Sex differences in the clinical presentation of early psychosis in a primary care setting.

Primary care is an important part of the help-seeking pathway for young people experiencing early psychosis, but sex differences in clinical presentation in these settings are unexplored. We aimed to identify sex differences in clinical presentation to primary care services in the 1-year period prior to a first diagnosis of psychotic disorder. We identified first-onset cases of non-affective psychotic disorder over a 10-year period (2005-2015) using health administrative data linked with electronic medical records (EMRs) from primary care (n = 465). Detailed information on encounters in the year prior to first diagnosis was abstracted, including psychiatric symptoms, other relevant behaviours, and diagnoses recorded by the family physician (FP). We used modified Poisson regression models to examine sex differences in the signs, symptoms, and diagnoses recorded by the FP, adjusting for various clinical and sociodemographic factors. Positive symptoms (PR = 0.76, 95%CI: 0.58, 0.98) and substance use (PR = 0.54, 95%CI: 0.40, 0.72) were less prevalent in the medical records of women. Visits by women were more likely to be assigned a diagnosis of depression or anxiety (PR = 1.18, 95%CI: 1.00, 1.38), personality disorder (PR = 5.49, 95%CI: 1.22, 24.62), psychological distress (PR = 11.29, 95%CI: 1.23, 103.91), and other mental or behavioral disorders (PR = 3.49, 95%CI: 1.14, 10.66) and less likely to be assigned a diagnosis of addiction (PR = 0.33, 95%CI: 0.13, 0.87). We identified evidence of sex differences in the clinical presentation of early psychosis and recorded diagnoses in the primary care EMR. Further research is needed to better understand sex differences in clinical presentation in the primary care context, which can facilitate better understanding, detection, and intervention for first-episode psychotic disorders.

Can Patients with Dementia Be Identified in Primary Care Electronic Medical Records Using Natural Language Processing?

Dementia and mild cognitive impairment can be underrecognized in primary care practice and research. Free-text fields in electronic medical records (EMRs) are a rich source of information which might support increased detection and enable a better understanding of populations at risk of dementia. We used natural language processing (NLP) to identify dementia-related features in EMRs and compared the performance of supervised machine learning models to classify patients with dementia. We assembled a cohort of primary care patients aged 66 + years in Ontario, Canada, from EMR notes collected until December 2016: 526 with dementia and 44,148 without dementia. We identified dementia-related features by applying published lists, clinician input, and NLP with word embeddings to free-text progress and consult notes and organized features into thematic groups. Using machine learning models, we compared the performance of features to detect dementia, overall and during time periods relative to dementia case ascertainment in health administrative databases. Over 900 dementia-related features were identified and grouped into eight themes (including symptoms, social, function, cognition). Using notes from all time periods, LASSO had the best performance (F1 score: 77.2%, sensitivity: 71.5%, specificity: 99.8%). Model performance was poor when notes written before case ascertainment were included (F1 score: 14.4%, sensitivity: 8.3%, specificity 99.9%) but improved as later notes were added. While similar models may eventually improve recognition of cognitive issues and dementia in primary care EMRs, our findings suggest that further research is needed to identify which additional EMR components might be useful to promote early detection of dementia. Supplementary Information: The online version contains supplementary material available at 10.1007/s41666-023-00125-6.

Evaluating the Impact of Statin Use on Influenza Vaccine Effectiveness and Influenza Infection in Older Adults.

BACKGROUND: Older adults are recommended to receive influenza vaccination annually, and many use statins. Statins have immunomodulatory properties that might modify influenza vaccine effectiveness (VE) and alter influenza infection risk. METHODS: Using the test-negative design and linked laboratory and health administrative databases in Ontario, Canada, we estimated VE against laboratory-confirmed influenza among community-dwelling statin users and nonusers aged ≥66 years during the 2010-2011 to 2018-2019 influenza seasons. We also estimated the odds ratio for influenza infection comparing statin users and nonusers by vaccination status. RESULTS: Among persons tested for influenza across the 9 seasons, 54 243 had continuous statin exposure before testing and 48 469 were deemed unexposed. The VE against laboratory-confirmed influenza was similar between statin users and nonusers (17% [95% confidence interval, 13%-20%] and 17% [13%-21%] respectively; test for interaction, P = .87). In both vaccinated and unvaccinated persons, statin users had higher odds of laboratory-confirmed influenza than nonusers (odds ratios for vaccinated and unvaccinated persons 1.15 [95% confidence interval, 1.10-1.21] and 1.15 [1.10-1.20], respectively). These findings were consistent by mean daily dose and statin type. VE did not differ between users and nonusers of other cardiovascular drugs, except for ß-blockers. We did not observe that vaccinated and unvaccinated users of these drugs had increased odds of influenza, except for unvaccinated ß-blocker users. CONCLUSIONS: Influenza VE did not differ between statin users and nonusers. Statin use was associated with increased odds of laboratory-confirmed influenza in vaccinated and unvaccinated persons, but these associations might be affected by residual confounding.

Developing a reference standard for pertussis by applying a stratified sampling strategy to electronic medical record data.

PURPOSE: Pertussis surveillance remains essential in Canada, but ascertainment bias limits the accuracy of surveillance data. Introducing other sources to improve detection has highlighted the importance of validation. However, challenges arise due to low prevalence, and oversampling suspected cases can introduce partial verification bias. The aim of this study was to build a reference standard for pertussis validation studies that provides adequate analytic precision and minimizes bias. METHODS: We used a stratified strategy to sample the reference standard from a primary care electronic medical record cohort. We incorporated abstractor notes into definite, possible, ruled-out, and no mention of pertussis classifications which were based on surveillance case definitions. RESULTS: We abstracted eight hundred records from the cohort of 404,922. There were 208 (26%) definite and 261 (32.6%) possible prevalent pertussis cases. Classifications demonstrated a wide variety of case severities. Abstraction reliability was moderate to substantial based on Cohen's kappa and raw percent agreement. CONCLUSIONS: When conducting validation studies for pertussis and other low prevalence diseases, this stratified sampling strategy can be used to develop a reference standard using limited resources. This approach mitigates verification and spectrum bias while providing sufficient precision and incorporating a range of case severities.

Validating pertussis data measures using electronic medical record data in Ontario, Canada 1986-2016.

Background: Pertussis is a reportable disease in many countries, but ascertainment bias has limited data accuracy. This study aims to validate pertussis data measures using a reference standard that incorporates different suspected case severities, allowing for the impact of case severity on accuracy and detection to be explored. Methods: We evaluated 25 pertussis detection algorithms in a primary care electronic medical record database between January 1, 1986 and December 30, 2016. We estimated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). We used sensitivity analyses to explore areas of uncertainty and evaluated reasons for lack of detection. Results: The algorithm including all data measures achieved the highest sensitivity at 20.6%. Sensitivity increased to 100% after reclassifying symptom-only cases as non-cases, but the PPV remained low. Age at first episode was significantly associated with detection in half of the tested scenarios, and false negatives often had some history of immunization. Conclusions: Sensitivity improved by reclassifying symptom-only cases but remained low unless multiple data sources were used. Results demonstrate a trade-off between PPV and sensitivity. EMRs can enhance detection through patient history and clinical note data. It is essential to improve case identification of older individuals with vaccination history to reduce ascertainment bias.

Development of algorithms to identify individuals with Neurofibromatosis type 1 within administrative data and electronic medical records in Ontario, Canada.

BACKGROUND: There is limited population-based data on Neurofibromatosis type 1 (NF1) in North America. We aimed to develop and validate algorithms using administrative health data and electronic medical records (EMRs) to identify individuals with NF1 in Ontario, Canada. METHODS: We conducted an electronic free-text search of 15 commonly-used terms related to NF1 in the Electronic Medical Records Primary Care Database. Records were reviewed by two trained abstractors who classified them as confirmed, possible, and not NF1. An investigator with clinical expertise performed final NF1 classification. Patients were classified as confirmed if there was a documented diagnosis, meeting NIH criteria. Patients were classified as possible if (1) NF1 was recorded in the cumulative patient profile, but no clinical information to support the diagnosis; (2) only one criterion for diagnosis (e.g. child of confirmed case) but no further data to confirm or rule out. We tested different combinations of outpatient and inpatient billing codes, and applied a free-text search algorithm to identify NF1 cases in administrative data and EMRs, respectively. RESULTS: Of 273,440 eligible patients, 2,058 had one or more NF1 terms in their medical records. The terms "NF", "café-au-lait", or "sheath tumour" were constrained to appear in combination with another NF1 term. This resulted in 837 patients: 37 with possible and 71 with confirmed NF1. The population prevalence ranged from 1 in 3851 (confirmed NF1) to 1 in 2532 (possible and confirmed NF1). Billing code algorithms had poor performance, with overall low PPV (highest being 71%). The accuracy of the free-text EMR algorithm in identifying patients with NF1 was: sensitivity 85% (95% CI 74-92%), specificity 100% (95% CI 100-100%), positive predictive value 80% (95% CI 69-88%), negative predictive value 100% (95% CI 100-100%), and false positive rate 20% (95% CI 11-33%). Of false positives, 53% were possible NF1. CONCLUSIONS: A free-text search algorithm within the EMR had high sensitivity, specificity and predictive values. Algorithms using billing codes had poor performance, likely due to the lack of NF-specific codes for outpatient visits. While NF1 ICD-9 and 10 codes are used for hospital admissions, only ~ 30% of confirmed NF1 cases had a hospitalization associated with an NF1 code.

Effectiveness of COVID-19 vaccines against symptomatic SARS-CoV-2 infection and severe outcomes with variants of concern in Ontario.

SARS-CoV-2 variants of concern (VOC) are more transmissible and may have the potential for increased disease severity and decreased vaccine effectiveness. We estimated the effectiveness of BNT162b2 (Pfizer-BioNTech Comirnaty), mRNA-1273 (Moderna Spikevax) and ChAdOx1 (AstraZeneca Vaxzevria) vaccines against symptomatic SARS-CoV-2 infection and COVID-19 hospitalization or death caused by the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) VOC in Ontario, Canada, using a test-negative design study. We identified 682,071 symptomatic community-dwelling individuals who were tested for SARS-CoV-2, and 15,269 individuals with a COVID-19 hospitalization or death. Effectiveness against symptomatic infection ≥7 d after two doses was 89-92% against Alpha, 87% against Beta, 88% against Gamma, 82-89% against Beta/Gamma and 87-95% against Delta across vaccine products. The corresponding estimates ≥14 d after one dose were lower. Effectiveness estimates against hospitalization or death were similar to or higher than against symptomatic infection. Effectiveness against symptomatic infection was generally lower for older adults (≥60 years) than for younger adults (<60 years) for most of the VOC-vaccine combinations. Our findings suggest that jurisdictions facing vaccine supply constraints may benefit from delaying the second dose in younger individuals to more rapidly achieve greater overall population protection; however, older adults would likely benefit most from minimizing the delay in receiving the second dose to achieve adequate protection against VOC.

Effectiveness of BNT162b2 and mRNA-1273 covid-19 vaccines against symptomatic SARS-CoV-2 infection and severe covid-19 outcomes in Ontario, Canada: test negative design study.

OBJECTIVE: To estimate the effectiveness of mRNA covid-19 vaccines against symptomatic infection and severe outcomes (hospital admission or death). DESIGN: Test negative design study. SETTING: Ontario, Canada between 14 December 2020 and 19 April 2021. PARTICIPANTS: 324 033 community dwelling people aged ≥16 years who had symptoms of covid-19 and were tested for SARS-CoV-2. INTERVENTIONS: BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccine. MAIN OUTCOME MEASURES: Laboratory confirmed SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) and hospital admissions and deaths associated with SARS-CoV-2 infection. Multivariable logistic regression was adjusted for personal and clinical characteristics associated with SARS-CoV-2 and vaccine receipt to estimate vaccine effectiveness against symptomatic infection and severe outcomes. RESULTS: Of 324 033 people with symptoms, 53 270 (16.4%) were positive for SARS-CoV-2 and 21 272 (6.6%) received at least one dose of vaccine. Among participants who tested positive, 2479 (4.7%) were admitted to hospital or died. Vaccine effectiveness against symptomatic infection observed ≥14 days after one dose was 60% (95% confidence interval 57% to 64%), increasing from 48% (41% to 54%) at 14-20 days after one dose to 71% (63% to 78%) at 35-41 days. Vaccine effectiveness observed ≥7 days after two doses was 91% (89% to 93%). Vaccine effectiveness against hospital admission or death observed ≥14 days after one dose was 70% (60% to 77%), increasing from 62% (44% to 75%) at 14-20 days to 91% (73% to 97%) at ≥35 days, whereas vaccine effectiveness observed ≥7 days after two doses was 98% (88% to 100%). For adults aged ≥70 years, vaccine effectiveness estimates were observed to be lower for intervals shortly after one dose but were comparable to those for younger people for all intervals after 28 days. After two doses, high vaccine effectiveness was observed against variants with the E484K mutation. CONCLUSIONS: Two doses of mRNA covid-19 vaccines were observed to be highly effective against symptomatic infection and severe outcomes. Vaccine effectiveness of one dose was observed to be lower, particularly for older adults shortly after the first dose.

Déterminants individuels et sociaux du test de dépistage du SRAS-CoV-2 et de l'obtention d'un résultat positif en Ontario, au Canada: une étude populationnelle.

CONTEXTE: Optimiser la réponse de la santé publique pour diminuer le fardeau de la COVID-19 nécessite la caractérisation de l'hétérogénéité du risque posé par la maladie à l'échelle de la population. Cependant, l'hétérogénéité du dépistage du SRAS-CoV-2 peut fausser les estimations selon le modèle d'étude analytique utilisé. Notre objectif était d'explorer les biais collisionneurs dans le cadre d'une vaste étude portant sur les déterminants de la maladie et d'évaluer les déterminants individuels, environnementaux et sociaux du dépistage et du diagnostic du SRAS-CoV-2 parmi les résidents de l'Ontario, au Canada. MÉTHODES: Nous avons exploré la présence potentielle de biais collisionneurs et caractérisé les déterminants individuels, environnementaux et sociaux de l'obtention d'un test de dépistage et d'un résultat positif à la présence de l'infection au SRAS-CoV-2 à l'aide d'analyses transversales parmi les 14,7 millions de personnes vivant dans la collectivité en Ontario, au Canada. Parmi les personnes ayant obtenu un diagnostic, nous avons utilisé des études analytiques distinctes afin de comparer les prédicteurs pour les personnes d'obtenir un résultat de test de dépistage positif plutôt que négatif, pour les personnes symptomatiques d'obtenir un résultat de test de dépistage positif plutôt que négatif et pour les personnes d'obtenir un résultat de test de dépistage positif plutôt que de ne pas obtenir un résultat positif (c.-à-d., obtenir un résultat de test de dépistage négatif ou ne pas obtenir de test de dépistage). Nos analyses comprennent des tests de dépistage réalisés entre le 1er mars et le 20 juin 2020. RÉSULTATS: Sur 14 695 579 personnes, nous avons constaté que 758 691 d'entre elles ont passé un test de dépistage du SRAS-CoV-2, parmi lesquelles 25 030 (3,3 %) ont obtenu un résultat positif. Plus la probabilité d'obtenir un test de dépistage s'éloignait de zéro, plus la variabilité généralement observée dans la probabilité d'un diagnostic était grande parmi les modèles d'études analytiques, particulièrement en ce qui a trait aux facteurs individuels. Nous avons constaté que la variabilité dans l'obtention d'un test de dépistage était moins importante en fonction des déterminants sociaux dans l'ensemble des études analytiques. Les facteurs tels que le fait d'habiter dans une région ayant une plus haute densité des ménages (rapport de cotes corrigé 1,86; intervalle de confiance [IC] à 95 % 1,75­1,98), une plus grande proportion de travailleurs essentiels (rapport de cotes corrigé 1,58; IC à 95 % 1,48­1,69), une population atteignant un plus faible niveau de scolarité (rapport de cotes corrigé 1,33; IC à 95 % 1,26­1,41) et une plus grande proportion d'immigrants récents (rapport de cotes corrigé 1,10; IC à 95 % 1,05­1,15), étaient systématiquement corrélés à une probabilité plus importante d'obtenir un diagnostic de SRAS-CoV-2, peu importe le modèle d'étude analytique employé. INTERPRÉTATION: Lorsque la capacité de dépister est limitée, nos résultats suggèrent que les facteurs de risque peuvent être estimés plus adéquatement en utilisant des comparateurs populationnels plutôt que des comparateurs de résultat négatif au test de dépistage. Optimiser la lutte contre la COVID-19 nécessite des investissements dans des interventions structurelles déployées de façon suffisante et adaptées à l'hétérogénéité des déterminants sociaux du risque, dont le surpeuplement des ménages, l'occupation professionnelle et le racisme structurel.

Individual and social determinants of SARS-CoV-2 testing and positivity in Ontario, Canada: a population-wide study.

BACKGROUND: Optimizing the public health response to reduce the burden of COVID-19 necessitates characterizing population-level heterogeneity of risks for the disease. However, heterogeneity in SARS-CoV-2 testing may introduce biased estimates depending on analytic design. We aimed to explore the potential for collider bias in a large study of disease determinants, and evaluate individual, environmental and social determinants associated with SARS-CoV-2 testing and diagnosis among residents of Ontario, Canada. METHODS: We explored the potential for collider bias and characterized individual, environmental and social determinants of being tested and testing positive for SARS-CoV-2 infection using cross-sectional analyses among 14.7 million community-dwelling people in Ontario, Canada. Among those with a diagnosis, we used separate analytic designs to compare predictors of people testing positive versus negative; symptomatic people testing positive versus testing negative; and people testing positive versus people not testing positive (i.e., testing negative or not being tested). Our analyses included tests conducted between Mar. 1 and June 20, 2020. RESULTS: Of 14 695 579 people, we found that 758 691 were tested for SARS-CoV-2, of whom 25 030 (3.3%) had a positive test result. The further the odds of testing from the null, the more variability we generally observed in the odds of diagnosis across analytic design, particularly among individual factors. We found that there was less variability in testing by social determinants across analytic designs. Residing in areas with the highest household density (adjusted odds ratio [OR] 1.86, 95% confidence interval [CI] 1.75-1.98), highest proportion of essential workers (adjusted OR 1.58, 95% CI 1.48-1.69), lowest educational attainment (adjusted OR 1.33, 95% CI 1.26-1.41) and highest proportion of recent immigrants (adjusted OR 1.10, 95% CI 1.05-1.15) were consistently related to increased odds of SARS-CoV-2 diagnosis regardless of analytic design. INTERPRETATION: Where testing is limited, our results suggest that risk factors may be better estimated using population comparators rather than test-negative comparators. Optimizing COVID-19 responses necessitates investment in and sufficient coverage of structural interventions tailored to heterogeneity in social determinants of risk, including household crowding, occupation and structural racism.

CRISPR screen identifies genes that sensitize AML cells to double-negative T-cell therapy.

Acute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted adoptive T-cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double-negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ∼30% of patients show resistance to DNT-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screen to identify genes that cause susceptibility of AML cells to DNT therapy. Inactivation of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression correlated strongly with the sensitivity of AML cells to DNT treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT therapy and suggest CD64 as a predictive marker for response in AML patients.

The Association Between High and Unnecessary Antibiotic Prescribing: A Cohort Study Using Family Physician Electronic Medical Records.

BACKGROUND: Approximately 25% of outpatient antibiotic prescriptions are unnecessary among family physicians in Canada. Minimizing unnecessary antibiotics is key for community antibiotic stewardship. However, unnecessary antibiotic prescribing is much harder to measure than total antibiotic prescribing. We investigated the association between total and unnecessary antibiotic use by family physicians and evaluated inter-physician variability in unnecessary antibiotic prescribing. METHODS: This was a cohort study based on electronic medical records of family physicians in Ontario, Canada, between April 2011 and March 2016. We used predefined expected antibiotic prescribing rates for 23 common primary care conditions to calculate unnecessary antibiotic prescribing rates. We used multilevel Poisson regression models to evaluate the association between total antibiotic volume (number of antibiotic prescriptions per patient visit), adjusted for multiple practice- and physician-level covariates, and unnecessary antibiotic prescribing. RESULTS: There were 499 570 physician-patient encounters resulting in 152 853 antibiotic prescriptions from 341 physicians. Substantial inter-physician variability was observed. In the fully adjusted model, we observed a significant association between total antibiotic volume and unnecessary prescribing rate (adjusted rate ratio 2.11 per 10% increase in total use; 95% CI 2.05-2.17), and none of the practice- and physician-level variables were associated with unnecessary prescribing rate. CONCLUSIONS: We demonstrated substantial inter-physician variability in unnecessary antibiotic prescribing in this cohort of family physicians. Total antibiotic use was strongly correlated with unnecessary antibiotic prescribing. Total antibiotic volume is a reasonable surrogate for unnecessary antibiotic use. These results can inform community antimicrobial stewardship efforts.

Unnecessary antibiotic prescribing in a Canadian primary care setting: a descriptive analysis using routinely collected electronic medical record data.

BACKGROUND: Unnecessary antibiotic use in the community in Canada is not well defined. Our objective was to quantify unnecessary antibiotic prescribing in a Canadian primary care setting. METHODS: We performed a descriptive analysis in Ontario from April 2011 to March 2016 using the Electronic Medical Records Primary Care database linked to other health administrative data sets at ICES. We determined antibiotic prescribing rates (per 100 patient-physician encounters) for 23 common conditions and estimated rates of unnecessary prescribing using predefined expected prescribing rates, both stratified by condition and patient age group. RESULTS: The study included 341 physicians, 204 313 patients and 499 570 encounters. The rate of unnecessary antibiotic prescribing for included conditions was 15.4% overall and was 17.6% for those less than 2 years of age, 18.6% for those aged 2-18, 14.5% for those aged 19-64 and 13.0% for those aged 65 or more. The highest unnecessary prescribing rates were observed for acute bronchitis (52.6%), acute sinusitis (48.4%) and acute otitis media (39.3%). The common cold, acute bronchitis, acute sinusitis and miscellaneous nonbacterial infections were responsible for 80% of the unnecessary antibiotic prescriptions. Of all antibiotics prescribed, 12.0% were for conditions for which they are never indicated, and 12.3% for conditions for which they are rarely indicated. In children, 25% of antibiotics were for conditions for which they are never indicated (e.g., common cold). INTERPRETATION: Antibiotics were prescribed unnecessarily for 15.4% of included encounters in a Canadian primary care setting. Almost one-quarter of antibiotics were prescribed for conditions for which they are rarely or never indicated. These findings should guide safe reductions in the use of antibiotics for the common cold, bronchitis and sinusitis.

Cellular immunotherapy for acute myeloid leukemia: How specific should it be?

Significant improvements in the survival of patients with hematological cancers following hematopoietic stem cell transplantation provide evidence supporting the potency of immune cell-mediated anti-leukemic effects. Studies focusing on immune cell-based cancer therapies have made significant breakthroughs in the last few years. Adoptive cellular therapy (ACT), and chimeric antigen receptor (CAR) T cell therapy, in particular, has significantly increased the survival of patients with B cell acute lymphoblastic leukemia and aggressive B cell lymphoma. Despite antigen-negative relapses and severe toxicities such as cytokine release syndrome after treatment, CAR-T cell therapies have been approved by the FDA in some conditions. Although a number of studies have tried to achieve similar results for acute myeloid leukemia (AML), clinical outcomes have not been as promising. In this review, we summarize recent and ongoing studies on cellular therapies for AML patients, with a focus on antigen-specific versus -nonspecific approaches.

IL-15 and IFN-γ signal through the ERK pathway to inhibit HCV replication, independent of type I IFN signaling.

Despite effective new treatments for Hepatitis C virus (HCV) infection, development of drug resistance, safety concerns and cost are remaining challenges. More importantly, there is no vaccine available against hepatitis C infection. Recent data suggest that there is a strong correlation between spontaneous HCV clearance and human NK cell function, particularly IFN-γ production. Further, IL-15 has innate antiviral activity and is also one of the main factors that activates NK cells to produce IFN-γ. To examine whether IL-15 and IFN-γ have direct antiviral activity against HCV, Huh7.5 cells were treated with either IFN-γ or IL-15 prior to HCV infection. Our data demonstrate that IFN-γ and IL-15 block HCV replication in vitro. Additionally, we show that IL-15 and IFN-γ do not induce anti-HCV effects through the type I interferon signaling pathway or nitric oxide (NO) production. Instead, IL-15 and IFN-γ provide protection against HCV via the ERK pathway. Treatment of Huh7.5 cells with a MEK/ERK inhibitor abrogated the anti-HCV effects of IL-15 and IFN-γ and overexpression of ERK1 prevented HCV replication compared to control transfection. Our in vitro data support the hypothesis that early production of IL-15 and activation of NK cells in the liver lead to control of HCV replication.

Combining Tumor Microenvironment Modulating Nanoparticles with Doxorubicin to Enhance Chemotherapeutic Efficacy and Boost Antitumor Immunity.

BACKGROUND: Tumor microenvironment (TME) and associated multiple factors are found to contribute to the failures in cancer therapies, including chemo- and immunotherapy. Here we report a new multimodal strategy that uses a bioreactive multifunctional hybrid polymer-lipid encapsulated manganese dioxide nanoparticle (PLMD NP) system to remodel the TME, suppress drug resistance factors, reverse immunosuppressive conditions, and enhance chemotherapy efficacy. METHODS: The influence of PLMD NPs on enhancing cellular uptake in EMT6 mouse breast cancer cells and tumor penetration of doxorubicin (DOX) in EMT6 orthotopic breast tumor mouse model was evaluated using confocal microscopy (n = 3-4). Immunohistochemistry was employed to examine the effect of PLMD NPs on downregulating hypoxia-induced drug resistance proteins and anticancer activity of DOX (n = 3-4). The efficacy of the combination therapy with PLMD NPS and DOX was assessed in murine EMT6 (n = 15-23) and 4T1 (n = 7) orthotopic breast tumor mouse models. Rechallenge and splenocyte transfer were performed to validate the stimulation of adaptive tumor immunity in the surviving mice. RESULTS: PLMD NPs enhanced intratumoral penetration and efficacy of DOX, and reduced intratumoral expression of P-glycoprotein, p53, and carbonic anhydrase IX by 74.5%, 38.0%, and 58.8% vs saline control, respectively. Combination treatment with PLMD NPs and DOX increased the number of tumor-infiltrated CD8+ T cells and resulted in up to 60.0% complete tumor regression. Of naïve mice (n = 7) that received splenocytes from the PLMD+DOX-treated surviving mice, 57.1% completely suppressed tumor growth whereas 100% of mice that received splenocytes from DOX-treated mice (n = 3) and the control group (n = 7) showed rapid tumor growth. CONCLUSIONS: The clinically suitable PLMD NPs can effectively downregulate TME-associated drug resistance and immunosuppression. The combination therapy with PLMD NPs and DOX is a multimodal and translational treatment approach for enhancing chemotherapeutic efficacy and boosting antitumor immunity.

Targeting chemotherapy-resistant leukemia by combining DNT cellular therapy with conventional chemotherapy.

BACKGROUND: While conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease. METHODS: Drug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity. RESULTS: Here, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity. CONCLUSIONS: Our results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy.

Allogeneic Human Double Negative T Cells as a Novel Immunotherapy for Acute Myeloid Leukemia and Its Underlying Mechanisms.

Purpose: To explore the potential of ex vivo expanded healthy donor-derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients.Experimental Design: Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry-based in vitro killing assays and xenograft models against AML patient blasts and healthy donor-derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays.Results: Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples in vitro, and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor-unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands' expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1-blocking antibodies.Conclusions: This study supports healthy donor-derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. Clin Cancer Res; 24(2); 370-82. ©2017 AACR.