Durable transgene expression and efficient re-administration after rAAV2.5T-mediated fCFTRΔR gene delivery to adult ferret lungs.

The dosing interval for effective recombinant adeno-associated virus (rAAV)-mediated gene therapy of cystic fibrosis lung disease remains unknown. Here, we assessed the durability of rAAV2.5T-fCFTRΔR-mediated transgene expression and neutralizing antibody (NAb) responses in lungs of adult wild-type ferrets. Within the first 3 months following rAAV2.5T-fCFTRΔR delivery to the lung, CFTRΔR transgene expression declined ∼5.6-fold and then remained stable to 5 months at ∼26% the level of endogenous CFTR. rAAV NAbs in the plasma and bronchoalveolar lavage fluid (BALF) peaked at 21 days, coinciding with peak ELISpot T cell responses to AAV capsid peptides, after which both responses declined and remained stable at 4-5 months post dosing. Administration of reporter vector rAAV2.5T-gLuc (gaussia luciferase) at 5 months following rAAV2.5T-fCFTRΔR dosing gave rise to similar levels of gLuc expression in the BALF as observed in age-matched reporter-only controls, demonstrating that residual BALF NAbs were functionally insignificant. Notably, the second vector administration led to a 2.6-fold greater ELISpot T cell response and ∼2.3-fold decline in fCFTRΔR mRNA and vector genomes derived from the initial rAAV2.5T-fCFTRΔR administration, suggesting selective destruction of transduced cells from the first vector dose. These findings provide insights into humoral and cellular immune response to rAAV that may be useful for optimizing gene therapy to the cystic fibrosis lung.

Hepatocyte growth factor is protective in early stage but bone-destructive in late stage of experimental periodontitis.

BACKGROUND AND OBJECTIVE: Clinical studies found high levels of hepatocyte growth factor (HGF) expression in patients with periodontitis. Studies suggest that HGF plays an important role in periodontitis, is involved in inflammation, and modulates alveolar bone integrity in periodontitis. This study aims to investigate the effects and mechanisms of HGF in the progression of experimental periodontitis. METHODS: We used silk thread ligation to induce periodontitis in HGF-overexpressing transgenic (HGF-Tg) and wild-type C57BL/6J mice. The effects of HGF overexpression on alveolar bone destruction were assessed by microcomputed tomography imaging at baseline and on days 7, 14, 21, and 28. We analyzed the cytokines (IL-6 and TNF-α) and lymphocytes in periodontitis tissues by enzyme-linked immunosorbent assay and flow cytometry. The effects of HGF on alveolar bone destruction were further tested by quantifying the systemic bone metabolism markers CTXI and PINP and by RNA sequencing for the signaling pathways involved in bone destruction. Western blotting and immunohistochemistry were performed to further elucidate the involved signaling pathways. RESULTS: We found that experimental periodontitis increased HGF production in periodontitis tissues; however, the effects of HGF overexpression were inconsistent with disease progression. In the early stage of periodontitis, periodontal inflammation and alveolar bone destruction were significantly lower in HGF-Tg mice than in wild-type mice. In the late stage, HGF-Tg mice showed higher inflammatory responses and progressively aggravated bone destruction with continued stimulation of inflammation. We identified the IL-17/RANKL/TRAF6 pathway as a signaling pathway involved in the HGF effects on the progression of periodontitis. CONCLUSION: HGF plays divergent effects in the progression of experimental periodontitis and accelerates osteoclastic activity and bone destruction in the late stage of inflammation.

Sonic Hedgehog Signaling Is Essential for Pulmonary Ionocyte Specification in Human and Ferret Airway Epithelia.

Pulmonary ionocytes express high levels of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that is critical for hydration of the airways and mucociliary clearance. However, the cellular mechanisms that govern ionocyte specification and function remain unclear. We observed that increased abundance of ionocytes in cystic fibrosis (CF) airway epithelium was associated with enhanced expression of Sonic Hedgehog (SHH) effectors. In this study, we evaluated whether the SHH pathway directly impacts ionocyte differentiation and CFTR function in airway epithelia. Pharmacological HPI1-mediated inhibition of SHH signaling component GLI1 significantly impaired human basal cell specification of ionocytes and ciliated cells but significantly enhanced specification of secretory cells. By contrast, activation of the SHH pathway effector smoothened (SMO) with the chemical agonist SAG significantly enhanced ionocyte specification. The abundance of CFTR+ BSND+ ionocytes under these conditions had a direct relationship with CFTR-mediated currents in differentiated air-liquid interface (ALI) airway cultures. These findings were corroborated in ferret ALI airway cultures generated from basal cells in which the genes encoding the SHH receptor PTCH1 or its intracellular effector SMO were genetically ablated using CRISPR-Cas9, causing aberrant activation or suppression of SHH signaling, respectively. These findings demonstrate that SHH signaling is directly involved in airway basal cell specification of CFTR-expressing pulmonary ionocytes and is likely responsible for enhanced ionocyte abundance in the CF proximal airways. Pharmacologic approaches to enhance ionocyte and reduce secretory cell specification after CFTR gene editing of basal cells may have utility in the treatment of CF.

Immunosuppression reduces rAAV2.5T neutralizing antibodies that limit efficacy following repeat dosing to ferret lungs.

The efficacy of redosing the recombinant adeno-associated virus (rAAV) vector rAAV2.5T to ferret lung is limited by AAV neutralizing antibody (NAb) responses. While immunosuppression strategies have allowed for systemic rAAV repeat dosing, their utility for rAAV lung-directed gene therapy is largely unexplored. To this end, we evaluated two immunosuppression (IS) strategies to improve repeat dosing of rAAV2.5T to ferret lungs: (1) a combination of three IS drugs (Tri-IS) with broad coverage against cellular and humoral responses (methylprednisolone [MP], azathioprine, and cyclosporine) and (2) MP alone, which is typically used in systemic rAAV applications. Repeat dosing utilized AAV2.5T-SP183-fCFTRΔR (recombinant ferret CFTR transgene), followed 28 days later by AAV2.5T-SP183-gLuc (for quantification of transgene expression). Both the Tri-IS and MP strategies significantly improved transgene expression following repeat dosing and reduced AAV2.5T NAb responses in the bronchioalveolar lavage fluid (BALF) and plasma, while AAV2.5T binding antibody subtypes and cellular immune responses by ELISpot were largely unchanged by IS. One exception was the reduction in plasma AAV2.5T binding immunoglobulin G (IgG) in both IS groups. Only the Tri-IS strategy significantly suppressed splenocyte expression of IFNA (interferon α [IFN-α]) and IL4. Our studies suggest that IS strategies may be useful in clinical application of rAAV targeting lung genetic diseases such as cystic fibrosis.

Study on the Application Value of the Content of Prostatic Exosomal Protein in the Treatment of Chronic Prostatitis.

BACKGROUND: To evaluate the application value of urinary prostatic exosomal protein (PSEP) in the treatment of chronic prostatitis (CP). METHODS: We evaluated 174 patients with chronic prostatitis (44 cases of NIH-II, 65 cases of NIH-IIIa, and 65 cases of NIH-IIIb) who had obvious symptoms of chronic prostatitis syndrome and met the diagnostic criteria of National Institutes of Health Prostatitis from May 2018 to February 2021. They were also evaluated according to the clinical treatment's effect after six weeks of treatment. Urine samples of CP patients were collected before treatment and after six weeks of treatment, and the level of PSEP in the urine samples of all patients, before and after treatment, was detected by the ELISA method to evaluate the application value of PSEP in the end of CP curative effect. RESULTS: After six weeks of treatment, the total CPSI score of CP patients decreased significantly, compared to patients before treatment. After six weeks of treatment, the PSEP content in the patients' urine was compared to before treatment. The PSEP levels of CP subgroups decreased significantly (p < 0.05): NIH-II group (1.55 ± 1.39 ng/mL vs. 3.09 ± 2.66 ng/mL); NIH-IIIa group (1.68 ± 1.06 ng/mL vs. 3.34 ± 2.69 ng/mL); and NIH-IIIb group (1.57 ± 1.17 ng/mL vs. 3.14 ± 2.81 ng/mL). CONCLUSIONS: The concentration of PSEP in the urine of CP patients has a good application value for evaluating clinical treatment's effect on chronic prostatitis, and its concentration level may affect the development and outcome of prostatitis.

An Overview of the Advances in Research on the Molecular Function and Specific Role of Circular RNA in Cardiovascular Diseases.

In recent years, the rate of residents suffering from cardiovascular disease (CVD), disability, and death has risen significantly. The latest report on CVD in China shows that it still has the highest mortality rate of all diseases in that country. Different from linear RNA, circular RNA (circRNA) is a covalently closed transcript, mainly through reverse splicing so that the 3'end and the 5'end are covalently connected to form a closed loop structure. It is structurally stable and abundant and has distinct tissue or cell specificity, and it is widely distributed in eukaryotes. Although circRNAs were discovered many years ago, researchers have only recently begun to slowly discover their extensive expression and regulatory functions in various biological processes. Studies have found that some circRNAs perform multiple functions in cells more used as RNA binding protein or microRNA sponge. In addition, accumulating evidence shows that the first change that occurs in patients with various metabolic diseases such as hypertension and cardiovascular disease is dysregulated circRNA expression. For cardiovascular and other related blood vessels, circRNA is one of the important causes of various complications. These findings contribute to a more comprehensive understanding and grasp of CVD, and the related molecular mechanisms of CVD should be further analyzed. Here, we review the new understanding of circRNAs in CVD and explain the role of these innovative biomarkers in the analysis and determination of other related cardiovascular events such as coronary heart disease. Thus, this study is aimed at providing new ideas and proposing more feasible medical research strategies based on circRNA.

Predicting prognosis and clinical features of the tumor microenvironment based on ferroptosis score in patients with breast cancer.

Ferroptosis genes have recently been reported to be involved in regulating the development of cancer, but their potential role in breast cancer (BRCA) is not fully understood. The purpose of this study is to systematically study the mechanism of ferroptosis in BRCA and its relationship with this cancer's prognosis, cell infiltration, gene mutation, and other clinical features. In this study, The Cancer Genome Atlas breast cancer (TCGA-BRCA) database (UCSC Xena) was used to mine the ferroptosis genes related to BRCA patients, and the genes with prognostic value were screened by Cox regression analysis, which were then used to construct a prognostic model for scoring prognostic molecular risk. The relationships between ferroptosis score and prognosis, molecular typing, and clinical characteristics of BRCA were also analyzed. A total of 176 ferroptosis genes related to BRCA were retrieved from the database, 22 of which were found to be significantly related to BRCA prognosis after screening by single-factor Cox regression analysis (p < 0.01). Unsupervised clustering of samples was performed using factoextra, and two subgroups (ferroptosis cluster A and ferroptosis cluster B) with significant differences in prognosis were identified. Subsequently, single-factor Cox regression analysis and random forest dimensionality reduction were used to screen characteristic genes to construct a ferroptosis score model, which included a high ferroptosis score group and a low ferroptosis score group. The results showed that there were significant differences in ferroptosis scores between the ferroptosis cluster A and B groups. The prognosis of patients with low ferroptosis scores was poor, and the overall survival (OS) rate of patients with high ferroptosis scores was significantly higher, indicating that the prognosis of the sample can be well characterized based on calculated ferroptosis scores. Ferroptosis scores differed significantly according to patient age, TP53 and PIK3CA gene mutations, different PAM50 molecular types, and clinical stages. Ferroptosis activation plays a non-negligible role in tumor occurrence and development. Evaluating the ferroptosis score within BRCA will help advance our understanding of the infiltrating properties of cells in the tumor microenvironment and may guide more effective immunotherapy strategies.

Ferret Lung Transplantation Models Differential Lymphoid Aggregate Morphology Between Restrictive and Obstructive Forms of Chronic Lung Allograft Dysfunction.

BACKGROUND: Long-term survival after lung transplantation remains limited by chronic lung allograft dysfunction (CLAD). CLAD has 2 histologic phenotypes, namely obliterative bronchiolitis (OB) and restrictive alveolar fibroelastosis (AFE), which have distinct clinical presentations, pathologies, and outcomes. Understanding of OB versus AFE pathogenesis would improve with better animal models. METHODS: We utilized a ferret orthotopic single-lung transplantation model to characterize allograft fibrosis as a histologic measure of CLAD. Native lobes and "No CLAD" allografts lacking aberrant histology were used as controls. We used morphometric analysis to evaluate the size and abundance of B-cell aggregates and tertiary lymphoid organs (TLOs) and their cell composition. Quantitative RNA expression of 47 target genes was performed simultaneously using a custom QuantiGene Plex Assay. RESULTS: Ferret lung allografts develop the full spectrum of human CLAD histology including OB and AFE subtypes. While both OB and AFE allografts developed TLOs, TLO size and number were greater with AFE histology. More activated germinal center cells marked by B-cell lymphoma 6 Transcription Repressor, (B-cell lymphoma 6) expression and fewer cells expressing forkhead box P3 correlated with AFE, congruent with greater diffuse immunoglobulin, plasma cell abundance, and complement 4d staining. Furthermore, forkhead box P3 RNA induction was significant in OB allografts specifically. RNA expression changes were seen in native lobes of animals with AFE but not OB when compared with No CLAD native lobes. CONCLUSIONS: The orthotopic ferret single-lung transplant model provides unique opportunities to better understand factors that dispose allografts to OB versus AFE. This will help develop potential immunomodulatory therapies and antifibrotic approaches for lung transplant patients.

Anti-inflammatory effect of HGF responses to oral traumatic ulcers using an HGF-Tg mouse model.

Hepatocyte growth factor (HGF) has been implicated in inhibiting diverse types of inflammation. Oral traumatic ulceration (OTU) is a common disease of the oral mucosa, and inflammation is the main process for ulcer healing. This study aimed to explore the expression of HGF in oral ulcers and its role in ulcer inflammation. The saliva of 14 recurrent alphous stomatitis (RAS) patients, 18 OTU patients and 17 healthy controls was collected. Traumatic ulcers of the left mucosa were observed in 42 wild-type (WT) and 42 HGF-overexpressing transgenic (HGF-Tg) mice. Histological scores, inflammatory cell expression and serum cytokine expression were measured and analyzed on the 5th day. The HGF protein level in ulcer-affected human saliva was 9.3-fold higher than that in healthy saliva. The HGF protein levels in RAS and OTU saliva were 14- and 5.7-fold higher, respectively, than those in healthy saliva. Traumatic ulcers enhanced HGF expression in ulcer-affected oral mucosa and in the blood of C57BL/6 mice by 1.21- and 1.40-fold, respectively. In HGF-Tg mouse traumatic ulcers, HGF expression was 1.34-fold higher than that in wild-type mice. HGF-Tg mice had lower weight loss, less ulcer area and lower histopathology scores than WT mice. The results from immunohistochemistry, flow cytometry and serum cytokine analysis showed that HGF-Tg animals presented fewer Ly6G-positive neutrophils and higher levels of circulating inflammatory cytokines. HGF overexpression alleviated weight loss, ulcer area and inflammation, suggesting the role of HGF in promoting the healing of oral ulcers.

Hepatocyte Growth Factor Overexpression Slows the Progression of 4NQO-Induced Oral Tumorigenesis.

OBJECTIVES: To investigate the role of hepatocyte growth factor (HGF)/c-Met signaling in oral malignant transformation. METHODS: We used immunohistochemistry to investigate HGF and c-Met expression in 53 oral squamous cell carcinoma (OSCC) specimens and 21 adjacent nontumor specimens and evaluated the associations between HGF and c-Met expression and clinicopathological parameters. Additionally, HGF-overexpression transgenic (HGF-Tg) and wild-type (Wt) mice were treated with 4-nitroquinoline-1-oxide (4NQO) to induce oral carcinogenesis for 16 weeks. At 16, 20, and 24 weeks, tongue lesions were collected for clinical observation; estimation of HGF, c-Met, and PCNA expression; apoptosis (TUNEL) assays; and RNA sequencing (RNA-seq). RESULTS: HGF and c-Met were positively expressed in 92.5% and 64% of OSCC samples, respectively. High HGF expression was significantly associated with smaller tumor size (p = 0.006) and inferior TNM stage (p = 0.032). No correlation between HGF and c-Met levels and other clinical parameters or prognosis was noted. In addition, HGF and c-Met expression was elevated in 4NQO-induced lesions of Wt mice. Compared with Wt mice, HGF-Tg mice have lower tumor incidence, number, volume, and lesion grade. In addition, the percentage of PCNA-positive cells in Wt mice was significantly higher than that in HGF-Tg mice at different time points. At 16 weeks, HGF-Tg mice exhibited less apoptotic cells compared with Wt mice (p < 0.000), and these levels gradually increased until the levels were greater than that of Wt mice at 24 weeks (p < 0.000). RNA-seq data revealed that 140 genes were upregulated and 137 genes were downregulated in HGF-Tg mice. KEGG enrichment analysis showed that upregulated differentially expressed genes (DEGs) are highly correlated with oxidative and metabolic signaling and that downregulated DEGs are related to MAPK and PI3K-AKT signaling. CONCLUSIONS: HGF and c-Met expression is upregulated in OSCC tissues and is associated with the occurrence and development of OSCC. HGF overexpression in normal oral epithelial tissue can inhibit 4NQO-induced tumorigenesis potentially through inhibiting proliferation and accelerating apoptosis via MAPK and PI3K-AKT signaling.

HGF can reduce accumulation of inflammation and regulate glucose homeostasis in T2D mice.

Hepatocyte growth factor (HGF) has been studied as a protective factor for the survival of islet ß cells and regulatory glucose transport and metabolism in many studies. The addition of exogenous HGF to cells or mice is the most common way to study HGF, but the persistence and stability of the administered HGF are unclear. In this experiment, wild-type C57BL6 (WT) mice and HGF-overexpressing transgenic (HGF-Tg) mice were divided into a normal diet (ND) group and an HFD group. The HGF protein level in the liver, kidney, spleen, pancreas, and VAT of HGF-Tg-ND mice was upregulated compared to that of WT-ND mice, and it was also upregulated in HGF-Tg-HFD mice compared to that in WT-HFD mice. In the ND group, though the HGF-Tg-ND mice showed higher fasted blood glucose levels and larger integrated density (IOD) of glucagon-positive cells than WT-ND mice, we found that HGF-Tg-ND mice can still maintain normal glucose tolerance based on an intraperitoneal glucose tolerance test (IPGTT) and an intraperitoneal insulin tolerance test (IPITT). In the HFD group, the HGF-Tg-HFD mice showed insulin sensitivity in IPGTT and IPITT and had larger areas and higher IOD values of islet ß cells and smaller areas and IOD values of islet α cells than the WT-HFD mice. HGF-Tg-HFD mice had lower level of serum insulin than WT-HFD mice. The HGF-Tg-HFD mice showed inhibited accumulation of CD4+ T cells, CD8+ T cells, Ly6G+ neutrophils, and F4/80+ macrophages in the blood and tissues and protected liver and kidney functions. Oil Red O-stained liver sections revealed that WT-HFD mice had larger areas and higher IOD values of Oil Red O-positive cells than HGF-Tg-HFD mice, and WT-HFD mice had higher score of NASH. PAS-stained kidney sections found WT-HFD has higher mesangial area/glomerular area × 100% than HGF-Tg-HFD mice. Comparative analyses demonstrated that HGF reduces the proportions of inflammatory cells in the blood and tissues, and protects liver and kidney tissues by regulating glucose homeostasis of type 2 diabetic mice.

The diagnostic value of urine heat shock protein 70 and prostatic exosomal protein in chronic prostatitis.

OBJECTIVE: To explore the diagnostic value of the levels of prostatic exosomal protein (PSEP) and heat shock protein 70 (HSP70) in the urine of patients with chronic prostatitis (CP). METHOD: Urine samples from 210 CP patients (70 cases of the USA National Institutes of Health Category II [NIH-II], 70 NIH-IIIa, and 70 NIH-IIIb patients) and 70 control subjects were collected between May 2018 and February 2020. The levels of PSEP and HSP70 in urine were detected by enzyme-linked immunosorbent assay. The differences in urine PSEP and HSP70 levels between the groups were analyzed, and receiver operating characteristic (ROC) curves were used to analyze the clinical value of PSEP and HSP70 in the diagnosis of CP. RESULTS: The PSEP levels of CP patients were significantly higher than those of the control group (p < 0.001), but there was no difference in PSEP levels among CP subgroups. The level of HSP70 in the urine of the NIH-II patients was significantly lower than the levels in the NIH-IIIa and NIH-IIIb subgroups and the control group, but there was no difference in HSP70 levels between the NIH-IIIa and NIH-IIIb subgroups and the control group. ROC curve analysis results showed that the area under the curve (AUC) of PSEP for the NIH-II, NIH-IIIa, and NIH-IIIb patients was 0.751, 0.776, and 0.731, respectively. The AUC of HSP70 in NIH-II patients was 0.784, and the AUC of combined detection of PSEP and HSP70 in NIH-II patients was 0.858. CONCLUSION: Urine PSEP can be used as a marker for the diagnosis of CP, but it cannot distinguish between the various types of CP, and HSP70 can be used as a diagnostic index for NIH-II classification.

Research on the circular RNA bioinformatics in patients with acute myocardial infarction.

OBJECTIVE: Through the detection of circular RNA (circRNA) using expression profiling chips, we searched for circRNAs related to acute myocardial infarction (AMI) and explored their relationship and possible mechanisms with AMI. METHOD: The study subjects included 3 AMI patients and 3 controls, and circRNA expression profiling analysis was performed using a microarray gene chip to identify circRNAs with large differences in expression between groups and to construct a circRNA-miRNA network. RESULTS: Compared with the control group, there were 650 differentially expressed circRNAs found in AMI patients (P < .05, fold change > 2), including 535 up-regulated circRNAs, such as hsa_circ_0050908, hsa_circRNA4010-22, hsa_circ_0081241, hsa_circ_0010551, hsa_circRNA4010-20, hsa_circRNA14702, hsa_circ_0115392, has_circRNA1825-44, has_circRNA8493-7, and hsa_circ_0025097. Furthermore, there were 115 down-regulated circRNAs, such as hsa_circ_0066439, hsa_circ_0054211, hsa_circ_0095920, hsa_circ_0122984, hsa_circ_0113067, hsa_circ_0039155, hsa_circRNA4014-45, hsa_circ_0122979, hsa_circ_0059665, and hsa_circ_0009319. The circRNAs hsa_circ_0066439, hsa_circ_0081241, and hsa_circ_0122984 can regulate multiple signal pathways to participate in the AMI process through hsa-miR-1254, hsa-miR-328-5p, and other miRNAs. In addition, the expression of circRNA-miRNA in peripheral blood is related to the network. Differentially expressed circRNAs are involved in chromatin organization, chromatin-modifying enzymes, signal transduction, lysine degradation, the mitogen-activated protein kinase (MAPK) signaling pathway, focal adhesion, and a variety of other pathways, such as myocardial infarction, coronary heart disease, hypertension, and other diseases. The gene ontology analysis results show that molecular function mainly involves binding and molecular structural activity, whereas the biological process mainly involves a single biological process, a cellular component for organization, and a cellular process, and the cellular component mainly involves a protein complex, an extracellular matrix, and a membrane. CONCLUSION: circRNA and microRNA interact to participate in the development of AMI. circRNA may be involved in the pathogenesis of AMI.

Repeat Dosing of AAV2.5T to Ferret Lungs Elicits an Antibody Response That Diminishes Transduction in an Age-Dependent Manner.

Readministration of recombinant adeno-associated virus (rAAV) may be necessary to treat cystic fibrosis (CF) lung disease using gene therapy. However, little is known about rAAV-mediated immune responses in the lung. Here, we demonstrate the suitability of the ferret for testing AAV2.5T-mediated CFTR delivery to the lung and characterization of neutralizing-antibody (NAb) responses. AAV2.5T-SP183-hCFTRΔR efficiently transduced both human and ferret airway epithelial cultures and complemented CFTR Cl- currents in CF airway cultures. Delivery of AAV2.5T-hCFTRΔR to neonatal and juvenile ferret lungs produced hCFTR mRNA at 200%-300% greater levels than endogenous fCFTR. Single-dose (AAV2.5T-SP183-gLuc) or repeat dosing (AAV2.5T-SP183-fCFTRΔR followed by AAV2.5T-SP183-gLuc) of AAV2.5T was performed in neonatal and juvenile ferrets. Repeat dosing significantly reduced transgene expression (11-fold) and increased bronchoalveolar lavage fluid (BALF) NAbs only in juvenile, but not neonatal, ferrets, despite near-equivalent plasma NAb responses in both age groups. Notably, both age groups demonstrated a reduction in BALF anti-capsid binding immunoglobulin (Ig) G, IgM, and IgA antibodies after repeat dosing. Unique to juvenile ferrets was a suppression of plasma anti-capsid-binding IgM after the second vector administration. Thus, age-dependent immune system maturation and isotype switching may affect the development of high-affinity lung NAbs after repeat dosing of AAV2.5T and may provide a path to blunt AAV-neutralizing responses in the lung.

Application value of NIPT for uncommon fetal chromosomal abnormalities.

OBJECTIVE: To investigate the clinical value of noninvasive prenatal testing (NIPT) for fetal chromosomal deletion, duplication, and sex chromosome abnormalities. METHODS: The study included 6239 pregnant women with singletons in the first and second trimester of pregnancy who received NIPT from December 2017 to June 2019. For pregnant women at high risk of deletion, duplication, and sex chromosome abnormalities indicated by NIPT, amniocentesis was recommended for karyotype analysis and chromosome copy number variation detection to verify the NIPT results and analyze chromosome abnormalities. Women at low risk and with no other abnormal results continued with their pregnancies. RESULTS: Among the 6239 pregnant women who received NIPT, there were 15 cases of chromosomal deletion (12 cases confirmed by amniocentesis), 16 cases of chromosomal duplication (9 cases confirmed by amniocentesis), and 17 cases of sex chromosome abnormalities (11 cases confirmed by amniocentesis). Of these cases, 32 were finally confirmed by amniotic fluid cell karyotype analysis. The coincidence rate was 66.7% (32/48). There were no abnormalities found for the remaining low risk pregnant women during follow-up. CONCLUSION: NIPT has good application value in predicting fetal chromosomal deletion, duplication, and sex chromosome abnormalities. It can improve the detection rate of fetal chromosomal abnormalities, but further prenatal diagnosis is needed.

Application of Non-Invasive Prenatal Tests in Serological Preclinical Screening for Women with Critical-Risk and Low-Risk Pregnancies but Abnormal Multiple of the Median Values.

BACKGROUND: To explore the clinical value of secondary screening using noninvasive prenatal testing (NIPT) for women with critical-risk and low-risk pregnancies who had multiple of the median (MoM) abnormalities in serological screening. METHODS: NIPT was used to analyze fetal free DNA in the peripheral blood of 2,325 women with critical-risk pregnancies and 239 women with low-risk pregnancies with MoM abnormalities in serological screening. Based on NIPT results, women with high-risk pregnancies were recommended for amniocentesis for fetal karyotype analysis. RESULTS: Among 2,325 women with critical-risk pregnancies as determined by serological screening, NIPT indicated 15 high-risk pregnancies (11 cases of trisomy 21 and 4 cases of trisomy 18). Of the 15 patients, 1 case refused prenatal diagnosis. The other 14 cases underwent invasive amniocentesis for fetal karyotype analysis, and 13 cases of fetal chromosomal abnormalities were diagnosed, including ten cases of trisomy 21 and three cases of trisomy 18. NIPT of 239 patients with low-risk pregnancies but abnormal MoM showed one case with a high risk of trisomy 21, which was diagnosed as a false positive by amniotic fluid karyotype analysis, and one case with a high risk of trisomy 13 (stillbirth) that was diagnosed by karyotype analysis. A case of gender chromosome abnormality was diagnosed as aneuploidy by karyotype analysis. CONCLUSIONS: The application of NIPT as a secondary screening for women with low- and critical-risk pregnancies as determined by serological screening but with MoM abnormalities will greatly reduce the number of invasive prenatal diagnosis procedures, significantly improve the rate and accuracy of fetal chromosomal abnormality detection.

Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease.

After more than two decades since clinical trials tested the first use of recombinant adeno-associated virus (rAAV) to treat cystic fibrosis (CF) lung disease, gene therapy for this disorder has undergone a tremendous resurgence. Fueling this enthusiasm has been an enhanced understanding of rAAV transduction biology and cellular processes that limit transduction of airway epithelia, the development of new rAAV serotypes and other vector systems with high-level tropism for airway epithelial cells, an improved understanding of CF lung pathogenesis and the cellular targets for gene therapy, and the development of new animal models that reproduce the human CF disease phenotype. These advances have created a preclinical path for both assessing the efficacy of gene therapies in the CF lung and interrogating the target cell types in the lung required for complementation of the CF disease state. Lessons learned from early gene therapy attempts with rAAV in the CF lung have guided thinking for the testing of next-generation vector systems. Although unknown questions still remain regarding the cellular targets in the lung that are required or sufficient to complement CF lung disease, the field is now well positioned to tackle these challenges. This review will highlight the role that next-generation CF animal models are playing in the preclinical development of gene therapies for CF lung disease and the knowledge gaps in disease pathophysiology that these models are attempting to fill.

The application of IL-10 and TNF-α in expressed prostatic secretions and prostatic exosomal protein in urine in the diagnosis of patients with chronic prostatitis.

BACKGROUND: The aim of this study was to investigate the expression of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) in expressed prostatic secretions (EPSs) of patients with chronic prostatitis (CP) and the expression of prostatic exosomal protein (PSEP) in urine, and to evaluate its correlation with the condition. METHODS: Urine samples from 310 patients with CP (101 National Institutes of Health [NIH] II, 112 NIH IIIa, and 97 NIH IIIb, classified according to the US National Institutes of Health) and 110 control group subjects were collected. The samples were tested for PSEP by enzyme-linked immunosorbent assay (ELISA). At the same time, EPSs in 60 patients from 310 patients with CP and 20 control group subjects were collected. The levels of IL-10 and TNF-α in the collected samples that EPS were determined by double antibody sandwich ELISA. SPSS 23.0 statistical software was used for statistical analysis of the measured data. RESULTS: The level of PSEP in patients with CP was significantly higher than that in the control group (P < .001). The levels of TNF-α and IL-10 in the EPS of patients with NIH II and NIH IIIa CP were higher than those of the patients with NIH IIIb and the control group (P < .001). There was a positive correlation between PSEP and IL-10 and TNF-α, while TNF-α and IL-10 were also positively correlated. CONCLUSION: PSEP, TNF-α, and IL-10 may serve as a basis for the classification diagnosis of CP. Their combination can provide more accurate diagnostic information for clinical CP typing.

Noninvasive prenatal testing detects microdeletion abnormalities of fetal chromosome 15.

OBJECTIVE: Noninvasive prenatal testing (NIPT) is widely used in clinical detection of fetal autosomal duplications or deletions. The aim of this study was to investigate the clinical application of NIPT for detection of chromosomal microdeletions. METHODS: Microdeletions of about 5 Mb in the long arm of chromosome 15 (q11.2-q12) were detected by NIPT and were confirmed by karyotype analysis and copy number variation (CNV) analysis based on high-throughput sequencing technology. RESULTS: The CNV results of prenatal diagnosis showed that there were approximately 4.96 Mb of microdeletions in 15q11.2-q13.1, which was consistent with the NIPT results. The karyotype analysis showed no abnormalities. CONCLUSION: In this study, the microdeletion fragment of fetal chromosome 15 was successfully detected and diagnosed using NIPT. This suggests that NIPT is an efficient method to gain genetic information about chromosomal abnormalities.

Upregulation of the long non-coding RNA CBR3-AS1 predicts tumor prognosis and contributes to breast cancer progression.

Breast cancer is the most common female malignancy and the major cause of cancer-related death in women. Long non-coding RNAs (lncRNAs), as oncogenic or tumor suppressor factor, involved in the development and progression of various cancers. In this study, we sought to investigate the function of lncRNA CBR3-AS1 in breast cancer. We evaluated the expression pattern of CBR3-AS1 in breast cancer tissues and cell lines, explored the correlation between CBR3-AS1 expression and the survival time of breast cancer patients, and probed the effect of CBR3-AS1 on tumor progression of breast cancer through loss-of-function and gain-of-function strategies. Our results showed that CBR3-AS1 was overexpressed in breast cancer tissues and cell lines and predicted the prognosis of breast cancer patients. And CBR3-AS1 exerted biological function as an oncogenic lncRNA, involved in the regulation of cell proliferation, colony formation, apoptosis and tumor growth in breast cancer. Taken together, CBR3-AS1 was up-regulated in breast cancer and promoted the risk of breast cancer. It may be a novel therapeutic target and potential prognostic marker for breast cancer.