Extensive Characterization of Polysorbate 80 Oxidative Degradation Under Stainless Steel Conditions.

Polysorbate-80 (PS-80) is a common surfactant used in biologics formulations. However, the tendency of oxidation to PS-80 when exposed to stainless steel surfaces brings various challenges during manufacturing processes, such as inconsistent shelf-life of PS-80 solutions, which can further impact the biologics and vaccines production. In this work, the root causes of PS-80 oxidation when in contact with stainless steel conditions were thoroughly investigated through the use of various complementary analytical techniques including U/HPLC-CAD, LC-MS, ICP-MS, peroxide assay, and EPR spectroscopy. The analytical tool kit used in this work successfully revealed a PS-80 degradation mechanism from the perspective of PS-80 content, PS-80 profile, iron content, peroxide production, and radical species. The combined datasets reveal that PS-80 oxidative degradation occurs in the presence of histidine and iron in addition to being combined with the hydroperoxides in PS-80 material. The oxidative pathway and potential degradants were identified by LC-MS. The PS-80 profile based on the U/HPLC-CAD assay provided an effective way to identify early-signs of PS-80 degradation. The results from a peroxide assay observed increased hydroperoxide along with PS-80 degradation. EPR spectra confirmed the presence of histidine-related radicals during PS-80 oxidation identifying how histidine is involved in the oxidation. All assays and findings introduced in this work will provide insight into how PS-80 oxidative degradation can be avoided, controlled, or detected. It will also provide valuable evaluations on techniques that can be used to identify PS-80 degradation related events that occur during the manufacturing process.

Dynamic alterations in the lung microbiota in a rat model of lipopolysaccharide-induced acute lung injury.

The lung microbiota have been found to be substantially altered in numerous pulmonary disorders, and crosstalk between the host pathophysiology and lung microbiota plays critical roles in the regulation of disease states. The aim of this study was to investigate dynamic changes in the lung microbiota during different stages of acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Rats receiving an intraperitoneal administration of lipopolysaccharide (LPS) were sacrificed at 12 and 48 h after injection, and the hematological parameters, serum cytokine levels, and histological characteristics of the lung tissue and lung microbiota were assessed. After LPS injection, along with fluctuations of systemic cytokine levels and the onset and regression of pulmonary edema, the diversity, components, and functionalities of the pulmonary microbiota underwent significant dynamic changes. The volatility of the α-diversity indices narrowed after LPS injection, and the indices significantly decreased 48 h later. The abundance of 18 genera and functionality of adenosine triphosphate-binding cassette (ABC) transporters, pentose phosphate, and bacterial chemotaxis pathways were found to significantly differ between specified time points. Several significant correlations between the components and functionalities of the lung microbiota and indicative symptoms of ALI/ARDS were also observed. Brevibacterium was correlated with cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-10, and IL-6 and with hematological percentage of neutrophils (NEU%); Wnt, Notch, and chronic myeloid leukemia signaling pathways were correlated with IL-1ß; mitogen-activated protein kinase (MAPK) signaling pathway-yeast was correlated with IL-10; and the pathways of ascorbate and aldarate metabolism and basal transcription factors were correlated with platelet-related indicators. The correlations between the lung microbiota and indicative symptoms of ALI/ARDS identified in this study support further investigation into the underlying mechanism of host-microbiota interactions during lung injury and repair.

Complete regression of pulmonary squamous carcinoma in IPF following gemcitabine plus cisplatin: a case report and literature review.

BACKGROUND: Lung cancer is one of the most common co-morbid conditions in patients with idiopathic pulmonary fibrosis (IPF) and negatively affects the prognosis of IPF; Current guidelines for the management of IPF do not give a clear statement on how to manage these patients, and traditional chemotherapy for lung cancer had a limited efficiency rate. Here, we present a rare case of primary lung squamous carcinoma in a patient with IPF whose tumor completely regressed following gemcitabine plus cisplatin therapy; the cancer was no longer detectable after 2 years upon follow-up. CASE PRESENTATION: Sixty-seven year-old male patient with IPF was admitted to hospital due to acute onset hemoptysis. In addition to a definite usual interstitial pneumonia (UIP) pattern, a chest CT scan showed a non-enhancing nodular opacity in the right upper lobe and an enhancing nodule in the right lower lobe. Bronchoscopic biopsy of the nodule in the right lower lobe revealed squamous lung cancer. After 2 cycles of chemotherapy with gemcitabine and cisplatin, the tumor in the right lower lobe was no longer detectable after 2 years of follow-up; however, the nodule in the right upper lobe had increased significantly. Finally, Mycobacterium tuberculosis (MTB) was cultured from the bronchoalveolar (BAL) sample submitted at the last evaluation, and the patient was confirmed to have active pulmonary TB. CONCLUSION: We report the first documented case of complete pulmonary squamous carcinoma regression in IPF following gemcitabine plus cisplatin. Traditional chemotherapy is considered inadequate to cause the resulting regression of the tumor. The concomitant active pulmonary tuberculosis possibly underlies the mechanism.

Notch promotes DNMT-mediated hypermethylation of Klotho leads to COPD-related inflammation.

AIM: Klotho expression significantly declines in alveolar macrophages and airway epithelial cells in chronic obstructive pulmonary disease (COPD) patients, and cigarette smoke extract dramatically inhibits the expression and secretion of α-Klotho. This suggests that the silencing of Klotho is the major factor promoting COPD related inflammatory responses. This study aims to investigate the mechanism of Klotho downregulation and its effect on the inflammatory cytokines secretion and cell apoptosis. METHODS: Expression of DNA methyltransferases (DNMTs) and Notch signaling activation were quantified in MH-S and 16HBE cells stimulated with cigarette smoke extract (CSE) solution. Specific inhibitors of DNMTs or Notch pathway were added together with CSE into treated and control cells. Inflammatory cytokines, cell viability and cell death were determined to explore the effect of Klotho on COPD related inflammation. RESULTS: CSE treatment statistically increased the level of DNMTs expression, Klotho promoter methylation, and activated the Notch signaling pathway. Notch signal activation played a critical role in the process of modification of Klotho promoter methylation. The inhibition of DNMTs and Notch pathway rescued Klotho levels and inhibited inflammation and cell apoptosis after CSE treatment. CONCLUSION: Notch-mediated Klotho hypermethylation inhibited Klotho expression, which promoted inflammatory response and cell apoptosis that were associated with the development of COPD.

Dual-Target Binding Ligands with Modulated Pharmacokinetics for Endoradiotherapy of Prostate Cancer.

Prostate-specific membrane antigen (PSMA)-targeted radiotherapy of prostate cancer (PCa) has emerged recently as a promising approach to the treatment of disseminated disease. A small number of ligands have been evaluated in patients, and although early tumor response is encouraging, relapse rate is high and these compounds localize to the parotid, salivary, and lacrimal glands as well as to the kidney, leading to dose-limiting toxicities and adverse events affecting quality of life. We envision that dual-target binding ligands displaying high affinity for PSMA and appropriate affinity for human serum albumin (HSA) may demonstrate a higher therapeutic index and be suitable for treatment of PCa by targeted α-therapy. Methods: Six novel urea-based ligands with varying affinities for PSMA and HSA were synthesized, labeled with 131I, and evaluated by in vitro binding and uptake assays in LNCaP cells. Four compounds were advanced for further evaluation in a preclinical model of PCa. The compounds were compared with MIP-1095, a PSMA ligand currently in clinical evaluation. Results: The compounds demonstrated affinity for PSMA on the order of 4-40 nM and affinity for HSA in the range of 1-53 µM. Compounds with relatively high affinity for HSA (≤2 µM) showed high and sustained blood-pool activity and reduced uptake in the kidneys. 131I-RPS-027, with a 50% inhibitory concentration (PSMA) of 15 nM and a dissociation constant (HSA) of 11.2 µM, cleared from the blood over the course of 48 h and showed good tumor uptake (10 percentage injected dose per gram) and retention and a greater than 5-fold decrease in kidney uptake relative to MIP-1095. The tumor-to-kidney ratio of 131I-RPS-027 was greater than 3:1 at 24 h after injection, increasing to 7:1 by 72 h. Conclusion: RPS-027 shows dual targeting to PSMA and albumin, resulting in a high tumor uptake, highly favorable tissue distribution, and promising therapeutic profile in a preclinical model of prostate cancer. In comparison to existing ligands proposed for targeted therapy of prostate cancer, RPS-027 has tumor-to-tissue ratios that predict a significant reduction in side effects during therapy. Using iodine/radioiodine as a surrogate for the radiohalogen 211At, we therefore propose dual-target binding ligands such as RPS-027 as next-generation radiopharmaceuticals for targeted α-therapy using 211At.

Imbalance between subpopulations of regulatory T cells in COPD.

BACKGROUND: Recent evidence indicates that human regulatory T cells (Tregs) are composed of three distinct subpopulations: CD25(++) CD45RA(+) resting Tregs (rTregs), CD25(+++) CD45RA(-) activated Tregs (aTregs), which are suppressive, and CD25(++) CD45RA(-) cytokine-secreting (Fr III) cells with pro-inflammatory capacity. OBJECTIVES: To evaluate the dynamic changes in circulating and pulmonary Treg subpopulations in smokers and patients with chronic obstructive pulmonary disease (COPD), and to explore their potential roles in COPD pathogenesis. METHODS: Blood samples were obtained from 57 never-smokers, 32 smokers with normal lung function and 66 patients with COPD. Bronchoalveolar lavage (BAL) samples were taken from 12 never-smokers, 12 smokers and 18 patients with COPD. The proportions of Treg subpopulations and activated CD8 T cells were evaluated using flow cytometry. RESULTS: In peripheral blood, increased proportions of rTregs, aTregs and Fr III cells were found in smokers compared with never-smokers, whereas patients with COPD showed decreased rTregs and aTregs, and significantly increased Fr III cells compared with smokers. The changes in Treg subpopulations, with an overall decrease in the (aTreg+rTreg):(Fr III) ratio, indicated that immune homeostasis favoured inflammation and correlated with enhanced CD8 T-cell activation (r=-0.399, p<0.001) and forced expiratory volume in 1 s (FEV1) % predicted value (r=0.435, p<0.001).The BAL (aTreg+rTreg):(Fr III) ratios displayed more robust correlations with FEV1% predicted value (r=0.741, p<0.01) and activation of effector T cells (r=-0.763, p<0.001). CONCLUSIONS: The imbalance between the anti-inflammatory subsets (aTreg+rTreg) and the pro-inflammatory subset (Fr III) of Tregs may play an important role in COPD progression.

[Effect of adenoviral E1A-involved apoptosis of rat alveolar epithelial cells and human lung adenocarcinoma epithelial cell line].

AIM: The relationship between latent adenovirus infection and apoptosis of airway epithelial cell have not been well documented.We want to illustrating the roles of adenovirus E1A protein on the apoptotic alveolar epithelial in response to TNF-alpha. METHODS: The expression vector for expressing adenovirus E1A protein was transfected into CCL149 and A549 cell respectively. Cell stably expressing E1A protein were selected by G418 resistance.All G418-resistant clones were indentified by RT-PCR and immunocytochemistry. The rate of apoptosis were measured by Hoeschest 33 258 and flow cytometry respectively. The apoptotic rate in response to 30 microg/L TNF-alpha was compared between E1A-positive clones and control clones both in A549 and CCL149. RESULTS: The rates of apoptosis were (2.63+/-0.8)%, (25.38+/-0.9)% respectively in E1A-positive CCL149 cell and (0.62+/- 0.3)%, (6.08+/-0.2)% respectively in E1A-negative CCL149 cell. The rates of apoptosis were (2.63+/-0.8)%, (25.38+/-0.9)% respectively in E1A-positive A549 cell and (0.62+/- 0.3)%, (6.08+/-0.2)% respectively in E1A-negative A549 cell. The rate of apoptosis were increased in E1A-positive cells compared with control with or without TNF-alpha stimulation. CONCLUSION: E1A sensitizes cell to TNF-alpha induced apoptosis of A549 cell and CCL149 cell.