Recognition of granulocyte-macrophage colony-stimulating factor by specific S100 proteins.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic myelopoietic growth factor and proinflammatory cytokine, clinically used for multiple indications and serving as a promising target for treatment of many disorders, including cancer, multiple sclerosis, rheumatoid arthritis, psoriasis, asthma, COVID-19. We have previously shown that dimeric Ca2+-bound forms of S100A6 and S100P proteins, members of the multifunctional S100 protein family, are specific to GM-CSF. To probe selectivity of these interactions, the affinity of recombinant human GM-CSF to dimeric Ca2+-loaded forms of 18 recombinant human S100 proteins was studied by surface plasmon resonance spectroscopy. Of them, only S100A4 protein specifically binds to GM-CSF with equilibrium dissociation constant, Kd, values of 0.3-2 µM, as confirmed by intrinsic fluorescence and chemical crosslinking data. Calcium removal prevents S100A4 binding to GM-CSF, whereas monomerization of S100A4/A6/P proteins disrupts S100A4/A6 interaction with GM-CSF and induces a slight decrease in S100P affinity for GM-CSF. Structural modelling indicates the presence in the GM-CSF molecule of a conserved S100A4/A6/P-binding site, consisting of the residues from its termini, helices I and III, some of which are involved in the interaction with GM-CSF receptors. The predicted involvement of the 'hinge' region and F89 residue of S100P in GM-CSF recognition was confirmed by mutagenesis. Examination of S100A4/A6/P ability to affect GM-CSF signaling showed that S100A4/A6 inhibit GM-CSF-induced suppression of viability of monocytic THP-1 cells. The ability of the S100 proteins to modulate GM-CSF activity is relevant to progression of various neoplasms and other diseases, according to bioinformatics analysis. The direct regulation of GM-CSF signaling by extracellular forms of the S100 proteins should be taken into account in the clinical use of GM-CSF and development of the therapeutic interventions targeting GM-CSF or its receptors.

Severe Hypocalcemia With Denosumab Among Older Female Dialysis-Dependent Patients.

Importance: Dialysis-dependent patients experience high rates of morbidity from fractures, yet little evidence is available on optimal treatment strategies. Chronic kidney disease-mineral and bone disorder is nearly universal in dialysis-dependent patients, complicating diagnosis and treatment of skeletal fragility. Objective: To examine the incidence and comparative risk of severe hypocalcemia with denosumab compared with oral bisphosphonates among dialysis-dependent patients treated for osteoporosis. Design, Setting, and Participants: Retrospective cohort study of female dialysis-dependent Medicare patients aged 65 years or older who initiated treatment with denosumab or oral bisphosphonates from 2013 to 2020. Clinical performance measures including monthly serum calcium were obtained through linkage to the Consolidated Renal Operations in a Web-Enabled Network database. Exposures: Denosumab, 60 mg, or oral bisphosphonates. Main Outcomes and Measures: Severe hypocalcemia was defined as total albumin-corrected serum calcium below 7.5 mg/dL (1.88 mmol/L) or a primary hospital or emergency department hypocalcemia diagnosis (emergent care). Very severe hypocalcemia (serum calcium below 6.5 mg/dL [1.63 mmol/L] or emergent care) was also assessed. Inverse probability of treatment-weighted cumulative incidence, weighted risk differences, and weighted risk ratios were calculated during the first 12 treatment weeks. Results: In the unweighted cohorts, 607 of 1523 denosumab-treated patients and 23 of 1281 oral bisphosphonate-treated patients developed severe hypocalcemia. The 12-week weighted cumulative incidence of severe hypocalcemia was 41.1% with denosumab vs 2.0% with oral bisphosphonates (weighted risk difference, 39.1% [95% CI, 36.3%-41.9%]; weighted risk ratio, 20.7 [95% CI, 13.2-41.2]). The 12-week weighted cumulative incidence of very severe hypocalcemia was also increased with denosumab (10.9%) vs oral bisphosphonates (0.4%) (weighted risk difference, 10.5% [95% CI, 8.8%-12.0%]; weighted risk ratio, 26.4 [95% CI, 9.7-449.5]). Conclusions and Relevance: Denosumab was associated with a markedly higher incidence of severe and very severe hypocalcemia in female dialysis-dependent patients aged 65 years or older compared with oral bisphosphonates. Given the complexity of diagnosing the underlying bone pathophysiology in dialysis-dependent patients, the high risk posed by denosumab in this population, and the complex strategies required to monitor and treat severe hypocalcemia, denosumab should be administered after careful patient selection and with plans for frequent monitoring.

Specific S100 Proteins Bind Tumor Necrosis Factor and Inhibit Its Activity.

Tumor necrosis factor (TNF) inhibitors (anti-TNFs) represent a cornerstone of the treatment of various immune-mediated inflammatory diseases and are among the most commercially successful therapeutic agents. Knowledge of TNF binding partners is critical for identification of the factors able to affect clinical efficacy of the anti-TNFs. Here, we report that among eighteen representatives of the multifunctional S100 protein family, only S100A11, S100A12 and S100A13 interact with the soluble form of TNF (sTNF) in vitro. The lowest equilibrium dissociation constants (Kd) for the complexes with monomeric sTNF determined using surface plasmon resonance spectroscopy range from 2 nM to 28 nM. The apparent Kd values for the complexes of multimeric sTNF with S100A11/A12 estimated from fluorimetric titrations are 0.1-0.3 µM. S100A12/A13 suppress the cytotoxic activity of sTNF against Huh-7 cells, as evidenced by the MTT assay. Structural modeling indicates that the sTNF-S100 interactions may interfere with the sTNF recognition by the therapeutic anti-TNFs. Bioinformatics analysis reveals dysregulation of TNF and S100A11/A12/A13 in numerous disorders. Overall, we have shown a novel potential regulatory role of the extracellular forms of specific S100 proteins that may affect the efficacy of anti-TNF treatment in various diseases.

The utility of low-iodine diet in preparation for thyroid cancer therapy with radioactive iodine-A cohort study.

Objective: A low-iodine diet (LID) of <50µ iodine/day is recommended as preparation for radioactive iodine (RAI) therapy in patients with differentiated thyroid cancer (DTC). The 24-h urinary iodine excretion (UIE) is utilized to evaluate the iodine-depleted status. The aim of this study was to test the association between UIE and progression-free survival (PFS). Patients and methods: In total, 70 patients with intermediate- or high-risk DTC, post-total thyroidectomy, adhered to 2 weeks of LID and had UIE measured before RAI therapy. A Cox regression model was performed to study the contribution of UIE to PFS. Results: The study group consisted of 68% (48/70) of women, aged 41.5 [IQR 31.0, 54.0] years, with tumor size 2.8 [IQR 1.8-4.5] cm, and presence of distant metastases in 22.9% (16/70) of patients. Patients were treated with 1-5 RAI dosages with the median cumulative activity of 150 [IQR 102-314] mCi (5.5 [IQR 3.8-11.6] GBq). During the follow-up of 3.7 [IQR 1.5-6.5] years, 21.4% (15/70) of patients had disease progression. The risk of progression was significantly higher in patients with UIE ≥200 µg/day at the time of RAI administration than in those with UIE <200 µg/day (HR 3.35, 95% CI 1.09-10.34, and p = 0.02). However, the multivariate Cox proportional hazards regression analysis adjusted for age, tumor size, and presence of distant metastases suggested that only distant metastases were independently significantly associated with the risk of progression (HR 5.80 (1.17-28.67), p = 0.03). Conclusions: Although UIE ≥200 µg/day might be associated with worse PFS in RAI-treated DTC patients, the presence of distant metastases is a strong independent predictor of progression. Less stringent LID might be sufficient to achieve a UIE of <200 µg/day.

NF-κB-dependent repression of Sox18 transcription factor requires the epigenetic regulators histone deacetylases 1 and 2 in acute lung injury.

In acute lung injury (ALI), the NF-κB-mediated downregulation of Sox18 gene expression leads to the disruption of the pulmonary endothelial barrier. Previous studies have suggested that the action of NF-κB as a transcriptional repressor also requires the action of class I histone deacetylases (HDACs). Thus, the purpose of this study was to investigate and further delineate the mechanism of Sox18 repression during lipopolysaccharide (LPS) induced ALI. Using selective inhibitors and specific siRNA-driven depletion of HDACs 1-3 in human lung microvascular endothelial cells (HLMVEC) we were able to demonstrate a critical role for HDACs 1 and 2 in the LPS-mediated repression of Sox18 gene expression and the loss of endothelial monolayer integrity. Moreover, our data demonstrate that HDAC1 associates with a transcription-repressive complex within the NF-κB-binding site of Sox18 promoter. Further, we were able to show that the selective inhibitor of HDAC1, tacedinaline, significantly reduced the endothelial permeability and injury associated with LPS challenge in the mouse lung. Taken together, our data demonstrate, for the first time, that transcription repressors HDACs 1 and 2 are involved in pathological mechanism of ALI and can be considered as therapeutic targets.

Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells.

OBJECTIVE: NFU1 is a mitochondrial iron-sulfur scaffold protein, involved in iron-sulfur assembly and transfer to complex II and LAS (lipoic acid synthase). Patients with the point mutation NFU1G208C and CRISPR/CAS9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-generated rats develop mitochondrial dysfunction leading to pulmonary arterial hypertension. However, the mechanistic understanding of pulmonary vascular proliferation due to a single mutation in NFU1 remains unresolved. Approach and Results: Quantitative proteomics of isolated mitochondria showed the entire phenotypic transformation of NFU1G206C rats with a disturbed mitochondrial proteomic landscape, involving significant changes in the expression of 208 mitochondrial proteins. The NFU1 mutation deranged the expression pattern of electron transport proteins, resulting in a significant decrease in mitochondrial respiration. Reduced reliance on mitochondrial respiration amplified glycolysis in pulmonary artery smooth muscle cell (PASMC) and activated GPD (glycerol-3-phosphate dehydrogenase), linking glycolysis to oxidative phosphorylation and lipid metabolism. Decreased PDH (pyruvate dehydrogenase) activity due to the lipoic acid shortage is compensated by increased fatty acid metabolism and oxidation. PASMC became dependent on extracellular fatty acid sources due to upregulated transporters such as CD36 (cluster of differentiation 36) and CPT (carnitine palmitoyltransferase)-1. Finally, the NFU1 mutation produced a dysregulated antioxidant system in the mitochondria, leading to increased reactive oxygen species levels. PASMC from NFU1 rats showed apoptosis resistance, increased anaplerosis, and attained a highly proliferative phenotype. Attenuation of mitochondrial reactive oxygen species by mitochondrial-targeted antioxidant significantly decreased PASMC proliferation. CONCLUSIONS: The alteration in iron-sulfur metabolism completely transforms the proteomic landscape of the mitochondria, leading toward metabolic plasticity and redistribution of energy sources to the acquisition of a proliferative phenotype by the PASMC.

Necrosis-Released HMGB1 (High Mobility Group Box 1) in the Progressive Pulmonary Arterial Hypertension Associated With Male Sex.

Damage-associated molecular patterns, such as HMGB1 (high mobility group box 1), play a well-recognized role in the development of pulmonary arterial hypertension (PAH), a progressive fatal disease of the pulmonary vasculature. However, the contribution of the particular type of vascular cells, type of cell death, or the form of released HMGB1 in PAH remains unclear. Moreover, although male patients with PAH show a higher level of circulating HMGB1, its involvement in the severe PAH phenotype reported in males is unknown. In this study, we aimed to investigate the sources and active forms of HMGB1 released from damaged vascular cells and their contribution to the progressive type of PAH in males. Our results showed that HMGB1 is released by either pulmonary artery human endothelial cells or human pulmonary artery smooth muscle cells that underwent necrotic cell death, although only human pulmonary artery smooth muscle cells produce HMGB1 during apoptosis. Moreover, only human pulmonary artery smooth muscle cell death induced a release of dimeric HMGB1, found to be mitochondrial reactive oxygen species dependent, and TLR4 (toll-like receptor 4) activation. The modified Sugen/Hypoxia rat model replicates the human sexual dimorphism in PAH severity (right ventricle systolic pressure in males versus females 54.7±2.3 versus 44.6±2 mm Hg). By using this model, we confirmed that necroptosis and necrosis are the primary sources of circulating HMGB1 in the male rats, although only necrosis increased circulation of HMGB1 dimers. Attenuation of necrosis but not apoptosis or necroptosis prevented TLR4 activation in males and blunted the sex differences in PAH severity. We conclude that necrosis, through the release of HMGB1 dimers, predisposes males to a progressive form of PAH.

Heme induces rapid endothelial barrier dysfunction via the MKK3/p38MAPK axis.

Several studies demonstrate that hemolysis and free heme in circulation cause endothelial barrier dysfunction and are associated with severe pathological conditions such as acute respiratory distress syndrome, acute chest syndrome, and sepsis. However, the precise molecular mechanisms involved in the pathology of heme-induced barrier disruption remain to be elucidated. In this study, we investigated the role of free heme in the endothelial barrier integrity and mechanisms of heme-mediated intracellular signaling of human lung microvascular endothelial cells (HLMVECs). Heme, in a dose-dependent manner, induced a rapid drop in the endothelial barrier integrity of HLMVECs. An investigation into barrier proteins revealed that heme primarily affected the tight junction proteins zona occludens-1, claudin-1, and claudin-5, which were significantly reduced after heme exposure. The p38MAPK/HSP27 pathway, involved in the regulation of endothelial cytoskeleton remodeling, was also significantly altered after heme treatment, both in HLMVECs and mice. By using a knockout (KO) mouse for MKK3, a key regulator of the p38MAPK pathway, we showed that this KO effectively decreased heme-induced endothelial barrier dysfunction. Taken together, our results indicate that targeting the p38MAPK pathway may represent a crucial treatment strategy in alleviating hemolytic diseases.

Sex-specific stress response and HMGB1 release in pulmonary endothelial cells.

Women are known to be associated with a higher susceptibility to pulmonary arterial hypertension (PAH). In contrast, male PAH patients have a worse survival prognosis. In this study, we investigated whether the contribution of sex goes beyond the effects of sex hormones by comparing the ability of isolated male and female pulmonary endothelial cells to respire, proliferate and tolerate the stress. Mouse lung endothelial cells (MLEC) were isolated from the lungs of male and female 3-week old mice. Male MLEC showed an increased basal mitochondrial respiration rate, elevated maximal respiration, a significantly greater level of mitochondrial polarization, and a higher rate of proliferation. Exposure of cells to hypoxia (2% of O2 for 24 hours) induced a strong apoptotic response in female but not male MLEC. In contrast, treatment with mitochondrial respiratory Complex III inhibitor Antimycin A (AA, 50µM) mediated severe necrosis specifically in male MLEC, while female cells again responded primarily by apoptosis. The same effect with female cells responding to the stress by apoptosis and male cells responding by necrosis was confirmed in starved pulmonary endothelial cells isolated from human donors. Elevated necrosis seen in male cells was associated with a significant release of damage-associated alarmin, HMGB1. No stimuli induced a significant elevation of HMGB1 secretion in females. We conclude that male cells appear to be protected against mild stress conditions, such as hypoxia, possibly due to increased mitochondrial respiration. In contrast, they are more sensitive to impaired mitochondrial function, to which they respond by necrotic death. Necrosis in male vascular cells releases a significant amount of HMGB1 that could contribute to the pro-inflammatory phenotype known to be associated with the male gender.

A Novel Risk Stratification System for Thyroid Nodules With Indeterminate Cytology-A Pilot Cohort Study.

Background: Thyroid ultrasound (US), fine needle aspiration biopsy (FNAB), and molecular testing have been widely used to stratify the risk of malignancy in thyroid nodules. The goal of this study was to investigate a novel diagnostic approach for cytologically indeterminate thyroid nodules (ITN) based upon a combination of US features and genetic alterations. Methods: We performed a pilot cohort study of patients with ITN (Bethesda III/IV), who underwent surgical treatment. Based on standardized sonographic patterns established by the American Thyroid Association (ATA), each ITN received an US score (XUS), ranging between 0 and 0.9 according to its risk of thyroid cancer (TC). DNA and RNA were extracted from pathologic material, available for all patients, and subjected to Oncomine™ Comprehensive Assay v2 (OCAv2) next-generation sequencing. Each genetic alteration was annotated based on its strength of association with TC and its sum served as the genomic classifier score (XGC). The total risk score (TRS) was the sum of XUS and XGC. ROC curves were generated to assess the diagnostic accuracy of XUS, XGC, and TRS. Results: The study cohort consisted of 50 patients (39 females and 11 males), aged 47.5 ± 14.8 years. Three patients were excluded due to molecular testing failure. Among the remaining 47 patients, 28 (59.6%) were diagnosed with TC. BRAFV600E was the most common mutation in papillary TC, PAX8-PPARG fusion was present in NIFTP, pathogenic variants of SLX4, ATM, and NRAS were found in Hürthle cell TC and RET mutations in medullary TC. The diagnostic accuracy of XGC and TRS was significantly higher compared with XUS (88 vs. 62.5%, p < 0.001; 85.2 vs. 62.5%, p < 0.001, respectively). However, this increased accuracy was due to significantly better sensitivity (80.7 vs. 34.6%, p < 0.001; 84.6 vs. 34.6%, p < 0.001, respectively) without improved specificity (94.7 vs. 90%, p = 0.55; 85.7 vs. 90%, p = 0.63, respectively). Conclusion: Molecular testing might not be necessary in ITN with high-risk US pattern (XUS = 0.9), as specificity of TC diagnosis based on Xus alone is sufficient and not improved with molecular testing. OCAv2 is useful in guiding the management of ITN with low-to-intermediate risk US features (XUS < 0.9), as it increases the accuracy of TC diagnosis.

Integrin Alpha V in Urine: A Novel Noninvasive Marker for Prostate Cancer Detection.

Prostate cancer (PCa) diagnosis based on patient urine analysis provides non-invasive and promising method as compared to biopsy and a prostate-specific antigen (PSA) test. This study was conceived to investigate whether Integrin alpha V (ITGAV) protein is present in urine and assess the urinary ITGAV diagnostic potential for PCa. MATERIALS AND METHODS: Urinary ITGAV expression was determined by Western blot analysis and quantified by ELISA in urine from men with PCa (n = 47), benign prostate hyperplasia (n = 42) and age-matched controls (n = 22). RESULTS: The level of ITGAV protein was significantly lower in PCa urine samples as compared to those in the control group (p < 0.00001). The decrease of ITGAV in urine was highly predictive of PCa with 91.5% sensitivity, 91.4% specificity, 0.93 area under the ROC curve, and its specificity was better than that of serum PSA. CONCLUSION: Urinary ITGAV provides a novel noninvasive biomarker with high specificity.

Rats with a Human Mutation of NFU1 Develop Pulmonary Hypertension.

NFU1 is a mitochondrial protein that is involved in the biosynthesis of iron-sulfur clusters, and its genetic modification is associated with disorders of mitochondrial energy metabolism. Patients with autosomal-recessive inheritance of the NFU1 mutation G208C have reduced activity of the respiratory chain Complex II and decreased levels of lipoic-acid-dependent enzymes, and develop pulmonary arterial hypertension (PAH) in ∼70% of cases. We investigated whether rats with a human mutation in NFU1 are also predisposed to PAH development. A point mutation in rat NFU1G206C (human G208C) was introduced through CRISPR/Cas9 genome editing. Hemodynamic data, tissue samples, and fresh mitochondria were collected and analyzed. NFU1G206C rats showed increased right ventricular pressure, right ventricular hypertrophy, and high levels of pulmonary artery remodeling. Computed tomography and angiography of the pulmonary vasculature indicated severe angioobliterative changes in NFU1G206C rats. Importantly, the penetrance of the PAH phenotype was found to be more prevalent in females than in males, replicating the established sex difference among patients with PAH. Male and female homozygote rats exhibited decreased expression and activity of mitochondrial Complex II, and markedly decreased pyruvate dehydrogenase activity and lipoate binding. The limited development of PAH in males correlated with the preserved levels of oligomeric NFU1, increased expression of ISCU (an alternative branch of the iron-sulfur assembly system), and increased complex IV activity. Thus, the male sex has additional plasticity to overcome the iron-sulfur cluster deficiency. Our work describes a novel, humanized rat model of NFU1 deficiency that showed mitochondrial dysfunction similar to that observed in patients and developed PAH with the same sex dimorphism.

Austalides V and W, new meroterpenoids from the fungus Aspergillus ustus and their antitumor activities.

Two new austalide meroterpenoids, named austalides V and W (1 and 2), were isolated from the fungus Aspergillus ustus VKM F-4692. Their structures were elucidated by extensive spectroscopic analysis and by comparison with related known compounds. The main structural feature of both compounds is a tetrahydrofuranyl ring (G), a structural fragment, first found in austalides. Austalides V (1) and W (2) were able to inhibit the propagation of prostate and bladder cancer cells; this biologic activity is possibly related to the inhibition of a number of key pathways regulating cell growth and migration.

Inositol monophosphatase 1 as a novel interacting partner of RAGE in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation end products (RAGE) plays an important role in PAH by promoting proliferation of pulmonary vascular cells. RAGE is also known to mediate activation of Akt signaling, although the particular molecular mechanism remains unknown. This study aimed to identify the interacting partner of RAGE that could facilitate RAGE-mediated Akt activation and vascular remodeling in PAH. The progressive angioproliferative PAH was induced in 24 female Sprague-Dawley rats ( n = 8/group) that were randomly assigned to develop PAH for 1, 2, or 5 wk [right ventricle systolic pressure (RVSP) 56.5 ± 3.2, 63.6 ± 1.6, and 111.1 ± 4.5 mmHg, respectively, vs. 22.9 ± 1.1 mmHg in controls]. PAH triggered early and late episodes of apoptosis in rat lungs accompanied by RAGE activation. Mass spectrometry analysis has identified IMPA1 as a novel PAH-specific interacting partner of RAGE. The proximity ligation assay (PLA) confirmed the formation of RAGE/IMPA1 complex in the pulmonary artery wall. Activation of IMPA1 in response to increased glucose 6-phosphate (G6P) is known to play a critical role in inositol synthesis and recycling. Indeed, we confirmed a threefold increase in G6P ( P = 0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), membrane translocation of PI3K, and a threefold increase in membrane Akt levels ( P = 0.02) and Akt phosphorylation. We conclude that the formation of the newly discovered RAGE-IMPA1 complex could be responsible for the stimulation of inositol pathways and activation of Akt signaling in PAH.

Adipose-derived mesenchymal stromal/stem cells in systemic sclerosis: Alterations in function and beneficial effect on lung fibrosis are regulated by caveolin-1.

The potential value of mesenchymal stromal/stem cell therapy in treating skin fibrosis in scleroderma (systemic sclerosis) and of the caveolin-1 scaffolding domain peptide in treating lung, skin, and heart fibrosis is known. To understand how these observations may relate to differences between mesenchymal stromal/stem cells from healthy subjects and subjects with fibrosis, we have characterized the fibrogenic and adipogenic potential of adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients, from mice with fibrotic lung and skin disease induced by systemic bleomycin treatment, and from healthy controls. Early passage systemic sclerosis adipose-derived mesenchymal stromal/stem cells have a profibrotic/anti-adipogenic phenotype compared to healthy adipose-derived mesenchymal stromal/stem cells (low caveolin-1, high α-smooth muscle actin, high HSP47, low pAKT, low capacity for adipogenic differentiation). This phenotype is mimicked by treating healthy adipose-derived mesenchymal stromal/stem cells with transforming growth factor beta or caveolin-1 small interfering RNA and is reversed in systemic sclerosis adipose-derived mesenchymal stromal/stem cells by treatment with caveolin-1 scaffolding domain peptide, but not scrambled caveolin-1 scaffolding domain peptide. Similar results were obtained with adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients and from bleomycin-treated mice, indicating the central role of caveolin-1 in mesenchymal stromal/stem cell differentiation in fibrotic disease.

The effect of lithium on the progression-free and overall survival in patients with metastatic differentiated thyroid cancer undergoing radioactive iodine therapy.

OBJECTIVE: Pretreatment with lithium (Li) is associated with an increased residence time of radioactive iodine (RAI) in differentiated thyroid cancer (DTC) metastases. There are no data translating this observation into long-term outcomes. The study goal was to compare the efficacy of three methods of preparation for RAI therapy in metastatic DTC-thyroid hormone withdrawal (THW), THW with pretreatment with Li (THW+Li), and recombinant human TSH (rhTSH). DESIGN/PATIENTS/MEASUREMENTS: We performed a cohort study comparing overall survival (OS) and progression-free survival (PFS) between the three groups: THW (n = 52), THW+Li (n = 41) and rhTSH (n = 42). Kaplan-Meier analyses were performed to compare OS and PFS between the groups. Cox proportional hazards regression model with a stepwise variable selection was performed to study the contribution of age, gender, histology, TNM status, a location of distant metastases and RAI dose. RESULTS: During the follow-up of median 5.1 (IQR = 3.0-8.1) years, 52% of patients had disease progression and 12.6% died. Although THW+Li group was characterized by the longest OS (P = 0.007), only age (HR 1.05, CI 1.01-1.09, P = 0.01) and widespread disease (HR 3.8, CI 1.2-11.8, P = 0.02) were found to affect OS in a multivariate model. There was no difference in PFS between the groups (P = 0.47). Presence of distant metastases limited to the lungs only was associated with longer PFS (PFS HR 0.35, CI 0.20-0.60, P = 0.0002). CONCLUSION: The older age is associated with shorter OS, while disease burden affects OS and PFS in patients with metastatic thyroid cancer. The method of preparation for RAI therapy does not affect the outcome.

Hemolysis-induced Lung Vascular Leakage Contributes to the Development of Pulmonary Hypertension.

Although hemolytic anemia-associated pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH) are more common than the prevalence of idiopathic PAH alone, the role of hemolysis in the development of PAH is poorly characterized. We hypothesized that hemolysis independently contributes to PAH pathogenesis via endothelial barrier dysfunction with resulting perivascular edema and inflammation. Plasma samples from patients with and without PAH (both confirmed by right heart catheterization) were used to measure free hemoglobin (Hb) and its correlation with PAH severity. A sugen (50 mg/kg)/hypoxia (3 wk)/normoxia (2 wk) rat model was used to elucidate the role of free Hb/heme pathways in PAH. Human lung microvascular endothelial cells were used to study heme-mediated endothelial barrier effects. Our data indicate that patients with PAH have increased levels of free Hb in plasma that correlate with PAH severity. There is also a significant accumulation of free Hb and depletion of haptoglobin in the rat model. In rats, perivascular edema was observed at early time points concomitant with increased infiltration of inflammatory cells. Heme-induced endothelial permeability in human lung microvascular endothelial cells involved activation of the p38/HSP27 pathway. Indeed, the rat model also exhibited increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling, there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF-1a during the first 2 weeks of PH regardless of hypoxic conditions. Our data suggest that hemolysis may play a significant role in PAH pathobiology.

Adenosine Production by Biomaterial-Supported Mesenchymal Stromal Cells Reduces the Innate Inflammatory Response in Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: During myocardial ischemia/reperfusion (MI/R) injury, there is extensive release of immunogenic metabolites that activate cells of the innate immune system. These include ATP and AMP, which upregulate chemotaxis, migration, and effector function of early infiltrating inflammatory cells. These cells subsequently drive further tissue devitalization. Mesenchymal stromal cells (MSCs) are a potential treatment modality for MI/R because of their powerful anti-inflammatory capabilities; however, the manner in which they regulate the acute inflammatory milieu requires further elucidation. CD73, an ecto-5'-nucleotidase, may be critical in regulating inflammation by converting pro-inflammatory AMP to anti-inflammatory adenosine. We hypothesized that MSC-mediated conversion of AMP into adenosine reduces inflammation in early MI/R, favoring a micro-environment that attenuates excessive innate immune cell activation and facilitates earlier cardiac recovery. METHODS AND RESULTS: Adult rats were subjected to 30 minutes of MI/R injury. MSCs were encapsulated within a hydrogel vehicle and implanted onto the myocardium. A subset of MSCs were pretreated with the CD73 inhibitor, α,ß-methylene adenosine diphosphate, before implantation. Using liquid chromatography/mass spectrometry, we found that MSCs increase myocardial adenosine availability following injury via CD73 activity. MSCs also reduce innate immune cell infiltration as measured by flow cytometry, and hydrogen peroxide formation as measured by Amplex Red assay. These effects were dependent on MSC-mediated CD73 activity. Finally, through echocardiography we found that CD73 activity on MSCs was critical to optimal protection of cardiac function following MI/R injury. CONCLUSIONS: MSC-mediated conversion of AMP to adenosine by CD73 exerts a powerful anti-inflammatory effect critical for cardiac recovery following MI/R injury.

Muc1 deficiency exacerbates pulmonary fibrosis in a mouse model of silicosis.

BACKGROUND: MUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin expressed on the apical surface of lung epithelial cells. However, in the lungs of patients with interstitial lung disease, MUC1 is aberrantly expressed in hyperplastic alveolar type II epithelial (ATII) cells and alveolar macrophages (AM), and elevated levels of extracellular MUC1 are found in bronchoalveolar lavage (BAL) fluid and the serum of these patients. While pro-fibrotic effects of extracellular MUC1 have recently been described in cultured fibroblasts, the contribution of MUC1 to the pathobiology of pulmonary fibrosis is unknown. In this study, we hypothesized that MUC1 deficiency would reduce susceptibility to pulmonary fibrosis in a mouse model of silicosis. METHODS: We employed human MUC1 transgenic mice, Muc1 deficient mice and wild-type mice on C57BL/6 background in these studies. Some mice received a one-time dose of crystalline silica instilled into their oropharynx in order to induce pulmonary fibrosis and assess the effects of Muc1 deficiency on fibrotic and inflammatory responses in the lung. RESULTS: As previously described in other mouse models of pulmonary fibrosis, we found that extracellular MUC1 levels were markedly increased in whole lung tissues, BALF and serum of human MUC1 transgenic mice after silica. We also detected an increase in total MUC1 levels in the lungs of these mice, indicating that production as well as release contributed to elevated levels after lung injury. Immunohistochemical staining revealed that increased MUC1 expression was mostly confined to ATII cells and AMs in areas of fibrotic remodeling, illustrating a pattern similar to the expression of MUC1 in human fibrotic lung tissues. However, contrary to our hypothesis, we found that Muc1 deficiency resulted in a worsening of fibrotic remodeling in the mouse lung as judged by an increase in number of silicotic nodules, an increase in lung collagen deposition and an increase in the severity of pulmonary inflammation. CONCLUSIONS: Altogether, our results indicate that Muc1 has anti-fibrotic properties in the mouse lung and suggest that elevated levels of MUC1 in patients with interstitial lung disease may serve a protective role, which aims to limit the severity of tissue remodeling in the lung.

Pseudomonas aeruginosa increases MUC1 expression in macrophages through the TLR4-p38 pathway.

Alveolar macrophages (AMs) play a critical role in the clearance of Pseudomonas aeruginosa (Pa) from the airways. However, hyper-activation of macrophages can impair bacterial clearance and contribute to morbidity and mortality. MUC1 mucin is a membrane-tethered, high molecular mass glycoprotein expressed on the apical surface of mucosal epithelial cells and some hematopoietic cells, including macrophages, where it counter-regulates inflammation. We recently reported that Pa up-regulates the expression of MUC1 in primary human AMs and THP-1 macrophages, and that increased MUC1 expression in these cells prevents hyper-activation of macrophages that appears to be important for host defense against severe pathology of Pa lung infection. The aims of this study were to elucidate the mechanism by which Pa increases MUC1 expression in macrophages. The results showed that: (a) Pa stimulation of THP-1 macrophages increased MUC1 expression both at transcriptional and protein levels in a dose-dependent manner; (b) Both Pa- and LPS-induced MUC1 expression in THP-1 cells were significantly diminished by an inhibitory peptide of TLR4; and (c) LPS-stimulated MUC1 expression was diminished at both the mRNA and protein levels by an inhibitor of the p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2, JNK, or IKK. We conclude that Pa-stimulated MUC1 expression in THP-1 macrophages is regulated mainly through the TLR4-p38 signaling pathway.