bluuebunny/crossref_metadata_2025 huggingface dataset:
P184 Developing a pathway for tocilizumab treatment in giant cell arteritis: a South London regional experience
Authors: Saha Pratyasha , Sandhu Virinderjit , Robinson Helena , Ezeonyeji Amara , Al-Shakarchi Israa , Chander Sumeet , Suresh Resmy , Kaul Arvind , Sofat Nidhi
Published: April 2020

Background

Tocilizumab is now approved by the National Institute of Health and Care Excellence (NICE) for up to a year in relapsing or refractory giant cell arteritis (GCA). The practicalities of developing a pathway for referral using a hub and spoke model were previously unknown. Here we discuss our novel experiences utilising tocilizumab for GCA in its first year of licensing, after the introduction of a new regional multi-disciplinary team referral pathway.

Methods

We assessed all patients started on tocilizumab for GCA between August 2018-May 2019. The central assessing hub is St George’s University Hospitals NHS Foundation Trust, a large tertiary rheumatology department in the South of England, serving a population of 1.3 million.

Results

As per Table 1 below. A total of 9 patients were identified: 6 female and 3 male, with an average age of 74.2 (range 63-80). 5 patients were referred internally from clinicians at St George’s Hospital, with the remainder from local district general hospitals. Steroid protocols between patients were varied, and two-thirds required a 3-day IV methylprednisolone course, including all 4 patients with visual symptoms. A third of patients were on concurrent methotrexate, a disease-modifying antirheumatic drug. 8 of 9 patients were on alendronate, vitamin D/calcium, and a gastroprotective agent, and 7 were on aspirin. Reported side effects from steroids were common, with weight gain, increased appetite and osteoporosis noted. All our 9 patients continue their tocilizumab injections, with one individual having a 3-month break for a routine hip operation, and another a 1-month hiatus due to temporary derangement in liver function tests. Tocilizumab proffered improved disease control and few side effects were noted. 3 patients have now been on tocilizumab for 12 months and raise interesting discussions about ongoing funding and treatment efficacy.

Conclusion

Our case series shows the development and delivery of an effective hub and spoke referral pathway for tocilizumab treatment in GCA. We show that steroid dosing could be reduced with tocilizumab, and that all subjects received full funding for treatment. Our referral pathway has encouraged the uptake of the IL-6 monoclonal antibody treatment for GCA and compliance with NICE guidelines.

Disclosures

P. Saha None. V. Sandhu None. H. Robinson None. A. Ezeonyeji None. I. Al-Shakarchi None. S. Chander None. R. Suresh None. A. Kaul None. N. Sofat None.
P186 Efficacy and safety of rituximab in the treatment of systemic vasculitis: experience at a single centre
Authors: Orr Carl , Verdiyeva Ayna , Ahmad Nadia , Ariff Hairulhadi , Luqmani Raashid
Published: April 2020

Background

Rituximab is a monoclonal chimeric antibody targeting the mature B cell molecule CD20, expressed on the cell surface. It is licenced and widely used in the treatment of autoimmune diseases, including the ANCA associated vasculitides and rheumatoid arthritis. The efficacy and safety of rituximab in rare diseases requires ongoing appraisal.

Methods

We interrogated the records of 62 consecutive patients treated with rituximab for small vessel systemic vasculitis at our unit over the last 3 years, using a contemporaneously maintained central database, as well as individual electronic patient records.

Results

The patient demographic data, and main results are presented in Table 1. 31 (50%) of the patients treated with rituximab were female, and the indication for treatment in most cases was ANCA associated vasculitis (77.4%). After a mean follow up period of 2.04 years, the mean reduction in BVAS was 1.64 (3.83). 5 severe adverse events were recorded, defined as death or hospitalisation potentially related to treatment with rituximab. 3 patients died of respiratory sepsis, 1 experienced a wound infection, and 1 experienced an ischaemic colon. Four patients were noted to have low immunoglobulins.

Conclusion

Rituximab is an effective treatment for many patients with systemic, small vessel vasculitis. Understanding the safety profile of this drug in the context of our cohort is challenging, given the significant morbidity associated with the underlying disease, as well as the age profile and concomitant immunosuppressive medications. Continued surveillance regarding efficacy and safety of rituximab in the small vessel systemic vasculitides remains invaluable.

Disclosures

C. Orr None. A. Verdiyeva None. N. Ahmad None. H. Ariff None. R. Luqmani None.
P212 The effect of body weight in response to subcutaneous tocilizumab in patients with RA
Authors: Davies Rebecca , Vivekanantham Arani , Lunt Mark , Watson Kath , Hyrich Kimme L , Group BSRBR-RA Contributors , Bluett James
Published: April 2020

Background

Tocilizumab is an IL-6 receptor humanised monoclonal antibody treatment option in rheumatoid arthritis (RA) who have not responded or are intolerant of disease modifying anti-rheumatic drugs (DMARDs) or other biologics. Tocilizumab was available initially as an intravenous preparation, dosed according to weight, and more recently as a subcutaneous (SC) preparation given at 162mg/weekly irrespective of body weight. Obesity is highly prevalent in RA and there has been concern that starting or switching patients to SC tocilizumab could reduce its effectiveness in those patients with a higher body weight. The objective of the current study is to investigate the relationship between body weight and DAS28 response at 6 months in tocilizumab naïve RA patients starting SC tocilizumab.

Methods

The study population comprised RA subjects recruited to the BSRBR-RA up to 30/11/2018 commencing SC tocilizumab for the first time. Patients had to be tocilizumab naïve and have at least one six monthly study follow-ups recorded after starting tocilizumab. Baseline characteristics at point of starting tocilizumab are described. Linear regression, fully adjusted for relevant confounders, was used to investigate the relationship between change in DAS28 score from baseline to six months and body weight per ten kilograms (kg), and in a separate analysis, as BMI category. Multiple imputation was used to handle missing data.

Results

Three hundred and fifteen patients starting SC tocilizumab were eligible for analysis. The median age was 60 years, majority were female, and had median disease duration of 11 years at baseline. Seventy percent had prior biologic exposure. Median weight was 76kg, and the majority of patients were categorised as normal weight (37%) or pre-obesity (31%) according to BMI. Median DAS28 score was 5.5 at start of treatment with median improvement after 6-months of 2.0 units. The fully adjusted linear regression model showed no association between body weight or BMI and change in DAS28 score at six months (Table 1).

Conclusion

Data from this study show that body weight does not appear to affect initial response to SC tocilizumab. This is reassuring given that patients are likely to be given subcutaneous tocilizumab due to ease of administration and reduced hospital costs.

Disclosures

R. Davies None. A. Vivekanantham None. M. Lunt None. K. Watson None. K.L. Hyrich None. J. Bluett None.
P240 Can ultrasound alone predict the need to treat ACPA positive individuals without synovitis?
Authors: Duquenne Laurence M , Mankia Kulveer , Montoya Leticia Garcia , Matteo Andrea Di , Nam Jacqueline , Emery Paul
Published: April 2020

Background

In anti-cyclic citrullinated peptide antibody-positive (ACPA+) individuals without clinical synovitis (at-risk), to define the critical ultrasound (US) features sufficiently predictive for inflammatory arthritis (IA) to enable logical initiation of therapy.

Methods

In a single centre prospective cohort, at risk ACPA+ individuals with a new musculoskeletal symptoms underwent an US scan of 38 joints and 18 tendons at first visit. The predictive value of US abnormalities (Power Doppler (PD), Grey Scale (GS), erosion or tenosynovitis (TSV)) for progression to IA was analysed and the best predictive joints determined by Multivariable Cox Regression, adjusted for confounders. The US results were combined with clinical symptoms/findings to produce predictive models.

Results

Consecutive at-risk ACPA+ individuals (n = 457, mean age 50.3 years old, 74.2% women) were followed up for median of 15.4 months (range 0.1-127.4), a complete dataset with follow-up of at least 6 months was available for 319 of them. 135 (29.5%) developed IA after a median of 11.3 months (range 0.1-111.7). The negative predictive value of a US scan without any abnormality was 82%. In multivariable Cox regression, both PD and TSV were predictive of progression, with respectively hazard ratios of 1.2 (9=0.026) and 1.13 (p = 0.025). All US abnormalities had a high specificity (spec) but only moderate sensitivity (sens), PD was the most specific with a spec/sens of 0.94/0.23, followed by TSV with a spec/sens of 0.91/0.26 but the best area under the curve (AUC) of 0.599 (P = 0.0015). The addition ACPA titre (high compared to low), but not GS, improved spec/sens up to 0.92/0.34 and AUC to 0.964 (p < 0.001). A selection of US and clinical data of 14 joints also improved prediction, with an AUC of 0.670 (p < 0.001) and a spec/sens of 0.65/0.62. A selection of the 34 most predictive features reached the same sens/spec as the ACR/EULAR 2010 classification criteria for RA, showing a spec/sens of 0.80/0.56.

Conclusion

In at-risk ACPA+ individuals, the presence of sub-clinical US abnormalities are highly specific for progression to IA. The only moderate sensitivity can be improved by using joints or features selection in combination with clinical examination. These results are the first step in providing guidance for which at-risk ACPA+ individuals to treat.

Disclosures

L.M. Duquenne None. K. Mankia None. L. Garcia Montoya None. A. Di Matteo None. J. Nam None. P. Emery None.
P252 Treatment pathways for patients with axSpA: a retrospective UK database analysis
Authors: Morgan Christopher L. I , White Abigail , Tomlinson Mark , Scott Amie , Tian Haijun
Published: April 2020

Background

This UK study aimed to identify a population with axial spondyloarthritis (axSpA) and to describe the presence of prior reported symptoms and treatment pathways associated with the condition.

Methods

The study was performed using a large routine primary care database from the UK, the Clinical Practice Research Datalink. Only patients whose record could be linked to Hospital Episode Statistics (HES) were included for analysis. Patients with an incident diagnosis of axSpA recorded from 2003-2017 were identified. The date of first axSpA diagnosis defined the index date and patients were observed until the end of database follow up. Recordings of axSpA-related symptoms (back pain, rheumatoid arthritis and fatigue) in the 12 months prior to the index date, before and after attendance at orthopaedic or rheumatology outpatient clinics, and after inpatient contact with either a primary or secondary diagnosis of axSpA were flagged. The proportion of patients prescribed tumour necrosis factor inhibitors (TNFis), corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs) or conventional systemic disease-modifying anti-rheumatic drugs (csDMARDs) subsequent to the index date and estimated first three lines of therapy were described.

Results

A total of 2,756 incident axSpA cases were identified. In the 12 months prior to the index date, 1,289 (46.8%) of these patients had at least one recorded symptom related to axSpA: back pain (1,195 [43.4%]), rheumatoid arthritis (140 [5.1%]) and fatigue (6 [0.2%]). Prior to the index date, 1,025 (37.2%) patients had been seen in an orthopaedic outpatient clinic and 1,274 (46.2%) in a rheumatology clinic. Subsequent to the index date, the respective figures were 815 (29.6%) and 1,641 (59.5%). In addition, 133 (4.8%) patients had a post-index inpatient admission with a primary diagnosis of axSpA and 895 (32.5%) had an inpatient admission with a secondary diagnosis. TNFis were prescribed for 97 (3.5%) patients, corticosteroids for 727 (26.4%), NSAIDs for 1,885 (68.4%) and csDMARDs for 461 (16.7%) patients. The most common first-line therapy was NSAIDs (1,579 [57.3%]) (Table).

Conclusion

This study describes the symptoms of and treatment pathways for axSpA patients within the UK. Issues regarding missing data within routine data should be considered when interpreting the results.

Disclosures

C.L.I. Morgan: Other; Employee of: Pharmatelligence. A. White: Shareholder/stock ownership; Novartis. Other; Employee of: Novartis. M. Tomlinson: Consultancies; Novartis. A. Scott: Consultancies; Novartis. H. Tian: Other; Employee of: Novartis.
P253 Secukinumab provides sustained improvements in tender and swollen joint counts: 5year results from the Phase 3 FUTURE 2 study
Authors: McInnes Iain , Chinoy Hector , Asquith Darren , White Abigail , Gaillez Corine
Published: April 2020

Background

Secukinumab, a fully human monoclonal antibody that neutralises interleukin-17A, demonstrated long-term efficacy and tolerability in psoriatic arthritis (PsA) patients in FUTURE 2. Tender joint count (TJC) and swollen joint count (SJC) scores are components of composite measures used to assess treatment response, which can include confounding factors. TJC and SJC were developed for rheumatoid arthritis, assessing 68 and 66 joints, respectively. Later, these scores evolved for use in PsA, which can affect small joints of the feet, assessing 78 and 76 joints. TJC and SJC measure synovitis, a key assessment of disease activity. Herein, we report the 5-year efficacy of secukinumab on reduction of 78 TJC and 76 SJC in FUTURE 2.

Methods

397 patients with active PsA were randomised to subcutaneous secukinumab loading dose (300, 150, 75 mg) or placebo at baseline, Weeks 1, 2, 3 and 4, and every 4 weeks thereafter. Placebo-treated patients were re-randomised to secukinumab 300 or 150 mg at Week 16 (non-responders) or 24 (responders). Secukinumab dose was escalated from 150 to 300 mg, and 75 to 150 or 300 mg starting at Week 128 and maintained thereafter, if active signs of disease were observed based on physician’s assessment. Data are reported as observed for secukinumab 300 and 150 mg (approved PsA doses) over 5 years (2 years of core study and 3-year extension).

Results

Baseline characteristics were comparable across treatment arms (Table 1). Overall, 62.5% (248/397) of all patients randomised completed 260 weeks of treatment. Of these, 48/65 (73.8%) and 49/66 (74.2%) patients randomised to secukinumab 300 and 150 mg achieved ACR20 at Week 260, respectively. Improvements in adjusted 78 TJC and 76 SJC change from baseline were sustained from Week 16 to Week 260 in all treatment groups (Table 1). At Week 260, mean change from baseline in adjusted 78 TJC/76 SJC was −12.8/−9.3 and −15.1/−8.5 for the secukinumab 300 mg and 150 mg group (including patients up-titrated to 300 mg), respectively. Numerical differences were comparable between secukinumab doses.

Conclusion

Secukinumab 300 and 150 mg provided sustained improvements in 78 TJC and 76 SJC scores over 5 years; numerical differences were comparable between secukinumab doses.

Disclosures

I. McInnes: Consultancies; AbbVie, Amgen, Bristol-Myers Squibb, Celgene, Janssen, Lilly, Novartis, Pfizer, UCB. Honoraria; AbbVie, Amgen, Bristol-Myers Squibb, Celgene, Janssen, Lilly, Novartis, Pfizer, UCB. Grants/research support; AbbVie, Amgen, Bristol-Myers Squibb, Celgene, Janssen, Lilly, Novartis, Pfizer, UCB. H. Chinoy: Honoraria; Novartis. D. Asquith: Shareholder/stock ownership; Novartis. Other; Employee of Novartis. A. White: Shareholder/stock ownership; Novartis. Other; Employee of Novartis. C. Gaillez: Shareholder/stock ownership; Novartis, Bristol-Myers Squibb. Other; Employee of Novartis.
Gender Equality and the EU's Economic Governance: The Strategic Use of Women’s Employment Within Labor Market Reforms in Italy
Authors: Chieregato Elisa
Published: April 2020

This article aims to evaluate the political implications of post-crisis governance in the European Union for national gender-equality strategies and policies. It presents a study of recent reforms of the labor market in Italy, where the response to the Eurozone crisis has increased concerns over the low rate of female employment. Through an analysis of policy documents and legislative reforms informed by discursive politics, the article argues that the Italian case provides evidence of the trend toward the further instrumentalization of gender equality, which has been strategically used in order to provide additional legitimation for labor market deregulation.
Indemnities and modern corporate structures
Authors: Watson Jack
Published: April 2020

This article considers the issue of trustee indemnity clauses and their interaction with the corporate structures which underlie many modern trusts. It considers the problems which can arise in relation to such clauses and the recent decision of First National Trustco (UK) Ltd v McQuitty [2020] EWCA Civ 107, in which the Court of Appeal gave guidance as to the construction of such clauses. Finally, it will conclude by addressing the wider impact of the decision on the ability of trustees to compel beneficiaries to make payment in respect of liabilities of underlying companies in order to ensure that taxes and other liabilities are paid.
11‐Keto‐a‐Boswellic Acid, a Novel Triterpenoid from Boswellia spp., with Cytotoxic Efficacy against Treatment‐Resistant Triple Negative Breast Cancer Cell Lines
Authors: Schmiech Michael , Lang Sophia Johanna , Paetz Christian , Ulrich Judith , Büchele Berthold , Syrovets Tatiana , Simmet Thomas
Published: April 2020

Triterpenoids including biologically active boswellic acids constitute about twenty percent of frankincense, the oleogum resin of Boswellia spp. trees from the Burseraceae family
[1–
2]. Frankincense as well as extracts thereof have been traditionally used by folk medicines, such as Ayurveda, African, Arab, and Chinese medicine, for the treatment of fever and pain. Several clinical pilot trials provided evidence for a potential therapeutic efficacy of frankincense preparations in various chronic inflammatory diseases. Moreover, preclinical studies have demonstrated that boswellic acids may be beneficial for treatment of human cancers because they also inhibit tumor growth in different animal models. We have performed a comparative analysis of more than 40 oleogum resins from different Boswellia species grown in different geographic locations
[1] and we have shown that frankincense extracts induce apoptosis in triple negative breast cancer cell lines in vitro and in vivo
[1–
2]. Moreover, the cytotoxic efficacy of the extracts correlated positively with the contents of boswellic acids
[2]. Boswellic acids exist as two structural isomers, α‐boswellic acids (oleanane‐type skeleton) and β‐boswellic acids (ursane‐type skeleton). In the current study, we have identified the novel oleanane‐type 11‐keto‐α‐boswellic acid (αKBA) in oleogum resins of different Boswellia species. The structure of αKBA was elucidated by 1D and 2D 1H and 13C NMR analysis. The contents of the individual boswellic acid isomers was quantified in different frankincense extracts by high performance reversed‐phase liquid chromatography coupled with tandem mass spectrometric detection (HPLC‐MS/MS) and the help of fluorinated stationary phases. We have compared the toxicities of different known boswellic acids and of the newly identified αKBA against tthe reatment‐resistant, triple negative human breast cancer cell lines MDA‐MB‐231 and CAL‐148. αKBA exhibited cytotoxic activity against the cancer cells and was less toxic to normal peripheral blood mononuclear cells (PBMC) and non‐malignant human breast epithelial cells indicating tumor selectivity. Structure‐activity‐relationship between the cytotoxic activities of different boswellic acid were analyzed. Hence, the study provides evidence for the existence of a novel biologically active boswellic acid with cytotoxic activity against triple negative human breast cancer cell lines in the oleogum resin of Boswellia spp. Additionally, it offers new clues for possible structural modifications of boswellic acids to increase their cytotoxicity against cancer cells.

Support or Funding Information

This work was supported by the Academic Center for Complementary and Integrative Medicine (AZKIM), State Ministry of Baden‐Württemberg for Sciences, Research and Arts.
Novel Treatment of Human Melanoma Cells that Express BRAF or NRAS Mutations by Antifungal Agents
Authors: Koh David W. , Jira Lukas R. , McKamey Shelby G.
Published: April 2020

Approximately 70% of all melanomas are characterized by mutations in the BRAF or NRAS genes. Although targeted therapy exists for BRAF mutations, this therapy is not commonly used in wild‐type melanoma, no such treatment is available for NRAS mutations, and resistance to all available treatments is increasing. These observations indicate that new strategies and targets are needed to improve melanoma therapy. We previously reported the potential for the antifungal agents, clotrimazole and econazole, to treat melanoma. Our objective here was to analyze the ability of these agents to treat human melanoma cell lines expressing the BRAF or NRAS mutations. Cell growth and cell death were investigated via flow cytometry, cell proliferation assays, and cell death assays after treatments.

Wild‐type human melanoma cell lines or those possessing the BRAF V600E mutation or the NRAS Q61R mutation were analyzed. A dose of 25 μM clotrimazole led to profound decreases in cell growth in wild‐type and NRAS mutant melanoma cells. Significant, but lesser effects were observed in BRAF mutants. However, treatment with 25 μM econazole caused substantial reductions in cell growth in BRAF mutant cells, as well as one line of wild‐type melanoma cells. These results were corroborated by cell death analyses, where clotrimazole treatment caused up to 50% cell death in wild‐type and NRAS mutant cells after 3 days. In BRAF mutants after 3 days, econazole treatment caused up to 45% cell death. Taken together, these preliminary studies indicated that clotrimazole was more effective in the treatment of wild‐type and NRAS mutant melanoma cells, while econazole demonstrated greater effects in melanoma cells expressing the BRAF mutation.

In conclusion, this study demonstrated the ability of antifungal agents to successfully treat human melanoma cell lines that possess BRAF and NRAS mutations. Since each agent displayed differential effects for each type of mutation, it is possible that these antifungal agents affect different molecular targets in each mutant melanoma cell line. These antifungal agents thus appear to be potential alternative treatments for wild‐type melanoma or melanoma characterized with the BRAF V600E or NRAS Q61R mutation.
Development of a Novel ω‐6 Polyunsaturated Fatty Acid Based Treatment Strategy for Non‐Melanoma Skin Cancer by Inhibiting Delta‐5‐Desaturase
Authors: Shah Harshit , Pang Lizhi , Qian Steven , Zhao Pinjing , Sathish Venkatachalem
Published: April 2020

Background

Cyclooxygenase‐2 (COX‐2) overexpression has been considered as one of the culprits for cancer progression, due to precancerous prostaglandin production from downstream ω‐6 polyunsaturated fatty acid (PUFA), arachidonic acid (AA), in the majority of the cancers including, Nonmelanoma skin cancer (NMSC). However, COX‐2 inhibitors have failed to provide protection against cancer in clinics. As per the recent studies from our lab, manipulation of ω‐6 PUFA metabolism by genetically inhibiting delta‐5‐desaturase (D5D), requires for metabolism of an upstream ω‐6 PUFA, dihomo gamma‐linolenic acid (DGLA), to AA, results in accumulation of DGLA which undergo peroxidation by COX‐2 to produce an anti‐cancer product, 8‐Hydroxyoctanoic acid (8‐HOA) in pancreatic and colon cancer model. However, this approach was associated with limitations, such as the degradation of D5D siRNA by endonuclease and the inability to cross the cell membrane. Hence in order to overcome the limitation and to get a sustained production of 8‐HOA inside the tumor, we developed a novel D5D inhibitor (D5Di) and hypothesized that D5Di could stimulate 8‐HOA production, which exerts anti‐cancer activity in NMSC.

Methods

In vitro studies like colony formation assay for proliferation analysis and PI Annexin V double staining assay for apoptosis analysis using A431 cell line and in vivo studies using xenograft mouse model evaluating the protective effect of D5Di against NMSC were performed. In order to dissect the molecular pathway, Western blot analysis, and immunohistochemistry studies were performed.

Results

D5Di treatment in the presence of DGLA resulted in a significant increase in apoptosis by 14.23±1.01% and reduction in survival fraction by 29.91±0.70%. Similar to the in vitro study, treatment with D5Di in animals receiving the DGLA supplement displayed a significant reduction in tumor size. The reduction in tumor size was due to a significantly high level of 8‐HOA (1.32±0.122μM) and a low PGE2/PGE1 ratio. In addition, an increase in cleaved PARP and reduction in Ki‐67 were observed by immunohistochemistry, which were indicating inhibitory effect of D5Di on tumor growth. Moreover, treatment with D5Di also caused downregulation of anti‐apoptotic factors such as procaspase‐3, procaspase‐9, and increased DNA damage marker, γH2Ax, both in vitro and in vivo.

Conclusion

These results concluded that the novel strategy of providing D5Di with DGLA could be a new therapeutic approach for advanced and high‐risk NMSC.

Support or Funding Information

This research work is supported by NIH R15CA195499.
Inhibition of BCRP in Multidrug Resistant Cancer using Novel Inhibitors
Authors: Park Chanyang , Mertz Sarah B. , Holcomb-Webb Kasi L. , Ammerman Lauren E. , Vogel Pia D. , Wise John G.
Published: April 2020

Multi‐drug resistance (MDR) occurs when cancer cells become resistant to a diverse array of chemotherapeutics and xenobiotics. Among the many mechanisms of MDR, one of the most prominent is the overexpression of ATP‐binding cassette (ABC) transporters. ABC transporters harness the energy from ATP hydrolysis to transport xenobiotics, drugs, and other toxic compounds out of the cell. When ABC transporters are overexpressed in cancer cells, their efflux activity can lower the intracellular concentration of medicinal compounds to sub‐therapeutic levels. One member of the ABC transporter family, the Breast Cancer Resistance Protein (BCRP, or ABCG2), confers MDR to a variety of cancers. Transient inhibition of BCRP should therefore restore sensitivity of resistant cells to chemotherapeutics. In previous work by our lab, several potential BCRP inhibitors were identified via computational methods. Using cell viability assays, we tested the potential BCRP inhibitors using a BCRP overexpressing cell line. Here we compare the effectiveness of the inhibitors at re‐sensitizing the cells to Mitoxantrone relative to the parental cell line which does not overexpress BCRP. The potential toxicities of the experimental compounds were also assessed using the non‐cancerous HFL1 cell line.
Exploring opioid receptor dependent biased agonism in the PreBötzinger Complex
Authors: Szujewski Caroline C. , Campbell Chrishon , Garcia Alfredo J.
Published: April 2020

Respiratory depression is a hallmark of opiate overdose and is mediated by activation of μ‐opioid receptors (MORs) to suppress the drive for breathing. Agonist binding to MOR can preferentially activate Gi or β‐arrestin‐2 (β‐arr2) signaling. Although a large body of literature suggests that activation of the β‐arr2 pathway is involved with respiratory depression, emerging evidence has challenged this perspective. This raises the question of whether the development of biased agonist opiates is valuable for mitigating adverse respiratory side effects. The preBötzinger complex (preBötC) is area in the respiratory network expressing MORs and is responsible for inspiratory rhythm generation. This ongoing study examines the role of MORs biased agonism on opioid modulation of rhythmogenesis in the preBötC. Extracellular population recordings were made in preBötC slices prepared from wildtype mice and β‐arr2 knock out mice (β‐arr2−/−). No differences were observed between genotypes in the degree of inhibition caused by the opiate agonist DAMGO. Additionally, no differences were observed with Endmorphin‐2 application, an opioid receptor agonist biased for β‐arr2 signaling. These findings indicate that β‐arr2 signaling in the preBötC is not responsible for the inhibition of rhythmogenesis caused by activation of MORs. Thus, developing biased agonist opiates to mitigate depressive respiratory side effects may not be an effective strategy.

Support or Funding Information

NIH T32DA043469, NIH PO 1 HL 144454, NIH R01 NS10742101
Effect of Hydrogen Sulfide‐Releasing Compounds on Proliferation of Human Colon Cancer HT29 Cells
Authors: Yakubu Momoh A. , Keita Djene , Ngele Kalu K. , Mbye Ya Fatou N. , Ohia Sunny E.
Published: April 2020

Both natural and synthetic compounds that release hydrogen sulfide (H2S) such as diallyl trisulfide and naproxen‐H2S hybrids growth and proliferation of human colon HT29 cells (Lai et al. J. Cell. Mol. Med 19: 474, 2014; Kodela et al. Drug Des. Dev. Ther. 9:4873, 2015). Aims of the present study were: (a) to investigate the pharmacological action of L‐cysteine (substrate for the biosynthesis of H2S) and NaHS (H2S‐releasing compound) on proliferation of HT29 cells at different time‐points, and (b) to study the role of enzymes (cystathionine β‐synthase, CBS and cystathionine γ‐lyase, CSE) and K+‐ATP channels on the response elicited by H2S‐producing compounds in HT29 cells. The colon adenoma HT29 cells were treated with different concentrations of L‐cysteine (1 μM – 1 mM) and NaHS (1 μM – 1 mM) for 24 and 48 hours, in the absence and presence of inhibitors of CBS and CSE, amino‐oxyacetic acid (AOA, 1 mM), an inhibitor of K+‐ATP channels, glibenclamide (GLB, 10 μM). After incubation, the anti‐proliferative response on cells caused by various agents was determined by MTT Assay. At equimolar concentrations (10 μM and 1 mM), both L‐cysteine and NaHS caused concentration‐dependent inhibitions of cell proliferation after incubation for 24 h, but increased cell growth at these same concentrations after 48 h of incubation. For instance, at 10 μM, both L‐cysteine and NaHS increased cell proliferation by 37% and 51% after 48 h of incubation. Pretreatment of cells with AOA (1 mM) had no significant (P>0.05) effect on the inhibitory response caused by L‐cysteine and NaHS after 24 h incubation. Interestingly, AOA blocked and even reversed the increase in proliferation caused by L‐cysteine (10 μM and 1 mM) and NaHS (10 μM and 1 mM) after 48 h incubation. After 24 h, both L‐cysteine and NaHS caused additional inhibitory effects on cell proliferation in HT 29 cells pretreated with GLB (10 μM). GLB blocked and even reversed the increase in proliferation caused by L‐cysteine and NaHS in HT29 cells after 48 h incubation. We conclude that H2S‐releasing compounds can elicit a time‐dependent inhibitory or excitatory action on the proliferation of HT29 cells. The inhibitory/excitatory effects caused by these compounds depends, at least in part, on endogenous biosynthesis of new gas and the activity of K+‐ATP channels in HT29 cells.

Support or Funding Information

This research is supported by Tittle III under the Award Number P031B090216;Title III, Part B, Historically Black Graduate Institutions (HBGI) (CFDA No. 84.031B).
Catecholamine‐Induced DNA Damage in Ovarian Cancer Cells
Authors: Acevedo Arelis , Caraballo-Lamboy Rocío , Armaiz-Peña Guillermo
Published: April 2020

Ovarian cancer is the fifth leading cause of mortality among women, responsible for 5% of all cancer deaths in women in the USA. Remarkable evidence exists of the relationship between the activation of the sympathetic nervous system and cancer progression. Interestingly, recent data suggest that adrenergic signaling alters genetic integrity of cancer cells by damaging the DNA and/or influencing the DNA Damage Response and Repair pathways. In addition, previous data suggest that increased adrenergic stimulation from stress released catecholamines can lead to genomic instability in various in vitro models. Therefore, we hypothesized that increased adrenergic activity will induce DNA damage in ovarian cancer cell lines. To test this, ovarian cancer cells were exposed to catecholamines (epinephrine and norepinephrine) for 1h and 24h; then, double‐stranded damaged was assessed by Neutral Comet Assays and γH2AX foci formation. Results show that norepinephrine significantly increased foci formation and induced higher levels of double‐stranded DNA breaks in SKOV3, OV90 and COV362 cells when compared to control group. Lastly, to determine if DNA damage is due to adrenergic stimuli, cells were pretreated with propranolol (non‐specific β‐blocker) and results showed a significant reduction in DNA damage levels. These results suggest that catecholamines can induce double‐stranded DNA damage in ovarian cancer cells.
Motor Neuron Pathology Leads to Respiratory Deficiency in Optn−/− Mice
Authors: McCall Angela L. , Dhindsa Justin S. , Pucci Logan A. , Strickland Laura M. , Kahn Amanda F. , Fusco Anna F. , Tseng Henry , ElMallah Mai K.
Published: April 2020

Amyotrophic Lateral Sclerosis (ALS) is a devastating and fatal neurodegenerative disease with no current cure. Respiratory failure is the leading cause of death in ALS. Death occurs3–5 years after diagnosis when patients with ALS ultimately succumb to inadequate ventilation, hypoxia, and respiratory failure. Several genes are associated with ALS. One of these genes encodes optineurin (OPTN) which is associated with neurodegeneration in both ALS and glaucoma. Optineurin has multiple roles in various biochemical pathways such as regulation of inflammation and autophagy. However, the exact mechanism by which loss of OPTN results in progressive neural degeneration and respiratory failure in ALS is still unclear.

In order to understand the impact of OPTN deficiency on respiratory function, our goal was to study the respiratory pathophysiology in the Optn−/− mouse. The hypothesis driving this work is that the Optn−/− mouse has respiratory insufficiency due to degeneration of the respiratory motor unit – motor neuron, nerve, neuromuscular junction and muscle. Physiological, histological and molecular outcome measures were used to assess the impact of OPTN on the respiratory system. We used whole body plethysmography (WBP) to assess breathing at baseline and during a respiratory challenge with hypercapnic and hypoxic conditions (FiCO2: 0.07, FiO2: 0.10; nitrogen balance). During the respiratory challenge, compared to the WT mice, Optn−/− mice had significantly lower tidal volume, minute ventilation, peak inspiratory flow, and peak expiratory flow starting at 6 months of age indicating weakened muscle strength. Furthermore, throughout the challenge period Optn−/− mice spent a greater amount of time in apnea indicating pathology in the respiratory control centers. The weakness and pathology in control of breathing during the respiratory challenge progresses as the mice age. At 1 year of age, Optn−/− mice have stunted growth accompanied by reduced diaphragm size compared to WT mice. Postmortem immunohistochemical studies of the medulla reveal Optn−/− mice have fewer hypoglossal motor neurons. Finally, the hypoglossal (XII) nerves of Optn−/− mice had significantly reduced g‐ratio which indicates pathology and decompaction of myelin sheaths. Within the XII nerve, there are an increase in both the number of mitochondria and area of mitochondria. These along with elevated levels of LC3‐II to LC3‐I indicate reduced mitophagy. In conclusion, the Optn−/− ALS mouse model displays motor neuron pathology, increased nerve demyelination and aberrant control of breathing leading to respiratory insufficiency.
Cardioprotective Effects of Melatonin, Berberine and Curcumin by Inducing the JAK2/STAT3 Signaling Pathway in Myocardial Ischemia Repercussion
Authors: Khan Naushad , Pandey Rajeev , Murthy Guru
Published: April 2020

Ischemic heart disease and stroke are the World’s major cause of deaths, accounting for a combined 15.2 million deaths in 2016 as per WHO. Following Cardiac Ischemia, we are often faced with a bigger problem of damage induced by myocardial ischemia/reperfusion injury. It has been observed that reperfused myocardium displays more severe injury than myocardium subjected to the same ischemic duration that had not been reperfused. This observation has led to deduce that reperfusion could result in Myocardial injury more severe than one that could have occurred by Ischemia alone. However certain mediators like melatonin, berberine and curcumin have shown to induce JAK2/STAT3 signaling pathway and have shown a protective effect against myocardial ischemia reperfusion. In the present work we will be mainly focusing on the correlation between Melatonin, Berberine & Curcumin and cardiac JAK2/STAT3 receptors in cardioprotection against myocardial ischemic reperfusion.

Support or Funding Information

Community Health and Research Center of Spartan (CHARCOS), Spartan Health Sciences University, School of Medicine, St. Lucia
Colonic Crypt‐Derived 3D Gut Organoid from Hypertensive Rat Maintain In vivo Characteristics
Authors: li Jing , Su Qing , Karas Marianthi M. , Richards Elaine M. , Raizada Mohan K.
Published: April 2020

Objective

Substantial evidence links gut dysbiosis to hypertension in both animal models and in patients with high blood pressure. Increased blood pressure and hypertension‐associated pathologies in normotensive animals transplanted with fecal microbiota from hypertensive animals, support the concept of microbiota‐gut communication as an important early signal initiating and establishing hypertension. We hypothesized that colonic cryp‐3D organoids would retain the characteristics of the in vivo gut epithelium form which they were derived, and would provide a model system to investigate epithelial‐microbiota communication in hypertension.

Methods

Colonic crypts were isolated from adult Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). 3D organoid cultures were established in organoid growth medium with Matrigel matrix and cultured for 3, 5 and 7 days without passage. The shapes and number of organoids were assessed by bright‐field microscopy using Trypan blue. Growth, differentiation and cell types in organoids were characterized with the use of antibodies (Ki67, Alpi, KRT20 and Muc2). Data were compared between WKY rats and SHR organoids.

Results

Reproducible 3D organoid cultures were successfully established from the crypts of adult WKY and SHR. They proliferated and differentiated, and predominately contained enterocytes and goblet cells. We observed significantly more organoids per crypt from normotensive vs hypertensive rats (WKY: 0.20 vs. SHR: 0.11; p<0.01). In vivo, WKY rats had 18% longer colons than SHR (0.07 cm/g vs. 0.059 cm/g; p<0.0001).

Conclusions

These observations demonstrate that 3D organoid cultures maintain characteristics of the colonic epithelium and show significant differences between WKY rats and SHR. Thus, these organoids represent an ideal ex vivo system to investigate the gut epithelium in hypertension, and to examine the effects of gut microbiome‐derived metabolites in blood pressure control and initiation and establishment of hypertension.

Support or Funding Information

HL132448
Mediator Kinase as a Transducer and Therapeutic Target in Oncogenic WNT/β‐ CATENIN Signaling
Authors: Barron Lindsey , Clark Allison , Burleson Marieke , Boyer Thomas
Published: April 2020

Colorectal cancer (CRC) is the second and third most commonly diagnosed cancer in women and men, respectively. Few treatment options exist for advanced stage disease, thus, the development of targeted therapies is crucial for treatment. The canonical wingless related integration site (WNT)/β‐CATENIN pathway is a driver in >90% of CRCs. The mediator kinase module (mediator complex subunit 12 (MED12), mediator complex subunit 13 (MED13), CYCLIN C, and cyclin dependent kinase 8 (CDK8)) is a key node of oncogenic activation within the WNT/β‐CATENIN pathway, since β‐CATENIN activates transcription through its direct physical interaction with MED12. Additionally, CDK8 is an oncoprotein required for β‐CATENIN‐dependent gene activation and CRC growth.

The objective of this study was to investigate the effects of genetic (disruption of MED12:CYCLIN C interface) and chemical inhibition of Mediator Kinase activity on CRC cell oncogenic properties and gene regulation. Oncogenic properties were assessed by colony formation and cell proliferation assays. Chromatin immunoprecipitation sequencing and RNA sequencing were used to evaluate gene regulation. Interestingly, genetic but not chemical inhibition led to a significant reduction in oncogenic properties. Superenhancer alterations and differential expression of superenhancer‐associated genes were observed with both genetic and chemical inhibition. However, little overlap was found for down‐regulated superenhancer‐associated genes.

Based on the significant reduction in oncogenic properties, genetic disruption of the MED12:CYCLIN C interface appears to be an attractive target for the development of targeted molecular therapies for CRC. The difference in effect on oncogenic properties with chemical versus genetic inhibition is likely explained by the differences in down‐regulated superenhancer‐associated genes. Concordantly, genes involved in the WNT/β‐CATENIN pathway were down‐regulated with genetic but not chemical inhibition.

Support or Funding Information

Lindsey Barron was supported by a CPRIT postdoctoral training fellowship, the CPRIT Research Training Award (RP170345) and is currently supported by an IRACDA Grant (K12GM111726).
Optimization of LC‐ESI‐MS Quantification of Ventromedial Hypothalamic Nucleus Estradiol Content by Central Composite Design
Authors: Bheemanapally Khaggeswar , Ibrahim Mostafa M.H. , Ali Md. Haider , Napit Prabhat R. , Briski Karen P.
Published: April 2020

Brain structures exhibit characteristic patterns of estrogen receptor expression and regulatory functions and local capacity to generate neuroestradiol, necessitating quantification of estradiol content of individual loci. Estradiol acts on ventromedial hypothalamic nucleus (VMN) substrates to control behavior and glucostasis. Current research utilized Analytical Quality of Design to optimize uHPLC‐electrospray ionization‐mass spectrometry (LC‐ESI‐MS) sensitivity for analysis of estradiol in VMN samples obtained by high‐resolution microdissection techniques. A Design of Experiments matrix was developed to assess effects of critical independent variables on estradiol chromatogram area. A second‐order polynomial equation was generated by statistical response surface methodology using central composite design to identify synergistic versus antagonistic factors and to predict singular/combinatory effects on response area. Estradiol, estrone, and estriol were used as optimizing compounds; ethynyl estradiol served as internal standard. Lysates of VMN tissue harvested by micropunch dissection were transferred to methanol‐water conditioned solid phase extraction syringe cartridges and treated with 2% formic acid. Estradiol content was extracted with methanol, then vacuum concentrated. Samples were treated with 2‐fluoro‐1‐methylpyridinium‐p‐toluenesulfonate (FMP‐TS) prior to application to a C18 column [0.2ml/min mobile phase (25:75, v/v) flow rate; 0.1% formic acid, acetonitrile], followed by spectrometric analysis of estradiol and internal standard responses at m/z 364.2 and 388.2, respectively. Optimized mass spectrometric vaporizer (320°C) and ion transfer tube (266 °C) temperatures and sheath (22 psig), auxiliary (5 psig), and sweep (0.7 psig) gas pressures resulted in maximum tissue estradiol response. Outcomes validate a novel combinatory methodology for estradiol quantification in precisely‐defined brain components.

Support or Funding Information

National Institutes of Health DK‐109382
Translation errors mediated by oxidized and methylated mRNA
Authors: Tanaka Mikiei , Tanaka Hikari , Chock P. Boon
Published: April 2020

Post translational modification of biomacromolecules play an important role in their normal physiological functions. However, under pathological conditions, extracellular toxic compounds are known to induce various covalent modification of the biomacromolecules to exert their toxic effects. It is known that oxidative damage and alkylation can cause mutation or strand break of genomic DNA leading to tumorigenesis. However, the consequences of these modifications to mRNA has not been well investigated. The aim of this study is to assess the protein synthesis activity mediated by the oxidized and methylated mRNA. Here, the synthesized protein was monitored by the luciferase activity of the in vitro synthesized mRNA encoding FLAG‐tagged luciferase gene. The synthesized mRNA was subjected to iron‐catalyzed oxidation (Fe‐ox) or monomethylation by methylmethane sulfonate (MMS). The data reveal that the modified mRNAs induces a decrease in luciferase activity both in the reticulocyte lysate and wheat germ lysate in in vitro translation system. Conversely, mRNA was synthesized in the presence of modified nucleotide, to generate mRNA that incorporated with modified base, N1‐mA, N6‐mA, N7‐mG, or 8‐oxo‐G. Our data reveal that N6‐mA and 8‐oxo‐G mRNA exhibit a reduced luciferase activity, however the reduction was modest. To ensure that the activity of the full‐‐length protein was not compromised by amino acid substitution caused by mistranslation, LC/MS method was used to analyze these products. Interestingly, the proteins generated with N1‐mA mRNA and N7‐mG mRNA exhibited full luciferase activity. In addition, Western blotting data showed an increase in the generation of fragmented peptides in the Fe‐ox and MMS modified mRNAs, and in the 8‐oxo‐G or N1‐m6A mRNA,suggesting that these modified nucleotides tend to induce translation errors.