Abstract: Indigenous people, including Māori in New Zealand, face many inequities in health and the determinants of health. Historically, the analysis and reporting of Indigenous health in the literature has usually taken a western medical view, often with a descriptive and deficit-oriented approach—ignoring the holistic nature of Indigenous health. This project takes a nondeficit approach and is interested in the factors that support the health and well-being of Indigenous people, including Māori. Flourishing is a recent and increasingly used term within the well-being literature; however, concepts, theories and determinants related to Indigenous flourishing are largely unknown. This scoping review aims to identify, describe and synthesise the nature and extent of the current empirical literature related to concepts, theories and determinants of Indigenous flourishing, in health and well-being contexts. Methods and analysis Scoping review methods and guidelines included in the framework developed by Arksey and O’Malley, and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping reviews, will be followed for best practice and reporting of this scoping review. The literature for this review will be identified by searching the following databases: Medline (OVID), EMBASE (Ovid), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, MAI journal, the Cochrane Library and Google Scholar. The research team has formulated a systematic search strategy, which will be restricted to articles published between January 1970 and May 2020 and published in the English language. Two reviewers will independently screen eligible studies for final study selection. A third reviewer will resolve any discrepancies that arise. Data from included studies will be extracted and included in thematic analysis, using a tool developed iteratively by the research team. Ethics and dissemination Ethical approval was not required for this review. Dissemination of results will include publication in peer-reviewed journal articles, presentation of results at conferences and interactive discussions with a project expert advisory group. This scoping review also informs a larger project, examining the long-term health and flourishing of Māori, the Indigenous people of New Zealand and their whānau (families).

Abstract: While immune checkpoint blockade therapy has improved progression-free survival in patients suffering from cancer over other treatments, 1–4 these typically elicit durable responses in only minority of patients, in part because of the highly immunosuppressive tumor microenvironment (TME). 5 6 Rational combinations with inflammatory cytokines can relieve some immunosuppression, 7 8 but systemic dosing of these proteins is impeded by severe immune-related adverse events (irAE). 9–14 One approach to focus the activity of immunostimulatory agents in tumors while lowering systemic toxicity is to administer these drugs intratumorally. However, intratumoral injection alone generally achieves limited persistence in the TME, as drugs quickly clear from the tumor via lymphatics and the tumor vasculature, rapidly leading to harmful accumulation in the circulation. 15 16 Thus, approaches to promote in vivo retention of intratumorally administered drugs are necessary to maximize local stimulation. Methods We engineered Interleukin-12 (IL-12) with a peptide tag containing multiple phosphoserine (pSer) residues, through in-cell phosphorylation during recombinant expression in mammalian cells. We then inoculated mice with B16F10, or Ag104A tumors, treated established tumors intratumorally with a single dose of IL-12 mixed with alum, and monitored the tumor size and weights over time. Immunophenotyping of tumors and draining lymph nodes (dLNs) was conducted at several timepoints after treatment. Tumors and serum were also collected to perform bead-based Luminex analysis of many cytokines (including IL-12 and IFN-γ). Results Cytokines with pSer tags bind tightly to the common vaccine adjuvant aluminum hydroxide (alum) via ligand exchange (72% pSer-IL-12 vs 3.5% IL-12, P 〈 0.0001). Alum particles form a physical depot at injection sites that is persistent over weeks. So, intratumoral injection of pSer-IL-12-loaded alum led to 〉 400-fold greater retention of protein relative to unanchored pSer-IL-12 with 2-fold lower serum ALT (a biomarker for IL-12 systemic toxicity). Further, a single dose of alum-tethered pSer-IL-12 induced 5-fold greater IFN-γ secretion (P=0.0031) at the tumor primarily by CD8+ T cells and doubled (P 〈 0.0001) the proportion of tumor antigen-carrying, CD86-expressing CD103+ DCs in dLN relative to free IL-12. Further, intratumoral alum/pSer-IL-12 therapy enhanced responses to checkpoint blockade (anti-PD1), leading to a cure rate of 52% in poorly immunogenic B16F10 tumors compared to 0% for free IL-12. Local intratumoral treatment of ipsilateral tumors in mice also led to clearance of large, untreated contralateral tumors in 9/15 animals for alum/pSer-IL-12 vs. 5/17 animals for unanchored IL-12 (P=0.04). Conclusions Thus, intratumoral treatment with alum-anchored cytokines presents a safe, tumor-agnostic approach to improve local and systemic anti-cancer immunity. References Wolchok JD, Chiarion-Sileni V, Gonzalez R, Rutkowski P, Grob JJ, Cowey CL, et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. New England Journal of Medicine 2017; 377 (14):1345–56. Ansell SM, Lesokhin AM, Borrello I, Halwani A, Scott EC, Gutierrez M, et al. PD-1 Blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma. New England Journal of Medicine [Internet] . 2015; 372 (4):311–9. Available from: http://www.nejm.org/doi/10.1056/NEJMoa1411087. Brahmer J, Reckamp KL, Baas P, Crinò L, Eberhardt WEE, Poddubskaya E, et al. Nivolumab versus docetaxel in advanced squamous-cell non–small-cell lung cancer. New England Journal of Medicine [Internet] . 2015; 373 (2):123–35. Available from: http://www.nejm.org/doi/10.1056/NEJMoa1504627. Bellmunt J, de Wit R, Vaughn DJ, Fradet Y, Lee J-L, Fong L, et al. Pembrolizumab as second-line therapy for advanced urothelial carcinoma. New England Journal of Medicine [Internet] . 2017; 376 (11):1015–26. Available from: http://www.nejm.org/doi/10.1056/NEJMoa1613683. Yi M, Jiao D, Xu H, Liu Q, Zhao W, Han X, et al. Biomarkers for predicting efficacy of PD-1/PD-L1 inhibitors [Internet]. Vol. 17, Molecular Cancer . BioMed Central Ltd.; 2018 [cited 2021 May 2]. p. 1–14. Available from: https://doi.org/10.1186/s12943-018-0864-3. Anderson KG, Stromnes IM, Greenberg PD. Obstacles posed by the tumor microenvironment to T cell activity: a case for synergistic therapies [Internet]. Vol. 31, Cancer Cell . Cell Press; 2017 [cited 2021 May 2]. p. 311–25. Available from: https://pubmed.ncbi.nlm.nih.gov/28292435/. Smyth MJ, Ngiow SF, Ribas A, Teng MWL. Combination cancer immunotherapies tailored to the tumour microenvironment [Internet]. Vol. 13, Nature Reviews Clinical Oncology. Nature Publishing Group; 2016;143–58. Available from: https://pubmed.ncbi.nlm.nih.gov/26598942/. Moynihan KD, Opel CF, Szeto GL, Tzeng A, Zhu EF, Engreitz JM, et al. Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses. Nature Medicine 2016; 22 (12):1402–10. Milling L, Zhang Y, Irvine DJ. Delivering safer immunotherapies for cancer. Advanced Drug Delivery Reviews [Internet] . 2017; 114 :79–101. Available from: http://dx.doi.org/10.1016/j.addr.2017.05.011. Lasek W, Zagożdżon R, Jakobisiak M. Interleukin 12: still a promising candidate for tumor immunotherapy? Cancer Immunology, Immunotherapy 2014; 63 (5):419–35. Kirchner GI, Franzke A, Buer J, Beil W, Probst-Kepper M, Wittke F, et al. Pharmacokinetics of recombinant human interleukin-2 in advanced renal cell carcinoma patients following subcutaneous application. British Journal of Clinical Pharmacology [ Internet ] 1998; 46 (1):5–10. Available from: /pmc/articles/PMC1873983/. June CH, Warshauer JT, Bluestone JA. Is autoimmunity the Achilles' heel of cancer immunotherapy? Nature Medicine [ Internet ] 2017; 23 (5):540–7. Available from: http://www.nature.com/articles/nm.4321. Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, Sosman JA, Dutcher JP, Vogelzang JLR. Effects of single-dose interleukin-12 exposure on interleukin-12–Associated toxicity and interferon-γ Production. Blood 1997;2541–8. Atkins MB, Robertson MJ, Gordon M, Lotze MT, DeCoste M, DuBois JS, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clinical Cancer Research 1997; 3 (3). van Herpen CML, van der Voort R, van der Laak JAWM, Klasen IS, de Graaf AO, van Kempen LCL, et al. Intratumoral rhIL-12 administration in head and neck squamous cell carcinoma patients induces B cell activation. International Journal of Cancer [ Internet ] 2008; 123 (10):2354–61. Available from: https://pubmed.ncbi.nlm.nih.gov/18729197/. Pfreundschuh MG, Tilman Steinmetz H, Tüschen R, Schenk V, Diehl V, Schaadt M. Phase I study of intratumoral application of recombinant human tumor necrosis factor. European Journal of Cancer and Clinical Oncology [ Internet ] 1989; 25 (2). Available from: https://pubmed.ncbi.nlm.nih.gov/2702990/ Ethics Approval All animal studies and procedures were carried out following federal, state and local guidelines under an institutional animal care and use committee-approved animal protocol (Protocol no. 0720-070-23) by the Committee of Animal Care at MIT.

Abstract: The global COVID-19 pandemic continues to have wide-ranging implications for health, including psychological well-being. A growing corpus of research reviews has emerged on the topic of psychological resilience in the context of the pandemic. However, this body of work has not been systematically reviewed for its quality, nor with respect to findings on the effectiveness of tools and strategies for psychological resilience. To this end, a meta-review protocol is proposed with the following objectives: (1) identify review work on the topic of psychological resilience during COVID-19; (2) assess the quality of this review work using A MeaSurement Tool to Assess systematic Reviews; (3) assess the risk of bias in this work; (4) generate a narrative summary of the key points, strengths and weaknesses; (5) identify the psychological resilience strategies that have been reviewed; (6) identify how these strategies have been evaluated for their effectiveness; (7) identify what outcomes were measured and (8) summarise the findings on strategies for psychological resilience so far, providing recommendations, if possible. Methods and analysis A systematic meta-review will be conducted in accordance with the Preferred Reporting Items for Systematic Reviews for Protocols and Joanna Briggs Institute umbrella review guidelines. Electronic searches of general databases, especially Web of Science, Scopus and PubMed, will be conducted. Only results from January 2020 onwards will be considered, coinciding with the COVID-19 pandemic. Only results in English will be included. Descriptive statistics, thematic analysis and narrative summaries describing the nature of the reviewed work and evaluation of psychological resilience strategies will be carried out. Ethics and dissemination Ethical approval is not needed for systematic review protocols. The results of the meta-review will be published in an international peer-reviewed journal. The raw and summarised data will be shared in the journal or other open venues. PROSPERO registration number CRD42021235288.

Abstract: The most frequent manifestation in adult Hypophosphatasia (HPP) is musculoskeletal pain. 1,2 The unspecific nature of its clinical presentation may prevent correct diagnosis. 3 Objectives: Identifying adult hypophosphatasia in the rheumatology unit. Methods: Over a period of 10 years 9,522 patients were screened in a rheumatological outpatient unit. Serum ALP levels ≤ 40 U/l were found in 524 patients. After screening for secondary causes, 73 patients were invited for clinical evaluation. Genetic testing was performed in 23 patients with suspected HPP. Logistic regression models were used to estimate the association of each clinical factor with HPP. Results: Mutations in the ALPL gene were observed in 57% of genetically screened patients. Arthralgia, fractures and pain were the leading symptoms in HPP patients. Chondrocalcinosis (OR 29.12; 95% CI 2.02-1593.52) and dental disease (OR 8.33; 95% CI 0.93-143.40) were associated with HPP independent of BMI. Onset of symptoms in HPP was at 35.1 (14.3) years, with a mean duration from symptoms to diagnosis of 14.4 (8.1) years. Bone mineral density (BMD) and trabecular bone score (TBS) as well as bone turnover markers were not indicative for HPP. Conclusion: HPP can mimic joint diseases. 4 Thus, in patients with uncertain rheumatologic complaints and low ALP, HPP should be considered as potential diagnosis. References: [1]Durrough C, Colazo JM, Simmons J, et al. Characterization of physical, functional, and cognitive performance in 15 adults with hypophosphatasia. Bone 2021;142:115695. [2]Seefried L, Kishnani PS, Moseley S, et al. Pharmacodynamics of asfotase alfa in adults with pediatric-onset hypophosphatasia. Bone 2021;142:115664. [3]Högler W, Langman C, Gomes da Silva H, et al. Diagnostic delay is common among patients with hypophosphatasia: initial findings from a longitudinal, prospective, global registry. BMC musculoskeletal disorders 2019;20(1):80. [4]Seefried L, Dahir K, Petryk A, et al. Burden of Illness in Adults With Hypophosphatasia: Data From the Global Hypophosphatasia Patient Registry. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 2020;35(11):2171-78. Disclosure of Interests: None declared.

Abstract: Deformational plagiocephaly (DP) is one of the most common cranial shape disorders in infancy. It is characterised by unilateral flattening of the skull due to head preference to one side. The literature suggests that DP is associated with comorbidities such as developmental delay, but the nature and prevalence of the comorbid impairments are still unclear and controversial. Therefore, our scoping review (ScR) aims to explore systematically the extent and nature of literature by identifying, mapping and categorising the most relevant comorbidities of DP in children up to the age of 2 years. Methods and analysis This protocol is based on the framework outlined by Arksey and O’Malley. A systematic search will be conducted to identify relevant full text studies from 1992 to 2021 using the databases of Cochrane, MEDLINE, Google Scholar, EMBASE, PubMed and University of Nicosia EBSCO. Two independent reviewers will screen abstracts and full articles in parallel, using specific inclusion and exclusion criteria. Specifically, this review will consider studies investigating DP and relevant comorbidities in children up to the age of 2 years of life without craniosynostosis, torticollis and any other diagnosed neurodevelopmental deficiency. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for ScR Checklist will be considered for results’ analysis and reporting. The results will be described in a narrative form in relation to the research question and in the context of the overall study purpose. Ethics and dissemination Research ethics approval is not required for this ScR since data will be retrieved from publicly available studies. Dissemination activities will include research findings’ submission for publication in a relevant peer-reviewed journal and presentation of the results at relevant conferences. Registration Our protocol was registered prospectively with the Open Science Framework ( https://osf.io/48am3/ ).

Abstract: IgG4-related disease (IgG4-RD) is an autoimmune disorder and frequently involve multiple organs. The respiratory tract is one of the most frequently involved sites. Objectives: This study aimed to compare the demographic and clinical characteristics of IgG4-related respiratory disease (IgG4-RRD) and non-IgG4-RRD patients in a large cohort. Methods: We carried out a retrospective study of 452 cases of IgG4-RD (104 IgG4-RRD patients and 348 non-IgG4-RRD patients) diagnosed at Peking University People’s Hospital between 2003 and 2020. Results: IgG4-RRD patients had an elder age of disease onset and diagnosis. Multiorgan involvement and hypocomplementemia were more common in IgG4-RRD. Besides, the level of ESR, eosinophilia, IgG and IgG4 were higher in IgG4-RRD patients. In IgG4-RRD group, salivary gland, lacrimal gland, lymph nodes, biliary system and kidney were more commonly involved than those in the non-IgG4-RRD group. Also, more numbers of organ involvement and biliary involvement were independent risk factors for the development of respiratory involvement in IgG4-RD patients. Conclusion: Our study revealed demographic, clinical, laboratory and imaging features of IgG4-RRD patients and the underlying differences in pathogenesis between the two phenotypes, which have important implications for the diagnosis and treatment of the disease. References: [1]Morales AT, Cignarella AG, Jabeen IS, Barkin JS, Mirsaeidi M. An update on IgG4-related lung disease. European journal of internal medicine. 2019;66:18-24. [2]Stone JH, Zen Y, Deshpande V. IgG4-related disease. The New England journal of medicine. 2012;366(6):539-51. [3]Vasaitis L. IgG4-related disease: A relatively new concept for clinicians. European journal of internal medicine. 2016;27:1-9. [4]Matsui S, Yamamoto H, Minamoto S, Waseda Y, Mishima M, Kubo K. Proposed diagnostic criteria for IgG4-related respiratory disease. Respiratory investigation. 2016;54(2):130-2. [5]Cao L, Chen YB, Zhao DH, Shi WF, Meng S, Xie LX. Pulmonary function tests findings and their diagnostic value in patients with IgG4-related disease. Journal of thoracic disease. 2017;9(3):547-54. [6]Wallace ZS, Perugino C, Matza M, Deshpande V, Sharma A, Stone JH. Immunoglobulin G4-related Disease. Clinics in chest medicine. 2019;40(3):583-97. [7]Matsui S. IgG4-related respiratory disease. Modern rheumatology. 2019;29(2):251-6. [8]Johansson SG, Hourihane JO, Bousquet J, Bruijnzeel-Koomen C, Dreborg S, Haahtela T, et al. A revised nomenclature for allergy. An EAACI position statement from the EAACI nomenclature task force. Allergy. 2001;56(9):813-24. [9]Fei Y, Shi J, Lin W, Chen Y, Feng R, Wu Q, et al. Intrathoracic Involvements of Immunoglobulin G4-Related Sclerosing Disease. Medicine. 2015;94(50):e2150. [10]Wallace ZS, Deshpande V, Mattoo H, Mahajan VS, Kulikova M, Pillai S, et al. IgG4-Related Disease: Clinical and Laboratory Features in One Hundred Twenty-Five Patients. Arthritis & rheumatology (Hoboken, NJ). 2015;67(9):2466-75. [11]Yamada K, Yamamoto M, Saeki T, Mizushima I, Matsui S, Fujisawa Y, et al. New clues to the nature of immunoglobulin G4-related disease: a retrospective Japanese multicenter study of baseline clinical features of 334 cases. Arthritis research & therapy. 2017;19(1):262. [12]Borges T, Silva S. IgG4-related disease: How to place it in the spectrum of immune-mediated and rheumatologic disorders? Modern rheumatology. 2020;30(4):609-16. [13]Liu Y, Xue M, Wang Z, Zeng Q, Ren L, Zhang Y, et al. Salivary gland involvement disparities in clinical characteristics of IgG4-related disease: a retrospective study of 428 patients. Rheumatology (Oxford, England). 2020;59(3):634-40. [14]Matsui S, Taki H, Shinoda K, Suzuki K, Hayashi R, Tobe K, et al. Respiratory involvement in IgG4-related Mikulicz’s disease. Modern rheumatology. 2012;22(1):31-9. Disclosure of Interests: None declared

Abstract: Post-stroke depression (PSD) is a severe complication of cerebrovascular stroke affecting about one-third of stroke survivors. Moreover, PSD is associated with functional recovery and quality of life (QOL) in stroke survivors. Screening for PSD is recommended. There are, however, differences in the literature on the impact of early screening on functional outcomes. In this systematic review, we synthesise the currently available literature regarding the associations between timing and setting of PSD screening and mortality, QOL and functional outcomes in stroke survivors. Methods and analysis We will systematically search electronic databases including PubMed, Embase, APA PsycINFO, Web of Science, Scopus and CINAHL from inception to August 2021. Four reviewers will screen the title and abstract and full-text level records identified in the search in a blinded fashion to determine the study eligibility. Any selection disagreements between the reviewers will be resolved by the study investigator. Data extraction of eligible studies will be conducted by two reviewers using a predefined template. We will complete the quality assessment of included articles independently by two reviewers using the Newcastle Ottawa Scale. Eventual discrepancies will be resolved by the principal investigator. Ethics and dissemination Due to the nature of the study design, ethical approval is not required. The systematic review and meta-analysis findings will be published and disseminated in a peer-reviewed journal. Our results will also be disseminated through posters and presentations at appropriate scientific conferences. PROSPERO registration number CRD42021235993.

Abstract: Melanoma and lung cancers have two of the highest response rates to immune checkpoint inhibitors (ICIs). 1 However, patients may respond unpredictably, partly due to heterogeneity in the quantity and quality of tumor-specific T cells. In this study, we performed an integrated transcriptomic analysis of anti-tumor CD8+ TIL from non-small cell lung cancer (NSCLC) and melanoma. Our goal was to study the global transcriptomic landscape of tumor-specific T cells and to compare their functional programming in lung cancer vs. melanoma. Methods TIL from 19 patients (15 NSCLC and 3 melanoma) were sequenced using combined single-cell (sc) RNA-seq/TCR-seq. All NSCLC patients received neoadjuvant anti-PD-1 (nivolumab, NCT02259621 ) whereas melanoma patients received a personal neoantigen vaccine ( NCT01970358 ). Neoantigen-, tumor-associated antigen-, and viral-specific CD8+ T cell clonotypes were identified using functional assays and were validated by TCR cloning as previously described. 2 3 Transcriptional profiles of antigen-specific T cells were identified using the TCRβ CDR3 as a barcode to link with the antigen specificity output from the functional assays. The prevalence, phenotype, and differentiation trajectory of tumor-specific T cells were compared between the two cancer types. Results A total of 175,826 CD8+ TIL were analyzed, of which 30,174 single cells were from the melanoma cohort and 145,652 were from the NSCLC cohort. Tumor-specific T cells were detected at variable frequencies among CD8+ TIL (median=1.2%, range 0.01%–35.8%) across nine patients, with melanoma having more clonal tumor-specific T cells as compared to NSCLC. CD8+ TIL from melanoma were more enriched in an activated tissue resident T cell (TRM) cluster characterized by upregulated expression of CXCL13, CRTAM, 4-1BB, XCL1/2, and FABP5, whereas those from NSCLC have a greater representation of a cytotoxic TRM cluster with an exhaustion signature (coexpression of GZMB, GZMH, PDCD1, and CTLA4). Distinct from EBV-specific T cells and flu-specific T cells, tumor-specific T cells primarily resided in TRM clusters in both cancers. More MANA-specific TIL from melanoma presented with an effector phenotype and were more proliferative as compared to those from NSCLC. To reveal the differentiation trajectory and regulatory programs of tumor-specific T cells upon tumor recognition and association with response to ICIs, pseudotime/velocity analysis of tumor-specific TIL is underway. Conclusions This is the first analysis to inform on the global transcriptomic landscape of tumor-specific CD8+ TIL in lung cancer and melanoma at single cell resolution. This provides a useful framework to study the underlying mechanisms of T cell exhaustion and dysfunction in human cancer. Trial Registration NCT02259621, NCT01970358 References Yarchoan M, Hopkins A, Jaffee EM. Tumor mutational burden and response rate to PD-1 inhibition. The New England Journal of Medicine 2017; 377 (25):2500. Caushi JX, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature 2021;1–7. Oliveira G, et al. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma. Nature 2021;1–7. Ethics Approval The melanoma clinical trial was approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB) ( NCT01970358 ). The NSCLC clinical trial was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU) and Memorial Sloan Kettering Cancer Center ( NCT02259621 ). All participants gave informed consent before taking part. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Abstract: FDA-approved immunotherapies for early and advanced stage bladder cancer have response rates of 15–65% in bladder cancer, suggesting that tumor-associated resistance mechanisms undermine their efficacy. Accordingly, there is an unmet need to identify accessible biomarkers that predict response. Urine, which is in direct contact with urothelial tumors, represents an easily accessible patient material that may reflect cellular and/or genetic signatures related to immune resistance. It has been demonstrated that urine from bladder cancer patients contains not only tumor cells, which are routinely assessed by clinical urinalyses, but also immune cells that previous studies suggest may reflect the tumor microenvironment (TME). 1 However, the concordance between cells in the urine and those in bladder tumors is unknown., Here, we characterized patient urine in an unbiased fashion by performing the first single-cell RNA sequencing (scRNAseq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) on matched bladder cancer patient urine, tumor, and peripheral blood. Methods Matched tumor tissue, urine, and peripheral blood were collected from bladder cancer patients (n=7) during surgery; either trans-urethral resection of bladder tumor or cystectomy. All three tissues were processed to single-cell suspensions and sequenced using the 10X Genomics platform (scRNAseq: 17 samples, CITE-seq: 3 samples). These sequencing approaches permitted quantification of both transcriptomic and surface protein expression of 54,469 cells total. 2 3 Analysis was performed using Seurat, Enrichr, and Monocle packages and platforms. 4 5 6 Results scRNAseq of urine from bladder cancer patients revealed several immune populations including CD4+ and CD8+ T cells, Treg cells, NK cells, B cells, neutrophils, dendritic cells, monocytes, and macrophages in addition to non-hematopoietic lineages including bladder epithelial cells, neuronal cells, prostate epithelial cells, fibroblasts, myofibroblasts, and endothelial cells. The composition and transcriptional profiles of urine immune cells were more similar to TME immune cells than to peripheral blood immune cells. Urine immune cells expressed gene signatures associated with hypoxia, anergy, pro-inflammation, and glucose deprivation that were more similar to tumor immune cells than those in the peripheral blood. Conclusions Our work represents the first scRNAseq and CITEseq profiling of cancer patient urine. Our study suggests several viable immune cells shed in bladder cancer patient urine that look more transcriptionally and phenotypically similar to the TME than peripheral blood cells. This important finding has several implications for future research and clinical applications as urine can be sampled non-invasively in scenarios when tumor resection may not be feasible. References Wong YNS, Joshi K, Khetrapal P, et al. Urine-derived lymphocytes as a non-invasive measure of the bladder tumor immune microenvironment. Journal of Experimental Medicine . 2018; 215 :2748–59. Zheng GXY, Terry JM, Belgrader P, et al. Massively parallel digital transcriptional profiling of single cells. Nature Communications 2017; 8. Stoeckius M, Hafemeister C, Stephenson W, et al. Simultaneous epitope and transcriptome measurement in single cells. Nature Methods 2017; 14 , 865–68. Butler A, Hoffman P, Smibert, P, et al. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nature Biotechnology 2018; 36 : 411–20. Xie Z, Bailey A, Kuleshov MV, et al. Gene set knowledge discovery with Enrichr. Current Protocols 2021. Trapnell C, Cacchiarelli D, Grimsby J, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nature Biotechnology 2014; 32 : 381–6. Ethics Approval The study was approved by Mount Sinai Institution’s Ethics Board, approval number 10–1180. Participants gave informed consent before taking part in the study.

Abstract: Hands play a part in the transmission of infections. Handwashing with soap sufficiently reduces the level of hand contamination and the spread of infections. As soap is not usually available due to cost, ash is often used as a zero-cost alternative to soap in the rural settings of developing countries. However, there is limited evidence on the effectiveness of ash to reduce microbial contamination of hands. This study is, therefore, designed to assess the effect of ash on microbial contamination of hands in the rural settings of northwest Ethiopia. Methods and analysis A two-arm clustered-randomised controlled trial will be employed. A total of 11 clusters per arm will be selected using simple random sampling technique. A total of 220 mothers or caregivers of under-5 children will be included in each arm. After providing health education on effective handwashing process, we will ask study subjects to do the usual activities. We will then take swab samples from the dominant hand before washing. After swabbing, participants will be asked to wash their hands with water only and with ash by following effective handwashing procedures. We will again take swab samples from the dominant hand after washing and drying. Finally, we will compare each intervention arm against the control. A generalised estimating equation (GEE) with robust SE estimation will be used to account the cluster nature of data. Ethics and dissemination Results will be published in peer-reviewed journal and presented at international conferences. The protocol is approved by the Institutional Review Board of the University of Gondar, Ethiopia. Trial registration number Pan African Clinical Trial Registry; PACTR202011855730652.