IL-17C regulates the innate immune function of epithelial cells in an autocrine manner.

Interleukin 17C (IL-17C) is a member of the IL-17 family that is selectively induced in epithelia by bacterial challenge and inflammatory stimuli. Here we show that IL-17C functioned in a unique autocrine manner, binding to a receptor complex consisting of the receptors IL-17RA and IL-17RE, which was preferentially expressed on tissue epithelial cells. IL-17C stimulated epithelial inflammatory responses, including the expression of proinflammatory cytokines, chemokines and antimicrobial peptides, which were similar to those induced by IL-17A and IL-17F. However, IL-17C was produced by distinct cellular sources, such as epithelial cells, in contrast to IL-17A, which was produced mainly by leukocytes, especially those of the T(H)17 subset of helper T cells. Whereas IL-17C promoted inflammation in an imiquimod-induced skin-inflammation model, it exerted protective functions in dextran sodium sulfate-induced colitis. Thus, IL-17C is an essential autocrine cytokine that regulates innate epithelial immune responses.

Context-dependent signaling defines roles of BMP9 and BMP10 in embryonic and postnatal development.

Many important signaling pathways rely on multiple ligands. It is unclear if this is a mechanism of safeguard via redundancy or if it serves other functional purposes. In this study, we report unique insight into this question by studying the activin receptor-like kinase 1 (ALK1) pathway. Despite its functional importance in vascular development, the physiological ligand or ligands for ALK1 remain to be determined. Using conventional knockout and specific antibodies against bone morphogenetic protein 9 (BMP9) or BMP10, we showed that BMP9 and BMP10 are the physiological, functionally equivalent ligands of ALK1 in vascular development. Timing of expression dictates the in vivo requisite role of each ligand, and concurrent expression results in redundancy. We generated mice (Bmp10(9/9)) in which the coding sequence of Bmp9 replaces that of Bmp10. Surprisingly, analysis of Bmp10(9/9) mice demonstrated that BMP10 has an exclusive function in cardiac development, which cannot be substituted by BMP9. Our study reveals context-dependent significance in having multiple ligands in a signaling pathway.

Discovery of a potent, selective, and tumor-suppressing antibody antagonist of adenosine A2A receptor.

New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries-two synthetic and two immunized-against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.

Furin-cleaved proprotein convertase subtilisin/kexin type 9 (PCSK9) is active and modulates low density lipoprotein receptor and serum cholesterol levels.

Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.

Dysmenorrhoea among Hong Kong university students: prevalence, impact, and management.

OBJECTIVE. To evaluate the prevalence of dysmenorrhoea, its impact, and management approaches in Hong Kong university students, and to compare between medical and non-medical students for any potential differences in coping strategies. DESIGN. Cross-sectional questionnaire survey. SETTING. The University of Hong Kong, Hong Kong. PARTICIPANTS. A total of 240 undergraduate (128 medical and 112 non-medical) students. MAIN OUTCOME MEASURES. Data on the presence and severity of dysmenorrhoea, its impact on daily life, management approaches, specific strategies, and their self-perceived effectiveness were obtained and analysed. RESULTS. In these subjects, the prevalence of dysmenorrhoea was 80% (95% confidence interval, 75-85%) with a mean (standard deviation) pain score of 5.0 (1.7). The most common impacts on daily life included reduced ability to concentrate and/or disturbance with study (75%) and changes in normal physical activity (60%). Only 6% sought medical advice, while 70% practised self-management. Pain scores and pain affecting normal physical activities were important predictive factors for self-management and for management based on pharmacological or non-pharmacological means. The commonest specific strategies used were a warm beverage (62%), paracetamol (57%), and sleeping (45%), while the most effective strategies were non-steroidal anti-inflammatory drugs (100%), traditional Chinese medicine (93%), and dietary/nutritional supplements (92%). Regarding the comparison of medical and non-medical students, the former used fewer pharmacological strategies among the various management approaches investigated. CONCLUSION. With data showing dysmenorrhoea as a very common condition having a significant impact in the Hong Kong community, primary care doctors should reassure young women with dysmenorrhoea that it is a common experience in the same age-group. Health education on the existence of effective treatment from medical practitioners could help women whose dysmenorrhoea was not controlled by self-management.

Functional GLP-1R antibodies identified from a synthetic GPCR-focused library demonstrate potent blood glucose control.

G protein-coupled receptors (GPCRs) are a group of seven-transmembrane receptor proteins that have proven to be successful drug targets. Antibodies are becoming an increasingly promising modality to target these receptors due to their unique properties, such as exquisite specificity, long half-life, and fewer side effects, and their improved pharmacokinetic and pharmacodynamic profiles compared to peptides and small molecules, which results from their more favorable biodistribution. To date, there are only two US Food and Drug Administration-approved GPCR antibody drugs, namely erenumab and mogamulizumab, and this highlights the challenges encountered in identifying functional antibodies against GPCRs. Utilizing Twist's precision DNA writing technologies, we have created a GPCR-focused phage display library with 1 × 1010 diversity. Specifically, we mined endogenous GPCR binding ligand and peptide sequences and incorporated these binding motifs into the heavy chain complementarity-determining region 3 in a synthetic antibody library. Glucagon-like peptide-1 receptor (GLP-1 R) is a class B GPCR that acts as the receptor for the incretin GLP-1, which is released to regulate insulin levels in response to food intake. GLP-1 R agonists have been widely used to increase insulin secretion to lower blood glucose levels for the treatment of type 1 and type 2 diabetes, whereas GLP-1 R antagonists have applications in the treatment of severe hypoglycemia associated with bariatric surgery and hyperinsulinomic hypoglycemia. Here we present the discovery and creation of both antagonistic and agonistic GLP-1 R antibodies by panning this GPCR-focused phage display library on a GLP-1 R-overexpressing Chinese hamster ovary cell line and demonstrate their in vitro and in vivo functional activity.

Counting Intraepithelial Lymphocytes: A Comparison Between Routine Staining and CD3 Immunohistochemistry.

Counting intraepithelial lymphocytes (IELs) is a key part of the assessment of duodenal biopsies. Immunohistochemistry (IHC) for CD3 can aid identification of lymphocytes in this context, but it is not evident that counts on hematoxylin and eosin (H&E) and CD3 are comparable. This study aimed to compare the IEL counts in duodenal biopsies using H&E stains and CD3 IHC, and to examine the interobserver variability. Thirty-five paired H&E and CD3 sections were reviewed by 6 pathologists who counted the number of IELs per 100 enterocytes. The counts were categorized into groups: normal (<25 lymphocytes), mildly raised (25-40 lymphocytes), and markedly raised (>40 lymphocytes). CD3 IHC was associated with significantly higher IEL counts than H&E. Four cases with normal H&E counts had raised counts with CD3. There was moderate agreement between observers for both H&E and CD3. Lack of concordance between CD3 and H&E IEL counts suggests that counts derived from the 2 methods may not be comparable to each other and should not be considered equivalent. There was no significant improvement in interobserver variability with CD3 IHC.

Heme oxygenase-1 inhibits pro-oxidant induced hypertrophy in HL-1 cardiomyocytes.

AIMS: Reactive oxygen species (ROS) activate multiple signaling pathways involved in cardiac hypertrophy. Since HO-1 exerts potent antioxidant effects, we hypothesized that this enzyme inhibits ROS-induced cardiomyocyte hypertrophy. METHODS: HL-1 cardiomyocytes were transduced with an adenovirus constitutively expressing HO-1 (AdHO-1) to increase basal HO-1 expression and then exposed to 200 microM hydrogen peroxide (H2O2). Hypertrophy was measured using 3H-leucine incorporation, planar morphometry and cell-size by forward-scatter flow-cytometry. The pro-oxidant effect of H2O2 was assessed by redox sensitive fluorophores. Inducing intracellular redox imbalance resulted in cardiomyocyte hypertrophy through transactivation of nuclear factor kappa B (NF-kappaB). RESULTS: Pre-emptive HO-1 overexpression attenuated the redox imbalance and reduced hypertrophic indices. This is the first time that HO-1 has directly been shown to inhibit oxidant-induced cardiomyocyte hypertrophy by a NF-kappaB-dependent mechanism. CONCLUSION: These results demonstrate that HO-1 inhibits pro-oxidant induced cardiomyocyte hypertrophy and suggest that HO-1 may yield therapeutic potential in treatment of.

Immune repertoire mining for rapid affinity optimization of mouse monoclonal antibodies.

Traditional hybridoma and B cell cloning antibody discovery platforms have inherent limits in immune repertoire sampling depth. One consequence is that monoclonal antibody (mAb) leads often lack the necessary affinity for therapeutic applications, thus requiring labor-intensive and time-consuming affinity in vitro engineering optimization steps. Here, we show that high-affinity variants of mouse-derived mAbs can be rapidly obtained by testing of somatic sequence variants obtained by deep sequencing of antibody variable regions in immune repertories from immunized mice, even with a relatively sparse sampling of sequence variants from large sequence datasets. Affinity improvements can be achieved for mAbs with a wide range of affinities. The optimized antibody variants derived from immune repertoire mining have no detectable in vitro off-target binding and have in vivo clearance comparable to the parental mAbs, essential properties in therapeutic antibody leads. As generation of antibody variants in vitro is replaced by mining of variants generated in vivo, the procedure can be applied to rapidly identify affinity-optimized mAb variants.

How prepared are pharmacists to provide over-the-counter naloxone? The role of previous education and new training opportunities.

INTRODUCTION: and Aims: Opioid overdose can be reversed with timely administration of naloxone. In Australia, naloxone was rescheduled from prescription only (S4) to pharmacist only over-the-counter (OTC, S3) in February 2016, increasing access for the general public. A key barrier to naloxone supply by pharmacists is a lack of knowledge, highlighting the role of pharmacist education. Community pharmacists' education, experience, and training preferences related to naloxone provision, overdose, and substance use disorder were examined. METHODS: Online survey data from a national sample of Australian pharmacists on their educational preferences regarding naloxone and overdose prevention, and prior training on substance use disorder (n = 595) was analyzed using bivariate and multivariate regression analysis. Data from qualitative semi-structured telephone interviews with pharmacists about OTC naloxone provision (n = 21) was analyzed using thematic analysis. RESULTS: Most pharmacists (81%, n = 479) were willing to be trained in opioid overdose prevention, with greater willingness to attend training associated with younger age, being female, fewer years of practice, not having attended previous education on substance use disorder, and higher confidence in issues relating to substance use disorder. Qualitative interviews confirmed community pharmacists' willingness to attend training but analysis revealed low awareness, knowledge, and confidence about naloxone and preventing opioid overdose. Most pharmacists preferred online training or webinars. DISCUSSION AND CONCLUSION: Most community pharmacists in Australia are willing to attend training on providing naloxone and preventing opioid overdose. There are opportunities to develop and expand the online presence of training, guidelines, and education materials to facilitate the expanded supply of OTC naloxone.

Why aren't Australian pharmacists supplying naloxone? Findings from a qualitative study.

BACKGROUND: Opioid overdose is a significant public health issue among people who use pharmaceutical opioids and/or heroin. One response to reducing overdose deaths is to expand public access to naloxone. The Australian Therapeutic Goods Administration down-scheduled naloxone from prescription only (S4) to pharmacist only over-the-counter (OTC, schedule 3) in February 2016. There is little research examining pharmacists' perspectives or experiences of this change. METHODS: Thirty-seven semi-structured interviews with Australian community pharmacists were conducted in 2016-2017 to investigate pharmacists' attitudes to and experiences of OTC naloxone. Transcripts were thematically analysed, guided by a broad interest in facilitators and barriers to OTC supply. RESULTS: Around half of the pharmacists were aware of the down-scheduling and only two had provided OTC naloxone. Core barriers to pharmacist provision of OTC naloxone included limited understanding of opioid overdose, confusion about the role and responsibilities of pharmacists in providing OTC naloxone, concerns about business, stigma related to people who inject drugs (PWID) and system-level challenges. CONCLUSION: Pharmacy provision of OTC naloxone offers an important opportunity to reduce overdose mortality. Our study suggests this opportunity is yet to be realised and highlights several individual- and structural-level impediments hindering the expansion of public access to naloxone via community pharmacies. There is a need to develop strategies to improve pharmacists' knowledge of OTC naloxone and opioid overdose as well as to address other logistical and cultural barriers that limit naloxone provision in pharmacy settings. These need to be addressed at the individual level (training) as well as the system level (information, regulation and supply).

Thromboembolism following cesarean section: a retrospective study.

OBJECTIVES: As thromboembolism (TE) continues to be one of the principal causes of death in obstetrical patients and as the postpartum period is associated with the highest risk for TE, we sought to determine the risk factors associated with TE following cesarean section (CS). METHODS: A retrospective analysis of patients who had CS at a large tertiary referral center was conducted. Patients were identified through hospital medical records and were contacted approximately 1 year following their CS. Medical records and a questionnaire were used to identify features that were potentially associated with TE. Univariate analysis was used to determine the risk associated with these characteristics. RESULTS: A total of 2206 patients had a CS, of which 1377 (62%) participated. Of the respondents, 137 patients received heparin (94% received a prophylactic dose, 6% received a therapeutic dose) and the remainder, 1233 patients, did not receive heparin. Seven patients (0.5%) developed a TE and 86% developed a TE within 7 days of CS. The odds ratio (OR) for TE for women with hypertension prior to pregnancy compared to patients who did not receive anticoagulation was 21.28 [95% confidence interval (CI) 4.64-90.13] and for patients who had varicose veins with superficial thrombophlebitis when compared to patients who had received heparin postpartum was 21.01 (95% CI 1.55-288.24). DISCUSSION: Hypertension and the presence of varicose veins were associated with TE following CS. Larger cohort analyses are required to confirm these associations so that risk scores incorporating these characteristics may accurately predict the occurrence of TE.