The study of the role of purified anti-mouse CD193 (CCR3) antibody in allergic rhinitis mouse animal models.

The pathogenesis of allergic asthma is similar to that of allergic rhinitis, with inflammation cells producing and releasing inflammatory mediators and cytokines closely related to CCR3.Based on the theory of "one airway, one disease", the use of CCR3 monoclonal antibody may have a similar effect on allergic rhinitis. However, there are few studies on CCR3 monoclonal antibody in allergic rhinitis. Therefore, the aim of this study was to investigate the effective concentration of CCR3 monoclonal antibody, to compare the effects of different methods of administration, and to examine the lung condition of allergic mice to investigate whether antibody treatment protects the lungs. In this study, we constructed a mouse model of allergic rhinitis and intraperitoneally injected different doses of CCR3 monoclonal antibody (5, 10, and 20 uL/mg) to observe its therapeutic effect: observing changes in tissue morphology of nasal mucosa, infiltration of inflammation, and using ELISA to detect changes in relevant inflammatory mediators and cytokines, studying the role of CCR3 mAb in inhibiting CCR3-related actions on the nasal mucosa of allergic rhinitis mice. Furthermore, In addition, the therapeutic effects of intraperitoneal injection (i.p.) and intranasal administration (i.n.) were studied on the basis of effective concentrations.

Experimental observation of the effect of immunotherapy on CD4+ T cells and Th1/Th2 cytokines in mice with allergic rhinitis.

The present study aims to investigate the effect of immunotherapy in a mouse model of allergic rhinitis (AR) and to explore the possible molecular mechanisms of action. An animal model of AR was established by sensitization and challenge of BALB/c mice with house dust mite (HDM) extract. The mice were injected subcutaneously with HDM for immunotherapy. AR nasal symptoms were evaluated according to the frequencies of nose rubbing and sneezing and the degree of rhinorrhea. The nasal mucosa and lung tissue architecture and inflammatory status by histological analysis; the infiltration of eosinophils in nasal lavage fluid (NALF) of mice was observed by Diff-Quik stain; ELISA-based quantification of serum HDM-specific IgE and TH1/TH2 cytokine concentration; and flow cytometry detected the number of serum CD4+/CD8+ cells to evaluate the mechanism of immunotherapy. It was found that after immunotherapy, the AR symptom score was reduced, the number of eosinophils in NALF was reduced, and the infiltration of inflammatory cells and tissue damage in the nasal mucosa and lung tissue were alleviated. Immunotherapy can increase the number of CD4+ T cells in the peripheral blood, increase the ratio of CD4+/CD8+ cells, increase the expression of Th1 cytokines such as IL-2 and IFN-γ, reduce the expression of Th2 cytokines such as IL-4 and IL-5. The results showed that repeated intraperitoneal injection of crude extract of HDM for sensitization, followed by nasal drops can effectively construct a mouse model of AR, and subcutaneous injection of immunotherapy in mice can reduce allergic inflammation in model mice and improve the inflammatory infiltration of the nasal cavity in allergic rhinitis. Immunotherapy can reduce the expression of inflammatory factors in AR, improve Th1/Th2 balance, and may play a role in the treatment of AR by improving the function of immune cells.

Hydroxysafflor Yellow A-Induced Osteoblast Differentiation and Proliferation of BM-MSCs by Up-Regulating Nuclear Vitamin D Receptor.

BACKGROUND: Vitamin D receptor (VDR) is critical for mineral and bone homeostasis since it plays an essential role in the osteoblast differentiation of bone marrow mesenchymal stem cells (BM-MSCs). Hydroxysafflor yellow A (HSYA) has the potential to promote bone mineralization and inhibit bone resorption, while its detailed mechanism needs to be elaborated. OBJECTIVE: This study intends to explore the action of HSYA on the proliferation and differentiation of BM-MSC and the underlying mechanism. METHODS: Different concentrations of HSYA to BM-MSC and CCK-8, and EdU were used to detect cell viability and proliferation. The alkaline phosphatase (ALP) was used to observe the differentiation ability of BM-MSC osteoblasts. The calcium uptake and mineralization of osteoblast-like cells were observed by alizarin red staining. The level of calcium ion uptake in cells was detected by flow cytometry. AutoDock was performed for molecular docking of HSYA to VDR protein. Immunofluorescence and western blotting were performed to detect the expression of VDR expression levels. Finally, the effect of VDR was verified by a VDR inhibitor. RESULTS: After treatment with HSYA, the proliferation and calcium uptake of BM-MSC were increased. The level of ALP increased significantly and reached its peak on the 12th day. HSYA promoted calcium uptake and calcium deposition, and mineralization of osteoblasts. The western blotting and immunofluorescence showed that HSYA increased the expression of VDR in the osteoblast-like cell's nucleus and upregulated Osteocalcin, S100 calcium-binding protein G, and CYP24A1. In addition, HYSA treatment increased the expression of osteopontin and the synthesis of osteogenic proteins, such as Type 1 collagen. After the addition of the VDR inhibitor, the effect of HSYA was weakened. CONCLUSION: HSYA could significantly promote the activity and proliferation of osteoblasts and increase the expression level of VDR in osteoblasts. HSYA may also improve calcium absorption by osteoblasts by regulating the synthesis of calciumbinding protein and vitamin D metabolic pathway-related proteins.

[Diagnosis a fetus with Coffin-Siris syndrome due to variant of SMARCA4 gene by whole exome sequencing].

OBJECTIVE: To explore the clinical phenotype and genetic basis for a fetus suspected for Coffin-Siris syndrome. METHODS: Chromosomal microarray analysis (CMA) and whole exome sequencing (WES) were carried out for the fetus. Candidate variant was verified by Sanger sequencing. RESULTS: Prenatal ultrasound at 23rd gestational week has revealed fetal ventriculomegaly. No abnormality was found by CMA, while WES revealed that the fetus has harbored a de novo heterozygous c.2851G>A (p.G951R) variant of the SMARCA4 gene, which was predicted to be pathogenic. CONCLUSION: Genetic testing should be considered for fetuses featuring progressive widening of lateral cerebral ventricles.

Role of Chemokines and Inflammatory Cells in Respiratory Allergy.

The idea of "one airway, one disease" has been gaining importance in the last decade. In the upper and lower airways, allergic mechanisms interact with each other. In the initial stage of respiratory allergic inflammation, allergens contact the respiratory epithelium, which produces chemokines and inflammatory factors, which cause allergic reactions by binding to the corresponding receptors and chemotactic various inflammatory cells to reach the epithelium and tissues. It also drives inflammatory cells to activate and produce more inflammatory factors, thus producing a cascade amplification effect. Inflammatory cell aggregation and activation are very complex and interact with each other in a lattice structure. By blocking the action of various chemokines, inflammatory cell aggregation is reduced, and ultimately the symptoms of respiratory allergy are alleviated. Chemokines can serve as cues for coordinated recruitment of immune cells into and out of tissues, as well as directing the spatial organization of immune cells within tissues and cellular interactions. Chemokines are critical in directing immune cell migration and thus have an important role in the direction of respiratory allergy: however, chemokines are also involved in the production and recruitment of immune cells that contribute to respiratory allergy. In this article, linking the upper and lower respiratory tracts. We review the role of the chemokine system in the respiratory immune response and discuss how respiratory disease modulates overall chemokines to shape the type and outcome of the immune response to the treatment of respiratory allergic disease so that we can further deepen our knowledge of chemokines in the direction of respiratory allergy. In the future, we can do drug research and development based on this network structure and explore new research directions.

[Enlarged multicystic dysplastic kidneys with oligohydramnios during infancy caused by NPHP3 gene mutation].

OBJECTIVE: To explore the clinical features and genomic abnorm ality of a fetus enlarged multicystic dysplastic kidneys with oligohydramnios caused by NPHP3 gene mutation. METHODS: The fetuse was found to have multicystic dysplastic kidneys with oligohydramnios upon ultrasonography during the second trimester. Following induced abortion, fetal tissue was collected for the extraction of DNA, chromosomal microarray analysis (CMA) and whole exome sequencing (WES). Sanger sequencing was used to verify the suspected variants in the family. RESULTS: Antenatal ultrasound examination at 19 weeks showed "polycystic" kidneys with Oligohydramnios. Delivery was by induced labour because of the critically low amniotic fluid volume. Testing of CMA was normal. WES showed a compound heterozygous mutation of c.1817G>A, p.W606X; c.432dupA, p.E145Rfs*18 mutations are novel mutations in this study. CONCLUSION: The research may further expand the NPHP3 gene mutation spectrum. Enlarged multicystic dysplastic kidneys with oligohydramnios caused by NPHP3 gene mutation at least include one or two splice site mutation, frameshift mutation or nonsense mutation foetal poor prognosis.

Gene knockdown of CCR3 reduces eosinophilic inflammation and the Th2 immune response by inhibiting the PI3K/AKT pathway in allergic rhinitis mice.

The CCR3 gene plays a critical role in allergic airway inflammation, such as allergic rhinitis (AR), and there is an inflammatory signal link between the nasal cavity and the CCR3 gene in bone marrow. However, the effects of the CCR3 gene in bone marrow cells on AR are not clear. The present study investigated the roles and underlying mechanisms of the bone marrow CCR3 gene in AR mice. Conditional knockout of the bone marrow CCR3 gene (CKO) in mice was generated using the Cre-LoxP recombination system, and offspring genotypes were identified using polymerase chain reaction (PCR). An ovalbumin-induced AR model was established in CKO and wild-type mice to measure eosinophilic inflammation and the Th2 immune response. The following mechanisms were explored using a specific PI3K/AKT pathway inhibitor (Ly294002). We successfully constructed and bred homozygous CKO mice and confirmed a significant increase in CCR3 expression and PI3K/AKT pathway activity in AR mice. Deficiency of the bone marrow CCR3 gene caused a remarkable reduction of CCR3 expression and the PI3K/AKT signaling pathway activity, inhibited histopathological lesions and eosinophil infiltration of the nasal cavity, and reduced the production of Th2 cytokines in serum, which led to the remission of allergic symptoms in AR mice. Ly294002 treatment also decreased these inflammatory indexes in a concentration-dependent manner and blocked inflammatory signals from CCR3, but it did not affect the high expression of CCR3 in AR mice. Collectively, our results suggest that conditional knockout of the bone marrow CCR3 gene can reduce eosinophilic inflammation and the Th2 immune response, which may be due to inhibition of the PI3K/AKT pathway.

[Analysis of TUBB2B gene variant in a fetus with complex cortical dysplasia with other brain malformations-7].

OBJECTIVE: To explore the genetic basis for a fetus with dysgenesis of corpus callosum and other brain malformations. METHODS: Whole exome sequencing was carried out for the fetus and its parents. Suspected pathogenic variants were verified by Sanger sequencing. RESULTS: A novel de novo missense variant c.758T>A (p.L253Q) of the TUBB2B gene was identified, which was unreported previously. Based on the guidelines from the American College of Medical Genetics, the c.758T>A variant was predicted to be likely pathogenic. Bioinformatics analysis predicted that the leucine at position 253 was highly conserved among various species, and the c.758T>A variant may impact the formation of hydrogen bonds between Leu253 and Asp249 and Met257 residues, which in turn may affect the combination of GTP/GDP and function of the TUBB2B protein. CONCLUSION: The c.758T>A variant of the TUBB2B gene probably underlay the fetal malformations in this Chinese family. Above discovery has enriched the spectrum of TUBB2B gene variants and provided a basis for genetic counseling and prenatal diagnosis.

CCR3 gene knockout in bone marrow cells ameliorates combined allergic rhinitis and asthma syndrome (CARAS) by reducing airway inflammatory cell infiltration and Th2 cytokines expression in mice model.

The present study aims to investigate the effects of CCR3 gene knockout in bone marrow cells (CCR3-KO) on the mouse model of combined allergic rhinitis and asthma syndrome (CARAS). It was found that CCR3-KO significantly reduced eosinophil (EOS) migration into the nasal (NALF) and bronchoalveolar (BALF) cavities of mice, and decreased Th2 cytokines (such as, IL-4, IL-5 and IL-13) levels in nasal mucosa and lung tissues. In addition, histological analysis showed that the damage degree of nasal mucosa structure in ovalbumin (OVA) modulated CCR3-KO mice was significantly less than that in OVA modulated Wild type (WT) mice, with reduced inflammatory cell infiltration and nasal mucus secretion. The infiltration of inflammatory cells in lung tissue was significantly reduced, and the proliferation of lung smooth muscle layer and extracellular matrix (ECM) production were decreased. Symptom analysis showed that CCR3-KO can reduced allergic rhinitis (AR) signals as nose scratching and sneezing. It was also found CCR3-KO reduce OVA-induced weight loss. The results showed that CCR3-KO could reduce the symptoms of allergic inflammation in CARAS mice by reducing airway inflammatory cell infiltration and down-regulating the expression of Th2 cytokines, and CCR3 gene could be used as a target gene for the treatment of CARAS.

Molecular genotyping of G6PD mutations for neonates in Ningbo area.

The aim of this study is to determine the cut-off value of glucose-6-phosphate dehydrogenase (G6PD) activity and the mutation spectrum of G6PD gene in neonates with G6PD deficiency at Ningbo. Around 82233 neonatal blood samples were measured to determine G6PD activity. The positive samples were further detected with gene analysis. A total of 445 neonates were confirmed as G6PD deficiency, and the incidence in Ningbo was 1/185. 17 types of G6PD gene mutations were found, including 11 single-site mutations and 6 double-site mutations. Considering the significant differences in G6PD activity, the cut-off value was detected to be 2.35 and 3.65 U/gHb for males and females, respectively. Significant differences in G6PD activities were noted and found to be varied from 4.61 to 6.02 U/gHb in different seasons (p < 0.0001). G6PD deficiency screening is a significant detection test for neonatal G6PD deficiency prevention. Our study highlights that the screening should be done using different cut-off values according to the sexes in different seasons.

[Study on newborn screening for Duchenne muscular dystrophy and diagnostic strategy].

OBJECTIVE: To establish a newborn screening system for Duchenne muscular dystrophy (DMD) through assessment of MM isoenzyme of creatine kinase (CK-MM) activity. METHODS: The CK-MM level was detected using dry blood spot filter paper from 10 252 male newborns. The results were grouped based on their gestational age, sampling time and intervals between the experiments. The threshold value for CK-MM necessitating genetic testing was determined. Next-generation sequencing (NGS) was carried out for those with a CK-MM value over the threshold, and the result was verified by multiplex ligation-dependent probe amplification (MLPA). RESULTS: Based on the result of non-parametric rank sum test, the median CK-MM concentration has increased with the gestational age, and was inversely correlated with the age of the newborns among unaffected specimens. CK-MM on dry blood spot filter paper can be stable for 14 days at 2-8℃. Statistical analysis of CK-MM value of the 10 252 neonates suggested that the threshold may be set as 700 ng/mL. Exonic deletions were found in 2 confirmed cases, whose CK-MM level was greater than 2000 ng/mL. CONCLUSION: Detection of CK-MM in dry blood spot filter paper has provided an effective method for newborn screening of DMD. This simple and inexpensive method can be used for large-scale screening, which is of great value to the early intervention and treatment of the disease.

Development of a digital breast phantom for photoacoustic computed tomography.

Photoacoustic (PA) imaging provides morphological and functional information about angiogenesis and thus is potentially suitable for breast cancer diagnosis. However, the development of PA breast imaging has been hindered by inadequate patients and a lack of ground truth images. Here, we report a digital breast phantom with realistic acoustic and optical properties, with which a digital PA-ultrasound imaging pipeline is developed to create a diverse pool of virtual patients with three types of masses: ductal carcinoma in situ, invasive breast cancer, and fibroadenoma. The experimental results demonstrate that our model is realistic, flexible, and can be potentially useful for accelerating the development of PA breast imaging technology.

Identification of Six Novel Variants of ACAD8 in Isobutyryl-CoA Dehydrogenase Deficiency With Increased C4 Carnitine Using Tandem Mass Spectrometry and NGS Sequencing.

Isobutyryl-CoA dehydrogenase deficiency (IBDHD, MIM: #611283) is a rare autosomal recessive hereditary disease, which is caused by genetic mutations of acyl-CoA dehydrogenase (ACAD) 8 and associated with valine catabolism. Here, tandem mass spectrometry (MS/MS) was applied to screen 302,993 neonates for inherited metabolic diseases (IMD) in Ningbo of China from 2017 to 2020. The results suggest that 198 newborns (0.7‰) were initially screened positive for IBDHD with C4-Carnitine, and 27 cases (0.1‰) were re-screened positive. Genetic diagnosis was performed on 21 of the 27 cases. Seven compound heterozygous variations, three biallelic variations, and one heterozygous variation of ACAD8 were found with a pathogenicity rate of 33.3% (7/21). In addition, seven biallelic variations, one heterozygous variation of acyl-CoA dehydrogenase short chain (ACADS), and one biallelic variation of acyl-CoA dehydrogenase short/branched chain (ACADSB) was detected. Further research showed that ACAD8 mutations of 11 IBDHD cases distributed in six different exons with total 14 mutation sites. Five of which were known suspected pathogenic sites (c.286G > A, c.553C > T, c.1000C > T, c.409G > A, c.500del) and six were novel mutation sites: c.911A > T, c.904C > T, c.826G > A, c.995T > C, c.1166G > A, c.1165C > T. This finding enriched the mutation spectrum of ACAD8 in IBDHD.

High-speed wide-field multi-parametric photoacoustic microscopy.

Capable of imaging blood perfusion, oxygenation, and flow simultaneously at the microscopic level, multi-parametric photoacoustic microscopy (PAM) has quickly emerged as a powerful tool for studying hemodynamic and metabolic changes due to physiological stimulations or pathological processes. However, the low scanning speed poised by the correlation-based blood flow measurement impedes its application in studying rapid microvascular responses. To address this challenge, we have developed a new, to the best of our knowledge, multi-parametric PAM system. By extending the optical scanning range with a cylindrically focused ultrasonic transducer (focal zone, 76µm×4.5mm) for simultaneous acquisition of 500 B-scans, the new system is 112 times faster than our previous multi-parametric system that uses a spherically focused transducer (focal diameter, 40 µm) and enables high-resolution imaging of blood perfusion, oxygenation, and flow over an area of 4.5×1mm2 at a frame rate of 1 Hz. We have demonstrated the feasibility of this system in the living mouse ear. Further development of this system into reflection mode will enable real-time cortex-wide imaging of hemodynamics and metabolism in the mouse brain.