ABT-263 exerts a protective effect on upper urinary tract damage by alleviating neurogenic bladder fibrosis.

This study investigated the mechanism of action of ABT-263 in the treatment of neurogenic bladder fibrosis (NBF)and its protective effects against upper urinary tract damage (UUTD). Sixty 12-week-old Sprague-Dawley (SD) rats were randomly divided into sham, sham + ABT-263 (50 mg/kg), NBF, NBF + ABT-263 (25 mg/kg, oral gavage), and NBF + ABT-263 (50 mg/kg, oral gavage) groups. After cystometry, bladder and kidney tissue samples were collected for hematoxylin and eosin (HE), Masson, and Sirius red staining, and Western Blotting (WB) and qPCR detection. Primary rat bladder fibroblasts were isolated, extracted, and cultured. After co-stimulation with TGF-ß1 (10 ng/mL) and ABT-263 (concentrations of 0, 0.1, 1, 10, and 100 µmol/L) for 24 h, cells were collected. Cell apoptosis was detected using CCK8, WB, immunofluorescence, and annexin/PI assays. Compared with the sham group, there was no significant difference in any physical parameters in the sham + ABT-263 (50 mg/kg) group. Compared with the NBF group, most of the markers involved in fibrosis were improved in the NBF + ABT-263 (25 mg/kg) and NBF + ABT-263 (50 mg/kg) groups, while the NBF + ABT-263 (50 mg/kg) group showed a significant improvement. When the concentration of ABT-263 was increased to 10 µmol/L, the apoptosis rate of primary bladder fibroblasts increased, and the expression of the anti-apoptotic protein BCL-xL began to decrease.ABT-263 plays an important role in relieving NBF and protecting against UUTD, which may be due to the promotion of myofibroblast apoptosis through the mitochondrial apoptosis pathway.

Alteration of N6-methyladenosine epitranscriptome profiles in bilateral ureteral obstruction-induced obstructive nephropathy in juvenile rats.

BACKGROUND: Urinary tract obstruction is a common cause of renal failure in children and infants, and the pathophysiological mechanisms of obstructive nephropathy are largely unclear. It has been reported that m6A modulation is involved in renal injury. However, whether m6A RNA modulation is associated with obstructive nephropathy has not yet been reported. The aim of this study was to investigate the m6A epitranscriptome profiles in the kidneys of bilateral ureteral obstruction (BUO) in young rats. METHODS: The total level of m6A in the kidneys was measured by liquid chromatography-tandem mass spectrometry. The mRNAs of related genes were detected by real-time PCR. Methylated RNA immunoprecipitation sequencing was performed to map the epitranscriptome-wide m6A profile. RESULTS: Global m6A levels were increased after BUO, and the mRNA expression levels of m6A methyltransferases and demethylases were significantly decreased in BUO group rat kidneys; the expression levels of EGFR and Brcal were significantly upregulated, while the mRNA expression levels of Notch1 were downregulated (P < 0.05). A total of 154 genes associated with 163 m6A peaks were identified. CONCLUSION: The m6A epitranscriptome was significantly altered in BUO rat kidneys, which is potentially implicated in the pathophysiological processes of obstructive nephropathy. IMPACT: The m6A RNA modification was associated with the process of renal injury in ureteral obstructive nephropathy by participating in multiple dimensions. The dysregulation of m6A methyltransferases and demethylases may be related to the pathophysiological changes of BUO-induced obstructive nephropathy. The m6A RNA modulation of the genes EGFR, Brca1, and Notch1 that were related to the regulation of aquaporin2 might be the potential mechanism for the polyuresis after ureteral obstruction.

[18F]-(2S,4R)4-Fluoroglutamine PET Imaging of Glutamine Metabolism in Murine Models of Hepatocellular Carcinoma (HCC).

Purpose: Quantitative in vivo [18F]-(2S,4R)4-fluoroglutamine ([18F]4-FGln or more simply [18F]FGln) metabolic kinetic parameters are compared with activity levels of glutamine metabolism in different types of hepatocellular carcinoma (HCC). Methods: For this study, we used two transgenic mouse models of HCC induced by protooncogenes, MYC, and MET. Biochemical data have shown that tumors induced by MYC have increased levels of glutamine metabolism compared to those induced by MET. One-hour dynamic [18F]FGln PET data were acquired and reconstructed for fasted MYC mice (n = 11 tumors from 7 animals), fasted MET mice (n = 8 tumors from 6 animals), fasted FVBN controls (n = 8 normal liver regions from 6 animals), nonfasted MYC mice (n = 16 tumors from 6 animals), and nonfasted FVBN controls (n = 8 normal liver regions from 3 animals). The influx rate constants (K 1) using the one-tissue compartment model were derived for each tumor with the left ventricular blood pool input function. Results: Influx rate constants were significantly higher for MYC tumors (K 1 = 0.374 ± 0.133) than for MET tumors (K 1 = 0.141 ± 0.058) under fasting conditions (P = 0.0002). Rate constants were also significantly lower for MET tumors (K 1 = 0.141 ± 0.135) than normal livers (K 1 = 0.332 ± 0.179) under fasting conditions (P = 0.0123). Fasting conditions tested for MYC tumors and normal livers did not result in any significant difference with P values > 0.005. Conclusion: Higher influx rate constants corresponded to elevated levels of glutamine metabolism as determined by biochemical assays. The data showed that there is a distinctive difference in glutamine metabolism between MYC and MET tumors. Our study has demonstrated the potential of [18F]FGln PET imaging as a tool to assess glutamine metabolism in HCC tumors in vivo with a caution that it may not be able to clearly distinguish HCC tumors from normal liver tissue.

METTL3/N6-methyladenosine/ miR-21-5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF-κB pathway activation.

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR-21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF-kB signalling pathway. However, whether miR-21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6-methyladenosine (m6 A) modification-dependent primary microRNA (pri-microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR-21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK-2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR-21-5p mimic or miR-21-5p inhibitor. We found that the levels of miR-21-5p and m6 A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF-kB pathway was activated by miR-21-5p, confirming that miR-21-5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m6 A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR-21-5p maturation. Our research is the first to demonstrate the role of the METTL3-m6 A-miR-21-5p-SPRY1/ERK/NF-kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.

SCF/c-kit signaling pathway participates in ICC damage in neurogenic bladder.

Neurogenic bladder (NB) is a type of double renal dysfunction caused by nerve lesions. The interstitial cells of Cajal (ICC) damage are involved in bladder dysfunction. The aim of this study is to investigate the effect of stem cell factor (SCF)/c-kit signaling pathway on ICC damage in NB model rats. Maximum cystometric capacity (MCC), bladder leak point pressures (BLPP), and bladder compliance (BC) were measured in sham-operated and NB model rats. Immunofluorescent staining for c-kit was performed to determine ICC count in rat bladder trigone. The morphology and ultrastructure changes of ICCs were observed under an electron microscope. The mRNA levels of c-kit and SCF in bladder tissues were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein levels of c-kit, SCF, p-JAK, p-STAT1, and p-STAT3 in bladder tissues were determined by western blot. ICC proliferation was detected by CCK-8 assay. NB resulted in changes in ultrastructure changes of ICCs and a decrease in the number of ICCs and in expression of c-kit, SCF, p-JAK, p-STAT1, and p-STAT3 in NB tissues. Inhibition of SCF/c-kit signaling pathway suppressed ICC proliferation by inhibiting JAK/STAT3 pathway. Moreover, inhibition of SCF/c-kit signaling pathway impaired the SCF-induced attenuation of ICC damage in NB model rats. Collectively, our data indicate that SCF/c-kit signaling pathway participates in ICC damage in NB.

Quantitative 99mTc Labeling Kit for HYNIC-Conjugated Single Chain Antibody Fragments Targeting Malignant Mesothelioma.

Single chain antibody fragment (scFv) is a promising agent for imaging and targeted therapy. The objective of the study is to evaluate a kit formulation for 99mTc labeling of scFv for tumor imaging. The scFv was engineered to contain a cysteine tag to accommodate the specific conjugation of HYNIC and subsequent 99mTc labeling. The labeling conditions were formulated to allow instantaneous one-pot quantitative labeling. The reproducibility of labeling was evaluated at various time points during kit storage at -20 °C. In vitro cell binding experiments and HPLC analysis were performed to assess binding affinity and radiolabel stability, respectively. In vivo tumor targeting study was performed in xenograft models with biodistribution studied at 1, 3, and 24 h post-injection. The optimized kit with 5 µg SnF2, pH 5.5, and 50 µg GH along with as low as 15 µg of HYNIC-cys-scFv provided high labeling yield (>95%), high specific activity (1.8 × 107 Ci/Mol), and robust reproducibility with shelf life up to 90 days when stored at -20 °C. The in vitro cell binding study showed the labeled scFv maintained the binding capability with an apparent KD of ∼27 nM. The animal study using tumor-bearing mice showed high tumor uptake at 16.9%ID/g 24 h post-injection along with rapid blood clearance (0.18%ID/g) and kidney excretion (44%ID/g), resulting in very high contrast (tumor/muscle >200:1). A kit formulation for 99mTc labeling of scFvs targeting mesothelioma was developed based on specific HYNIC conjugation and GH (Glucoheptonate) as a coligand, producing not only high specific activity, but also improved tumor uptake. This convenient one-pot labeling method has the potential for translation into clinical use and is applicable to other scFvs as well.

Application of clean intermittent catheterization for neurogenic bladder in infants less than 1 year old.

BACKGROUND: Neurogenic bladder (NB) is a common pediatric urological disease caused by a variety of neurological pathologies. Clean intermittent catheterization (CIC) has been the preferred method to empty bladder. OBJECTIVE: To investigate the effect of CIC on preserving bladder and upper urinary tract function in infants less than 1 year old with NB. METHODS: A retrospective analysis was conducted on 76 infants with NB. Patients were divided into two groups according to treatment initiation: the early CIC group (ECG) (<1 year old) and the late CIC group (LCG) (>3 years old). RESULTS: Bladder compliance (BC), safe bladder capacity (SBC) and maximum cystometric capacity (MCC) were significantly higher in the ECG than those in the LCG at 6 years of follow-up respectively (P <  0.05). The frequencies of vesicoureteral reflux (VUR) and urinary tract infection (UTI) in the ECG were significantly lower than those in the LCG (P <  0.05) at 6 years of follow-up. Two and nine patients exhibited mild renal damage in the ECG and LCG, respectively, resulting in a significant difference (P <  0.05) at 6 years of follow-up. CONCLUSION: Early CIC plays an important role in preserving bladder function and preventing UTI and renal deterioration in infants with NB, especially in the first year of life.

The value of synchro-cystourethrometry for evaluating the relationship between urethral instability and overactive bladder.

PURPOSE: To investigate the correlation between urethral instability (URI) and overactive bladder (OAB) in children. METHODS: We retrospectively investigated 126 children with OAB and 36 children without OAB using synchro-cystourethrometry. The prevalence of detrusor overactivity (DO) and URI, and the diagnostic sensitivity of DO alone and DO combined with URI, was compared. The OAB children with URI voluntarily received transcutaneous electrical pudendal nerve stimulation with anisodamine (stimulation group, SG) or anisodamine alone (non-stimulation group, NSG). The effectiveness of treatment was evaluated. Average voided volume (AVV), maximum voided volume (MVV), and number of voids per day (NV) were collected and analyzed. RESULTS: In OAB children, the prevalence of DO and URI was 51.6 and 32.5%, respectively. The prevalence of URI was 5.6% in controls. The prevalence of URI was significantly higher in OAB children. The diagnostic sensitivity and Youden index of DO combined with URI were higher than DO alone. In SG, 45.7% of children were cured, with a ≥ 50% improvement rate of 82.9%, while no child was cured, with a ≥ 50% improvement rate of 36.8% in NSG. A significant increase in AVV and MVV together, with a decrease in NV, was seen in SG. There was a significant difference in visual analogue scale values between SG and NSG (P < 0.01). CONCLUSIONS: Urethral instability plays an essential role in the pathogenesis and progression of OAB in children. Synchro-cystourethrometry is a useful urodynamic technology to precisely diagnose OAB, and transcutaneous electrical pudendal nerve stimulation may be an effective treatment for OAB children induced by URI.

Novel human single chain antibody fragments that are rapidly internalizing effectively target epithelioid and sarcomatoid mesotheliomas.

Human antibodies targeting all subtypes of mesothelioma could be useful to image and treat this deadly disease. Here we report tumor targeting of a novel internalizing human single chain antibody fragment (scFv) labeled with (99m)Tc ((99m)Tc-M40) in murine models of mesothelioma of both epithelioid (M28) and sarcomatoid (VAMT-1) origins. (99m)Tc-M40 was taken up rapidly and specifically by both subtype tumor cells in vitro, with 68% to 92% internalized within 1 hour. The specificity of binding was evidenced by blocking (up to 95%) with 10-fold excess of unlabeled M40. In animal studies, tumors of both subtypes were clearly visualized by SPECT/CT as early as 1 hour postinjection of (99m)Tc-M40. Tumor uptake measured as percent of injected dose per gram tissue (%ID/g) at 3 hours was 4.38 and 5.84 for M28 and VAMT-1 tumors, respectively, significantly greater than all organs or tissues studied (liver, 2.62%ID/g; other organs or tissues <1.7%ID/g), except the kidneys (130.7%ID/g), giving tumor-to-blood ratios of 5:1 and 7:1 and tumor-to-muscle ratios of 45:1 and 60:1, for M28 and VAMT-1, respectively. The target-mediated uptake was confirmed by a nearly 70% reduction in tumor activity following administration of 10-fold excess of unlabeled scFv. Taken together, these results indicate that M40 can rapidly and specifically target epithelioid and sarcomatoid tumor cells, demonstrating the potential of this agent as a versatile targeting ligand for imaging and therapy of all subtypes of mesothelioma.

The effect of internalizing human single chain antibody fragment on liposome targeting to epithelioid and sarcomatoid mesothelioma.

Immunoliposomes (ILs) anchored with internalizing human antibodies capable of targeting all subtypes of mesothelioma can be useful for targeted imaging and therapy of this malignant disease. The objectives of this study were to evaluate both the in vitro and in vivo tumor targeted internalization of novel internalizing human single chain antibody (scFv) anchored ILs on both epithelioid (M28) and sarcomatoid (VAMT-1) subtypes of human mesothelioma. ILs were prepared by post-insertion of mesothelioma-targeting human scFv (M1) onto preformed liposomes and radiolabeled with (111)In ((111)In-IL-M1), along with control non-targeted liposomes ((111)In-CL). Incubation of (111)In-IL-M1 with M28, VAMT-1, and a control non-tumorigenic cell line (BPH-1) at 37 °C for 24 h revealed efficient binding and rapid internalization of ILs into both subtypes of tumor cells but not into the BPH-1 cells; internalization accounted for approximately 81-94% of total cell accumulation in mesothelioma cells compared to 37-55% in control cells. In tumor-bearing mice intravenous (i.v.) injection of (111)In-IL-M1 led to remarkable tumor accumulation: 4% and 4.7% injected dose per gram (% ID/g) for M28 and VAMT-1 tumors, respectively, 48 h after injection. Furthermore, tumor uptake of (111)In-IL-M1 in live xenograft animal models was verified by single photon emission computed tomography (SPECT/CT). In contrast, i.v. injection of (111)In-CL in tumor-bearing mice revealed very low uptake in both subtypes of mesothelioma, 48 h after injection. In conclusion, M1 scFv-anchored ILs showed selective tumor targeting and rapid internalization into both epithelioid and sarcomatoid subtypes of human mesothelioma, demonstrating its potential as a promising vector for enhanced tumor drug targeting.

Targeting prostate cancer cells in vivo using a rapidly internalizing novel human single-chain antibody fragment.

UNLABELLED: Human antibodies targeting prostate cancer cell surface epitopes may be useful for imaging and therapy. The objective of this study was to evaluate the tumor targeting of an internalizing human antibody fragment, a small-size platform, to provide high contrast in a mouse model of human prostate carcinoma. METHODS: A prostate tumor-targeting single-chain antibody fragment (scFv), UA20, along with a nonbinding control scFv, N3M2, were labeled with (99m)Tc and evaluated for binding and rapid internalization into human prostate tumor cells in vitro and tumor homing in vivo using xenograft models. For the in vitro studies, the labeled UA20 scFv was incubated at 37 degrees C for 1 h with metastatic prostate cancer cells (DU145) to assess the total cellular uptake versus intracellular uptake. For the animal studies, labeled UA20 and N3M2 scFvs were administered to athymic mice implanted subcutaneously with DU145 cells. Mice were imaged with small-animal SPECT/CT with concomitant biodistribution at 1 and 3 h after injection. RESULTS: The UA20 scFv was labeled in 55%-65% yield and remained stable in phosphate buffer within 24 h. The labeled UA20 scFv was taken up specifically by prostate tumor cells. Internalization was rapid, because incubation at 37 degrees C for less than 1 h resulted in 93% internalization of total cell-associated scFvs. In animal studies, SPECT/CT showed significant tumor uptake as early as 1 h after injection. At 3 h after injection, tumor uptake was 4.4 percentage injected dose per gram (%ID/g), significantly greater than all organs or tissues studied (liver, 2.7 %ID/g; other organs or tissues, <1 %ID/g), except the kidneys (81.4 %ID/g), giving tumor-to-blood and tumor-to-muscle ratios of 12:1 and 70:1, respectively. In contrast, the control antibody exhibited a tumor uptake of only 0.26 %ID/g, similar to that of muscle and fat. Tumor-specific targeting was evidenced by reduced tumor uptake of nearly 70% on administration of a 10-fold excess of unlabeled UA20 scFv. Kidney uptake was nonspecific, consistent with the route of excretion by scFvs. CONCLUSION: The UA20 scFv showed rapid and specific internalization in prostate tumor cells in vitro and accumulation in prostate tumor xenografts in vivo, demonstrating the potential for future development for prostate cancer imaging and targeted therapy.

Identification of MCAM/CD146 as the target antigen of a human monoclonal antibody that recognizes both epithelioid and sarcomatoid types of mesothelioma.

The prognosis for patients diagnosed with mesothelioma is generally poor, and currently available treatments are usually ineffective. Therapies that specifically target tumor cells hold much promise for the treatment of cancers that are resistant to current approaches. We have previously selected phage antibody display libraries on mesothelioma cell lines to identify a panel of internalizing human single chain (scFv) antibodies that target mesothelioma-associated, clinically represented cell surface antigens and further exploited the internalizing function of these scFvs to specifically deliver lethal doses of liposome-encapsulated small molecule drugs to both epithelioid and sarcomatous subtypes of mesothelioma cells. Here, we report the identification of MCAM/MUC18/CD146 as the surface antigen bound by one of the mesothelioma-targeting scFvs using a novel cloning strategy based on yeast surface human proteome display. Immunohistochemical analysis of mesothelioma tissue microarrays confirmed that MCAM is widely expressed by both epithelioid and sarcomatous types of mesothelioma tumor cells in situ but not by normal mesothelial cells. In addition, quantum dot-labeled anti-MCAM scFv targets primary meosthelioma cells in tumor fragment spheroids cultured ex vivo. As the first step in evaluating the therapeutic potential of MCAM-targeting antibodies, we performed single-photon emission computed tomography studies using the anti-MCAM scFv and found that it recognizes mesothelioma organotypic xenografts in vivo. The combination of phage antibody library selection on tumor cells and rapid target antigen identification by screening the yeast surface-displayed human proteome could be a powerful method for mapping the targetable tumor cell surface epitope space.

Biodistributions of 177Lu- and 111In-labeled 7E11 antibodies to prostate-specific membrane antigen in xenograft model of prostate cancer and potential use of 111In-7E11 as a pre-therapeutic agent for 177Lu-7E11 radioimmunotherapy.

INTRODUCTION: Prostate-specific membrane antigen is a transmembrane glycoprotein highly expressed in many prostate cancers and can be targeted with radiolabeled antibodies for diagnosis and treatment of this disease. To serve as a radioimmunotherapeutic agent, a kinetically inert conjugate is desired to maximize tumor uptake and tumor radiation dose with minimal nonspecific exposure to bone marrow and other major organs. MATERIALS AND METHODS: In this study, we assessed the pharmacokinetics and biodistribution of the 7E11 monoclonal antibody (MAb) radiolabeled with the lutetium-177 ((177)Lu)-tetraazacyclododecanetetraacetic acid conjugate system ((177)Lu-7E11) versus those of the 7E11 MAb radiolabeled with the indium-111 ((111)In)/glycyl-tyrosyl-(N,-diethylenetriaminepentaacetic acid)/lysine hydrochloride conjugate system ((111)In-7E11, also known as ProstaScint) to determine the feasibility of using (111)In-7E11 as a pre-therapeutic agent for (177)Lu-7E11 radioimmunotherapy. Pharmacokinetic and biodistribution studies of (177)Lu-7E11 in lymph node cancer of the prostate (LNCaP) xenograft mice were performed at 2, 8, 12, 24, 72, and 168 h after radiopharmaceutical administration. For (111)In-7E11, pharmacokinetic and biodistribution studies were performed at 8, 24, and 72 h. Parallel studies of (177)Lu-7E11 in non-tumor-bearing mice at 8, 24, and 72 h post-injection served as controls. Gamma scintigraphy was performed, followed by autoradiography and tissue counting, to demonstrate and quantify the distributions of radioconjugated MAb in the tumor and normal tissues. RESULTS AND DISCUSSION: Both (177)Lu- and (111)In-7E11 conjugates demonstrated an early blood pool phase in which uptake was dominated by the blood, lung, spleen and liver, followed by uptake and retention of the radiolabeled antibody in the tumor which was most prominent at 24 h. Total accumulation of radioconjugated MAb in tumor at 24 h was greater in the case of (177)Lu-7E11 in comparison to that of (111)In-7E11. Continued accumulation in tumor was observed for the entire time course studied for both (177)Lu-7E11 and (111)In-7E11. The liver was the only major organ demonstrating a significant difference in accumulation between the two conjugates. In conclusion, pharmacokinetic and biodistribution studies of (177)Lu-7E11 in LNCaP xenograft mouse models support its potential application as a radioimmunotherapeutic agent targeting prostate cancer, and the distribution and tumor uptake of (111)In-7E11 appear to be similar to those of (177)Lu-7E11, supporting its use as a pre-therapeutic tool to assess the potential accumulation of (177)Lu-7E11 radioimmunotherapeutic at sites of prostate cancer. However, the different accumulation patterns of the (111)In and (177)Lu immunoconjugates in liver will likely prevent the use of (111)In-7E11 as a true dosimetry tool for (177)Lu-7E11 radioimmunotherapy.