Search

Background Extracellular vesicles (EVs) and their mimics from mesenchymal stem cells (MSCs) are promising drug carriers to improve cancer treatment, but their application is hindered by donor variations and expansion limitations of conventional tissue-derived MSCs. To circumvent these issues, we made EV-mimicking nanovesicles from standardized MSCs derived from human induced pluripotent stem cells (iPSCs) with a theoretically limitless expandability, and examined the targeting capacity of these nanovesicles to prostate cancer. Methods Nanovesicles are made from intact iPSC-MSCs through serial extrusion. The selective uptake of fluorescently labeled nanovesicles by prostate cancer cells vs. non-tumor cells was examined with flow cytometry. For in vivo tracing, nanovesicles were labeled with fluorescent dye DiR or renilla luciferase. In mice carrying subcutaneous or bone metastatic PC3 prostate cancer, the biodistribution of systemically infused nanovesicles was examined with in vivo and ex vivo imaging of DiR and luminescent signals. A chemotherapeutic drug, docetaxel, was loaded into nanovesicles during extrusion. The cytotoxicities of nanovesicle-encapsulated docetaxel on docetaxel-sensitive and -resistant prostate cancer cells and non-tumor cells were examined in comparison with free docetaxel. Therapeutic effects of nanovesicle-encapsulated docetaxel were examined in mice carrying subcutaneous or bone metastatic prostate cancer by monitoring tumor growth in comparison with free docetaxel. Results iPSC-MSC nanovesicles are more selectively taken up by prostate cancer cells vs. non-tumor cells in vitro compared with EVs, membrane-only EV-mimetic nanoghosts and liposomes, which is not affected by storage for up to 6 weeks. In mouse models of subcutaneous and bone metastatic PC3 prostate cancer, systemically infused nanovesicles accumulate in tumor regions with significantly higher selectivity than liposomes. The loading of docetaxel into nanovesicles was efficient and did not affect the selective uptake of nanovesicles by prostate cancer cells. The cytotoxicities of nanovesicle-encapsulated docetaxel are significantly stronger on docetaxel-resistant prostate cancer cells and weaker on non-tumor cells than free docetaxel. In mouse models of subcutaneous and bone metastatic prostate cancer, nanovesicle-encapsulated docetaxel significantly decreased the tumor growth and toxicity to white blood cells compared with free docetaxel. Conclusions Our data indicate that EV-mimicking iPSC-MSC nanovesicles are promising to improve the treatment of metastatic prostate cancer.

Management of a long proximal ureteral stricture is challenging. Lingual mucosal graft ureteroplasty (LMGU) is a novel minimally invasive technique for ureteral reconstruction that avoids the morbidity of bowel interposition or autotransplantation.To evaluate the long-term effectiveness of LMGU for managing long, complex proximal ureteral strictures in a multi-institutional cohort of patients.This retrospective study involved data for 41 patients treated with LMGU at three centers between June 2015 and January 2021.LMGU was performed using either an onlay ureteroplasty in which the diseased ureter was incised ventrally and repaired with a lingual mucosal graft (LMG) to widen the ureteral lumen, or an augmented anastomotic technique in which the obliterated segment of the ureter was excised and reanastomosed primarily on dorsal side, and an LMG was placed on the ventral side.Pre-, intra-, and postoperative variables and outcomes were assessed. A descriptive statistical analysis was performed.Of 41patients, 40 were operated with laparoscopic procedures and one with a robot. Twenty-four (59%) patients underwent an onlay ureteroplasty, and 17 (41%) underwent an augmented anastomotic ureteroplasty. The reconstructed ureter was wrapped with omentum in 90% of cases. The median (range) stricture length was 4.8 cm (2.0-8.0), operative time was 166 min (98-306), and estimated blood loss was 65 ml (15-220). No open conversions and intraoperative complications occurred. At a median follow-up of 35 mo (range 13-80), the overall success rate was 97.6% (40/41).LMGU is a safe, feasible, and effective long-term technique for managing long, complex proximal ureteral strictures.We reported a novel technique for long proximal complex ureteral strictures using an onlay lingual mucosal graft (LMG). Our 6-yr outcomes demonstrate that onlay LMG ureteroplasty is a safe, feasible, and effective long-term procedure for ureteral reconstruction.

Purpose Duchenne muscular dystrophy (DMD) is currently the most commonly diagnosed form of muscular dystrophy due to mutations in the dystrophin gene. However, its pathological process remains unknown and there is a lack of specific molecular biomarkers. The aim of our study is to explore key regulatory connections underlying the progression of DMD. Materials and methods The gene expression profile dataset GSE38417 of DMD was obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between DMD patients and healthy controls were screened using geo2R, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analyses. Then a protein-protein interaction (PPI) network and sub-network of modules were constructed. To investigate the regulatory network underlying DMD, a global triple network including miRNAs, mRNAs and transcription factors (TFs) was constructed. Results A total of 1811 DEGs were found between the DMD and control groups, among which HERC5, SKP2 and FBXW5 were defined as hub genes with a degree of connectivity >35 in the PPI network. Furthermore, the five TFs ZNF362, ATAT1, SPI1, TCF12 and ABCF2, as well as the eight miRNAs miR-124a, miR-200b/200c/429, miR-19a/b, miR-23a/b, miR-182, miR-144, miR-498 and miR-18a/b were identified as playing crucial roles in the molecular pathogenesis of DMD. Conclusions This paper provides a comprehensive perspective on the miRNA-TF-mRNA co-regulatory network underlying DMD, although the bioinformatic findings need further validation in future studies.

SUMMARY Spinal cord injury (SCI) is one of the most devastating neural injuries without effective therapeutic solutions. Astrocytes are the predominant component of the scar. Understanding the complex contributions of reactive astrocytes to SCI pathophysiologies is fundamentally important for developing therapeutic strategies. We have studied the molecular changes in the injury environment and the astrocyte-specific responses by astrocyte purification from injured spinal cords from acute to chronic stages. In addition to protein-coding genes, we have systematically analyzed the expression profiles of long non-coding RNAs (lncRNAs) (>200 bp), which are regulatory RNAs that play important roles in the CNS. We have identified a highly conserved lncRNA, Zeb2os, and demonstrated using functional assays that it plays an important role in reactive astrogliosis through the Zeb2os/Zeb2/Stat3 axis. These studies provide valuable insights into the molecular basis of reactive astrogliosis and fill the knowledge gap regarding the function(s) of lncRNAs in astrogliosis and SCI., Graphical Abstract, In Brief Wei et al. comprehensively investigate the coding and long non-coding gene expression changes and astrocyte-specific responses in injured spinal cord tissue and purified astrocytes from acute to chronic stages. Bioinformatic and functional analysis identify a conserved lncRNA Zeb2os that plays an essential role in reactive astrogliosis through the Zeb2os/Zeb2/Stat3 axis.

Aberrant activation of Notch signaling plays an oncogenic role in cancer development. Jagged1 (JAG1) is an important Notch ligand that triggers Notch signaling through cell-cell interactions. JAG1 overexpression has been reported in many different types of cancer and correlates with a poor clinical prognosis. JAG1/Notch signaling controls oncogenic processes in different cell types and cellular contexts. Furthermore, JAG1/Notch signaling cascades activate a number of oncogenic factors that regulate cellular functions such as proliferation, metastasis, drug-resistance, and angiogenesis. To suppress the severe toxicity of pan-Notch inhibitors, JAG1 is attracting increasing attention as a source of therapeutic targets for cancers. In this review, the oncogenic role of JAG1/Notch signaling in cancer is discussed, as well as implications of strategies to inhibit JAG1/Notch signaling activity.

In the present study, we investigated the differentially expressed genes (DEGs), pathways and immune cell infiltration characteristics of pediatric and adult ulcerative colitis (UC). We conducted DEG analysis using the microarray dataset GSE87473 containing 19 pediatric and 87 adult UC samples downloaded from the Gene Expression Omnibus. Gene ontology and pathway enrichment analyses were conducted using Metascape. We constructed the protein–protein interaction (PPI) network and the drug–target interaction network of DEGs and identified hub modules and genes using Cytoscape and analyzed immune cell infiltration in pediatric and adult UC using CIBERSORT. In total, 1700 DEGs were screened from the dataset. These genes were enriched mainly in inter-cellular items relating to cell junctions, cell adhesion, actin cytoskeleton and transmembrane receptor signaling pathways and intra-cellular items relating to the splicing, metabolism and localization of RNA. CDC42, POLR2A, RAC1, PIK3R1, MAPK1 and SRC were identified as hub DEGs. Immune cell infiltration analysis revealed higher proportions of naive B cells, resting memory T helper cells, regulatory T cells, monocytes, M0 macrophages and activated mast cells in pediatric UC, along with lower proportions of memory B cells, follicular helper T cells, γδ T cells, M2 macrophages, and activated dendritic cells. Our study suggested that hub genes CDC42, POLR2A, RAC1, PIK3R1, MAPK1 and SRC and immune cells including B cells, T cells, monocytes, macrophages and mast cells play vital roles in the pathological differences between pediatric and adult UC and may serve as potential biomarkers in the diagnosis and treatment of UC.

Urinary brain-derived neurotrophic factor (BDNF), an ubiquitous neurotrophin, was found to rise in patients with benign prostatic hyperplasia (BPH). We hypothesized that the urinary level of BDNF could be a potential biomarker for lower urinary tract symptoms (LUTS) in patients with BPH. Totally, 76 patients with BPH-caused LUTS and 32 male control subjects without BPH were enrolled. International Prostate Symptom Score (IPSS) was applied to assess the symptom severity of LUTS. Urodynamic tests were performed for the diagnosis of underlying detrusor overactivity (DO) in the patients with BPH. Urine samples were collected from all subjects. Urinary BDNF levels were measured using enzyme-linked immunosorbent assays and normalized by urinary creatinine (Cr) levels. Seventy-six BPH patients were divided into moderate LUTS group (n=51, 7IPSS≤20) and severe LUTS group (n=25, IPSS20) according to the IPSS. Of the 76 BPH patients, DO was present in 34 (44.7%) according to the urodynamic test. The urinary BDNF/Cr levels were significantly higher in BPH patients with moderate LUTS (8.29±3.635, P0.0001) and severe LUTS (11.8±6.44, P0.0001) than normal controls (1.71±0.555). Patients with severe LUTS tended to have higher urinary BDNF/Cr levels than patients with moderate LUTS (11.8±6.44 vs. 8.29±3.635, P=0.000). The conditions of BPH with LUTS correlated with elevated urinary BDNF levels, and urinary BDNF levels were even higher in BPH-DO patients. The results of this study have provided evidence to suggest that urinary BDNF level test could evaluate the severity of LUTS in BPH patients, and BDNF level can be used as a biomarker for the diagnosis of DO in BPH patients.

Background: Q fever endocarditis, a chronic illness caused by Coxiella burnetii, can be fatal if misdiagnosed or left untreated. Despite a relatively high positive rate of Q fever serology in healthy individuals in the mainland of China, very few cases of Q fever endocarditis have been reported. This study summarized cases of Q fever endocarditis among blood culture negative endocarditis (BCNE) patients and discussed factors attributing to the low diagnostic rate. Methods: We identified confirmed cases of Q fever endocarditis among 637 consecutive patients with infective endocarditis (IE) in the Peking Union Medical College Hospital between 2006 and 2016. The clinical findings for each confirmed case were recorded. BCNE patients were also examined and each BCNE patient’s Q fever risk factors were identified. The risk factors and presence of Q fever serologic testing between BCNE patients suspected and unsuspected of Q fever were compared using the Chi-squared or Chi-squared with Yates’ correction for continuity. Results: Among the IE patients examined, there were 147 BCNE patients, of whom only 11 patients (7.5%) were suspected of Q fever and undergone serological testing for C. burnetii. Six out of 11 suspected cases were diagnosed as Q fever endocarditis. For the remaining 136 BCNE patients, none of them was suspected of Q fever nor underwent relevant testing. Risk factors for Q fever endocarditis were comparable between suspected and unsuspected patients, with the most common risk factors being valvulopathy in both groups. However, significantly more patients had consulted the Infectious Diseases Division and undergone comprehensive diagnostic tests in the suspected group than the unsuspected group (100% vs. 63%, P = 0.03). Conclusions: Q fever endocarditis is a serious yet treatable condition. Lacking awareness of the disease may prevent BCNE patients from being identified, despite having Q fever risk factors. Increasing awareness and guideline adherence are crucial in avoiding misdiagnosing and missed diagnosing of the disease. Key words: Blood Culture; Endocarditis; Q Fever

Immune checkpoint blockade of the inhibitory immune receptors PD-L1, PD-1 and CTLA-4 has emerged as a successful treatment strategy for several advanced cancers. Here we demonstrate that miR-424(322) regulates the PD-L1/PD-1 and CD80/CTLA-4 pathways in chemoresistant ovarian cancer. miR-424(322) is inversely correlated with PD-L1, PD-1, CD80 and CTLA-4 expression. High levels of miR-424(322) in the tumours are positively correlated with the progression-free survival of ovarian cancer patients. Mechanistic investigations demonstrated that miR-424(322) inhibited PD-L1 and CD80 expression through direct binding to the 3′-untranslated region. Restoration of miR-424(322) expression reverses chemoresistance, which is accompanied by blockage of the PD-L1 immune checkpoint. The synergistic effect of chemotherapy and immunotherapy is associated with the proliferation of functional cytotoxic CD8+ T cells and the inhibition of myeloid-derived suppressive cells and regulatory T cells. Collectively, our data suggest a biological and functional interaction between PD-L1 and chemoresistance through the microRNA regulatory cascade., Resistance to chemotherapy occurs in many ovarian cancer cases. Here, the authors show that mir-424(322) expression restores the sensitivity of ovarian cancer cells to chemotherapy by blocking the PD-L1 immune checkpoint, and find that combining immunotherapy and chemotherapy has a synergistic effect.

Nanoparticles made from membrane of mesenchymal stem cells (MSCs) showed active targeting capacities to prostate and lung cancers, but further studies are hindered by limited expandability and donor variations of tissue-derived MSCs. We have derived MSCs with an unlimited supply and uniform homing capacity to triple-negative breast cancer (TNBC) from human induced pluripotent stem cells (iPSCs). By breaking down intact iPSC-MSCs, we efficiently developed nanovesicles that selectively accumulated in primary and metastatic TNBC after systemic infusion in mouse models. When loaded with a chemotherapeutic drug doxorubicin, iPSC-MSC nanovesicles showed superior cytotoxic effects on doxorubicin-resistant TNBC cells, and significantly decreased the incidence and burden of metastases in mouse models of spontaneous and experimental metastatic TNBC compared with free or liposomal doxorubicin. These nanovesicles showed no detectable immunogenicity or toxicity, and are stable after storage. Our data indicate that iPSC-MSC nanovesicles are promising to improve TNBC treatment as a standardized targeting platform.

The aim of study was to evaluate the association between serum DHEAS levels and depression with a case-control study together with a meta-analysis. Radioimmunoassay (RIA) was performed to measure the serum DHEAS levels of all participants before and after treatment. Depression Patients were divided into mild depression and severe depression based on Hamilton depression scale (HAMD24) and received 5-hydroxytryptamine (5-HT) and citalopram (20mg/d) for 8 weeks. Case-control studies related to our study theme were enrolled for meta-analysis and Comprehensive Meta-analysis 2.0 (CMA 2.0) was used for statistical analysis. After treatment, DHEAS levels in depression patients were significantly increased, while before and after treatment, DHEAS levels were all lower in depression patients than in controls (all P0.001); further analysis on age revealed that DHEAS levels were decreased with the rising of age. Meta-analysis results suggested that serum DHEAS levels (ng/mL) were significantly higher in healthy controls compared to depression patients (SMD=0.777, 95%CI=0.156-1.399, P=0.014). In conclusion, our study suggests that serum DHEAS levels are associated with the development of depression and it decreased with the rising of age.

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications.

The authors' previous study demonstrated that Golgi phosphoprotein 3 (GOLPH3) was significantly overexpressed in esophageal squamous cell carcinoma (ESCC), correlating with poor patient survival. In the present study, GOLPH3 stable overexpression and knockdown KYSE‑140 cell lines were constructed. Cell proliferation, colony formation, cell cycle progression and tumorigenesis assays were performed. The results revealed that GOLPH3 promoted ESCC cell growth and proliferation. The effects of GOLPH3 on the mechanistic target of rapamycin (mTOR) and Wnt/β‑catenin signaling pathways were investigated using western blot analyis and dual‑luciferase reporter assays, and were observed to be activated in cells with GOLPH3 overexpression. Furthermore, overexpression of GOLPH3 resulted in the downregulation of p21 protein, upregulation of cyclin D1 and increased retinoblastoma‑associated protein phosphorylation, consequently leading to accelerated cell cycle progression. In addition, GOLPH3 knockdown resulted in reversed effects. The results of the current study suggest that GOLPH3 serves an important role in promoting tumorigenicity of ESCC via mTOR and Wnt/β‑catenin signaling pathway activation.

Lipoprotein(a) [Lp(a)], a low-density lipoprotein-like particle, is recognized as an independent risk factor for atherosclerosis, cardiovascular diseases, and diabetic vascular diseases. Our recent studies revealed that the single nucleotide polymorphisms (SNPs) of peroxisome proliferator-activated receptors (PPARα/δ/γ) gene are involved in the regulation of lipid storage and metabolism. In order to investigate the relationships between the SNPs of PPARα/γ gene and plasma levels of Lp(a), 644 participants were randomly selected from Chinese Han population in the present study. As the results shown, Lp(a) was significantly associated with L162V (rs1800206) in PPARα. Compared with those subjects with widetype (LL), significantly higher Lp(a) concentration was determined in the individuals with mutant (LV + VV) (mean difference: 49.07 mg/l, 95% CI 23.32-74.82 mg/l, p = 0.0002). Moreover, with generalized multifactor dimensionality reduction analysis, our present results indicated that there was a significant association between plasma Lp(a) level and gene-gene interaction among the polymorphisms rs1800206, rs135539 in PPARα and rs10865710, rs1805192, and rs4684847 in PPARγ. Therefore, our presented study indicated that PPARα/γ polymorphisms should be involved in the regulation of plasma Lp(a) in independently and/or in an interactive manner, suggesting that PPARα/γ gene may influence the risk of hypertension, cardiovascular diseases, and dyslipidemia by regulating Lp(a) level.

The safety and effectiveness of a novel Chinese one-shot dilation technique based on stimulated diuresis for percutaneous nephrolithotomy (PCNL) were investigated. After the feasibility of the Chinese one-shot dilation based on stimulated diuresis was verified by an animal study, this technique was applied in the clinical practice. A total of 67 patients in our department underwent the modified PCNL from July 2014 to June 2015. After the renal infundibulum was distended by stimulated diuresis, the kidney was punctured under the ultrasonographic guidance via the fornix of the target calyx. The working channel was dilated using a special designed pencil-shaped fascial dilator. The successful access rate, nephrostomy tract creation time, pre- and postoperative hemoglobin values and serum creatinine concentrations, stone-free rate and complications were recorded and analyzed. The renal infundibulum was successfully distended in all of the patients by the diuresis treatment. Under the ultrasonographic guidance, the successful access rate was 100% and the mean tract creation time was 2.0 min (range: 1.5-5.0 min). The stone-free rate right after surgery was 91.0%. Although the postoperative hemoglobin was significantly reduced (P0.01), transfusion was not clinically necessary. There was no significant difference in serum creatinine concentrations before and after operation (P0.05). No severe complication occurred during or after the PCNL. It was suggested that this Chinese one-shot dilation technique based on stimulated diuresis is an efficient and safe innovation for PCNL, and is even helpful for those patients with non-dilated pelvicaliceal systems.

We present our initial experience and 9-month outcomes of the novel technique of laparoscopic onlay lingual mucosal graft ureteroplasty for proximal ureteral stricture. In June 2015, transperitoneal laparoscopic onlay lingual mucosal graft ureteroplasty was performed on a male patient with proximal stricture of the left ureter. The patient complained with left frank pain. Severe hydronephrosis and proximal ureteral dilatation were noted through ultrasonography and CT scan. The length of upper ureteral stricture was 30 mm including 10-mm occlusion. A 46 mm in length and 15 mm in width lingual mucosa graft was harvested from the ventral of the tongue and placed in the strictured ureter as a ventral onlay for laparoscopic ureteroplasty. Operative time, intraoperative, and postoperative complications were well recorded. Follow-up was performed with renal ultrasound, CT scan, and nuclear scan renography as well as clinical assessment of symptoms. The new technique was performed successful without intraoperative and postoperative complications. Neither hydronephrosis nor proximal ureteral dilatation in the left side was found through ultrasonography 3, 6, 9 months and CT scan 6 month postoperatively. The left renal function, glomerular filtration rate, had a recovery from 9.6 ml/min preoperatively to 14.0 ml/min at 6-month follow-up, and the patient has no complaints about the donor site and flank pain. To our knowledge, we present the initial experience with laparoscopic onlay lingual mucosal graft ureteroplasty for proximal ureteral stricture. With 9-month outcomes, the new technique appears to be an excellent option for proximal ureteral stricture.

MicroRNAs (miRNAs) are a group of endogenous, non-coding, 18-24 nucleotide length single-strand RNAs that mediate gene expression at the post-transcriptional level through mRNA degradation or translational repression. They are involved in regulating diverse cellular biological processes such as cell cycle, differentiation and apoptosis. Deregulation of miRNAs affects normal biological processes leading to malignancies, including oral squamous cell carcinoma (OSCC). Recent studies have identified aberrant miRNA expression profiles in OSCC tissues and/or cell lines compared with matched normal controls, the mechanisms of which are becoming unveiled. In addition, a small number of dysregulated miRNAs have been implicated either as oncogenes or tumor suppressors, affecting the initiation and progression of OSCC through the regulation of proliferation, apoptosis, metastasis and chemoresistance. Also, these missexpressed miRNAs have been shown to have potential as novel diagnostic, prognostic and therapeutic tools, which are expected to advance the clinical management of OSCC in the near future.

Previously (Shan et al, 2005), we reported that adenoviral vector-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to minipig parotid glands following irradiation (IR) transiently restored salivary flow to near normal levels. This study evaluated a serotype 2, adeno-associated viral (AAV2) vector for extended correction of IR (single dose; 20 Gy)-induced, parotid salivary hypofunction in minipigs. At 16 weeks following the IR parotid salivary flow decreased by 85-90%. AAV2hAQP1 administration at week 17 transduced only duct cells and resulted in a dose-dependent increase in salivary flow to approximately 35% of pre-IR levels (to approximately 1 ml per 10 min) after 8 weeks (peak response). Administration of a control AAV2 vector or saline was without effect. Little change was observed in clinical chemistry and hematology values after AAV2hAQP1 delivery. Vector-treated animals generated high anti-AAV2 neutralizing antibody titers by week 4 (approximately 1:1600) and significant elevations in salivary (approximately 15%), but not serum, granulocyte macrophage colony-stimulating factor levels. Following vector administration, salivary [Na(+)] was dramatically increased, from approximately 10 to approximately 55 mM (at 4 weeks) and finally to 39 mM (8 weeks). The findings demonstrate that localized delivery of AAV2hAQP1 to IR-damaged parotid glands leads to increased fluid secretion from surviving duct cells, and may be useful in providing extended relief of salivary hypofunction in previously irradiated patients.

Background Gene transfer to salivary glands for use in treating both systemic and upper gastrointestinal tract diseases shows considerable potential. Numerous studies in rodents demonstrate that salivary glands can secrete transgenic secretory proteins either into saliva, primarily via the regulated secretory pathway (RSP), or into the bloodstream, primarily by the constitutive secretory pathway (CSP). The purpose of the present study was to assess the sorting characteristics of human growth hormone (hGH), a RSP protein, and human erythropoietin (hEpo), a CSP protein, in a large animal model of salivary gland gene transfer, the miniature pig. Methods Recombinant serotype 5 adenoviral (Ad5; 1011 particles/gland) vectors encoding either hGH (AdCMVhGH) or hEpo (AdCMVhEpo) were administered to both parotid glands of male miniature pigs by intraductal cannulation. The secretion of hGH or hEpo was measured in both saliva and serum on days 3, 7 and 14 following administration. Detailed serum chemistry and hematological analyses were performed, and the presence of serum antibodies to hGH and hEpo was measured. For AdCMVhEpo-treated minipigs vector distribution in multiple tissues was determined by quantitative polymerase chain reaction (QPCR). Results The RSP protein hGH was secreted entirely into saliva, while the CSP protein hEpo was secreted into both saliva and serum. Most hEpo was found in saliva, but serum hEpo levels were sufficient to significantly increase hematocrit levels in treated animals by ∼10%. Expression of both transgenes was maximal on day 3 and declined to near background by day 14. The amount of vector found in the targeted glands was 100× more than in other tissues. Conclusions Secretion of transgenic hGH from minipig parotid glands occurred principally into saliva via the RSP, as seen in rodents, while hEpo was secreted into both saliva and serum, the latter presumably via the CSP. Even though hEpo secretion into the bloodstream was not to the extent previously observed in rodents, serum hEpo levels were considerable and the hEpo was biologically active. Ad5 vector distribution was highly restricted to the parotid glands with little vector detected elsewhere. While the results in this large animal model support the established notion that salivary gland gene transfer can be used for treating systemic single protein deficiency disorders, they also highlight differences in transgenic CSP protein sorting between rodents and miniature pigs. Copyright © 2007 John Wiley & Sons, Ltd.

To investigate the association between ten single nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptors (PPARα, β, γ) with apolipoprotein A I/apolipoprotein B100 (ApoA I/ApoB100) ratio and the additional role of a gene-gene interactions among the 10 SNPs.Participants were recruited under the framework of the Prevention of Multiple Metabolic Disorders and Metabolic Syndrome in Jiangsu Province (PMMJS) cohort population survey in the urban community of Jiangsu province of China.A total of 630 subjects were randomly selected and no individual was related.Ten SNPs (rs135539, rs4253778, rs1800206, rs2016520, rs9794, rs10865710, rs1805192, rs709158, rs3856806 and rs4684847) were selected from the HapMap database,which covered PPARα, PPARβ and PPARγ. A linear regression model was used to analyze the relations between ten SNPs in the PPARs and ApoA I/ApoB100 ratio level. Mean difference and 95% CI were calculated. Interactions were explored by using the method of Generalized Multifactor Dimensionality Reduction (GMDR).After adjusting for age, gender, smoking status, alcohol consumption, occupational physical activity, high-fat diet as well as low-fiber diet, both rs1800206 and rs3856806 were significantly associated with a decreased level of ApoA I/ApoB100 ratio, mean difference (95% CI) values were -1.19 (-1.88 to -0.50) and -0.77 (-1.40 to -0.14). Whereas rs4253778 was significantly associated with an increased level of ApoA I/ApoB100 ratio, Mean difference (95% CI) values was 0.80 (0.08 to 1.52). GMDR analysis showed a significant gene-gene interaction among rs4253778, rs1800206 of PPARα, rs9794, rs2016520 of PPARβ and rs10865710, rs3856806, rs709158, rs1805192 of PPARγ for eight-dimension models (P = 0.01), in which prediction accuracy was 0.624 and cross-validation consistency was 7/10.The rs1800206 of PPARα and rs3856806 of PPARγ are significantly associated with a decreased level of ApoA I/ApoB100 ratio while rs4253778 of PPARα is associated with an increased level of ApoA I/ApoB100 ratio. There is a gene-gene interaction between multiple SNPs.

A total of 1,345 Ixodes persulcatus ticks collected from northeastern China were investigated for the presence of Anaplasma phagocytophila and Borrelia burgdorferi by a nested polymerase chain reaction (PCR). Sixty-two (4.6%) ticks were positive for A. phagocytophila and 454 (33.8%) were positive for B. burgdorferi. Seven (0.5%) were coinfected with both agents. Sequence analysis of 919-basepair PCR amplicons revealed three types of A. phagocytophila. Type 1 was identical to the published sequences of A. phagocytophilas responsible for human granulocytic ehrlichiosis (HGE). The other two variants differed from the HGE agent sequence at one and four positions, respectively. These findings imply that infection with A. phagocytophila poses a potential health threat to both humans and animals in northeastern China, and that ehrlichiosis should be considered in the differential diagnosis of febrile patients with a history of tick bite, particularly when clinical manifestations are atypical for Lyme disease.

The aim of this study was to investigate the association between three single-nucleotide polymorphisms (SNP) of in the peroxisome proliferator-activated receptor (PPAR) α gene and the level of lipoprotein (a) [Lp(a)].Participants were recruited under the framework of a cohort populations survey from the PMMJS (Prevention of Multiple Metabolic Disorders and MS in Jiangsu Province) which was conducted in the urban community of Jiangsu province from 1999 to 2007. 644 subjects (234 males, 410 females) were randomly selected and genotyped for three polymorphisms which were used as genetic marker for PPARα gene (rs1800206, rs4253778 and rs135539). Data related to individual polymorphism and haplotype were available for analysis. χ² test was used to determine if the whole population was in Hardy-Weinberg genetic equilibrium. Linear regression models were used to analyze the association between SNPs in PPARα gene and the level of Lp(a). Associations between PPARα haplotypes and serum Lp(a) levels were analyzed by the SNPstats software.In the dominant model, after factors as sex, age, smoking, alcohol and BMI were adjusted, the presence of the V162 allele of L162V appeared associated with a high level of Lp(a) (mean difference was 57.70 mg/L (95% CI: 32.03-83.37 mg/L), P0.001. Data from the haplotype analysis revealed that A-G-V and C-G-V haplotype (established by 1AC, 7GC L162V) were significantly associated with a higher level of Lp(a) (P = 0.012 0 and 0.009 7).Results from our study might help to clarify the role of PPARα gene in regulation of Lp(a) and the evaluation of its polymorphisms and haplotypes which were characterized as genetic factors for Lp(a).

To investigate the association of ten single nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptors (PPARα, δ, γ) with lipid accumulation product (LAP) and the additional role of a gene-gene interactions among the 10 SNPs.Participants were recruited under the framework of the PMMJS (Prevention of Multiple Metabolic Disorders and Metabolic Syndrome in Jiangsu province) cohort populations survey in the urban community of Jiangsu province of China. A total of 820 subjects were randomly selected and no individual was related. Ten SNPs (rs135539, rs4253778, rs1800206, rs2016520, rs9794, rs10865710, rs1805192, rs709158, rs3856806 and rs4684847) were selected from the HapMap database, which covered PPARα, PPARδ and PPARγ. A linear regression model was used to analyze the relations between ten SNPs in the PPARs and LAP. Mean difference (Difference) and 95% confident interval (95%CI)were calculated. Interactions were explored by using the method of Generalized Multifactor Dimensionality Reduction (GMDR).After adjusting the factors as age, gender, smoking status, occupational physical activity, educational level, high-fat diet as well as low-fiber diet, both rs1800206, s1805192 and rs3856806 were significantly associated with the increased level of LAP. Difference (95% CI) values were 32.47 (22.62-42.31), 12.97 (4.61-21.33) and 12.49 (4.24-20.75). Whereas rs2016520 was significantly associated with the decreased level of LAP. Difference (95%CI) values was -14.67 ( -22.97--6.55). GMDR analysis showed a significant gene-gene interaction among rs135539, rs1800206 of PPARα, rs2016520 of PPARδ and rs10865710, rs3856806, rs709158, rs1805192, rs4684847 of PPARγ for eight-dimension models (P0.05), in which prediction accuracy was 0.5902 and cross-validation consistency was 10/10.The rs1800206 of PPARα and rs1805192, rs3856806 of PPARγ were significantly associated with the increased level of LAP; rs2016520 of PPARδ was associated with the decreased level of LAP. There was a gene-gene interaction between multiple SNPs.

Purpose: To examine the effects and mechanisms of transient activation of the Hedgehog pathway on rescuing radiotherapy-induced hyposalivation in survivors of head and neck cancer. Experimental Design: Mouse salivary glands and cultured human salivary epithelial cells were irradiated by a single 15-Gy dose. The Hedgehog pathway was transiently activated in mouse salivary glands, by briefly overexpressing the Sonic hedgehog (Shh) transgene or administrating smoothened agonist, and in human salivary epithelial cells, by infecting with adenovirus encoding Gli1. The activity of Hedgehog signaling was examined by the expression of the Ptch1-lacZ reporter and endogenous Hedgehog target genes. The salivary flow rate was measured following pilocarpine stimulation. Salivary stem/progenitor cells (SSPC), parasympathetic innervation, and expression of related genes were examined by flow cytometry, salisphere assay, immunohistochemistry, quantitative reverse transcription PCR, Western blotting, and ELISA. Results: Irradiation does not activate Hedgehog signaling in mouse salivary glands. Transient Shh overexpression activated the Hedgehog pathway in ductal epithelia and, after irradiation, rescued salivary function in male mice, which is related with preservation of functional SSPCs and parasympathetic innervation. The preservation of SSPCs was likely mediated by the rescue of signaling activities of the Bmi1 and Chrm1–HB-EGF pathways. The preservation of parasympathetic innervation was associated with the rescue of the expression of neurotrophic factors such as Bdnf and Nrtn. The expression of genes related with maintenance of SSPCs and parasympathetic innervation in female salivary glands and cultured human salivary epithelial cells was similarly affected by irradiation and transient Hedgehog activation. Conclusions: These findings suggest that transient activation of the Hedgehog pathway has the potential to restore salivary gland function after irradiation-induced dysfunction. Clin Cancer Res; 20(1); 140–50. ©2013 AACR.

Bone marrow mesenchymal stem cells (BM-MSCs) have multiple therapeutic potentials for regenerative, anti-inflammatory, and immunomodulatory purposes and also show promise as vehicles for gene therapy of various metastatic cancers based on their tumor-tropic capacity. However, BM-MSCs are also a source of carcinoma-associated fibroblasts (CAFs) and may promote growth and metastasis of cancer. Transforming growth factor β (TGF-β) signaling is required to induce CAF differentiation of mouse BM-MSCs in vivo and can induce expression of some CAF markers in human BM-MSCs in vitro. To determine whether inhibiting TGF-β signaling in human BM-MSCs can block their differentiation to CAFs induced by tumor microenvironments and the consequent protumor effects, we transduced human BM-MSCs with a lentiviral vector encoding bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a decoy TGF-β receptor. BAMBI transduction significantly inhibited TGF-β/Smad signaling and expression of CAF markers in human BM-MSCs treated with TGF-β1 or tumor-conditioned medium or cocultured with cancer cells, but did not alter the stem cell properties and the tumor-tropic property of MSCs. In addition, BAMBI transduction disrupted the cytokine network mediating the interaction between MSCs and breast cancer cells. Consequently, BAMBI transduction abolished protumor effects of BM-MSCs in vitro and in an orthotopic breast cancer xenograft model, and instead significantly inhibited growth and metastasis of coinoculated cancer. These results indicated that TGF-β signaling is essential for differentiation of human BM-MSCs to CAFs in tumor microenvironments and the consequent protumor effects, and inhibiting TGF-β/Smad pathway may improve the safety of MSC-based therapies in cancer patients.

Regenerative therapy of the salivary gland (SG) is a promising therapeutic approach for irreversible hyposalivation in patients with head and neck cancer treated by radiotherapy. However, little is known about the molecular regulators of stem/progenitor cell activity and regenerative processes in the SG. Wnt/β-catenin signaling regulates the function of many adult stem cell populations, but its role in SG development and regeneration is unknown. Using BAT-gal Wnt reporter transgenic mice, we demonstrate that in the submandibular glands (SMGs) of newborn mice Wnt/β-catenin signaling is active in a few cells at the basal layer of intercalated ducts, the putative location of salivary gland stem/progenitor cells (SGPCs). Wnt activity decreases as mice age, but is markedly enhanced in SG ducts during regeneration of adult SMG after ligation of the main secretory duct. The Hedgehog (Hh) pathway is also activated after duct ligation. Inhibition of epithelial β-catenin signaling in young Keratin5-rtTA/tetO-Dkk1 mice impairs the postnatal development of SMG, particularly affecting maturation of granular convoluted tubules. Conversely, forced activation of epithelial β-catenin signaling in adult Keratin5-rtTA/tetO-Cre/Ctnnb1((Ex3)fl) mice promotes proliferation of ductal cells, expansion of the SGPC compartment, and ectopic activation of Hh signaling. Taken together, these results indicate that Wnt/β-catenin signaling regulates the activity of SGPCs during postnatal development and regeneration upstream of the Hh pathway, and suggest the potential of modulating Wnt/β-catenin and/or Hh pathways for functional restoration of SGs after irradiation.

Background Previously, using an adenoviral vector, we showed that miniature pigs could provide a valuable and affordable large animal model for pre-clinical gene therapy studies to correct parotid gland radiation damage. However, adenoviral vectors lead to short-term transgene expression and, ideally, a more stable correction is required. In the present study, we examined the suitability of using a serotype 2 adeno-associated viral (AAV2) vector to mediate more stable gene transfer in the parotid glands of these animals. Methods Heparan sulfate proteoglycan was detected by immunohistochemistry. β-galactosidase expression was determined histochemically. An AAV2 vector encoding human erythropoietin (hEpo) was administered via Stensen's duct. Salivary and serum hEpo levels were measured using an enzyme-linked immunosorbent assay. Serum chemistry and hematological analyses were performed and serum antibodies to hEpo were measured throughout the study. Vector distribution was determined by a quantitative polymerase chain reaction. Results Transgene expression was vector dose-dependent, with high levels of hEpo being detected for up to 32 weeks (i.e. the longest time studied). hEpo reached maximal levels during weeks 4–8, but declined to approximately 25% of these values by week 32. Haematocrits were elevated from week 2. Transduced animals exhibited low serum anti-hEpo antibodies (1 : 8–1 : 16). Vector biodistribution at animal sacrifice revealed that most copies were in the targeted parotid gland, with few being detected elsewhere. No consistent adverse changes in serum chemistry or hematology parameters were seen. Conclusions AAV2 vectors mediate extended gene transfer to miniature pig parotid glands and should be useful for testing pre-clinical gene therapy strategies aiming to correct salivary gland radiation damage. Copyright © 2009 John Wiley & Sons, Ltd.

This study was aimed to compare HHGV678 with imatinib (IM) in growth inhibition of Bcr-Abl wild type and IM-resistant cell lines, investigate the possibility of replacing IM with HHGV678 in treatment of chronic myeloid leukemia (CML) and IM-resistant CML patients. Viability of two Bcr-Abl wild type cell lines (K562 and 32Dp210) and 16 IM-resistant cell lines (K562R and 15 Bcr-Abl point mutant cell lines) treated with HHGV678 and IM was analyzed by MTT. The apoptosis of those cells was identified by flow cytometry with Annexin V staining and DNA ladder analysis. Western blot was applied for detecting the expression of Bcr-Abl and phosphotyrosine protein levels. The results indicated that HHGV678 significantly inhibited the growth of two Bcr-Abl wild types and IM-resistant cell lines in dose-dependent manner except cell line of T315I point mutant. IC(50) results showed that the growth inhibition of HHGV678 was 15.5 and 28-fold higher than that of IM in K562, 32Dp210 and 1.4 to 124.3-fold higher than that of IM in 15 IM-resistant cell lines respectively. Compared with IM, HHGV678 more significantly inhibited phosphotyrosine kinase protein of the cells mentioned above at different concentrations. With most importance, HHGV678 of 10.0 micromol/L induced cell apoptosis of 40.06% and 33.32% in K562R and 32Dp210(T315I) cell lines, which were much higher than that of IM (19.77% and 10.68%). It is concluded that HHGV678 is more effective than IM in the growth inhibition of Bcr-Abl wild type cell lines and IM-resistant cell lines, especially in strongest IM-resistant cell lines. Further studies are needed to show whether HHGV678 may be a novel targeting drug in treatment of CML and IM-resistant CML patients.

In total, 676 Dermacentor silvarum Olenev (Acari: Ixodidae) from a forest area of Jilin Province in northeastern China were examined by polymerase chain reaction for the presence of spotted fever group (SFG) Rickettsia. The overall positive rate was 10.7%, with a 95% confidence interval from 8.3 to 13.0%. The SFG Rickettsia infection was more prevalent in adults than in nymphs, and in fed ticks obtained from domestic animals than in those collected on vegetation. Sequence analysis of the partial outer membrane protein A gene confirmed the existence of R. sibirica and discovered a novel rickettsial agent in this area, the sequence of which was identical to that of DnS14 genotype Rickettsia previously reported in the former Soviet Union.

To develop a real-time quantitative polymerase chain reaction(PCR) assay for detecting Rickettsia rickettsii.The primers and TaqMan-MGB probe were designed according to the ompB gene of R. rickettsii. A DNA fragment of ompB gene amplified from R. rickettsii by PCR was used as a standard template for the development of the method.5 copies of ompB fragments of R. rickettsii were detected. The genomic DNA of R. rickettsii was detected by the developed quantitative PCR assay. However, the genomic DNA from another rickettsial or bacterial agent was not determined. Through this developed method, the positive results were obtained from the animals and cells, artificially infected with R. rickettsii.The real-time quantitative PCR assay seemed to be highly sensitive and specific which might be used to rapidly detect R. rickettsia DNA in various samples and to early diagnose patients infected by R. rickettsii.

1. Antunes-Lopes T, Pinto R, Carvalho-Barros S (2011) Urinary levels of Brain Derived Neurotrophic Factor (BDNF) in women with overactive bladder (OAB) syndrome correlate with the severity of symptoms. Eur Urol Suppl 10(2):277–278 2. Liu HT, Kuo HC (2008) Urinary nerve growth factor level could be a potential biomarker for diagnosis of overactive bladder. J Urol 179(6):2270–2274 3. Kim JC, Park EY, Hong SH, Seo SI, Park YH et al (2005) Changes of urinary nerve growth factor and prostaglandins in male patients with overactive bladder symptom. Int J Urol 12(10):875–880 Editor