands, two different AP-1 consensus sequences, GR and MR DNA-binding domain mutants, and siRNA knockdown or overexpression of core AP-1 family members. Both GR and MR repressed an NFκB reporter without influencing p65 or p50 binding to DNA. Likewise, neither GR nor MR affected AP-1 binding, but repression or activation of AP-1 reporters occurred in a ligand-, AP-1 consensus sequence-, and AP-1 family member-specific manner. Notably, aldosterone interactions with both GR and MR demonstrated a potential to activate AP-1. DNA-binding domain mutations that eliminated the ability of GR and MR to cis-activate a hormone response element-driven reporter variably affected the strength and polarity of these responses. Importantly, MR modulation of NFκB and AP-1 signaling was consistent with a trans-mechanism, and AP-1 effects were confirmed for specific gene targets in primary human cells. Steroid nuclear receptor trans-effects on inflammatory signaling are context-dependent and influenced by nuclear receptor conformation, DNA sequence, and the expression of heterologous binding partners. Aldosterone activation of AP-1 may contribute to its proinflammatory effects in the vasculature.

The tumor lysis syndrome.

TO THE EDITOR: We recently admitted a patient to our intensive care unit with methemoglobinemia and severe hemolytic anemia after he received a single dose of rasburicase. The patient was 55-year-old black man with chronic lymphocytic leukemia in whom the tumor lysis syndrome developed after rituximab and bendamustine treatment despite saline and allopurinol prophylaxis. Within 6 hours after receiving rasburicase at dose of 0.2 mg per kilogram of body weight, he became hypoxic, with a methemoglobin concentration of 12.2% (Table 1). He subsequently had acute intravascular hemolysis, with the hemoglobin level decreasing from 13.1 to 4.5 g per deciliter, the lactate dehydrogenase level increasing from 158 to 1229 U per liter, and the haptoglobin level decreasing from 130 to 10 mg per deciliter. An elevated plasma oxyhemoglobin level (30.9 mg per deciliter [4.8 μmol per liter]; reference range, 0.0 to 12.4 mg per deciliter [0.0 to 1.9 μmol per liter]) was accompanied by acute pulmonary hypertension (tricuspid regurgitant jet velocity of 3.5 m per second; reference range, 1.7 to 2.8).1,2 He was found to have a glucose-6-phosphate dehydrogenase (G6PD) deficiency. Rasburicase causes oxidative stress by releasing hydrogen peroxide during the conversion of uric acid to allantoin.3 Although not specifically mentioned in the review by Howard et al. (May 12 issue),4 the Food and Drug Administration recommends that patients from populations where G6PD deficiency is common undergo testing before treatment with rasburicase.5 Table 1 Biochemical Evidence of Methemoglobinemia and Intravascular Hemolysis after Rasburicase Administration.*The Authors Reply: The false negative rate of sentinel-lymph-node biopsy has been variably defined with the use of two approaches. In early studies, complete lymph-node dissection was performed immediately after sentinel-lymph-node biopsy to identify microscopic metastases in “nonsentinel lymph nodes” within the same basin; with the use of these data, an “immediate false negative rate” was determined. An alternative approach is based on later identification of nodal metastases after sentinel-node biopsy following an interval of observation (the “delayed false negative rate”). The letters by Thomas and by Nieweg and Veenstra suggest that this latter assessment better reflects the accuracy of sentinel-lymph-node biopsy and that the former approach (described in the article) paints a more favorable picture; we submit that the first method, which reflects sentinel-node status at the time of the procedure, is most relevant. Sources of nodal failure after a negative sentinel-lymph-node biopsy may be classified as technical (there was failure to remove a sentinel node containing disease), pathological (a sentinel node containing metastasis was removed, but the metastasis was not detected by means of routine histologic techniques), or biologic (there was synchronous microscopic in-transit disease or subsequent metastasis from a clinical locoregional recurrence).1 Pathological failure, which probably represents the bulk of nodal failures in many early studies, has been largely resolved by the use of enhanced pathological analysis. The relatively high “false negative” rates cited by Thomas probably reflect such early studies. The concept of microscopic “false positivity” is promulgated by Thomas and by de Giorgi et al. to explain why patients with positive sentinelnode-biopsy specimens fare better than those in whom palpable nodal disease develops and why the comparison of such cohorts in the MSLT-1 trial should be invalid.2 Data providing support for this concept, however, are limited. In contrast, data from MSLT-1 provide support for the clinical relevance of microscopic disease. Specifically, among patients randomly assigned to nodal observation, the incidence of clinical nodal failure and distribution of relevant prognostic factors for primary tumors were identical to the incidence and distribution in the combined group of patients with positive sentinel-lymph-node biopsy specimens and those patients with negative sentinel-lymph-node biopsy specimens in whom nodal disease subsequently developed.2,3

Spironolactone-induced Degradation of the TFIIH Core Complex XPB Subunit Suppresses NF-κB and AP-1 Signaling.

Aims Spironolactone (SPL) improves endothelial dysfunction and survival in heart failure. Immune modulation, including poorly understood mineralocorticoid receptor (MR)-independent effects of SPL might contribute to these benefits and possibly be useful in other inflammatory cardiovascular diseases such as pulmonary arterial hypertension. Methods and results Using human embryonic kidney cells (HEK 293) expressing specific nuclear receptors, SPL suppressed NF-κB and AP-1 reporter activity independent of MR and other recognized nuclear receptor partners. NF-κB and AP-1 DNA binding were not affected by SPL and protein synthesis blockade did not interfere with SPL-induced suppression of inflammatory signalling. In contrast, proteasome blockade to inhibit degradation of xeroderma pigmentosum group B complementing protein (XPB), a subunit of the general transcription factor TFIIH, or XPB overexpression both prevented SPL-mediated suppression of inflammation. Similar to HEK 293 cells, a proteasome inhibitor blocked XPB loss and SPL suppression of AP-1 induced target genes in human pulmonary artery endothelial cells (PAECs). Unlike SPL, eplerenone (EPL) did not cause XPB degradation and failed to similarly suppress inflammatory signalling. SPL combined with siRNA XPB knockdown further reduced XPB protein levels and had the greatest effect on PAEC inflammatory gene transcription. Using chromatin-immunoprecipitation, PAEC target gene susceptibility to SPL was associated with low basal RNA polymerase II (RNAPII) occupancy and TNFα-induced RNAPII and XPB recruitment. XP patient-derived fibroblasts carrying an N-terminal but not C-terminal XPB mutations were insensitive to both SPL-mediated XPB degradation and TNFα-induced target gene suppression. Importantly, SPL treatment decreased whole lung XPB protein levels in a monocrotaline rat model of pulmonary hypertension and reduced inflammatory markers in an observational cohort of PAH patients. Conclusion SPL has important anti-inflammatory effects independent of aldosterone and MR, not shared with EPL. Drug-induced, proteasome-dependent XPB degradation may be a useful therapeutic approach in cardiovascular diseases driven by inflammation.