Ultrasound-guided intramural inoculation of orthotopic bladder cancer xenografts: a novel high-precision approach

Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23–37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice

Inflammasome and IL-1β-Mediated Disorders

The NLRP3 inflammasome is an intracellular complex that regulates the release of proinflammatory cytokines such as interleukin-1β in response to exogenous pathogens and endogenous danger signals. Evidence from studies involving human genetics, human ex vivo mononuclear cell responses, and in vivo and in vitro murine models confirms the importance of the inflammasome and interleukin-1β in the pathogenesis of several inherited and complex diseases. The availability of several effective interleukin-1β targeted therapies has allowed for successful proof-of-concept studies in several of these disorders. However, many other diseases are likely to be mediated by the inflammasome and interleukin-1β, providing additional targets in the future

Aberrant methylation of Polo-like kinase CpG islands in Plk4 heterozygous mice

<p>Abstract</p> <p>Background</p> <p>Hepatocellular carcinoma (HCC), one of the most common cancers world-wide occurs twice as often in men compared to women. Predisposing conditions such as alcoholism, chronic viral hepatitis, aflatoxin B1 ingestion, and cirrhosis all contribute to the development of HCC.</p> <p>Methods</p> <p>We used a combination of methylation specific PCR and bisulfite sequencing, qReal-Time PCR (qPCR), and Western blot analysis to examine epigenetic changes for the <it>Polo-like kinases </it>(<it>Plks</it>) during the development of hepatocellular carcinoma (HCC) in <it>Plk4 </it>heterozygous mice and murine embryonic fibroblasts (MEFs).</p> <p>Results</p> <p>Here we report that the promoter methylation of <it>Plk4 </it>CpG islands increases with age, was more prevalent in males and that <it>Plk4 </it>epigenetic modification and subsequent downregulation of expression was associated with the development of HCC in <it>Plk4 </it>mutant mice. Interestingly, the opposite occurs with another Plk family member, <it>Plk1 </it>which was typically hypermethylated in normal liver tissue but became hypomethylated and upregulated in liver tumours. Furthermore, upon alcohol exposure murine embryonic fibroblasts exhibited increased <it>Plk4 </it>hypermethylation and downregulation along with increased centrosome numbers and multinucleation.</p> <p>Conclusions</p> <p>These results suggest that aberrant <it>Plk </it>methylation is correlated with the development of HCC in mice.</p

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Background: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. Results: We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. Conclusions: Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases

Concurrent Oral 1 - Therapy of rheumatic disease: OP4. Effectiveness of Rituximab in Rheumatoid Arthritis: Results from the British Society for Rheumatology Biologics Register (BSRBR)

Background: Rituximab (RTX) in combination with methotrexate (MTX) has been licensed since 2006 for the management of severe active rheumatoid arthritis (RA) in patients who have failed at least one anti-tumour necrosis factor (anti-TNF) therapy. Published clinical trials have demonstrated the efficacy of RTX in improving both clinical symptoms and patients' physical function. This study aimed to assess the effectiveness of RTX in RA patients treated in routine clinical practice by examining clinical and patient reported outcomes six months after receiving a first course of RTX. Methods: The analysis involved 550 RA patients registered with the BSRBR, who were starting RTX and were followed up for at least 6 months. Change in Disease Activity Score (DAS28) and European League Against Rheumatism (EULAR) response were used to assess the clinical response while change in Health Assessment Questionnaire (HAQ) score was used to assess the physical function of the patients 6 months after starting RTX. The change in DAS28 and HAQ was compared between seronegative and seropositive patients and anti-TNF naïve patients versus anti-TNF failures. The response was also compared between patients receiving RTX in combination with MTX, other non-biologic disease modifying anti-rheumatic drugs (nbDMARDs) or no nbDMARDs. Results: The mean (s.d.) age of the cohort was 59 (12) years and 78% of the patients were females. The patients had a mean (s.d.) of 15 (10) years of disease duration. 16% were biologic naïve while 84% were anti-TNF failures. 32% of the patients were seronegative and 68% were seropositive. The mean (95% CI) DAS28 at baseline was 6.2 (6.1, 6.3) which decreased to 4.8 (4.7, 4.9) at 6 months of follow up. 16% were EULAR good responders, 43% were moderate responders and 41% were non responders. The mean (95% CI) change in HAQ was −0.1 (−0.2, −0.1) (Table 1). The mean change in DAS28 was similar in seropositive and seronegative patients (p = 0.18) while the anti-TNF naïve patients showed a greater reduction in DAS28 scores than anti-TNF failures (p = 0.05). Patients receiving RTX in combination with MTX showed similar changes in DAS28 and HAQ compared to patients receiving RTX alone or with other nbDMARDs. Conclusions: RTX has proven to be effective in the routine clinical practice. Anti-TNF naïve patients seem to benefit more from RTX treatment than anti-TNF failures. Disclosure statement: The authors have declared no conflicts of interes

Search for rare or forbidden decays of the D0 meson

We present a search for nine lepton-number-violating and three lepton-flavor-violating neutral charm decays of the type D0→h'−h−ℓ'+ℓ+ and D0→h'−h+ℓ'±ℓ∓, where h and h′ represent a K or π meson and ℓ and ℓ′ an electron or muon. The analysis is based on 468 fb−1 of e+e− annihilation data collected at or close to the Υ(4S) resonance with the BABAR detector at the SLAC National Accelerator Laboratory. No significant signal is observed for any of the twelve modes, and we establish 90% confidence level upper limits on the branching fractions in the range (1.0–30.6)×10−7. The limits are between 1 and 3 orders of magnitude more stringent than previous measurements.publishedVersio

Light meson spectroscopy from Dalitz plot analyses of ηc decays to η0 K+K− , η0 π + π − , and ηπ + π − produced in two-photon interactions

We study the processes γγ→ηc→η′K+K−, η′π+π−, and ηπ+π− using a data sample of 519  fb−1 recorded with the BABAR detector operating at the SLAC PEP-II asymmetric-energy e+e− collider at center-of-mass energies at and near the Υ(nS) (n=2, 3, 4) resonances. This is the first observation of the decay ηc→η′K+K− and we measure the branching fraction Γ(ηc→η′K+K−)/(Γ(ηc→η′π+π−)=0.644±0.039stat±0.032sys. Significant interference is observed between γγ→ηc→ηπ+π− and the nonresonant two-photon process γγ→ηπ+π−. A Dalitz plot analysis is performed of ηc decays to η′K+K−, η′π+π−, and ηπ+π−. Combined with our previous analysis of ηc→K¯Kπ, we measure the K∗0(1430) parameters and the ratio between its η′K and πK couplings. The decay ηc→η′π+π− is dominated by the f0(2100) resonance, also observed in J/ψ radiative decays. A new a0(1700)→ηπ resonance is observed in the ηc→ηπ+π− channel. We also compare ηc decays to η and η′ final states in association with scalar mesons as they relate to the identification of the scalar glueball.publishedVersio

Measurements of the absolute branching fractions of B± →k±Xc c

A study of the two-body decays B±→Xc¯cK±, where Xc¯c refers to one charmonium state, is reported by the BABAR Collaboration using a data sample of 424 fb−1. The absolute determination of branching fractions for these decays are significantly improved compared to previous BABAR measurements. Evidence is found for the decay B+→X(3872)K+ at the 3σ level. The absolute branching fraction B[B+→X(3872)K+]=[2.1±0.6(stat)±0.3(syst)]×10−4 is measured for the first time. It follows that B[X(3872)→J/ψπ+π−]=(4.1±1.3)%, supporting the hypothesis of a molecular component for this resonance.publishedVersio

The Association of Dietary Intake of Purine-Rich Vegetables, Sugar-Sweetened Beverages and Dairy with Plasma Urate, in a Cross-Sectional Study

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