High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.

IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

Eliminating rote postoperative radiographs for surgically managed pediatric supracondylar humerus fractures.

BACKGROUND: Use of postoperative radiographs after surgical management of supracondylar humerus (SCH) fractures is often based on rote practice rather than evidence. The purpose of this study was to determine the frequency with which 3-week postoperative radiographs at the time of pin removal altered management plans in pediatric SCH fractures that were intraoperatively stable after closed reduction and percutaneous pinning (CRPP). METHODS: We prospectively recruited pediatric patients with SCH fractures managed by CRPP at our institution from June 2020 until June 2022, and reviewed retrospective data on pediatric SCH fractures managed surgically at our institution between April 2008 and March 2015. Patients were assessed for post-CRPP fracture alignment and stability. For prospective patients, we asked clinicians to document their management decision at the 3-week follow-up visit before evaluating the postoperative radiographs. Our primary outcome was change in management because of radiographic findings. RESULTS: Overall, 1066 patients in the retrospective data and 446 prospectively recruited patients met the inclusion criteria. In the prospective group, radiographic findings altered management for 2 patients (0.4%). One patient had slow callus formation and 1 patient was identified as having cubitus varus. Altered management included prolonged immobilization or additional radiographic follow-up. Radiographic findings altered management in 0 (0%) of 175 type II fractures, in 2 (0.9%) of 221 type III fractures, and in 0 (0%) of 44 type IV fractures. We obtained similar findings from retrospective data. CONCLUSION: Rote use of 3-week postoperative radiographs after surgical management of SCH fractures that are intraoperatively stable has minimal utility. Eliminating rote postoperative radiographs for SCH fractures can decrease the time and financial burdens on families and health care systems without affecting patient outcomes.

Engaging medical students in cardiac surgery: a focus on equity, diversity, and inclusion.

PURPOSE OF REVIEW: The Coronavirus Disease 2019 pandemic prohibited Canadian medical students from in-person observerships. This may be particularly detrimental to under-represented groups that may consider surgical subspecialties. To address the unprecedented need for alternative surgical career exploration and diversity within the profession, The University of Toronto Cardiac Surgery Interest Group and Division of Cardiac Surgery collaborated on virtual experiential programming. RECENT FINDINGS: Medical students were invited to virtual (1) observerships of a cardiac bypass case, (2) mentorship sessions with surgeons, (3) resident teaching sessions, (4) multidisciplinary case-based Heart Team discussions to further their understanding of the scope of Cardiac surgery, and (5) a virtual coronary anastomosis training program. Additionally, a comprehensive virtual program was spearheaded to increase interest in Cardiac surgery among low-income Black high school students. SUMMARY: Trainee response to the virtual education, mentorship, and skill acquisition was positive. Trainees reported high levels of interest in the profession, particularly among females and under-represented minorities, supporting the principles of equity diversity, and inclusion in Cardiac surgery.

Country of birth and non-small cell lung cancer incidence, treatment, and outcomes in New South Wales, Australia: a population-based linkage study.

OBJECTIVE: To compare treatment within 12 months of diagnosis, and survival by country of birth for people diagnosed with invasive non-small cell lung cancer (NSCLC) in New South Wales (NSW), Australia. DESIGN, PATIENTS, AND SETTING: A population-based cohort study of NSW residents diagnosed with NSCLC in 2003-2016 using de-identified linked data from the NSW Cancer Registry, NSW Admitted Patient Data collection, Emergency Departments, Medicare Benefits and Pharmaceutical Benefits Scheme, and National Death Index. MAIN OUTCOME MEASURES: Odds of receiving any treatment, surgery, systemic therapy, or radiotherapy respectively, in the 12 months following diagnosis were calculated using multivariable logistic regression. The hazard of death (all-cause) at one- and five-years following diagnosis was calculated using multivariable proportional hazards regression. RESULTS: 27,114 People were recorded with NSCLC in the 14-year study period. Higher percentages of older males from European countries applied in the earlier years, with a shift to younger people from South East Asia, New Zealand, and the Middle East. Adjusted analyses indicated that, compared with the Australian born, people from European countries were more likely to receive treatment, and, specifically surgery. Also, people from Asian countries were more likely to receive systemic therapy but less likely to receive radiotherapy. Survival at one- and five-years following diagnosis was higher for people born in countries other than Australia, New Zealand the United Kingdom and Germany. CONCLUSIONS: Variations exist in treatment and survival by country of birth in NSW. This may be affected by differences in factors not recorded in the NSW Registry, including use of general health services, family histories, underlying health conditions, other intrinsic factors, and cultural, social, and behavioural influences.

Female breast cancer in New South Wales, Australia, by country of birth: implications for health-service delivery.

BACKGROUND: NSW has a multicultural population with increasing migration from South East Asia, the Western Pacific and Eastern Mediterranean. OBJECTIVE: To compare cancer stage, treatment (first 12 months) and survival for 12 country of birth (COB) categories recorded on the population-based NSW Cancer Registry. DESIGN: Historic cohort study of invasive breast cancers diagnosed in 2003-2016. PATIENTS: Data for 48,909 women (18+ ages) analysed using linked cancer registry, hospital inpatient and Medicare and pharmaceutical benefits claims data. MEASUREMENT: Comparisons by COB using multivariate logistic regression and proportional hazards regression with follow-up of vital status to April 30th, 2020. RESULTS: Compared with the Australia-born, women born in China, the Philippines, Vietnam and Lebanon were younger at diagnosis, whereas those from the United Kingdom, Germany, Italy and Greece were older. Women born in China, the Philippines, Vietnam, Greece and Italy lived in less advantaged areas. Adjusted analyses indicated that: (1) stage at diagnosis was less localised for women born in Germany, Greece, Italy and Lebanon; (2) a lower proportion reported comorbidity for those born in China, the Philippines and Vietnam; (3) surgery type varied, with mastectomy more likely for women born in China, the Philippines and Vietnam, and less likely for women born in Italy, Greece and Lebanon; (4) radiotherapy was more likely where breast conserving surgery was more common (Greece, Italy, and Lebanon) and the United Kingdom; and (5) systemic drug therapy was less common for women born in China and Germany. Five-year survival in NSW was high by international standards and increasing. Adjusted analyses indicate that, compared with the Australian born, survival from death from cancer at 5 years from diagnosis was higher for women born in China, the Philippines, Vietnam, Italy, the United Kingdom and Greece. CONCLUSIONS: There is diversity by COB of stage, treatment and survival. Reasons for survival differences may include cultural factors and healthier migrant populations with lower comorbidity, and potentially, less complete death recording in Australia if some women return to their birth countries for treatment and end-of-life care. More research is needed to explore the cultural and clinical factors that health services need to accommodate.

Defining the dose-volume criteria for laryngeal sparing in locally advanced oropharyngeal cancer utilizing split-field IMRT, whole-field IMRT and VMAT.

PURPOSE: To determine the optimal dose-volume constraint for laryngeal sparing using three commonly employed intensity modulated radiation therapy (IMRT) approaches in patients with oropharyngeal cancer treated to the bilateral neck. MATERIALS AND METHODS: Thirty patients with stage II-IVA oropharynx cancers received definitive radiotherapy with split-field IMRT (SF-IMRT) to the bilateral neck between 2008 and 2013. Each case was re-planned using whole-field IMRT (WF-IMRT) and volumetric modulated arc therapy (VMAT) and plan quality metrics and dose to laryngeal structures was evaluated. Two larynx volumes were defined and compared on the current study: the Radiation Therapy Oncology Group (RTOG) larynx as defined per the RTOG 1016 protocol and the MDACC larynx defined as the components of the larynx bounded by the superior and inferior extent of the thyroid cartilage. RESULTS: Target coverage, conformity, and heterogeneity indices were similar in all techniques. The RTOG larynx mean dose was lower with WF-IMRT than SF-IMRT (22.1 vs 25.8 Gy; P < 0.01). The MDACC larynx mean dose was 17.5 Gy ± 5.4 Gy with no differences between the 3 techniques. WF-IMRT and VMAT plans were associated with lower mean doses to the supraglottic larynx (42.1 vs 41.2 vs 54.8 Gy; P < 0.01) and esophagus (18.1 vs 18.2 vs 36 Gy; P < 0.01). CONCLUSIONS: Modern whole field techniques can provide effective laryngeal sparing in patients receiving radiotherapy to the bilateral neck for advanced oropharyngeal cancers. SUMMARY: We evaluated laryngeal dose in patients with locally advanced oropharyngeal cancer treated to the bilateral neck using split-field IMRT (SF-IMRT), whole-field IMRT (WF-IMRT) and volumetric arc therapy (VMAT). All three techniques provided good sparing of laryngeal structures and were able to achieve a mean larynx dose < 33 Gy. There were no significant differences in dose to target structures or non-laryngeal organs at risk among techniques.

Patterns of surgical care for women with ovarian cancer in New South Wales.

BACKGROUND: Little is known about the delivery of surgical services and outcomes for women with ovarian cancer across New South Wales (NSW). AIM: The study objective was to provide a descriptive analysis of the proportion of women who had surgery for ovarian cancer in NSW in specialist gynaecological oncology hospitals and compare outcomes for women attending specialist and non-specialist services in NSW. MATERIALS AND METHODS: This study is a retrospective analysis of women with primary ovarian, fallopian tube or peritoneal cancer from 2009 to 2012. Data were analysed from the NSW Cancer Registry, NSW Admitted Patient Data Collection and Register of Births Deaths and Marriages. Treating hospitals were characterised as public specialist, public non-specialist and private. Morbidity and mortality outcomes are reported. RESULTS: The study included 1106 women. Fifty-seven hospitals performed surgery: seven public specialist, 27 private and 23 public non-specialist hospitals. The highest proportion of surgery was performed in public specialist hospitals (61%). There was considerable variation in the utilisation of public specialist hospitals between local health districts. There was no significant difference in outcomes related to the type of hospital where surgery was performed. CONCLUSIONS: Although the majority of women are having surgery in a specialist gynaecological oncology public hospital across NSW, many are not. Women living in regional and remote NSW were less likely to have their surgery in a specialist hospital. This is the first step in understanding where women in NSW are currently receiving their surgical care, as well as the outcomes related to this.

Residency spiral concussion curriculum design.

BACKGROUND: Resident-focused concussion curricula that measure learner behaviours are currently unavailable. We sought to fill this gap by developing and iteratively implementing a Spiral Integrated Concussion Curriculum (SICC). APPROACH: Programme elements of the concussion curriculum include academic half-days (AHDs) and three half-day clinics for first- and second-year family medicine residents. Our SICC utilises social cognitive learning principles, the constructivism paradigm and utilisation-focused evaluation. EVALUATION: A mixed-method evaluation with a pre-/post-test design and interviews was utilised. Surveys and knowledge tests were used to measure knowledge and confidence pre-AHD and 6 months post-AHD. Interviews at 6 months explored programme perception and behaviour change. Of the 141 programme attendees, 114 (80%) participated in the pre-intervention knowledge test and 33 completed the pre- and post-AHD test. Immediate pre-/post-testing demonstrated statistically significant improvement in knowledge (p = 0.042). At 6 months post-AHD, residents in Cycle 1 (n = 5) had a knowledge decrease of 3.33% (p > 0.05). Residents in Cycle 2 (n = 7) had a knowledge increase of 11.6% (p > 0.05). Both cycles of residents had an increase in confidence (Cycle 1: 65.0% [p = 0.025]; Cycle 2: 62.8% [p = 0.0014]). Residents (5 out of 6) reported positive behavioural changes at 6 months. Valued programme elements included concussion diagnosis and management, the self-study guide resource and the organised structure. IMPLICATIONS: The SICC enriched these residents' learning and fostered sustained knowledge improvement and behavioural change at 6 months post-intervention. This approach may provide a workable design for future competency-based curriculum development.

Overexpression of Krüppel-Like Factor 9 Enhances the Antitumor Properties of Paclitaxel in Malignant Melanoma-Derived Cell Lines.

Over the past decade, the treatment of metastatic melanoma has improved significantly due to the development of innovative therapies, such as drugs that target the BRAF/MAPK kinase pathway and the PD-1 pathway. However, these therapies do not work for all patients, highlighting the need for additional research on the pathophysiology of melanoma. Paclitaxel is a chemotherapeutic agent used when first-line treatments are unsuccessful; however, its efficacy is limited. Since Krüppel-like factor 9 (KLF9) (antioxidant repressor) is downregulated in melanoma, we propose that restoring KLF9 levels may sensitize malignant melanoma to chemotherapeutic agents, such as paclitaxel. We used adenovirus overexpression and siRNA technologies to assess the role of KLF9 in mediating the response of malignant melanoma-derived cell lines RPMI-7951 and A375 to paclitaxel treatment. We found that increasing KLF9 levels potentiates the effectiveness of paclitaxel, as shown by apoptotic parameters such as decreased cell viability, pro-caspase-3 activation, increased number of annexin V-positive cells, and reduction in nuclear proliferation marker (KI67). These results suggest that KLF9 may be a potential target for improving chemotherapeutic response in melanoma.

Colorectal cancer and country of birth in New South Wales, Australia: All-of-population data for prioritising health service delivery and research.

INTRODUCTION: Cancer care and outcomes differ across cultural groups in Australia. Quantifying these differences facilitates prioritisation and targeting of services and research. All-of-population data are needed by health agencies to understand and fulfil their cancer-control responsibilities. Compiling these data can be challenging while maintaining privacy. We have used data linkage to gain population-wide colorectal cancer data on stage (degree of spread), treatment, and survival in New South Wales (NSW), Australia, by country of birth (COB), and consider service implications. METHODS: We studied colon and rectal cancers diagnosed in 2003-2016 and recorded on the NSW Cancer Registry (n = 41,575), plus linked hospital data and data from Australian Medical and Pharmaceutical Benefits payments, other treatment data and death records. Outcomes for 12 COB categories were analysed using multiple logistic and proportional hazards regression, with Australia as the reference category. RESULTS: Compared with Australian born, the adjusted odds ratio for distant spread of colon cancer was higher for people born in Lebanon and the United Kingdom. Treatment was less common for people born in China (surgery), Germany (systemic), Italy (surgery), New Zealand (any treatment) and Vietnam (all treatments), while treatment for rectal cancer was more common for people born in Italy (surgery), United Kingdom (radiotherapy, systemic therapy), and Vietnam (surgery), and less frequent for people born in China (radiotherapy). Adjusted 5-year survival was higher for people born in China, Italy, Vietnam, Greece (colon), Lebanon (colon) and other non-English speaking countries. More advanced stage was negatively related to having surgery and survival. CONCLUSIONS: This study illustrates how linked data can enable comparisons of multiple outcomes for colorectal cancer by country of birth across an entire population. Results disclose "big picture" variations in population characteristics, stage, treatment and survival. This will enable better targeting and prioritisation of services and inform research priorities to address disparities.

Tolerance to FVIII: Role of the Immune Metabolic Enzymes Indoleamine 2,3 Dyoxigenase-1 and Heme Oxygenase-1.

The occurrence of neutralizing anti-FVIII antibodies is a major complication in the treatment of patients affected by hemophilia A. The immune response to FVIII is a complex, multi-factorial process that has been extensively studied for the past two decades. The reasons why only a proportion of hemophilic patients treated with FVIII concentrates develop a clinically significant immune response is incompletely understood. The "danger theory" has been proposed as a possible explanation to interpret the findings of some observational clinical studies highlighting the possible detrimental impact of inflammatory stimuli at the time of replacement therapy on inhibitor development. The host immune system is often challenged to react to FVIII under steady state or inflammatory conditions (e.g., bleeding, infections) although fine tuning of mechanisms of immune tolerance can control this reactivity and promote long-term unresponsiveness to the therapeutically administered factor. Recent studies have provided evidence that multiple interactions involving central and peripheral mechanisms of tolerance are integrated by the host immune system with the environmental conditions at the time of FVIII exposure and influence the balance between immunity and tolerance to FVIII. Here we review evidences showing the involvement of two key immunoregulatory oxygenase enzymes (IDO1, HO-1) that have been studied in hemophilia patients and pre-clinical models, showing that the ability of the host immune system to induce such regulatory proteins under inflammatory conditions can play important roles in the balance between immunity and tolerance to exogenous FVIII.

Inducible reprogramming of human T cells into Treg cells by a conditionally active form of FOXP3.

FOXP3 is required for the development of Treg and its expression is often used as a surrogate marker of functional suppression. However, it is now known that activated human T effector cells can also express FOXP3 without acquiring regulatory activity. To more closely examine the requirements for FOXP3 to reprogram human T cells into Treg, we developed a conditionally active form of FOXP3 and show here that full acquisition of Treg phenotype and function is strictly dependent on the amount of active FOXP3 a T cell expresses. In addition, the phenotypic and functional alterations induced by FOXP3 are only fully manifested following prolonged induction of protein activity. Induction of FOXP3 activity does not upregulate EBI3 or p35 mRNA, providing evidence that secretion of IL-35 does not substantially contribute to the suppressive mechanism of human Treg. These data represent the first formal evidence that FOXP3 acts as a quantitative regulator rather than a simple molecular switch for Treg.

ROS-deficient monocytes have aberrant gene expression that correlates with inflammatory disorders of chronic granulomatous disease.

Chronic granulomatous disease is an immunodeficiency caused by an inability to produce reactive oxygen species. While the mechanism of hyper-sensitivity to infection is well understood in CGD, the basis for debilitating inflammatory disorders that arise in the absence of evident infection has not been fully explained. Herein it is demonstrated that resting and TLR-activated monocytes from individuals with CGD expressed significantly higher levels of inflammatory mediators than control cells; the expression in CGD cells resembled normal cells stimulated with lipopolysaccharide. The lack of acute illness, infection or circulating endotoxin in the blood of the CGD patients at the time of sampling was consistent with infection-free inflammation. The enhanced expression of inflammatory mediators correlated with elevated expression of NF-kappaB and was dependent on ERK1/2 signalling. The results are consistent with the hypothesis that ROS are anti-inflammatory mediators that control gene expression and potentially limit the development of sterile inflammatory disorders.

Metabolic heterogeneity of idiopathic pulmonary fibrosis: a metabolomic study.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown cause characterised by progressive fibrotic formation in lung tissue. We hypothesise that disrupted metabolic pathways in IPF contribute to disease pathogenesis. METHODS: Metabolomics of human IPF was performed using mass spectroscopy (IPF lung=8; donor lung=8). Gene expression of key metabolic enzymes was measured using microarrays. Of the 108 metabolites whose levels were found altered, 48 were significantly increased, whereas 60 were significantly decreased in IPF samples compared with normal controls. RESULTS: Specific metabolic pathways mediating the IPF remodelling were found with a downregulated sphingolipid metabolic pathway but an upregulated arginine pathway in IPF. In addition, disrupted glycolysis, mitochondrial beta-oxidation and tricarboxylic acid cycle, altered bile acid, haem and glutamate/aspartate metabolism were found in IPF samples compared with control. CONCLUSIONS: Our results show alterations in metabolic pathways for energy consumption during lung structural remodelling, which may contribute to IPF pathogenesis. We believe that this is the first report of simultaneously and systemically measuring changes of metabolites involving nine metabolic pathways in human severe IPF lungs. The measurement of the metabolites may serve in the future diagnosis and prognosis of IPF.

Gold nanoparticles supported on mesoporous silica: origin of high activity and role of Au NPs in selective oxidation of cyclohexane.

Homogeneous immobilization of gold nanoparticles (Au NPs) on mesoporous silica has been achieved by using a one-pot synthesis method in the presence of organosilane mercapto-propyl-trimethoxysilane (MPTMS). The resultant Au NPs exhibited an excellent catalytic activity in the solvent-free selective oxidation of cyclohexane using molecular oxygen. By establishing the structure-performance relationship, the origin of the high activity of mesoporous supported Au catalyst was identified to be due to the presence of low-coordinated Au (0) sites with high dispersion. Au NPs were confirmed to play a critical role in the catalytic oxidation of cyclohexane by promoting the activation of O2 molecules and accelerating the formation of surface-active oxygen species.

Insights into the electrocatalysis of nitrobenzene using chemically-modified carbon nanotube electrodes.

The electrochemical behavior of nitrobenzene and its derivatives at chemically-functionalized multi-wall carbon nanotubes (MWNTs) modified electrodes was studied. Experimental results showed that hydroxyl-containing MWNTs exhibited the highest electrocatalytic activity among the used MWNTs because of its weak capacitive features and oxygen-containing functional groups. The cycle voltammetrys of nitrobenzene derivatives on the MWNTs modified electrodes can be easily tuned by changing the substituted groups of nitrobenzene. Based on the experimental data, the electrochemical reaction mechanisms of nitrobenzene and its derivatives on the MWNTs modified electrodes have been discussed and analyzed.

Experimental mouse models of T cell-dependent inflammatory bowel disease.

Inflammatory bowel diseases (IBD) represent idiopathic chronic inflammatory disorders of the intestinal tract that are associated with aberrant immune responses against intestinal bacteria. Here, we describe two T cell-dependent models of experimental murine IBD. In the "T cell transfer" model, lymphopenic (scid or Rag (-/-) ) mice develop colitis upon adoptive transfer of naïve CD4(+) T cells. This model has also been extensively employed to identify mechanisms through which CD4(+)CD25(+) regulatory T cells suppress intestinal inflammation in vivo. We also describe a model of T cell-dependent IBD in immunocompetent mice, induced by infection with the intestinal bacterium Helicobacter hepaticus and concomitant treatment with a blocking αIL-10R mAb, which leads to the development of chronic inflammation of the caecum and colon (typhlocolitis). Both models reproduce many facets of human IBD pathology, including epithelial hyperplasia, goblet cell depletion, and leukocyte infiltration. These models provide reliable and tractable systems for the analyses of the induction and regulation of chronic inflammation in the gut.