Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

Endoscopic Injection of Low Dose Triamcinolone: A Simple, Minimally Invasive, and Effective Therapy for Interstitial Cystitis With Hunner Lesions.

OBJECTIVE: To investigate the efficacy of low dose triamcinolone injection for effectiveness and durability in patients with interstitial cystitis/bladder pain syndrome (IC/BPS) with Hunner lesions (HL). MATERIALS AND METHODS: Clinical data from patients with HL who underwent endoscopic submucosal injection of triamcinolone were reviewed. Demographics, pre- and postoperative pain and nocturia scores, and long-term clinical outcomes were assessed. Duration of response was estimated by time to repeat procedure. Kaplan-Meier estimator was used to evaluate time to repeat procedure. RESULTS: A total of 36 patients who received injections of triamcinolone between 2011 and 2015 were included. Median age ± standard deviation of patients was 61.5 ± 12.0 years; 28 (77.8%) were female patients and 8 (22.2%) were male patients. Twenty six patients (72.2%) received only 1 set of injections, 8 (22.2%) received 2 sets of injections, and 2 (5.56%) received 3 or more sets of injections. Average time between injections in those receiving more than 1 set of injections was 344.9 days (median: 313.5, range: 77-714). Preprocedural pain scores were 8.3 ± 1.2 (mean ± standard deviation) on Likert pain scale (0-10), and mean postprocedural pain scores at approximately 1 month were 3.8 ± 2.2, P <.001. Mean preprocedural nocturia bother scores was 7.5 ± 2.0 and mean postprocedural nocturia bother scores was 5.1 ± 2.5, P <.001. CONCLUSION: Endoscopic submucosal injection of low dose triamcinolone in patients with IC/BPS with HL is an effective and durable adjunct to existing treatment modalities. This approach is associated with low morbidity and can be performed on an outpatient basis.

Crosstalk between the immune system and neural pathways in interstitial cystitis/bladder pain syndrome.

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a condition causing intense pelvic pain and urinary symptoms. While it is thought to affect millions of people and significantly impair quality of life, difficulty with diagnosis and a lack of reliably effective treatment options leave much progress to be made in managing this condition. We describe what is currently known about the immunological and neurological basis of this disease, focusing on the interactions between the immune and nervous system. Evidence for immune involvement in IC/BPS comes from its high co-occurrence with known autoimmune diseases, altered cytokine profiles, and immune cell infiltration in patients. These cytokines have the ability to cross-talk with the nervous system via NGF signaling, resulting in hyper-sensitization of pain receptors, causing them to release substance P and creating a positive feedback loop of neuroinflammation. While it seems that the crosstalk between the immune and nervous system in IC is understood, much of the information comes from studying other diseases or from animal models, and it remains to be confirmed in patients with the disease. Identifying biomarkers and confirming the mechanism of IC/BPS are ultimately important for selecting drug targets and for improving the lives of patients with this disease.

Activation of GPER-1 estradiol receptor downregulates production of testosterone in isolated rat Leydig cells and adult human testis.

PURPOSE: Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis. MATERIALS AND METHODS: Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. RESULTS: GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20-30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs. CONCLUSIONS: Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

A novel sorting technology allows for highly efficient selection of sperm without chromatin damage.

Sperm chromatin damage has been associated with male infertility, increased risk for spontaneous abortion, and poor embryo development. Available methods for detecting chromatin damage render the sperm no longer suitable for clinical use. Early apoptotic events resulting in chromatin damage are associated with increased permeability of the cell membrane to large ions. We propose the use of a large fluorescent organic cation, proprietary fluorochrome (PF-1), for fluorescence-activated cell sorting (FACS) for negative selection of sperm without chromatin damage. Sperm with chromatin damage are PF-1 positive. Performance of cell sorting by PF-1 was verified with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) after FACS on PF-1(+) and PF-1(-) subpopulations. Whereas 19.5% of PF-1 positive sperm were TUNEL positive only 1.5% sperm in the PF-1(-) fraction were TUNEL positive (p < 0.00001). TUNEL values below 1.9% were considered background fluorescence. Post-sorting motility and vitality were 49.4% (SD: 12.5) and 65.0% (SD: 14.99), respectively. Proprietary fluorochrome activated sperm sorting may decrease or most likely eliminate all of TUNEL positive sperm without adverse effects on viability, providing a new therapeutic avenue for men with a high percentage of TUNEL positive sperm. Further research is needed to determine if the reduction in TUNEL positive sperm using PF-1 will improve in vitro fertilization (IVF) outcomes.