Oceanobacillus picturae alleviates cadmium stress and promotes growth in soybean seedlings.

Cadmium (Cd) is a heavy metal that significantly impacts human health and the environment. Microorganisms play a crucial role in reducing heavy metal stress in plants; however, the mechanisms by which microorganisms enhance plant tolerance to Cd stress and the interplay between plants and microorganisms under such stress remain unclear. In this study, Oceanobacillus picturae (O. picturae) was isolated for interaction with soybean seedlings under Cd stress. Results indicated that Cd treatment alone markedly inhibited soybean seedling growth. Conversely, inoculation with O. picturae significantly improved growth indices such as plant height, root length, and fresh weight, while also promoting recovery in soil physiological indicators and pH. Metabolomic and transcriptomic analyses identified 157 genes related to aspartic acid, cysteine, and flavonoid biosynthesis pathways. Sixty-three microbial species were significantly associated with metabolites in these pathways, including pathogenic, adversity-resistant, and bioconductive bacteria. This research experimentally demonstrates, for the first time, the growth-promoting effect of the O. picturae strain on soybean seedlings under non-stress conditions. It also highlights its role in enhancing root growth and reducing Cd accumulation in the roots under Cd stress. Additionally, through the utilization of untargeted metabolomics, metagenomics, and transcriptomics for a multi-omics analysis, we investigated the impact of O. picturae on the soil microbiome and its correlation with differential gene expression in plants. This innovative approach unveils the molecular mechanisms underlying O. picturae's promotion of root growth and adaptation to Cd stress.

A facile electrochemical immunosensor based on EDTA-Pb2+ complexation reaction.

The sensitivity of electrochemical (EC) sensors has been improved through the development of multiple approaches. However, the majority of EC sensors were limited in their practical application by high costs or tedious procedures. Herein, based on ethylenediaminetetraacetic acid (EDTA)-Pb2+ complexation reaction, a facile and affordable immunosensor was designed. Pb2+-magnesium silicate hydrate was served as the sensing substrate. The immunorecognition process was carried out in the Eppendorf tube, and antibody-functionalized Pb2+-polydopamine was utilized as immunoprobe. In the tube, the quantitative and appropriate excess of EDTA was introduced to complex with Pb2+ on the immunoprobes. The remaining EDTA was added to the sensing substrate surface to coordinate with some Pb2+ in it. This leaded to the reduction of the EC signal of Pb2+, which was related to the antigen concentration. Using prostate-specific antigen as the model analyte, the sensitive detection was realized with a low limit of detection (30.49 fg mL-1). Remarkably, the assay results were available within 24 min, sensibly faster than the most existing EC sensors.

Hydrogen sulfide inhibits alveolar type II cell senescence and limits pulmonary fibrosis via promoting MDM2-mediated p53 degradation.

AIM: Senescence of alveolar type II (AT2) cells is an important driver of pulmonary fibrosis. This study aimed to investigate whether and how dysregulation of hydrogen sulfide (H2 S) production affected AT2 cell senescence, and then explored the effect of H2 S on the communication between AT2 and fibroblasts. METHODS: ICR mice were intratracheally administered with bleomycin (3 mg/kg). Sodium hydrosulfide (NaHS, 28 µmol/kg/d) was intraperitoneally injected for 2 weeks. The H2 S-generating enzyme cystathionine-ß-synthase (CBS) knockout heterozygous (CBS+/- ) mice were used as a low H2 S production model. RESULTS: Analysis of microarray datasets revealed downregulation of H2 S-generating enzymes in lung tissues of patients with pulmonary fibrosis. Decreased H2 S production was correlated with higher levels of cell senescence markers p53 and p21 in bleomycin-induced lung fibrosis. CBS+/- mice exhibited increased levels of p53 and p21. The numbers of AT2 cells positive for p53 and p21 were increased in CBS+/- mice as compared to control mice. H2 S donor NaHS attenuated bleomycin-induced AT2 cell senescence both in vivo and in vitro. H2 S donor suppressed bleomycin-induced senescence-associated secretory phenotype (SASP) of AT2 cells via inhibiting p53/p21 pathway, consequently suppressing proliferation and myofibroblast transdifferentiation of fibroblasts. Mechanically, H2 S suppressed p53 expression by enhancing the mouse double-minute 2 homologue (MDM2)-mediated ubiquitination and degradation of p53. CONCLUSION: H2 S inactivated p53-p21 pathway, consequently suppressing AT2 cell senescence as well as cell communication between senescent AT2 cells and fibroblasts. Aberrant H2 S synthesis may contribute to the development of pulmonary fibrosis through promoting the activation loop involving senescent AT2 cells and activated fibroblasts.

A comparison of pulsed radiofrequency and radiofrequency denervation for lumbar facet joint pain.

BACKGROUND: Lumbar facet joint pain is a common disorder. The main symptom is chronic lumbar pain, which can reduce quality of life. Radiofrequency has often been used to treat lumbar facet joint pain. However, the effectiveness of this technique has been controversial. This study was conducted to compare the effectiveness of pulsed radiofrequency (PRF) and radiofrequency denervation (RD) for lumbar facet joint pain. METHODS: One hundred and forty-two patients with lumbar facet joint pain were allocated to two treatment groups: PRF group (N = 72) and RD group (N = 70). Patients enrolled in the study were assessed using a visual analogue scale (VAS), Roland-Morris questionnaire (RMQ), Oswestry disability index (ODI) and Short-Form 36 (SF-36) questionnaire before therapy, 3 months and 12 months later. RESULTS: There were no significant differences in VAS, RMQ score, ODI score and SF-36 score at 3 months (p > 0.05). Significant differences in pain control were observed in both groups at 12 months (3.09 ± 1.72 vs. 2.37 ± 1.22, p = 0.006). There was a significant difference in RMQ score (11.58 ± 3.58 vs. 8.17 ± 2.34, p < 0.001) and ODI score (43.65 ± 11.01 vs. 35.42 ± 11.32, p < 0.001) at 12 months. The total SF-36 score was higher in the RD group than in the PRF group at 12 months (58.45 ± 6.97 vs. 69.36 ± 6.43, p < 0.001). In terms of complications, skin numbness occurred in three patients. Mild pain such as burning and pinking at the puncture site in two patients. One patient experienced a decrease in back muscle strength and back muscle fatigue. These complications disappeared in 3 weeks without any treatment. There were no serious adverse events in the PRF group. CONCLUSION: Radiofrequency is an effective and safe treatment option for patients with lumbar facet joint pain. RD could provide good and lasting pain relief, with significant improvement in lumbar function and quality of life at long-term follow-up.

Geometric complement heterogeneous information and random forest for predicting lncRNA-disease associations.

More and more evidences have showed that the unnatural expression of long non-coding RNA (lncRNA) is relevant to varieties of human diseases. Therefore, accurate identification of disease-related lncRNAs can help to understand lncRNA expression at the molecular level and to explore more effective treatments for diseases. Plenty of lncRNA-disease association prediction models have been raised but it is still a challenge to recognize unknown lncRNA-disease associations. In this work, we have proposed a computational model for predicting lncRNA-disease associations based on geometric complement heterogeneous information and random forest. Firstly, geometric complement heterogeneous information was used to integrate lncRNA-miRNA interactions and miRNA-disease associations verified by experiments. Secondly, lncRNA and disease features consisted of their respective similarity coefficients were fused into input feature space. Thirdly, an autoencoder was adopted to project raw high-dimensional features into low-dimension space to learn representation for lncRNAs and diseases. Finally, the low-dimensional lncRNA and disease features were fused into input feature space to train a random forest classifier for lncRNA-disease association prediction. Under five-fold cross-validation, the AUC (area under the receiver operating characteristic curve) is 0.9897 and the AUPR (area under the precision-recall curve) is 0.7040, indicating that the performance of our model is better than several state-of-the-art lncRNA-disease association prediction models. In addition, case studies on colon and stomach cancer indicate that our model has a good ability to predict disease-related lncRNAs.

A clustering-based sampling method for miRNA-disease association prediction.

More and more studies have proved that microRNAs (miRNAs) play a critical role in gene expression regulation, and the irregular expression of miRNAs tends to be associated with a variety of complex human diseases. Because of the high cost and low efficiency of identifying disease-associated miRNAs through biological experiments, scholars have focused on predicting potential disease-associated miRNAs by computational methods. Considering that the existing methods are flawed in constructing negative sample set, we proposed a clustering-based sampling method for miRNA-disease association prediction (CSMDA). Firstly, we integrated multiple similarity information of miRNA and disease to represent miRNA-disease pairs. Secondly, we performed a clustering-based sampling method to avoid introducing potential positive samples when constructing negative sample set. Thirdly, we employed a random forest-based feature selection method to reduce noise and redundant information in the high-dimensional feature space. Finally, we implemented an ensemble learning framework for predicting miRNA-disease associations by soft voting. The Precision, Recall, F1-score, AUROC and AUPR of the CSMDA achieved 0.9676, 0.9545, 0.9610, 0.9928, and 0.9940, respectively, under five-fold cross-validation. Besides, case study on three cancers showed that the top 20 potentially associated miRNAs predicted by the CSMDA were confirmed by the dbDEMC database or literatures. The above results demonstrate that the CSMDA can predict potential disease-associated miRNAs more accurately.

MC4R Deficiency Causes Dysregulation of Postsynaptic Excitatory Synaptic Transmission as a Crucial Culprit for Obesity.

Melanocortin 4 receptor (MC4R) in the paraventricular nucleus of the hypothalamus (PVH) shows bidirectional characterization in modulating food intake and energy homeostasis. We demonstrate that MC4R knockdown (KD) in the PVH can attenuate AMPA receptor (AMPAR)-mediated postsynaptic responses by altering the phosphorylation of AMPAR GluA1 subunit through the protein kinase A (PKA)-dependent signaling cascade and simultaneously lead to rapid body weight gain. Furthermore, PKA KD in the PVH engendered similar electrophysiological and behavioral phenotypes as in MC4R KD mice. Importantly, we observed that the reduction of AMPAR GluA1 expression not only led to attenuated synaptic responses but also caused body weight gain, suggesting that the aberration of synaptic responses may be one of the crucial pathogeny of obesity. Our study provides the synaptic and molecular explanations of how body weight is regulated by MC4R in the PVH.

Upregulation of FOXO1 contributes to lipopolysaccharide-induced pulmonary endothelial injury by induction of autophagy.

Background: Autophagy is activated during the pathogenesis of endothelial dysfunction and sepsis-associated acute lung injury (ALI). This study aimed to investigate whether autophagy affected endothelial barrier dysfunction and lung injury in a murine model of lipopolysaccharide (LPS)-induced ALI, and then further clarify whether forkhead box O1 (FOXO1), an autophagy-related transcriptional factor, contributed to autophagy activation and ALI induced by LPS. Methods: Male C57BL/6 mice were treated with LPS (30 mg/kg), and then were allocated to a control group and an LPS group with or without FOXO1 inhibitor (AS1842856) treatment, respectively. Primary cultured mouse lung vascular endothelial cells (MLVECs) were treated with LPS, autophagy inhibitor 3-methyladenine (3-MA), AS1842856, and small interfering RNA (siRNA) targeting autophagy-related gene 5 (ATG5) or FOXO1. Endothelial autophagic flux was assessed by transfection of MLVECs with red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (RFP-GFP-LC3) adenovirus. Endothelial permeability was analyzed by the diffusion of fluorescein isothiocyanate-carboxymethyl (FITC)-dextran through the endothelial monolayer. Evans blue albumin tracer was used to measure the pulmonary transvascular permeability, and hematoxylin and eosin (H&E) staining was used to observe pathological changes in the lung tissues. Immunofluorescence staining was also used to detect the expression of zonula occludens-1 (ZO-1) and FOXO1. Results: This study found autophagy induction in lung tissues of endotoxemic mice and LPS-treated MLVECs, as evidenced by elevated expression of light chain 3 II (LC3-II) and Unc-51-like kinase (ULK1) and autophagic flux. LPS treatment decreased vascular endothelial (VE)-cadherin and ZO-1 expression and increased endothelial permeability in MLVECs, which were significantly alleviated by autophagy inhibitor 3-MA and ATG5 siRNA. It was found that both phosphorylated FOXO1 and FOXO1 were upregulated in the lung tissues of endotoxemic mice and LPS-treated MLVECs. Both FOXO1 inhibitor AS1842856 and FOXO1 siRNA suppressed LPS-induced autophagy and endothelial cell injury in MLVECs. Moreover, FOXO1 inhibition profoundly alleviated autophagy, lung endothelial hyperpermeability, and ALI in endotoxemic mice. Conclusions: This work demonstrated that FOXO1 upregulation is an important contributor to LPS-induced autophagy in pulmonary VE cells. The detrimental effects of FOXO1 in endotoxemia-associated endothelial dysfunction and ALI are partly due to its potent pro-autophagic property. Inhibition of FOXO1 may be a potential therapeutic option for the treatment of ALI.

Preparation and related properties of melanin iron supplement.

In this study, BM-Fe (black sesame melanin-iron complex) was prepared and characterized. The results showed that the carboxyl hydroxyl group of BSM (black sesame melanin) participated in the chelation of iron ions. EDS (energy dispersive spectroscopy) and XPS (X-ray photoelectron spectroscopy) results confirmed the presence of iron ions in BM-Fe. The results of DLS (dynamic light scattering) showed that the average particle sizes of BSM and BM-Fe were 844.9 nm and 294.3 nm, respectively, indicating that the structure of BM-Fe with a smaller particle size was formed after the binding of iron ions with the active group on BSM. Finally, the in vitro iron dissolution, iron ion identification, in vitro iron ion reduction, antioxidant activity, cytotoxicity and moisture resistance properties of BM-Fe and FST (ferrous sulfate tablets, a commonly used iron supplement) were comprehensively compared. The results showed that BSM combined with iron instead of physically mixing, and BM-Fe was easily reduced in the gastrointestinal environment. BM-Fe had good bioavailability and retained the excellent characteristics (such as oxidation resistance and biocompatibility) of BSM, and had the potential to be applied in the treatment of iron-deficiency-related diseases. In summary, BM-Fe prepared in this study not only retained the excellent characteristics of BSM but also had a good effect on iron supplementation, high bioavailability and low side effects. Comprehensive analysis showed that the performance of BM-Fe prepared in this study was similar to or even better than that of the control (FST). Thus, BM-Fe is expected to become a new comprehensive multi-functional iron supplement and has a broad developmental prospect.

Lidocaine attenuates CFA-induced inflammatory pain in rats by regulating the MAPK/ERK/NF-κB signaling pathway.

Lidocaine is a commonly used local anesthetic that also confers analgesic effects, resistance to hyperalgesia and anti-inflammatory properties. The present study aimed to explore the effects of lidocaine on complete Freund's adjuvant (CFA)-induced inflammatory pain. In the present study, rats were subcutaneously injected with CFA to investigate the molecular mechanisms associated with lidocaine in an inflammation-induced pain model. Firstly, CFA was subcutaneously injected into the paws of Sprague-Dawley rats, following which lidocaine or saline and the ERK agonist recombinant human epidermal growth factor (rh-EGF) were injected via the tail vein. Rat behavior was then assessed at 0 and 4 h, 1, 4, 7 and 14 days after CFA treatment. Proinflammatory cytokine levels in the serum were measured using ELISA. Western blotting was performed to detect the protein levels of phosphorylated (p)-ERK1/2, ERK1/2 and NF-κB subunits p-p65 and p65. Reverse transcription-quantitative PCR was used to measure the mRNA expression of ERK1/2 and p65 in rat spinal cord tissues. The results showed that injection of CFA significantly reduced the mechanical withdrawal threshold, thermal withdrawal latency and the frequency of responses to cold stimulation in rats, whilst promoting tumor necrosis factor-α, interleukin (IL)-1ß, IL-6 levels in addition to ERK1/2, p65 protein phosphorylation. These effects were alleviated by lidocaine treatment. Furthermore, treatment with rh-EGF reversed the protective effects of lidocaine on inflammatory pain caused by CFA. In conclusion, lidocaine inhibits the inflammatory response and pain through the MAPK/ERK/NF-κB pathway in a rat model of pain induced by CFA.

PET/CT Imaging of 89Zr-N-sucDf-Pembrolizumab in Healthy Cynomolgus Monkeys.

PURPOSE: Programmed cell death-1 receptor (PD-1) and its ligand (PD-L1) are the targets for immunotherapy in many cancer types. Although PD-1 blockade has therapeutic effects, the efficacy differs between patients. Factors contributing to this variability are PD-L1 expression levels and immune cells present in tumors. However, it is not well understood how PD-1 expression in the tumor microenvironment impacts immunotherapy response. Thus, imaging of PD-1-expressing immune cells is of interest. This study aims to evaluate the biodistribution of Zirconium-89 (89Zr)-labeled pembrolizumab, a humanized IgG4 kappa monoclonal antibody targeting PD-1, in healthy cynomolgus monkeys as a translational model of tracking PD-1-positive immune cells. PROCEDURES: Pembrolizumab was conjugated with the tetrafluorophenol-N-succinyl desferal-Fe(III) ester (TFP-N-sucDf) and subsequently radiolabeled with 89Zr. Four cynomolgus monkeys with no previous exposure to humanized monoclonal antibodies received tracer only or tracer co-injected with pembrolizumab intravenously over 5 min. Thereafter, a static whole-body positron emission tomography (PET) scan was acquired with 10 min per bed position on days 0, 2, 5, and 7. Image-derived standardized uptake values (SUVmean) were quantified by region of interest (ROI) analysis. RESULTS: 89Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (> 99 %) and acceptable molar activity (> 7 MBq/nmol). In animals dosed with tracer only, 89Zr-N-sucDf-pembrolizumab distribution in lymphoid tissues such as mesenteric lymph nodes, spleen, and tonsils increased over time. Except for the liver, low radiotracer distribution was observed in all non-lymphoid tissue including the lung, muscle, brain, heart, and kidney. When a large excess of pembrolizumab was co-administered with a radiotracer, accumulation in the lymph nodes, spleen, and tonsils was reduced, suggestive of target-mediated accumulation. CONCLUSIONS: 89Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89Zr-N-sucDf-pembrolizumab may be useful in tracking the distribution of a subset of immune cells in non-human primates and humans. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02760225.

Colorimetric determination of cysteine by a paper-based assay system using aspartic acid modified gold nanoparticles.

A method is described for cysteine (Cys) determination on paper-based analytical devices using aspartic acid modified gold nanoparticles (Asp-AuNPs). The Asp-AuNPs were characterized by their size, zeta potential, and UV-visible absorption spectrum. After the addition of Cys, it will interact with Asp-AuNPs selectively and leads to the aggregation of Asp-AuNPs. A color change from red to blue can be observed on the paper-based analytical devices. The results were recorded using a cell phone and subsequently analyzed using the Photoshop software. The ratiometric color intensity at red channel and blue channel (Red/Blue) increased linearly in the range 99.9-998.7 µM for Cys (R = 0.9984), and the limit of detection was 1.0 µM. The effects of assay conditions have been investigated and are discussed. The Cys concentration was determined as (0.27 ± 0.02 mM) in human plasma, and the recovery was from 99.2 to 101.1%. Graphical abstract Schematic representation of the paper-based assay system using aspartic acid modified gold nanoparticles (Asp-AuNPs). The ratiometric color intensity method was used for the cysteine (Cys) determination.

STAT3-induced upregulation of circCCDC66 facilitates the progression of non-small cell lung cancer by targeting miR-33a-5p/KPNA4 axis.

Emerging evidence has manifested the critical effect of abnormally expressed circular RNAs (circRNAs) on the initiation and progression of non-small cell lung cancer (NSCLC). Although circRNA circCCDC66 has been revealed to elicit facilitating impact on cell growth and metastasis in colon cancer, the potential biological function and regulatory mechanism of it in NSCLC still require to be explored. In this study, circCCDC66 in NSCLC cells was highly expressed. Downregulation of circCCDC66 impaired cell proliferation, migration and invasion whereas boosted cell apoptosis in NSCLC. Data from molecular mechanism assays testified that circCCDC66 bound with miR-33a-5p in NSCLC cells. And miR-33a-5p inhibition could rescue the suppressive effect of circCCDC66 knockdown on NSCLC progression. In addition, karyopherin subunit alpha 4 (KPNA4) in NSCLC cells was proofed to be directly targeted by miR-33a-5p. Moreover, through rescued-function assays, we observed that upregulating KPNA4 expression could countervail the restraining function of silenced circCCDC66 on NSCLC progression. Furthermore, signal transducer and activator of transcription 3 (STAT3) was validated to activate CCDC66 transcription and thereby promote circCCDC66 expression in NSCLC cells. Briefly, STAT3-induced circCCDC66 upregulation accelerates NSCLC progression via miR-33a-5p/KPNA4 axis, suggesting circCCDC66 as a promising biomarker in NSCLC treatment.

A successful bronchial thermoplasty procedure in a "very severe" asthma patient with rare complications: a case report.

Introduction: Bronchial thermoplasty (BT) is a unique bronchoscopic treatment for severe asthma that utilizes radiofrequency ablation to reduce smooth muscle in the bronchial walls. Current studies mainly focused on uncontrolled severe asthma with a forced expiratory volume in 1 second (FEV1) above 60% and associated complications, no human studies have performed on "very severe" asthma as well as its complications. Case study: We present a 60-year-old male with more than 15 years history of very severe asthma, who underwent BT. His FEV1 was only 20.4% predicted, which would have excluded him from all prior clinical trials of BT. The first BT procedure occurred without an issue. After the second BT procedure, he experienced severe dyspnea due to an infection with a non-flu respiratory virus. This illness was complicated by the formation of a pulmonary cyst. During recovering from the third procedure, he developed stomach stones. This is mainly related with taking large amounts of hawthorn previously, also cannot exclude the role of thermal energy injury on gastrointestinal nerve function. Results: Despite these unexpected complications, his quality of life greatly improved after BT, yet his lung function did not improve. Conclusion: This case is the first to describe BT procedures in patient with this level of lung function compromise, although accompanied with rare complications; our report indicates BT may be an opportunity and choice for the "very severe" asthma patients.

Upregulated circular RNA circ_0007534 indicates an unfavorable prognosis in pancreatic ductal adenocarcinoma and regulates cell proliferation, apoptosis, and invasion by sponging miR-625 and miR-892b.

Circular RNAs (circRNAs) have been regarded as critical regulators of human diseases and biological markers in some types of malignancies, including pancreatic ductal adenocarcinoma (PDAC). Recently, circ_0007534 has been identified as a novel cancer-related circRNA. Nevertheless, its clinical relevance, functional roles, and mechanism have not been studied in PDAC. In the current study, real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of circ_0007534 in 60-paired PDAC tissue samples and different cell lines. Loss-of-function and gain-of-function assays were performed to detect cell proliferation, apoptosis, and metastatic properties affected by circ_0007534. An animal study was also carried out. The luciferase reporter assay was performed to uncover the underlying mechanism of circ_0007534. As a result, circ_0007534 was overexpressed not only in PDAC tissues but also in a panel of PDAC cell lines, and this overexpression is closely associated with advanced tumor stage and positive lymph node invasion. In addition, circ_0007534 may be regarded as an independent prognostic factor for patients with PDAC. For the part of functional assays, circ_0007534 significantly increased cell proliferation, migratory, and invasive potential of PDAC cells. Circ_0007534 could inhibit cell apoptosis partly via a Bcl-2/caspase-3 pathway. The xenograft study further confirmed the cell growth promoting the role of circ_0007534. Mechanistically, miR-625 and miR-892b were sponged by circ_0007534. The oncogenic functions of circ_0007534 is partly dependent on its regulation of miR-625 and miR-892b. In conclusion, our study illuminates a novel circRNA that confers an oncogenic function in PDAC.

Development of Anti-CD74 Antibody-Drug Conjugates to Target Glucocorticoids to Immune Cells.

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

[Value of serum CRISPLD2 levels for the diagnosis and prognosis evaluation of sepsis patients].

OBJECTIVE: To investigate the value of cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) in diagnosis and prognosis in patients with sepsis. METHODS: Clinical data of patients admitted to intensive care unit (ICU) of the First Hospital of Hebei Medical University from December 2014 to December 2016 were retrospectively analyzed. According to the severity of sepsis, the patients were divided into three groups: sepsis patients, severe sepsis patients and septic shock patients, and 100 healthy persons were enrolled as control group. Levels of serum CRISPLD2, procalcitonin (PCT) and C-reactive protein (CRP), acute physiology and chronic health evaluation II (APACHE II) score, sequential organ failure assessment (SOFA) score, and 28-day prognosis were recorded. Analysis of the correlation between CRISPLD2 and PCT, CRP, APACHE II score, SOFA score was done. The receiver operating characteristic (ROC) curve was plotted for the CRISPLD2 value for the diagnosis and prognosis in patients with sepsis. RESULTS: A total of 115 patients with sepsis were enrolled in this study, including 52 sepsis, 48 severe sepsis, and 15 septic shock; 29 patients died after 28 days, 28-days mortality rate was 25.2%. There was no significant difference in CRISPLD2 between sepsis and healthy control group (mg/L: 204.1±74.5 vs. 211.3±12.0, P > 0.05); the level of CRISPLD2 in septic shock group was significantly lower than that in sepsis group and severe sepsis group (mg/L: 139.0±55.0 vs. 240.2±89.6, 233.0±8.9, both P < 0.05). The level of PCT, CRP and APACHE II score, SOFA score in sepsis patients were significantly higher than those in healthy control group, and increased with the severity of sepsis. There was no statistically significant difference in CRISPLD2 level between the dead and the survival of sepsis, and the levels of PCT and CRP in death group were significantly higher. The levels of CRISPLD2 were significantly negative correlated with the levels of PCT, CRP, APACHE II score and SOFA score (r values were -0.089, -0.431, -0.115, -0.201, respectively, all P < 0.05). It was shown by ROC curve analysis that the area under ROC curve (AUC) and 95% confidence interval (95%CI) of CRISPLD2, PCT, CRP for diagnosis of sepsis were 0.907 (0.871-0.944), 0.922 (0.886-0.958), 0.916 (0.878-0.954) respectively, all P = 0.000; when the cut-off value of CRISPLD2 > 216.0 mg/L, the sensitivity was 96.7%, and the specificity was 92.6%, which power lied between PCT and CRP. The AUC of CRISPLD2 for prognosis was significantly lower than that of PCT [0.617 (0.507-0.727) vs. 0.786 (0.668-0.903), P < 0.01]; when the cut-off value of CRISPLD2 was 103.5 mg/L, the sensitivity was 100%, and the specificity was 25.6%. CONCLUSIONS: CRISPLD2 is a potential biomarker in sepsis, but cannot predict the prognosis of patients with sepsis.

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice.

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies.