A novel inhalable nanobody targeting IL-4Rα for the treatment of asthma.

BACKGROUND: Inhalable biologics represent a promising approach to improve the efficacy and safety of asthma treatment. Although several mAbs targeting IL-4 receptor α chain (IL-4Rα) have been approved or are undergoing clinical trials, the development of inhalable mAbs targeting IL-4Rα presents significant challenges. OBJECTIVE: Capitalizing on the distinctive advantages of nanobodies (Nbs) in maintaining efficacy during storage and administration, we sought to develop a novel inhalable IL-4Rα Nb for effectively treating asthma. METHODS: Three IL-4Rα immunized Nb libraries were used to generate specific and functional IL-4Rα Nbs. LQ036, a bivalent Nb comprising 2 HuNb103 units, was constructed with a high affinity and specificity for human IL-4Rα. The efficacy, pharmacokinetics, and safety of inhaled LQ036 were evaluated in B-hIL4/hIL4RA humanized mice. RESULTS: LQ036 inhibited secreted embryonic alkaline phosphatase reporter activity, inhibited TF-1 cell proliferation, and suppressed phosphorylated signal transducer and activator of transduction 6 in T cells from patients with asthma. Crystal structure analysis revealed a binding region similar to dupilumab but with higher affinity, leading to better efficacy in blocking the signaling pathway. HuNb103 competed with IL-4 and IL-13 for IL-4Rα binding. Additionally, LQ036 significantly inhibited ovalbumin-specific IgE levels in serum, CCL17 levels in bronchoalveolar lavage fluid, bronchial mucous cell hyperplasia, and airway goblet cell hyperplasia in B-hIL4/hIL4RA humanized mice. Inhaled LQ036 exhibited favorable pharmacokinetics, safety, and tissue distribution, with higher concentrations observed in the lungs and bronchi. CONCLUSIONS: These findings from preclinical studies establish the safety and efficacy of inhaled LQ036, underscoring its potential as a pioneering inhalable biologic therapy for asthma.

High performance production process development and scale-up of an anti-TSLP nanobody.

Nanobodies (Nbs) represent a class of single-domain antibodies with great potential application value across diverse biotechnology fields, including therapy and diagnostics. Thymic Stromal Lymphopoietin (TSLP) is an epithelial cell-derived cytokine, playing a crucial role in the regulation of type 2 immune responses at barrier surfaces such as skin and the respiratory/gastrointestinal tract. In this study, a method for the expression and purification of anti-TSLP nanobody (Nb3341) was established at 7 L scale and subsequently scaled up to 100 L scale. Key parameters, including induction temperature, methanol feed and induction pH were identified as key factors by Plackett-Burman design (PBD) and were optimized in 7 L bioreactor, yielding optimal values of 24 °C, 8.5 mL/L/h and 6.5, respectively. Furthermore, Diamond Mix-A and Diamond MMC were demonstrated to be the optimal capture and polishing resins. The expression and purification process of Nb3341 at 100L scale resulted in 22.97 g/L titer, 98.7% SEC-HPLC purity, 95.7% AEX-HPLC purity, 4 ppm of HCP content and 1 pg/mg of HCD residue. The parameters of the scaling-up process were consistent with the results of the optimized process, further demonstrating the feasibility and stability of this method. This study provides a highly promising and competitive approach for transitioning from laboratory-scale to commercial production-scale of nanobodies.

MR defecography in assessing stress urinary incontinence with or without symptomatic pelvic organ prolapse.

PURPOSE: Utilize magnetic resonance defecography (MRD) to analyze the primary pelvic floor dysfunctions in patients with stress urinary incontinence (SUI) associated with pelvic organ prolapse (POP), and in SUI patients with asymptomatic POP. METHOD: We performed MRD in both SUI and POP subjects. As a primary analysis, the functional MR parameters were compared between the isolated POP and POP combined SUI groups. As a secondary analysis, the functional MR data were compared between the POP combined SUI and the SUI with asymptomatic POP (isolated SUI) groups. RESULTS: MRD noted the main characteristics of SUI combined moderate or severe POP, including the shorter closed urethra length (1.87 cm vs. 2.50 cm, p < 0.001), more prevalent urethral hypermobility (112.31° vs. 85.67°, p = 0.003), bladder neck funneling (48.28% vs. 20.51%, p = 0.020), lower position of vesicourethral junction (2.11 cm vs. 1.67 cm, p = 0.030), and more severe prolapse of the posterior bladder wall (6.26 cm vs. 4.35 cm, p = 0.008). The isolated SUI patients showed the shortest length of the closed urethra (1.56 cm vs. 1.87 cm, p = 0.029), a larger vesicourethral angle (153.80° vs. 107.58°, p < 0.001), the more positive bladder funneling (84.85% vs. 48.28%, p = 0.002) and a special urethral opening sign (45.45% vs. 3.45%, p < 0.001). CONCLUSIONS: Patients with SUI accompanying POP primarily exhibit excessive urethral mobility and a shortened urethral closure. SUI patients with asymptomatic POP mainly show dysfunction of the urethra and bladder neck, characterized by the opening of the urethra and bladder neck and a shortened urethral closure.

Preparation and Luminescence Properties of Broadband Orange-Emitting Persistent ScBaZn3GaO7:Bi3+ Phosphor.

This article describes a new kind of afterglow material, ScBaZn3GaO7:Bi3+, which was synthesized through a high-temperature solid-phase method. Its crystal structure, photoluminescent characteristics, and afterglow characteristics were studied and analyzed. Upon excitation at 344 nm, ScBaZn3GaO7:Bi3+ exhibits broadband emission with a central wavelength located at 571 nm (fwhm = 172.98 nm). The sample exhibits an internal quantum efficiency of 65.1%. The bright yellow persistent luminescence of the ScBaZn3GaO7:Bi3+ sample was observed after 365 nm irradiation. Thermoluminescence spectroscopy revealed four primary traps within ScBaZn3GaO7:Bi3+, with depths of 0.676, 0.794, 0.882, and 0.972 eV. The traps located at energy levels of 0.676 and 0.794 eV were identified as the key contributors to the sample's afterglow. Finally, the ScBaZn3GaO7:Bi3+ sample was combined with a UV-LED chip to fabricate a high-power warm white-light-emitting diode (WLED) device, indicating the potential application prospect of ScBaZn3GaO7:Bi3+ phosphor in single-phase warm WLEDs.

Posterior circulation ischemic stroke: radiomics-based machine learning approach to identify onset time from magnetic resonance imaging.

PURPOSE: Posterior circulation ischemic stroke (PCIS) possesses unique features. However, previous studies have primarily or exclusively relied on anterior circulation stroke cases to build machine learning (ML) models for predicting onset time. To date, there is no research reporting the effectiveness and stability of ML in identifying PCIS onset time. We aimed to build diffusion-weighted imaging-based ML models to identify the onset time of PCIS patients. METHODS: Consecutive PCIS patients within 24 h of definite symptom onset were included (112 in the training set and 49 in the independent test set). Images were processed as follows: volume of interest segmentation, image feature extraction, and feature selection. Five ML models, naïve Bayes, logistic regression, tree ensemble, k-nearest neighbor, and random forest, were built based on the training set to estimate the stroke onset time (binary classification: ≤ 4.5 h or > 4.5 h). Relative standard deviations (RSD), receiver operating characteristic (ROC) curves, and the calibration plot was performed to evaluate the stability and performance of the five models. RESULTS: The random forest model had the best performance in the test set, with the highest area under the curve (AUC, 0.840; 95% CI: 0.706, 0.974). This model also achieved the highest accuracy, sensitivity, specificity, positive predictive value, and negative predictive value (83.7%, 64.3%, 91.4%, 75.0%, and 86.5%, respectively). Furthermore, the model had high stability (RSD = 0.0094). CONCLUSION: The PCIS case-based ML model was effective for estimating the symptom onset time and achieved considerably high specificity and stability.

[Medium to long-term efficacy of tension-free vaginal tape procedure in the treatment of female recurrent stress urinary incontinence].

OBJECTIVE: To investigate the effectiveness and clinical efficacy of tension-free vaginal tape (TVT) surgery in treating female recurrent stress urinary incontinence (rSUI). METHODS: A retrospective analysis was conducted on 24 patients who experienced recurrence of mid-urethral sling failure and were treated with TVT surgery at Beijing Chao-Yang Hospital from January 2016 to June 2020. Basic patient information was collected. The International Consultation on Incontinence questionnaire-short form (ICI-Q-SF) was used to record urinary incontinence symptom scores preoperatively, 1-year postoperatively, and more than three years postoperatively. The changes in various ICI-Q-SF scores and total scores were compared. Additionally, clinical symptom severity of urinary incontinence was recorded and compared preoperatively and more than three years postoperatively. RESULTS: Among the 24 patients included in the follow-up, one patient was lost to follow-up due to death from a cerebrovascular accident one year postoperatively, leaving 23 patients with a follow-up period ranging from 3.9 to 7.3 years, with an ave-rage follow-up time of (5.2±1.1) years. Preoperatively, the median ICI-Q-SF total score was 20.0 (16.0, 21.0); at the 1-year follow-up, the median ICI-Q-SF total score was 5.0 (1.5, 7.8) (P < 0.001); at an average follow-up of five years, the median ICI-Q-SF total score was 6.0 (3.0, 9.0), still showing a statistically significant difference compared with preoperative scores (P < 0.001). The individual ICI-Q-SF scores were significantly lower at the 1-year and average five-year follow-ups compared with preoperative scores (P < 0.001). Regarding the severity of urinary incontinence, all the patients had moderate to severe urinary incontinence preoperatively; Five years postoperatively, 87.0% (20/23) of the patients had no or only mild urinary incontinence, and 13.0% (3/23) had recurrent moderate to severe urinary incontinence (P < 0.001). CONCLUSION: TVT surgery is effective in treating female recurrent stress urinary incontinence, with an average 5-year cure and improvement rate of 87.0%.

Discovery of Natural Potent HMG-CoA Reductase Degraders for Lowering Cholesterol.

Exploitation of key protected wild plant resources makes great sense, but their limited populations become the major barrier. A particular strategy for breaking this barrier was inspired by the exploration of a resource-saving fungal endophyte Penicillium sp. DG23, which inhabits the key protected wild plant Schisandra macrocarpa. Chemical studies on the cultures of this strain afforded eight novel indole diterpenoids, schipenindolenes A-H (1-8), belonging to six diverse skeleton types. Importantly, semisyntheses suggested some key nonenzymatic reactions constructing these molecules and provided targeted compounds, in particular schipenindolene A (Spid A, 1) with low natural abundance. Remarkably, Spid A was the most potent HMG-CoA reductase (HMGCR) degrader among the indole diterpenoid family. It degraded statin-induced accumulation of HMGCR protein, decreased cholesterol levels and acted synergistically with statin to further lower cholesterol. Mechanistically, transcriptomic and proteomic profiling suggested that Spid A potentially activated the endoplasmic reticulum-associated degradation (ERAD) pathway to enhance the degradation of HMGCR, while simultaneously inhibiting the statin-activated expression of many key enzymes in the cholesterol and fatty acid synthesis pathways, thereby strengthening the efficacy of statins and potentially reducing the side effects of statins. Collectively, this study suggests the potential of Spid A for treating cardiovascular disease.

TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer.

BACKGROUND: Pancreatic cancer is a highly aggressive malignancy with limited treatment options and a poor prognosis. Trophoblast cell surface antigen 2 (TROP2), a cell surface antigen overexpressed in the tumors of more than half of pancreatic cancer patients, has been identified as a potential target for antibody-drug conjugates (ADCs). Almost all reported TROP2-targeted ADCs are of the IgG type and have been poorly studied in pancreatic cancer. Here, we aimed to develop a novel nanobody-drug conjugate (NDC) targeting TROP2 for the treatment of pancreatic cancer. RESULTS: In this study, we developed a novel TROP2-targeted NDC, HuNbTROP2-HSA-MMAE, for the treatment of TROP2-positive pancreatic cancer. HuNbTROP2-HSA-MMAE is characterized by the use of nanobodies against TROP2 and human serum albumin (HSA) and has a drug-antibody ratio of 1. HuNbTROP2-HSA-MMAE exhibited specific binding to TROP2 and was internalized into tumor cells with high endocytosis efficiency within 5 h, followed by intracellular translocation to lysosomes and release of MMAE to induce cell apoptosis in TROP2-positive pancreatic cancer cells through the caspase-3/9 pathway. In a xenograft model of pancreatic cancer, doses of 0.2 mg/kg and 1 mg/kg HuNbTROP2-HSA-MMAE demonstrated significant antitumor effects, and a dose of 5 mg/kg even eradicated the tumor. CONCLUSION: HuNbTROP2-HSA-MMAE has desirable affinity, internalization efficiency and antitumor activity. It holds significant promise as a potential therapeutic option for the treatment of TROP2-positive pancreatic cancer.

Perfusate Neutrophil Gelatinase-Associated Lipocalin, Kidney Injury Molecular-1, Liver-Type Fatty Acid Binding Protein, and Interleukin-18 as Potential Biomarkers to Predict Delayed Graft Function and Long-Term Prognosis in Kidney Transplant Recipients: A Single-Center Retrospective Study.

BACKGROUND Delayed graft function (DGF) caused by ischemia-reperfusion injury is a common pathophysiological process that should be monitored by specific biomarkers in addition to serum creatinine. Thus, this single-center retrospective study aimed to investigate the association between levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecular-1 (KIM-1), liver-type fatty acid binding protein (L-FABP), and interleukin-18 (IL-18) in DGF associated with acute kidney injury in kidney transplant recipients (KTRs) and estimated glomerular filtration rate (eGFR) at 3 years post-transplant. MATERIAL AND METHODS A total of 102 KTRs [14(13.7%) of DGF and 88(86.3%) of NON-DGF] were enrolled. DGF was defined as "dialysis is needed within 1 week after kidney transplantation". NGAL, KIM-1, L-FABP, and IL-18 were obtained from perfusate samples of donation-after-cardiac-death (DCD) kidneys, and measured by ELISA. RESULTS Compared to the NON-DGF group, KTRs in the DGF group had a statistically significant increase in levels of NGAL (P<0.001) and KIM-1 (P<0.001). Multiple logistic regression analyses showed that NGAL (OR=1.204, 95% CI 1.057-1.372, P=0.005) and KIM-1 (OR=1.248, CI=1.065-1.463, P=0.006) could be regarded as independent risk factors. The accuracy of NGAL and KIM-1 was 83.3% and 82.1%, respectively, calculated using the area under the receiver operating characteristic curve. Furthermore, the eGFR at 3 years post-transplant had a moderate negative correlation with NGAL (r=-0.208, P=0.036) and KIM-1 (r=-0.260, P=0.008). CONCLUSIONS Our results support those from previous studies showing that perfusate levels of NGAL and KIM-1 are associated with DGF in KTRs and also with reduced eGFR at 3 years post-transplant.

Preclinical development of a long-acting trivalent bispecific nanobody targeting IL-5 for the treatment of eosinophilic asthma.

BACKGROUND: Eosinophilic asthma is a common subtype of severe asthma with high morbidity and mortality. The cytokine IL-5 has been shown to be a key driver of the development and progression of disease. Although approved monoclonal antibodies (mAbs) targeting IL-5/IL-5R have shown good safety and efficacy, some patients have inadequate responses and frequent dosing results in medication nonadherence. RESULTS: We constructed a novel trivalent bispecific nanobody (Nb) consisting of 3 VHHs that bind to 2 different epitopes of IL-5 and 1 epitope of albumin derived from immunized phage display libraries. This trivalent IL-5-HSA Nb exhibited similar IL-5/IL-5R blocking activities to mepolizumab (Nucala), an approved targeting IL-5 mAb. Surprisingly, this trivalent Nb was 58 times more active than mepolizumab in inhibiting TF-1-cell proliferation. In primate studies, the trivalent IL-5-HSA Nb showed excellent pharmacokinetic properties, and peripheral blood eosinophil levels remained significantly suppressed for two months after a single dose. In addition, the trivalent IL-5-HSA Nb could be produced on a large scale in a P. pastoris X-33 yeast system with high purity and good thermal stability. CONCLUSIONS: These findings suggest that the trivalent bispecific IL-5-HSA Nb has the potential to be a next-generation therapeutic agent targeting IL-5 for the treatment of severe eosinophilic asthma.

Assessment of the Ovarian Reserve by Serum Anti-Müllerian Hormone in Rheumatoid Arthritis Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: The ovarian reserve has been reported to be diminished in patients with rheumatoid arthritis. However, these results are still controversial. Anti-Müllerian hormone (AMH) is considered a reliable biomarker for the ovarian reserve. We thus performed a meta-analysis to evaluate the AMH levels and the effect of DMARDs on the ovarian reserve in rheumatoid arthritis patients. METHODS: PubMed, EMBASE, the Cochrane Library, and 2 Chinese databases (CNKI and Wanfang database), up to September 2021, were searched for relevant studies. The Newcastle-Ottawa scale (NOS) was used to assess the quality of the included studies. Pooled standard mean difference (SMD) with 95% confidence intervals (CIs) were determined with the random-effects model. The heterogeneity was described by I2 statistic and p value from the Cochrane Q test. RESULTS: Eight eligible studies (679 patients and 1,460 controls) were included in the meta-analysis. Compared with healthy control, the AMH levels in RA patients were significantly lower with the pooled SMD of -0.40 (95% CI: -0.66 to -0.14). However, in comparison of AMH with and without DMARD treatment, there was no significant difference with the pooled SMD of -0.1 (95% CI: -0.39 to 0.19). CONCLUSION: The results indicated that there was an increased risk of ovarian failure in RA patients and which is not related to DMARD treatment.

Microbial communities of ascocarps and soils in a natural habitat of Tuber indicum.

Truffles are the fruiting bodies of hypogeous fungi in the genus Tuber. Some truffle species usually grow in an area devoid of vegetation, called brûlé, but limited knowledge is available on the microbial composition and structure of them. Here, we investigated the bacterial and fungal communities of Tuber indicum ascocarps and soils inside and outside a characteristic brûlé from a poplar plantation with no truffle production history in northeastern China using a high-throughput sequencing approach. A predominance of members of the bacterial phylum Proteobacteria was observed in all samples. Members of Bacillus were the main genera in the ascocarps, while members of Lysobacter and unidentified Acidobacteria were more abundant in the soil. In addition, members of Gibberella, Fusarium, and Absidia were the dominant fungi in the ascocarps, while members of Tuber were enriched in the ascocarps and soils inside the brûlé. Some mycorrhization helper bacteria (Rhizobium) and ectomycorrhiza-associated bacteria (Lysobacter) were detected, indicating their potential roles in the complex development of underground fruiting bodies and brûlé formation. These findings may contribute to the protection and cultivation of truffles.

High Speed Railway Fastener Defect Detection by Using Improved YoLoX-Nano Model.

Rails play a vital role in the bearing and guidance of high-speed trains, and the normal condition of rail components is the guarantee of the operation and maintenance safety. Fasteners are critical components for fixing the rails, so it is particularly important to detect whether they are in a normal state or not. The current rail-fastener detection models have some drawbacks, including poor generalization ability, large model volume and low detection efficiency. In view of this, an improved YoLoX-Nano rail-fastener-defect-detection method is proposed in this paper. The CA attention mechanism is added to the three output feature maps of CSPDarknet and the enhanced feature extraction part of the Path Aggregation Feature Pyramid Network (PAFPN); the Adaptively Spatial Feature Fusion (ASFF) is added after the PAFPN output feature map, which enables the semantic information of the high-level features and the fine-grained features of the bottom layer to be further enhanced. The improved YoLoX-Nano model has improved the AP value by 27.42% on fractured fasteners, 15.88% on displacement fasteners and 12.96% on normal fasteners. Moreover, the mAP value is improved by 18.75%, and it is 14.75% higher than the two-stage model Faster-RCNN on mAP. In addition, compared with YoLov7-tiny, the improved YoLoX-Nano model achieves 13.56% improvement on mAP. Although the improved model increases a certain amount of calculation, the detection speed of the improved model has been increased by 30.54 fps and by 32.33 fps when compared with that of the Single-Shot Multi-Box Detector (SSD) model and the You Only Look Once v3 (YoLov3) model, reaching 54.35 fps. The improved YoLoX-Nano model enables accurate and rapid identification of the defects of rail fasteners, which can meet the needs of real-time detection. Furthermore, it has advantages in lightweight deployment of terminals for rail-fastener detection, thus providing some reference for image recognition and detection in other fields.

Naringenin inhibits autophagy and epithelial-mesenchymal transition of human lens epithelial cells by regulating the Smad2/3 pathway.

Cataract is the number one cause of blindness in the world. Fibrosis of the lens is the main cause of cataract. Pathological epithelial-mesenchymal transition (EMT) plays an important role in the development of fibrotic cataract. Inhibition of EMT may be an effective treatment for fibrosis of lens epithelial cells. Naringin (NRG) is one of the major citrus flavonoids, which has many pharmacological properties, including anti-inflammatory and cardioprotective. However, the effect of NRG on cataract induced by abnormal fibrosis of LECs is not clear. Herein, we found NRG inhibited transforming growth factor ß2 (TGFß2)-induced SRA01/04 cell viability. Additionally, NRG inhibited TGFß2-induced cell migration and EMT. We further noticed that NRG inhibited autophagy and Smad2/3 phosphorylation in LECs. We therefore thought Naringenin inhibited autophagy and EMT of human LECs by regulating the Smad2/3 pathway. NRG could therefore serve as a promising drug for cataract treatment.

A novel bispecific nanobody with PD-L1/TIGIT dual immune checkpoint blockade.

Cancer immunotherapy have changed the paradigm of cancer treatment, but there remains a great need for improvement given that less patients with tumors respond to the treatment of PD-1/PD-L1 blockade. TIGIT (also called T cell immunoreceptor with Ig and ITIM domains), a novel immune checkpoint molecule, has been shown a promising target for drug development of immunotherapy. Here we report generation and characterization of a multivalent bispecific antibody (BsAb) that co-targets PD-L1 and TIGIT. The BsAb consists of tetravalent anti-PD-L1 Fc-fusion nanobody (Nb) and tetravalent anti-TIGIT Nb. The parental anti-PD-L1 Nb showed high specificity and affinity to primate PD-L1, the enhanced T cell activity in vitro and anti-tumor activity in vivo. Similarly, the parental anti-TIGIT Nb showed the high specificity and affinity to primate TIGIT and the enhanced T cell activity. Furthermore, we demonstrated that the BsAb retained high blocking activity towards PD-1/PD-L1 or TIGIT/CD155 interaction. The BsAb synergistically enhanced T cell activities in vitro compared to two parental Nbs. Taken together, we obtained a multivalent BsAb blocking biological function of PD-L1 and TIGIT and it is worthy to further study the anti-tumor activities of this BsAb in vivo.

EGb761 Protects Brain Microvascular Endothelial Cells Against Oxygen-Glucose Deprivation-Induced Injury Through lncRNA Rmst/miR-150 Axis.

In the present study, we aimed to illustrate the roles and working mechanisms of long non-coding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (Rmst) and EGb761 in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). OGD exposure augmented the level of Rmst while reduced the expression of miR-150 in bEnd.3 cells. MiR-150 could directly bind to Rmst in bEnd.3 cells. Rmst silencing abrogated the inhibitory influences on the proliferation and migration and the promoting impact on the apoptosis of bEnd.3 cells caused by OGD exposure. Rmst overexpression intensified OGD-induced injury in bEnd.3 cells. OGD induced the injury of bEnd.3 cells through Rmst/miR-150 axis. EGb761 attenuated the damage in bEnd.3 cells induced by OGD through targeting Rmst/miR-150 axis. EGb761 might be an effective therapeutic agent to protect brain microvascular endothelial cells from hypoxia-ischemia induced injury.

Reduction-sensitive poly(ethylene glycol)-polypeptide conjugate micelles for highly efficient intracellular delivery and enhanced antitumor efficacy of hydroxycamptothecin.

The non-specific biodistribution of traditional chemotherapeutic drugs against tumors is the key factor that causes systemic toxicity and hinders their clinical application. In this study, a reduction-sensitive polymer conjugate micelle was manufactured to achieve tumor-specific targeting, reduce toxic side-effects and improve anti-tumor activity of a natural anti-cancer drug, hydroxycamptothecin (HCPT). Therefore, HCPT was conjugated with methoxy-poly(ethylene glycol)-poly(ß-benzyl-L-aspartate) (mPEG-PBLA) by a disulfide bond or succinate bond for the first time to obtain the mPEG-PBLA-SS-HCPT (PPSH) and mPEG-PBLA-CC-HCPT (PPCH) that would form micelles after high-speed agitation and dialysis. The PPSH micelles showed an average particle size of 126.3 nm, a low polydispersity index of 0.209, and a negative surface charge of -21.1 mV zeta potential. Transmission electron microscopy showed the PPSH micelles to have spherical morphology. PPSH had a low critical micelle concentration of 1.29 µg ml-1 with high dilution stability, storage stability and reproducibility. Moreover, the particle size of the PPSH micelles had no significant change after incubation with rat plasma for 72 h, probably resulting in high long circulation in the blood. The PPSH micelles showed significant reduction sensitivity to glutathione. Their sizes increased by 403.2 nm after 24 h post-incubation, and 87.6% drug release was achieved 48 h post-incubation with 40 mM glutathione solutions. The PPSH micelles showed stronger inhibition of HepG2 cells in vitro and growth of H-22 tumor in vivo than the PPCH and HCPT solutions after intravenous injection. The accumulation of PPSH micelles in the tumor tissue contributed to the high anti-tumor effect with little side-effect on the normal tissues. The reduction-sensitive PPSH micelles were a promising carrier of HCPT and other poorly soluble anti-cancer drugs.

Preclinical development of a novel CD47 nanobody with less toxicity and enhanced anti-cancer therapeutic potential.

BACKGROUND: CD47, the integrin-related protein, plays an important role in immune resistance and escape of tumor cells. Antibodies blocking the CD47/SIRPα signal pathway can effectively stimulate macrophage-mediated phagocytosis of tumor cells, which becomes a promising approach for tumor immunotherapy. Nanobodies (Nbs) derived from camelid animals are emerging as a new force in antibody therapy. RESULTS: HuNb1-IgG4, an innovative anti-CD47 nanobody, was developed with high affinity and specificity. It effectively enhanced macrophage-mediated phagocytosis of tumor cells in vitro and showed potent anti-ovarian and anti-lymphoma activity in vivo. Importantly, HuNb1-IgG4 did not induce the agglutination of human red blood cells (RBCs) in vitro and exhibited high safety for hematopoietic system in cynomolgus monkey. In addition, HuNb1-IgG4 could be produced on a large scale in CHO-S cells with high activity and good stability. Also, we established anti-CD47/CD20 bispecific antibody (BsAb) consisted of HuNb1 and Rituximab, showing more preference binding to tumor cells and more potent anti-lymphoma activity compared to HuNb1-IgG4. CONCLUSIONS: Both of HuNb1-IgG4 and anti-CD47/CD20 BsAb are potent antagonists of CD47/SIRPα pathway and promising candidates for clinical trials.

Neonatal hand, foot, and mouth disease due to coxsackievirus A6 in Shanghai.

BACKGROUND: Evidence of hand, foot, and mouth disease (HFMD) in neonates is limited. The aim of this study was to evaluate the clinical symptoms, pathogens, possible transmission routes, and prognosis of neonatal HFMD in Shanghai. METHODS: This was a case-control study based on the HFMD registry surveillance system. All neonates and infected family members were enrolled between 2016 and 2017 in Shanghai. Neonates with HFMD were followed for at least half a year. Detailed questionnaires, medical history, and physical examination were recorded. Routine blood examination, liver and renal function, immunophenotypes of peripheral blood lymphocytes (CD3, CD4, and CD8 T-cells; NK cells), immunoglobulin (Ig) M, IgG, and IgA, and cytokine interleukin (IL-1ß, IL-2R, IL-6, IL-8, IL-10, and TNF-α) levels were measured. All rectal swab specimens were collected and genotyped for enterovirus, and phylogenetic analysis based on the VP1 sequences of coxsackievirus A6 (CV-A6) was performed to investigate molecular and evolutionary characteristics. T-test or nonparametric test was used to evaluate the differences. Logistic analysis was applied to calculate the risk of clinical manifestations in the group of HFMD neonates and their paired siblings. RESULTS: There were 16 neonates among the 12,608 diagnosed patients with HFMD, accounting for 0.13%. All neonatal infections were transmitted by other members of the family, mainly the elder siblings, and were caused by CV-A6. CV-A6 was the emerging and predominant causative agent of HFMD in Shanghai. None of the neonates with HFMD experienced fever, onychomadesis, or severe complications. However, two elder sibling patients showed lethargy, and one developed hypoperfusion. In the elder siblings with HFMD, the proportion of white blood cells was generally higher than in neonates with HFMD. The immunologic function of the neonates with HFMD was basically normal. The levels of inflammatory markers were higher in both neonates and elder siblings with HFMD compared to age-matched controls. The clinical symptoms receded about 1 week after onset. None of the neonates had sequelae. CONCLUSIONS: In our study, CV-A6 infection in neonates was benign, but had the character of family clustering. Due to the two-child policy in China, elder siblings may be the main route of HFMD transmission.

Collateral Circulation in Moyamoya Disease: A New Grading System.

Background and Purpose- Predicting the risk of stroke and determining intervention indications are highly important for patients with Moyamoya disease (MMD). Here, we evaluated a novel MMD grading system based on collateral circulation and Suzuki stage to evaluate symptoms and predict prognosis. Methods- In total, 301 idiopathic MMD patients were retrospectively analyzed between 2014 and 2016. A collateral circulation grading system with scores ranging from 1 to 12 was established: the anatomic extent of pial collateral blood flow from posterior cerebral artery to middle cerebral artery and anterior cerebral artery was scored from 1 to 6; perforator collateral and internal cerebral artery flow were scored as 6 to 1, which corresponded to Suzuki stages 1 to 6. Dynamic susceptibility contrast-magnetic resonance imaging was used to evaluate hemodynamic status. We assessed the association between the grading system and clinical characteristics. Results- We analyzed 364 symptomatic hemispheres of 301 patients (146 males, 28±16 years). Ischemic patients who presented with infarction were more likely to score <8 points (P<0.001), whereas those with ischemia symptoms (transient ischemic attack and headache) were more likely to score >8 points. Hemorrhagic patients who presented with intraparenchymal hemorrhage were more likely to score <8 points, whereas those who presented with intraventricular hemorrhage were more likely to score >8 points (P<0.001). According to dynamic susceptibility contrast-magnetic resonance imaging, lower scores were correlated with more severe time to peak delay (P<0.001) and worse relative cerebral blood volume ratio (P=0.016) and cerebral flow ratio (P=0.002). Encephaloduroarteriosynangiosis was performed in 348 symptomatic hemispheres. Patients who had collateral scores <4 points were more likely to have a postoperative stroke and a worse prognosis during the follow-up. Conclusions- This new MMD collateral grading system correlated well with clinical symptoms, hemodynamic status, and therapeutic prognosis and may facilitate risk stratification and prognosis predictions in patients with MMD.