T cell-redirecting antibody for treatment of solid tumors via targeting mesothelin.

T cell engaging bispecific antibodies (TCBs) have recently become significant in cancer treatment. In this study we developed MSLN490, a novel TCB designed to target mesothelin (MSLN), a glycosylphosphatidylinositol (GPI)-linked glycoprotein highly expressed in various cancers, and evaluated its efficacy against solid tumors. CDR walking and phage display techniques were used to improve affinity of the parental antibody M912, resulting in a pool of antibodies with different affinities to MSLN. From this pool, various bispecific antibodies (BsAbs) were assembled. Notably, MSLN490 with its IgG-[L]-scFv structure displayed remarkable anti-tumor activity against MSLN-expressing tumors (EC50: 0.16 pM in HT-29-hMSLN cells). Furthermore, MSLN490 remained effective even in the presence of non-membrane-anchored MSLN (soluble MSLN). Moreover, the anti-tumor activity of MSLN490 was enhanced when combined with either Atezolizumab or TAA × CD28 BsAbs. Notably, a synergistic effect was observed between MSLN490 and paclitaxel, as paclitaxel disrupted the immunosuppressive microenvironment within solid tumors, enhancing immune cells infiltration and improved anti-tumor efficacy. Overall, MSLN490 exhibits robust anti-tumor activity, resilience to soluble MSLN interference, and enhanced anti-tumor effects when combined with other therapies, offering a promising future for the treatment of a variety of solid tumors. This study provides a strong foundation for further exploration of MSLN490's clinical potential.

A Review of Sensing Technologies for Indoor Autonomous Mobile Robots.

As a fundamental issue in robotics academia and industry, indoor autonomous mobile robots (AMRs) have been extensively studied. For AMRs, it is crucial to obtain information about their working environment and themselves, which can be realized through sensors and the extraction of corresponding information from the measurements of these sensors. The application of sensing technologies can enable mobile robots to perform localization, mapping, target or obstacle recognition, and motion tasks, etc. This paper reviews sensing technologies for autonomous mobile robots in indoor scenes. The benefits and potential problems of using a single sensor in application are analyzed and compared, and the basic principles and popular algorithms used in processing these sensor data are introduced. In addition, some mainstream technologies of multi-sensor fusion are introduced. Finally, this paper discusses the future development trends in the sensing technology for autonomous mobile robots in indoor scenes, as well as the challenges in the practical application environments.

Long-term passaging of pseudo-typed SARS-CoV-2 reveals the breadth of monoclonal and bispecific antibody cocktails.

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses challenges to the effectiveness of neutralizing antibodies. Rational design of antibody cocktails is a realizable approach addressing viral immune evasion. However, evaluating the breadth of antibody cocktails is essential for understanding the development potential. Here, based on a replication competent vesicular stomatitis virus model that incorporates the spike of SARS-CoV-2 (VSV-SARS-CoV-2), we evaluated the breadth of a number of antibody cocktails consisting of monoclonal antibodies and bispecific antibodies by long-term passaging the virus in the presence of the cocktails. Results from over two-month passaging of the virus showed that 9E12 + 10D4 + 2G1 and 7B9-9D11 + 2G1 from these cocktails were highly resistant to random mutation, and there was no breakthrough after 30 rounds of passaging. As a control, antibody REGN10933 was broken through in the third passage. Next generation sequencing was performed and several critical mutations related to viral evasion were identified. These mutations caused a decrease in neutralization efficiency, but the reduced replication rate and ACE2 susceptibility of the mutant virus suggested that they might not have the potential to become epidemic strains. The 9E12 + 10D4 + 2G1 and 7B9-9D11 + 2G1 cocktails that picked from the VSV-SARS-CoV-2 system efficiently neutralized all current variants of concern and variants of interest including the most recent variants Delta and Omicron, as well as SARS-CoV-1. Our results highlight the feasibility of using the VSV-SARS-CoV-2 system to develop SARS-CoV-2 antibody cocktails and provide a reference for the clinical selection of therapeutic strategies to address the mutational escape of SARS-CoV-2.

Tumour inhibitory activity on pancreatic cancer by bispecific nanobody targeting PD-L1 and CXCR4.

BACKGROUND: Antibodies and derivative drugs targeting immune checkpoints have been approved for the treatment of several malignancies, but there are fewer responses in patients with pancreatic cancer. Here, we designed a nanobody molecule with bi-targeting on PD-L1 and CXCR4, as both targets are overexpressed in many cancer cells and play important roles in tumorigenesis. We characterized the biochemical and anti-tumour activities of the bispecific nanobodies in vitro and in vivo. METHODS: A nanobody molecule was designed and constructed. The nanobody sequences targeting PD-L1 and CXCR4 were linked by the (G4S)3 flexible peptide to construct the anti-PD-L1/CXCR4 bispecific nanobody. The bispecific nanobody was expressed in E. coli cells and purified by affinity chromatography. The purified nanobody was biochemically characterized by mass spectrometry, Western blotting and flow cytometry to confirm the molecule and its association with both PD-L1 and CXCR4. The biological function of the nanobody and its anti-tumour effects were examined by an in vitro tumour cell-killing assay and in vivo tumour inhibition in mouse xenograft models. RESULTS: A novel anti-PD-L1/CXCR4 bispecific nanobody was designed, constructed and characterized. The molecule specifically bound to two targets on the surface of human cancer cells and inhibited CXCL12-induced Jurkat cell migration. The bispecific nanobody increased the level of IFN-γ secreted by T-cell activation. The cytotoxicity of human peripheral blood mononuclear cells (hPBMCs) against pancreatic cancer cells was enhanced by the molecule in combination with IL-2. In a human pancreatic cancer xenograft model, the anti-PD-L1/CXCR4 nanobody markedly inhibited tumour growth and was superior to the combo-treatment by anti-PD-L1 nanobody and anti-CXCR4 nanobody or treatment with atezolizumab as a positive control. Immunofluorescence and immunohistochemical staining of xenograft tumours showed that the anti-tumour effects were associated with the inhibition of angiogenesis and the infiltration of immune cells. CONCLUSION: These results clearly revealed that the anti-PD-L1/CXCR4 bispecific nanobody exerted anti-tumour efficacy in vitro and inhibited tumour growth in vivo. This agent can be further developed as a therapeutic reagent to treat human pancreatic cancer by simultaneously blocking two critical targets.

Effects of less invasive surfactant administration versus intubation-surfactant-extubation on bronchopulmonary dysplasia in preterm infants with respiratory distress syndrome: a single-center, retrospective study from China.

BACKGROUND: This study evaluated the effects of less invasive surfactant administration (LISA) and intubation-surfactant-extubation (InSurE) on bronchopulmonary dysplasia (BPD) in preterm infants with respiratory distress syndrome (RDS). METHODS: Neonates with respiratory distress syndrome requiring surfactant, with gestational age < 32 weeks and birth weight < 1500 g admitted to our neonatal intensive care unit from January 2018 to December 2019, were retrospectively analyzed. LISA and InSurE were used independently. The incidence of BPD at 36 weeks postmenstrual age, pre-discharge mortality, and need for mechanical ventilation (MV) within 72 h of birth were compared between LISA and InSurE group. Secondary outcomes including necrotizing enterocolitis requiring surgery, retinopathy of prematurity ≥ stage 3, patent ductus arteriosus requiring medical therapy or surgery, and length of hospitalization were analyzed. RESULTS: Among the 148 included neonates, there were 46 and 102 infants in LISA group and InSurE group, respectively. There were no significant differences in BPD incidence, the severity of BPD at 36 weeks postmenstrual age, and the rate of MV within the first 72 h after birth between the two groups (P > 0.05, respectively). The incidences of necrotizing enterocolitis requiring surgery, retinopathy of prematurity ≥ stage 3, patent ductus arteriosus requiring medical therapy or surgery, and length of hospitalization did not differ significantly between the two groups (P > 0.05, respectively). CONCLUSIONS: For surfactant administration among preterm infants with respiratory distress syndrome, LISA did not decrease bronchopulmonary dysplasia and severity of BPD at 36 weeks postmenstrual age. The benefits of LISA would require further evaluations.

[Proteomics analysis of Astragalus polysaccharide on TLR4-activated lung cancer cell-derived exosomes].

Astragalus polysaccharide(APS), one of the main active components of Astragali Radix, plays an anti-tumor effect by regulating the inflammatory microenvironment of tumors. Exosomes are small extracellular vesicles with a diameter ranging from 50 to 200 nm and carry several biological components from parental cells such as nucleic acids and proteins. When combined with recipient cells, they play an important role in intercellular communication and immune response. In this study, exosomes released from H460 cells at the inflammatory state or with APS addition activated by Toll-like receptor 4(TLR4) were extracted by ultracentrifugation and characterized by Western blot, transmission electron microscopy, and nanoparticle tracking analysis. The exosomal proteins derived from H460 cells in the three groups were further analyzed by label-free proteomics, and 897, 800, and 911 proteins were identified in the three groups(Con, LPS, and APS groups), 88% of which belonged to the ExoCarta exosome protein database. Difference statistical analysis showed that the expression of 111 proteins was changed in the LPS group and the APS group(P<0.05). The biological information analysis of the differential proteins was carried out. The molecular functions, biological processes, and signaling pathways related to the differential proteins mainly involved viral processes, protein binding, and bacterial invasion of proteasome and epithelial cells. Key differential proteins mainly included plasminogen activator inhibitor-1, laminin α5, laminin α1, and CD44, indicating that tumor cells underwent systemic changes in different states and were reflected in exosomes in the inflammatory microenvironment. The analysis results also suggested that APS might affect the inflammatory microenvironment through the TLR4/MyD88/NF-κB signaling pathway or the regulation of the extracellular matrix. This study is conducive to a better understanding of the mechanism of tumor development in the inflammatory state and the exploration of the anti-inflammatory effect of APS at the exosome level.

Hyaluronan-fullerene/AIEgen nanogel as CD44-targeted delivery of tirapazamine for synergistic photodynamic-hypoxia activated therapy.

The therapeutic effect of oxygen-concentration-dependent photodynamic therapy (PDT) can be diminished in the hypoxic environment of solid tumours, the effective solution to this problem is utilising hypoxic-activated bioreduction therapy (BRT). In this research, a biocompatible HA-C60/TPENH2nanogel which can specifically bind to CD44 receptor was developed for highly efficient PDT-BRT synergistic therapy. The nanogel was degradable in acidic microenvironments of tumours and facilitated the release of biological reduction prodrug tirapazamine (TPZ). Importantly, HA-C60/TPENH2nanogel produced reactive oxygen species and consumed oxygen content in the cell to activate TPZ, leading to higher cytotoxicity than the free TPZ did. The intracellular observation of nanogel indicated that the HA-C60/TPENH2nanogel was self-fluorescence for cell imaging. This study applied PDT-BRT to design smart HA-based nanogel with targeted delivery, pH response, and AIEgen feature for efficient cancer therapy.

Nano-Hydroxyapatite Particle Brushes via Direct Initiator Tethering and Surface-Initiated Atom Transfer Radical Polymerization for Dual Responsive Pickering Emulsion.

Well-defined polymer-grafted solid inorganic nanoparticles (NPs) are imperative for practical applications in various fields based on the prerequisite of facile initiator immobilization. Direct atom transfer radical polymerization (ATRP) initiator-tethered solid NPs are described using 2-bromo-2-methylpropionic acid as a tetherable initiator. To illustrate the versatility of the proposed strategy, nano-hydroxyapatite (n-HAP) nanocrystals (NCs) were selected to demonstrate the morphology-controlled synthesis of 2-bromo-2-methylpropionate (2-BrMP) group-immobilized n-HAP (n-HAP-Br) NCs. When water was employed as the sole solvent, the continually introduced 2-BrMP groups altered the surface hydrophobic capacity of the n-HAP-Br NC and thus led to unavoidable aggregation of n-HAP-Br NCs. The synthesis of individually dispersed n-HAP-Br NCs was achieved by rational adjusting polarity of the aqueous medium through adding a portion of water-miscible organic solvents. The type and concentration of added water-miscible organic solvents had critical effects on the morphology and particle size of n-HAP-Br NCs. To verify the efficiency of the tethered initiator, n-HAP-g-poly2-(dimethylamino) ethyl methacrylate (n-HAP-g-PDMAEMA), n-HAP-g-polyacrylonitrile (n-HAP-g-PAN), and n-HAP-g-polymethyl methacrylate (n-HAP-g-PMMA) were fabricated by surface-initiated ATRP (SI-ATRP). Acting as a solid particle emulsifier, the designed n-HAP-g-PDMAEMA-stabilized Pickering emulsion displayed dual pH and temperature response with reversible behaviors. This work presents a versatile and simple way for the fabrication of initiator-immobilized solid NPs (e.g., n-HAP NCs, gibbsite nanoplatelets, and γ-FeOOH nanofibers) ready for polymer grafting and thus enables promising performance in widespread applications.

[Remote monitoring of neonatal jaundice in newborns with ABO hemolytic disease].

OBJECTIVE: To explore the feasibility of remote monitoring of neonatal jaundice in newborns with ABO hemolytic disease. METHODS: Forty six neonates of gestational age >35 weeks with ABO hemolytic disease admitted to Women's Hospital, Zhejiang University School of Medicine from January 20th, 2020 to February 29th, 2020 were enrolled in the study (study group). The newborns were followed up at home after discharge, the transcutaneous bilirubin (TCB) levels were measured by parents using the provided device and the results were sent to the doctor by smart phone using the installed APP. Fifty six newborns with ABO hemolytic disease admitted in 2018 who received conventional outpatient follow-up after discharge served as the control group. The demographic characteristics, total serum bilirubin (TSB) level during hospitalization, number of outpatient visit and rate of re-admission due to rebound hyperbilirubinemia were compared between the two groups. RESULTS: There were no significant differences between the two groups in gestational age, birth weight, delivery mode, gender, length of the first hospitalization, TSB level before phototherapy and before discharge, and the managements during the first hospitalization (all P>0.05). Compared with the control group, TSB level before readmission [(265±16) µmol/L vs. (295±15) µmol/L] and the number of outpatient visits (1.3±0.8 vs. 3.8±0.5) were significantly lower in the study group (all P<0.01), while the rate of readmission (17.4%vs. 12.5%) and the weight at the time of readmission[(3398±452) g vs. (3477±324) g] were not significantly different (all P>0.05). No cases of acute bilirubin encephalopathy occurred in both groups. CONCLUSIONS: The remote follow-up for neonatal jaundice at home can effectively reduce the number of outpatient visits without increasing the risk of readmission and severe neonatal hyperbilirubinemia for newborns with ABO hemolytic disease.

Unexpectedly High Adsorption Capacity of Esterified Hydroxyapatite for Heavy Metal Removal.

Nanohydroxyapatite (n-HAP) as an environmentally friendly adsorbent of heavy metal ions still requires the rational design of the pore structure and surface characteristic for enhancing their adsorption capacity toward heavy metal ions. A novel one-step strategy was developed to regulate the pore structure and surface characteristic of esterified HAP (n-EHAP) nanocrystals (NCs) for enhancing the adsorption capacity by incorporation of 2-bromo-2-methylpropionate (2-BrMP) groups on the surface of n-EHAP NCs. When using water as the sole solvent, the aggregation of n-EHAP NCs became unavoidable because of incorporation of hydrophobic 2-BrMP groups on n-HAP particle surfaces. The synthesis of uniform and individual n-EHAP NCs was achieved by rational adjustment of the aqueous dispersion medium to avoid agglomeration and precipitation, which was induced by the changing surface characteristic of n-EHAP NCs during the continuing incorporation of hydrophobic 2-BrMP groups in the water/acetone system. The successful incorporation of hydrophobic 2-BrMP groups on the surface of n-EHAP NCs was characterized by X-ray powder diffraction, field-emission scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and liquid nitrogen adsorption isotherms. To explore the potential application in water treatment, a series of systematically designed batch experiments were conducted to determine the influences of the adsorbent dosage, solution pH, and contact time on the adsorption behavior of n-EHAP NCs. Experimental results indicated that the addition of water-soluble acetone greatly promoted the formation of individual n-EHAP NCs without aggregation, and furthermore, the successful incorporation of hydrophobic 2-BrMP groups led to formation of porously structured n-EHAP NCs with a higher surface area and an increasing micro-/mesopore ratio. Compared with pristine n-HAP, n-EHAP NCs exhibited lower crystallinity with smaller crystallite size and demonstrated an ultrahigh adsorption capacity for Pb(II) in acidic solution with a record of close to 2400 mg/g. The improved performance of n-EHAP NCs originated from both the suitable porous structure with a higher micro-/mesoporosity ratio and the existing tethered 2-BrMP group-induced the ester bond, providing more adsorption active affinity sites for heavy metal ions. The highly efficient adsorption (99.99%) was further achieved using tap water spiked with traces of Pb(II) (63 ppb). The presented findings promise the application of n-EHAP NCs in water treatment as an alternative, low-cost, and ecofriendly adsorbent for environmental remediation.

Extravascular modified lipoproteins: a role in the propagation of diabetic retinopathy in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: We aimed to determine whether plasma lipoproteins, after leakage into the retina and modification by glycation and oxidation, contribute to the development of diabetic retinopathy in a mouse model of type 1 diabetes. METHODS: To simulate permeation of plasma lipoproteins into retinal tissues, streptozotocin-induced mouse models of diabetes and non-diabetic mice were challenged with intravitreal injection of human 'highly-oxidised glycated' low-density lipoprotein (HOG-LDL), native- (N-) LDL, or the vehicle PBS. Retinal histology, electroretinography (ERG) and biochemical markers were assessed over the subsequent 14 days. RESULTS: Intravitreal administration of N-LDL and PBS had no effect on retinal structure or function in either diabetic or non-diabetic animals. In non-diabetic mice, HOG-LDL elicited a transient inflammatory response without altering retinal function, but in diabetic mice it caused severe, progressive retinal injury, with abnormal morphology, ERG changes, vascular leakage, vascular endothelial growth factor overexpression, gliosis, endoplasmic reticulum stress, and propensity to apoptosis. CONCLUSIONS/INTERPRETATION: Diabetes confers susceptibility to retinal injury imposed by intravitreal injection of modified LDL. The data add to the existing evidence that extravasated, modified plasma lipoproteins contribute to the propagation of diabetic retinopathy. Intravitreal delivery of HOG-LDL simulates a stress known to be present, in addition to hyperglycaemia, in human diabetic retinopathy once blood-retinal barriers are compromised.

Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy.

AIMS/HYPOTHESIS: Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects. METHODS: Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein-LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 µmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis. RESULTS: Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100-200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25-200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis. CONCLUSIONS: Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

Exogenous IL-1Ra attenuates intestinal mucositis induced by oxaliplatin and 5-fluorouracil through suppression of p53-dependent apoptosis.

Chemotherapy-induced intestinal mucositis (CIM) is a major dose-limiting side effect of many chemoagents, resulting in weight loss, diarrhea, and even death. The current treatments for CIM are palliative and have limited benefit. Interleukin-1 receptor antagonist is a natural antagonist of interleukin-1. Our previous studies showed the protective effect of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) on the intestine in mice after 5-fluorouracil chemotherapy. In this study, we further evaluated rhIL-1Ra in the treatment of CIM induced by different chemoagents and their combination. Normal as well as tumor-bearing mice were administered oxaliplatin (L-OHP), 5-fluorouracil, or their combination to induce intestinal mucositis and mortality. rhIL-1Ra administered after the chemotherapy, but not after the onset of diarrhea, significantly improved mouse survival, attenuated body weight loss, and reduced the incidence, severity, and duration of diarrhea. Histological examination showed that rhIL-1Ra-treated mice had a relatively intact mucosa structure, more proliferating crypt cells, and higher acid mucin content than the vehicle-treated mice. rhIL-1Ra suppressed crypt apoptosis by reducing the levels of proapoptotic proteins in wild-type, but not in IL-1RI or p53 mice. In addition, rhIL-1Ra was as effective as octreotide acetate in the treatment of chemotherapy-induced diarrhea, but with the advantage of reducing the epithelial apoptosis, the major cause of CIM. Importantly, the tumor sensitivity to chemotherapy was not affected by rhIL-1Ra. Thus, our data strongly suggest that rhIL-1Ra may be useful for the treatment of intestinal mucositis and improving the quality of life for cancer patients on chemotherapy.

A pilot study of less invasive surfactant administration in very preterm infants in a Chinese tertiary center.

BACKGROUND: Less invasive surfactant administration (LISA) to spontaneously breathing preterm infants has been reported to reduce the duration of mechanical ventilation and the incidence of bronchopulmonary dysplasia (BPD) in previous study. The objective of this study was to explore the feasibility and potential benefits of LISA in early preterm infants on nasal continuous positive airway pressure (nCPAP) compared to conventional endotracheal instillation. METHODS: All infants with respiratory distress born at 28-32 weeks' gestational age from January 2012 to December 2012 (n=90), who were eligible for exogenous pulmonary surfactant (PS) therapy were randomized to receive PS by intubation with an endotracheal tube (Intubation group, n=43), or by intubation using a catheter while on nCPAP (LISA group, n=47). Respiratory indices were recorded every 30 seconds during PS administration, and every 1 hour thereafter for the first day. The rate of mechanical ventilation (MV) in the first 72 hours, mean duration of both MV and nCPAP, mean duration of oxygen requirement and neonatal outcomes were recorded. RESULTS: PS was successfully administered in 43 (100%) out of 43 babies using the conventional approach and in 46 (97%) out of 47 babies using LISA. The duration of both MV and nCPAP was significantly shorter in LISA group, when compared with intubation group. However, there were no significant differences in both the rate of MV in the first 72 hours and mean duration of oxygen requirement. There were also no differences in the mortality or in the incidence of bronchopulmonary dysplasia, intraventricular hemorrhage, retinopathy of prematurity and necrotizing enterocolitis, or in the duration of respiratory support. CONCLUSIONS: LISA in spontaneously breathing infants on nCPAP is an alternative therapy for PS delivery, avoiding intubation with an endotracheal tube. The method is feasible and potentially effective, and deserves further clinical trials. TRIAL REGISTRATION: Current Controlled Trials ChiCTR-ICR-15006001. Registered 20 February 2015.

Development of a cell-based qualitative assay for detection of neutralizing anti-human interleukin-1 receptor antagonist (hIL-1Ra) antibodies in rats.

To determine the incidence of the positive neutralizing anti-human interleukin receptor antagonist (anti-IL-1Ra), a novel assay based on the proliferation of human melanoma A375.S2 cells was developed and validated. In the presence of a growth-limiting concentration of IL-1ß, A375.S2 cells were able to regain proliferation following the addition of IL-1Ra in a concentration-dependent manner. This dose-response effect enabled the validation of a standard curve for calculation of the concentration of IL-1Ra or, inversely, the concentration of neutralizing anti-IL-1Ra antibodies in cell culture medium or sera. The assay used CCK-8 as an indicator of proliferation. The dose-response relationship between rhIL-1Ra (dose range of 5-75 ng/ml rhIL-1Ra) and A375.S2 cell proliferation was sigmoidal and fitted a four-parameter logistic model. The percent coefficients of variation (%CVs) of quality control samples were 12.5 and 11.9% for intra-assay repeatability and 14.5 and 19.5% for inter-assay repeatability, while the total accuracy was in the range of 97.2-103.6%. For the neutralization assay, the optimal sample dilution factor was found to be 40-fold and the reasonable standard for positive and negative decision was calculated to be 59.4% neutralization rate. The %CVs of quality control samples were 12.7 and 24.0% for intra-assay repeatability and 11.6 and 30.0% for inter-assay repeatability. Analysis using the assay showed that rats could produce neutralizing anti-IL-1Ra antibodies after repeated intramuscular injection with rhIL-1Ra, and this response was not significantly dependent on the dose injected.

Immune complex formation in human diabetic retina enhances toxicity of oxidized LDL towards retinal capillary pericytes.

Recently it has been shown that levels of circulating oxidized LDL immune complexes (ox-LDL-ICs) predict the development of diabetic retinopathy (DR). This study aimed to investigate whether ox-LDL-ICs are actually present in the diabetic retina, and to define their effects on human retinal pericytes versus ox-LDL. In retinal sections from people with type 2 diabetes, costaining for ox-LDL and IgG was present, proportionate to DR severity, and detectable even in the absence of clinical DR. In contrast, no such staining was observed in retinas from nondiabetic subjects. In vitro, human retinal pericytes were treated with native LDL, ox-LDL, and ox-LDL-IC (0-200 mg protein/l), and measures of viability, receptor expression, apoptosis, endoplasmic reticulum (ER) and oxidative stresses, and cytokine secretion were evaluated. Ox-LDL-IC exhibited greater cytotoxicity than ox-LDL toward retinal pericytes. Acting through the scavenger (CD36) and IgG (CD64) receptors, low concentrations of ox-LDL-IC triggered apoptosis mediated by oxidative and ER stresses, and enhanced inflammatory cytokine secretion. The data suggest that IC formation in the diabetic retina enhances the injurious effects of ox-LDL. These findings offer new insights into pathogenic mechanisms of DR, and may lead to new preventive measures and treatments.

Activated expression of the chemokine Mig after chemotherapy contributes to chemotherapy-induced bone marrow suppression and lethal toxicity.

Alterations in gene expression after chemotherapy may potentially help to identify mediators that induce suppression or regeneration in bone marrow. This paper reports our observation that the expression of the chemokine monokine induced by IFN-γ (Mig) and its receptor CXCR3 was significantly activated in mice after treatment with the chemotherapeutic agent 5-fluorouracil (5-FU). The neutralization of antibodies against the activated Mig increased the survival rate and accelerated BM recovery after chemotherapy. In addition, elevation of Mig plasma levels after 5-FU treatment corresponded with increased mortality. The cell cycle-inhibiting effect of the prophylactic administration of Mig protected hematopoietic progenitor cells (HPCs) from 1-ß-d-arabinofuranosylcytosine in spleen colony assays and enhanced the irradiated recipients' survival. In CXCR3(-/-) mice, Mig did not propagate BM suppression, indicating that the suppressive effect of Mig is dependent on CXCR3. On the one hand, Mig stimulated p70 S6K and Erk1/2 pathways in mesenchymal stroma cells, inhibiting mesenchymal stroma cell-dependent HPC expansion. Moreover, Mig suppressed the STAT5 pathway in HPCs, inhibiting leukocyte differentiation. Our results strongly suggest that Mig contributes to the acute lethal toxicity arising from 5-FU administration. Neutralization of Mig may offer new strategies to alleviate BM toxicity with potentially dramatic implications for chemotherapy.

How important is differentiation in the therapeutic effect of mesenchymal stromal cells in liver disease?

BACKGROUND AIMS: The protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stromal cells (MSCs) have been well established. Previous data have shown that MSCs and their derived HLCs were able to engraft injured liver and alleviate injuries induced by carbon tetrachloride. The goal of the current study was to determine the differences of MSCs and their derived HLCs in terms of therapeutic functions in liver diseases. METHODS: After hepatic differentiation of umbilical cord-derived MSCs in vitro, we detected both MSC and HLC expressions of adhesion molecules and chemokine receptor CXCR4 by flow cytometry; immunosuppressive potential and hepatocyte growth factor expression were determined by means of enzyme-linked immunosorbent assay. We compared the therapeutic effect for fulminant hepatic failure in a mouse model. RESULTS: MSC-derived-HLCs expressed lower levels of hepatocyte growth factor, accompanied by impaired immunosuppression in comparison with MSCs. Furthermore, undifferentiated MSCs showed rescuing potentials superior to those in HLCs for the treatment of fulminant hepatic failure. CONCLUSIONS: After differentiation, HLCs lost several major properties in comparison with undifferentiated MSCs, which are beneficial for their application in liver diseases. Undifferentiated MSCs may be more appropriate than are HLCs for the treatment of liver diseases.

IL-1Ra selectively protects intestinal crypt epithelial cells, but not tumor cells, from chemotoxicity via p53-mediated upregulation of p21(WAF1) and p27(KIP1.).

Chemotherapy-induced intestinal mucositis (CIM) is a major dose-limiting side effect, resulting from the nonspecific cytoablative actions of chemoagents, including 5-fluorouracil (5-FU) and irinotecan (CPT-11). Preventive strategies are urgently needed for the predictable CIM. Previously, we have demonstrated an important role of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) in the prevention of cyclophosphamide-induced mucositis in mice. In this study, the preventive role of rhIL-1Ra was further evaluated in 5-FU- and CPT-11-induced mucositis mouse models. rhIL-1Ra pretreatment reduced the incidence, severity, and duration of chemotherapy-induced diarrhea, through attenuating crypt apoptosis and improving crypt survival in wild-type mice, but not in IL-1RI(-/-), p53(-/-), and p21(-/-) mice. Further studies demonstrated that rhIL-1Ra promoted the cell cycle arrest of intestinal crypt epithelia (ICE) through elevating the cellular level of p21(WAF1) and p27(KIP1), which was abolished in IL-1RI(-/-) and p53(-/-) mice, and in p21(WAF1) and p27(KIP1) silenced IEC-6 cells. Importantly, the tumor growth and sensitivity to chemotherapy were not affected by rhIL-1Ra in cultures of tumor cell lines and in a syngeneic tumor-transplantation mouse model. The present study demonstrated that rhIL-1Ra effectively and specifically protected ICE from chemotoxicity through reversible reduction of the basal level of IL-1 signaling to promote normal cell cycle arrest, but not tumor cells. Our findings support the clinical development of rhIL-1Ra in the prevention of CIM.

The therapeutic effect of rhMK on osteoarthritis in mice, induced by destabilization of the medial meniscus.

Osteoarthritis (OA) is a worldwide disease in aged people, causing not only physical suffering to the patients themselves, but also a great burden on their families and on society. Here we used a mouse OA model induced by destabilization of the medial meniscus (DMM), and studied the therapeutic effect of recombinant human midkine (rhMK) on this OA model. Our results indicated that the DMM surgery induced mechanical allodynia and locomotor activity obstacles, together with cartilage injury in the C57BL/6 mice. The rhMK treatment mitigated the OA related mechanical allodynia, improved locomotor activity capacity, and prevented degradation of the cartilage. Considering the safety issue of rhMK used as a biologic, we also inspected the main organs in the rhMK treated mice throughout the process and found no pathological change. These results suggest that rhMK could be used as a biologic to treat OA or OA related pain.