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© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. We present a silica nanoparticle (SNP) functionalized with polyphosphate (polyP) that accelerates the natural clotting process of the body. SNPs initiate the contact pathway of the blood-clotting system; short-chain polyP accelerates the common pathway by the rapid formation of thrombin, which enhances the overall blood-clotting system, both by accelerating fibrin generation and by facilitating the regulatory anticoagulation mechanisms essential for hemostasis. Analysis of the clotting properties of bare SNPs, bare polyP, and polyP-functionalized SNPs in plasma demonstrated that the attachment of polyP to SNPs to form polyP-SNPs creates a substantially enhanced synergistic effect that lowers clotting time and increases thrombin production at low concentrations. PolyP-SNP even retains its clotting function at ambient temperature. The polyP-SNP system has the potential to significantly improve trauma-treatment protocols and outcomes in hospital and prehospital settings.

Purpose: Nadofaragene firadenovec-vncg is a nonreplicating adenoviral vector-based gene therapy for bacillus Calmette-Guérin (BCG)-unresponsive carcinoma in situ (CIS) with/without high-grade Ta/T1. We report outcomes following 5 years of planned follow-up., Materials and Methods: This open-label phase 3 trial (NCT02773849) enrolled patients with BCG-unresponsive nonmuscle-invasive bladder cancer in 2 cohorts: CIS ± Ta/T1 (CIS; n = 107) and Ta/T1 without CIS (Ta/T1 cohort; n = 50). Patients received 75 mL (3 × 10 11 vp/mL) nadofaragene firadenovec intravesically once every 3 months with cystoscopy and cytology assessments, with continued treatment offered to those remaining high grade recurrence-free (HGRF)., Results: One hundred fifty-seven patients were enrolled from 33 US sites (n = 151 included in efficacy analyses). Median follow-up was 50.8 months (interquartile range 39.1-60.0), with 27% receiving ≥ 5 instillations and 7.6% receiving treatment for ≥ 57 months. Of patients with CIS 5.8% (95% CI 2.2-12.2) were HGRF at month 57, and 15% (95% CI 6.1-27.8) of patients with high-grade Ta/T1 were HGRF at month 57. Kaplan-Meier-estimated HGRF survival at 57 months was 13% (95% CI 6.9-21.5) and 33% (95% CI 19.5-46.6) in the CIS and Ta/T1 cohorts, respectively. Cystectomy-free survival at month 60 was 49% (95% CI 40.0-57.1): 43% (95% CI 32.2-53.7) in the CIS cohort and 59% (95% CI 43.1-71.4) in the Ta/T1 cohort. Overall survival at 60 months was 80% (71.0, 86.0): 76% (64.6-84.5) and 86% (70.9-93.5) in the CIS and Ta/T1 cohorts, respectively. Only 5 patients (4 with CIS and 1 with Ta/T1) experienced clinical progression to muscle-invasive disease., Conclusions: At 60 months, nadofaragene firadenovec-vncg allowed bladder preservation in nearly half of the patients and proved to be a safe option for BCG-unresponsive nonmuscle-invasive bladder cancer.

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment., Competing Interests: C.P.D. received personal compensation from FKD Therapies, Oy for consulting and advisory services, including serving as the Independent Chairman of steering committee for the phase 3 nadofaragene firadenovec (rAd-IFNα/Syn3) trial., (© 2022 The Author(s).)

A recent phase 3 trial of intravesical nadofaragene firadenovec reported a promising complete response rate for patients with bacillus Calmette-Guérin-unresponsive non-muscle-invasive bladder cancer. This study examined the ability of antiadenovirus antibody levels to predict the durability of therapeutic response to nadofaragene firadenovec. A standardized and validated quantitative assay was used to prospectively assess baseline and post-treatment serum antibody levels among 91 patients from the phase 3 trial, of whom 47 (52%) were high-grade recurrence free at 12 mo (responders). While baseline titers did not predict treatment response, 3-mo titer >800 was associated with a higher likelihood of durable response (p = 0.026). Peak post-treatment titers >800 were noted in 42 (89%) responders versus 26 (59%) nonresponders (p = 0.001; assay sensitivity, 89%; negative predictive value, 78%). Moreover, 22 (47%) responders compared with eight (18%) nonresponders had a combination of peak post-treatment titers >800 and peak antibody fold change >8 (p = 0.004; assay specificity, 82%; positive predictive value, 73%). A majority of responders continued to have post-treatment antibody titers >800 after the first 6 mo of therapy. In conclusion, serum antiadenovirus antibody quantification may serve as a novel predictive marker for nadofaragene firadenovec response durability. Future studies will focus on large-scale validation and clinical utility of the assay. PATIENT SUMMARY: This study reports on a planned secondary analysis of a phase 3 multicenter clinical trial that established the benefit of nadofaragene firadenovec, a novel intravesical gene therapeutic, for the treatment of patients with bacillus Calmette-Guérin (BCG)-unresponsive high-risk non-muscle-invasive bladder cancer. Prospective assessment of serum anti-human adenovirus type-5 antibody levels of patients in this trial indicated that a combination of post-treatment titers and fold change from baseline can predict treatment efficacy. While this merits additional validation, our findings suggest that serum antiadenovirus antibody levels can serve as an important predictive marker for the durability of therapeutic response to nadofaragene firadenovec., (Copyright © 2021 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: BCG is the most effective therapy for high-risk non-muscle-invasive bladder cancer. Nadofaragene firadenovec (also known as rAd-IFNa/Syn3) is a replication-deficient recombinant adenovirus that delivers human interferon alfa-2b cDNA into the bladder epithelium, and a novel intravesical therapy for BCG-unresponsive non-muscle-invasive bladder cancer. We aimed to evaluate its efficacy in patients with BCG-unresponsive non-muscle-invasive bladder cancer., Methods: In this phase 3, multicentre, open-label, repeat-dose study done in 33 centres (hospitals and clinics) in the USA, we recruited patients aged 18 years or older, with BCG-unresponsive non-muscle-invasive bladder cancer and an Eastern Cooperative Oncology Group status of 2 or less. Patients were excluded if they had upper urinary tract disease, urothelial carcinoma within the prostatic urethra, lymphovascular invasion, micropapillary disease, or hydronephrosis. Eligible patients received a single intravesical 75 mL dose of nadofaragene firadenovec (3 × 10 11 viral particles per mL). Repeat dosing at months 3, 6, and 9 was done in the absence of high-grade recurrence. The primary endpoint was complete response at any time in patients with carcinoma in situ (with or without a high-grade Ta or T1 tumour). The null hypothesis specified a complete response rate of less than 27% in this cohort. Efficacy analyses were done on the per-protocol population, to include only patients strictly meeting the BCG-unresponsive definition. Safety analyses were done in all patients who received at least one dose of treatment. The study is ongoing, with a planned 4-year treatment and monitoring phase. This study is registered with ClinicalTrials.gov, NCT02773849., Findings: Between Sept 19, 2016, and May 24, 2019, 198 patients were assessed for eligibility. 41 patients were excluded, and 157 were enrolled and received at least one dose of the study drug. Six patients did not meet the definition of BCG-unresponsive non-muscle-invasive bladder cancer and were therefore excluded from efficacy analyses; the remaining 151 patients were included in the per-protocol efficacy analyses. 55 (53·4%) of 103 patients with carcinoma in situ (with or without a high-grade Ta or T1 tumour) had a complete response within 3 months of the first dose and this response was maintained in 25 (45·5%) of 55 patients at 12 months. Micturition urgency was the most common grade 3-4 study drug-related adverse event (two [1%] of 157 patients, both grade 3), and there were no treatment-related deaths., Interpretation: Intravesical nadofaragene firadenovec was efficacious, with a favourable benefit:risk ratio, in patients with BCG-unresponsive non-muscle-invasive bladder cancer. This represents a novel treatment option in a therapeutically challenging disease state., Funding: FKD Therapies Oy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gene expression plasticity is central for macrophages' timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages are a distinct macrophage type, possessing immunoregulatory, anti-inflammatory, and angiogenic properties. Due to these features, regulatory macrophages are considered as a potential cell therapy product to treat clinical conditions, e.g., non-healing diabetic foot ulcers. In this study we characterized two differently manufactured clinically relevant regulatory macrophages, programmable cells of monocytic origin and comparator macrophages (M1, M2a and M0) using flow-cytometry, RT-qPCR, phagocytosis and secretome measurements, and RNA-Seq. We demonstrate that conventional phenotyping had a limited potential to discriminate different types of macrophages which was ameliorated when global transcriptome characterization by RNA-Seq was employed. Using this approach we confirmed that macrophage manufacturing processes can result in a highly reproducible cell phenotype. At the same time, minor changes introduced in manufacturing resulted in phenotypically and functionally distinct regulatory macrophage types. Additionally, we have identified a novel constellation of process specific biomarkers, which will support further clinical product development.

The interest in lentiviral vectors (LVs) has increased prominently for gene therapy applications, but few have reached the later stages of clinical trials. The main challenge has remained in scaling up the manufacturing process for the fragile vector to obtain high titers for in vivo usage. We have previously scaled up the LV production to iCELLis 500, being able to produce up to 180 L of harvest material in one run with perfusion. The following challenge considers the purification and concentration of the product to meet titer and purity requirements for clinical use. We have developed a downstream process, beginning with clarification, buffer exchange, and concentration, by tangential flow filtration. This is followed by a purification step using single membrane-based anion exchange chromatography and final formulation with tangential flow filtration. Different materials and conditions were compared to optimize the process, especially for the chromatography step that has been the bottleneck in lentiviral vector purification scale-up. The final infectious titer of the lentiviral vector product manufactured using the optimized scale-up process was determined to be 1.97 × 10 9 transducing units (TU)/mL, which can be considered as a high titer for lentiviral vectors., (© 2020 The Author(s).)

Background and Purpose: BCG unresponsive bladder cancer is an inherently resistant disease state for which the preferred treatment is radical cystectomy. To date, no effective intravesical therapies exist for patients who possess these resistant tumors. For this reason, many research groups are actively investigating/testing novel therapeutic agents to aid in bladder preservation for this patient population. This review article describes our 15-year experience developing and testing IFN-based gene therapy., Methods: A comprehensive review was performed of all studies pertaining to IFN-based gene therapy for non-muscle invasive bladder cancer from 2003 to 2018., Results and Conclusions: Over the past two decades, gene therapy has evolved into a powerful tool in our fight against cancer. After overcoming the initial barriers associated with gene delivery to the bladder, we have made significant strides forward in developing this novel therapeutic strategy for the treatment of this inherently resistant disease state. Our results to date are very encouraging; however, much work lies ahead to better understand and optimize this novel approach for treating non-muscle invasive bladder.

The therapeutic efficacy of a lentiviral vector (LV) expressing the herpes simplex virus thymidine kinase (HSV-TK) was studied in an immunocompetent rat glioblastoma model. Intraperitoneal ganciclovir injections (50 mg/kg/day) were administered for 14 consecutive days, resulting in reduced tumor volumes as monitored by MRI. Survival analyses revealed a significant improvement among the LV-expressing HSV-TK (LV-TK)/ganciclovir-treated animals when compared to non-treated control rats. However, a limiting factor in the use of LV has been the suboptimal small-scale production in flasks. Our aim during the translation phase, prior to entering the final pre-clinical and early clinical phases, was to develop a scalable, robust, and disposable manufacturing process for LV-TKs. We also aimed to minimize future process changes and enable production upscaling to make the process suitable for larger patient populations. The upstream process relies on fixed-bed iCELLis technology and transient plasmid transfection. This is the first time iCELLis 500 commercial-scale bioreactor was used for LV production. A testing strategy to determine the pharmacological activity of LV-TK drug product by measuring cell viability was developed, and the specificity of the potency assay was also proven. In this paper we focus on upstream process development while showing analytical development and the proof-of-concept of LV-TK functionality., (© 2019 The Authors.)

Type I interferon (IFN-I) has potent anti-tumor effects against urothelial carcinoma (UC) and may be an alternative treatment option for patients who do not respond to Bacillus Calmette-Guerin. However, the mechanisms that mediate the IFN-I-stimulated immune responses against UC have yet to be elucidated. Herein, we evaluated the anti-tumor mechanisms of IFN-I in UC in human patients and in mice. Patient tumors from a Phase I clinical trial with adenoviral interferon-α (Ad-IFNα/Syn3) showed increased expression of T cell and checkpoint markers following treatment with Ad-IFNα/Syn3 by RNAseq and immunohistochemistry analysis in 25% of patients. In mice, peritumoral injections of poly(I:C) into MB49 UC tumors was used to incite an IFN-driven inflammatory response that significantly inhibited tumor growth. IFN-I engaged both innate and adaptive cells, seen in increased intratumoral CD8 T cells, NK cells, and CD11b + Ly6G + cells, but tumor inhibition was not reliant on any one immune cell type. Nonetheless, poly(I:C)-mediated tumor regression and change in the myeloid cell landscape was dependent on IL-6. Mice were also treated with poly(I:C) in combination with anti-PD-1 monoclonal antibody (mAb) to assess for additional benefit to tumor growth and animal survival. When used in combination with anti-PD-1 mAb, IFN-I stimulation prolonged survival, coinciding with inhibition of angiogenesis and enriched gene signatures of metabolism, extracellular matrix organization, and MAPK/AKT signaling. Altogether, these findings suggest IFN-I's immune-driven antitumor response in UC is mediated by IL-6 and a collaboration of immune cells, and its use in combination with checkpoint blockade therapy can increase clinical benefit.

While treatment with surgery, radiotherapy and/or chemotherapy may prolong life for patients with glioblastoma, recurrence is inevitable. What is still being discovered is how much these treatments and recurrence of disease affect the molecular profiles of these tumors and how these tumors adapt to withstand these treatment pressures. Understanding such changes will uncover pathways used by the tumor to evade destruction and will elucidate new targets for treatment development. Nineteen matched pre-treatment and post-treatment glioblastoma tumors were subjected to gene expression profiling (Fluidigm, TaqMan assays), MGMT promoter methylation analysis (pyrosequencing) and protein expression analysis of the DNA repair pathways, known to be involved in temozolomide resistance (immunohistochemistry). Gene expression profiling to molecularly subtype tumors revealed that 26% of recurrent post-treatment specimens did not match their primary diagnostic specimen subtype. Post-treatment specimens had molecular changes which correlated with known resistance mechanisms including increased expression of APEX1 ( p < 0.05) and altered MGMT methylation status. In addition, genes associated with immune suppression, invasion and aggression ( GPNMB, CCL5, and KLRC1 ) and polarization toward an M2 phenotype ( CD163 and MSR1 ) were up-regulated in post-treatment tumors, demonstrating an overall change in the tumor microenvironment favoring aggressive tumor growth and disease recurrence. This was confirmed by in vitro studies that determined that glioma cell migration was enhanced in the presence of M2 polarized macrophage conditioned media. Further, M2 macrophage-modulated migration was markedly enhanced in post-treatment (temozolomide resistant) glioma cells. These findings highlight the ability of glioblastomas to evade not only the toxic onslaught of therapy but also to evade the immune system suggesting that immune-altering therapies may be of value in treating this terrible disease.

Purpose Many patients with high-risk non-muscle-invasive bladder cancer (NMIBC) are either refractory to bacillus Calmette-Guerin (BCG) treatment or may experience disease relapse. We assessed the efficacy and safety of recombinant adenovirus interferon alfa with Syn3 (rAd-IFNα/Syn3), a replication-deficient recombinant adenovirus gene transfer vector, for patients with high-grade (HG) BCG-refractory or relapsed NMIBC. Methods In this open-label, multicenter (n = 13), parallel-arm, phase II study ( ClinicalTrials.gov identifier: NCT01687244), 43 patients with HG BCG-refractory or relapsed NMIBC received intravesical rAd-IFNα/Syn3 (randomly assigned 1:1 to 1 × 10 11 viral particles (vp)/mL or 3 × 10 11 vp/mL). Patients who responded at months 3, 6, and 9 were retreated at months 4, 7, and 10. The primary end point was 12-month HG recurrence-free survival (RFS). All patients who received at least one dose were included in efficacy and safety analyses. Results Forty patients received rAd-IFNα/Syn3 (1 × 10 11 vp/mL, n = 21; 3 × 10 11 vp/mL, n = 19) between November 5, 2012, and April 8, 2015. Fourteen patients (35.0%; 90% CI, 22.6% to 49.2%) remained free of HG recurrence 12 months after initial treatment. Comparable 12-month HG RFS was noted for both doses. Of these 14 patients, two experienced recurrence at 21 and 28 months, respectively, after treatment initiation, and one died as a result of an upper tract tumor at 17 months without a recurrence. rAd-IFNα/Syn3 was well tolerated; no grade four or five adverse events (AEs) occurred, and no patient discontinued treatment because of an adverse event. The most frequently reported drug-related AEs were micturition urgency (n = 16; 40%), dysuria (n = 16; 40%), fatigue (n = 13; 32.5%), pollakiuria (n = 11; 28%), and hematuria and nocturia (n = 10 each; 25%). Conclusion rAd-IFNα/Syn3 was well tolerated. It demonstrated promising efficacy for patients with HG NMIBC after BCG therapy who were unable or unwilling to undergo radical cystectomy.

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.

We present a silica nanoparticle (SNP) functionalized with polyphosphate (polyP) that accelerates the natural clotting process of the body. SNPs initiate the contact pathway of the blood-clotting system; short-chain polyP accelerates the common pathway by the rapid formation of thrombin, which enhances the overall blood-clotting system, both by accelerating fibrin generation and by facilitating the regulatory anticoagulation mechanisms essential for hemostasis. Analysis of the clotting properties of bare SNPs, bare polyP, and polyP-functionalized SNPs in plasma demonstrated that the attachment of polyP to SNPs to form polyP-SNPs creates a substantially enhanced synergistic effect that lowers clotting time and increases thrombin production at low concentrations. PolyP-SNP even retains its clotting function at ambient temperature. The polyP-SNP system has the potential to significantly improve trauma-treatment protocols and outcomes in hospital and prehospital settings., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glioblastomas, (grade 4 astrocytomas), are aggressive primary brain tumors characterized by histopathological heterogeneity. High-resolution sequencing technologies have shown that these tumors also feature significant inter-tumoral molecular heterogeneity. Molecular subtyping of these tumors has revealed several predictive and prognostic biomarkers. However, intra-tumoral heterogeneity may undermine the use of single biopsy analysis for determining tumor genotype and has implications for potential targeted therapies. The clinical relevance and theories of tumoral molecular heterogeneity in glioblastoma are discussed.

Background: Broad or general consent given by cancer patients for their tissue, blood, and clinical information to be stored in institutional biorepositories is fundamental to enable future ethical translational cancer research. The decision to consent for biobanking will contribute to the development of advanced diagnostic and prognostic tests, as well as new therapies to improve patient outcomes. While the rate of patient participation in biobanking programs is generally reported as high worldwide, few studies have investigated factors that may influence this decision. Biobanking at our medical research institute, an associated public (government-run) university hospital, and private hospital has been established for over 20 years, with collection of certain tumor types embedded in the research culture of these institutions. In this study, we investigated factors that may influence a cancer patient's decision to give broad consent for biobanking of their specimens., Methods: Data on patient consent were collected over a 6-month period from both government and private hospitals associated with our medical research institute. Factors considered included gender, patient age at surgery, type of malignancy (breast, duodenal, cervical, endometrial, gastric, liver, esophageal, ovarian, pancreatic, pelvic, uterine, or vulval), type of institution where surgery was performed, and timing of consent., Results: Of 171 cancer patients, 159 (93%) gave broad consent for biobanking of their tissue and blood specimens for future cancer research projects receiving ethical and scientific approval. None of the factors analyzed was shown to influence a patient's decision to contribute biological specimens and clinical data to a biorepository for future medical research., Conclusion: Biobanking for future ethically and scientifically approved research projects in an established institution is an initiative that receives strong support from patients undergoing cancer surgery, independent of factors including gender, age, type of tumor, type of institution where surgery was performed, or timing of consent.

miR-210 is a key regulator of response to hypoxia. Pheochromocytomas (PCs) and paragangliomas (PGLs) with germline SDHx or VHL mutations have pseudohypoxic gene expression signatures. We hypothesised that PC/PGLs containing SDHx or VHL mutations, and succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumours (GISTs), would overexpress miR-210 relative to non-SDH or -VHL-mutated counterparts. miR-210 was analysed by quantitative PCR in i) 39 PC/PGLs, according to genotype (one SDHA, five SDHB, seven VHL, three NF1, seven RET, 15 sporadic, one unknown) and pathology (18 benign, eight atypical, 11 malignant, two unknown); ii) 18 GISTs, according to SDHB immunoreactivity (nine SDH-deficient and nine SDH-proficient) and iii) two novel SDHB-mutant neurosphere cell lines. miR-210 was higher in SDHx- or VHL-mutated PC/PGLs (7.6-fold) compared with tumours without SDHx or VHL mutations (P=0.0016). miR-210 was higher in malignant than in unequivocally benign PC/PGLs (P=0.05), but significance was lost when benign and atypical tumours were combined (P=0.08). In multivariate analysis, elevated miR-210 was significantly associated with SDHx or VHL mutation, but not with malignancy. In GISTs, miR-210 was higher in SDH-deficient (median 2.58) compared with SDH-proficient tumours (median 0.60; P=0.0078). miR-210 was higher in patient-derived neurosphere cell lines containing SDHB mutations (6.5-fold increase) compared with normal controls, in normoxic conditions (P<0.01). Furthermore, siRNA-knockdown of SDHB in HEK293 cells increased miR-210 by 2.7-fold (P=0.001) under normoxia. Overall, our results suggest that SDH deficiency in PC, PGL and GISTs induces miR-210 expression and substantiates the role of aberrant hypoxic-type cellular responses in the development of these tumours.

Background: MicroRNA 132 (miR-132) is dysregulated in a range of human malignancies; however, its role in glioma has not been reported. The aim of this study was to profile miR-132 expression in a cohort of patients with primary glioblastoma multiforme (GBM) treated with the Stupp regimen and to correlate microRNA levels with patient outcome., Methods: miR-132 levels relative to RNU44 were assessed by quantitative reverse transcription-polymerase chain reaction in 43 GBMs and normal brain tissue. The cohort comprised patients less than 72 years of age with Eastern Cooperative Oncology Group (ECOG) scores between 0 and 2 who had undergone 6-week concomitant radiation and temozolomide followed by adjuvant temozolomide. Survival data were available for all cases. Tumors were characterized for O6-methylguanine-DNA methyltransferase (MGMT) methylation and isocitrate dehydrogenase (IDH) 1/2 mutation status. Associations between miR-132 expression and clinical indicators were analyzed., Results: Tumor miR-132 levels ranged from 0.07- to 40.4-fold increase (mean = 5.5-fold increase) relative to normal brain. High-level miR-132 (above the mean) independently predicted for a significantly shorter overall survival (P = .008). miR-132 was a stronger prognostic indicator than ECOG score (P = .012) and age at diagnosis (P = .026) but did not correlate with MGMT methylation status or extent of tumor resection. Cox regression analysis confirmed high miR-132 as the strongest predictor of outcome (P = .010) with a hazard ratio of 2.8., Conclusions: This study identified high miR-132 expression as a biomarker of poor prognosis in patients with primary GBM treated with the Stupp regimen.

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an alpha-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic alpha-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally "unwound." The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics.

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression.

Purpose: Human ultraviolet light (UV) filters, such as kynurenine (Kyn), readily deaminate to reactive unsaturated ketones that covalently modify proteins in older human lenses. The aim of this study was to examine in vitro rates of formation and decomposition of the three major Kyn-amino acid adducts and possible consequences for the lens., Methods: The t-Boc-protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC., Results: At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in beta-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys., Conclusions: These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.

Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.

Purpose: Posttranslational modification by UV filters is a key event in human lenses that appears to be largely responsible for normal age-dependent yellowing. It has been proposed that subsequent reactions of these covalently bound UV filters may also be involved in the genesis of age-related nuclear cataract. To examine this hypothesis, the levels of kynurenine-lysine and kynurenine-histidine were measured in both normal and cataractous human lenses., Methods: Proteins isolated from the nuclei of normal lenses and lenses with and types I to IV nuclear cataract were hydrolyzed in 6 M HCl, and the levels of kynurenine-lysine and kynurenine-histidine were determined by HPLC., Results: The content of kynurenine-lysine and kynurenine-histidine decreased substantially with the progression of age-related nuclear cataract. On average, levels of both kynurenine adducts were four times lower in advanced cataract (type IV) than in normal lenses. Simple autoxidation of the derivatives did not appear to be responsible for this decrease, because incubation in the presence of oxygen or H(2)O(2) did not affect adduct stability., Conclusions: Although protein-bound kynurenine accumulates over time in normal lenses, the levels attached to the proteins decrease significantly with the progression of age-related nuclear cataract. This finding suggests that in cataract there is a breakdown of the protein-bound adducts. Such further reactions of bound UV filters may contribute to the etiology of age-related nuclear cataract.

The physiochemical and healing characteristics of the Plasma-TFE synthetic artery (Atrium Medical Corp., Amherst, NH) were studied and compared to a similarly constructed polyethylene terephthalate graft without Plasma-TFE treatment. Plasma-TFE treatment is a method of coating a graft's surface with tetrafluoroethylene and was found to alter significantly the surface chemistry and energy of the graft. However, results of this study suggested that Plasma-TFE treatment had no effect on tissue penetration and neointima formation after implantation into the abdominal aorta of the dog for 8 weeks.

The diagnosis and management of surgical diseases of the esophagus are discussed. Esophageal foreign bodies, strictures, esophagobronchial fistulas, diverticula, gastroesophageal intussusception, hiatal hernias, and megaesophagus are included in the discussion.