Machine learning-based detection of adventitious microbes in T-cell therapy cultures using long-read sequencing.

Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP <71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated, and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimized experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 colony-forming units (CFU)/mL using metagenomic classification. Machine learning can be coupled with long-read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP <71> organisms to 10 CFU/mL. IMPORTANCE This research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long-read nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.

Impact of weight loss on treatment interruption and unplanned hospital admission in head and neck cancer patients undergoing curative (chemo)-radiotherapy in Hong Kong.

PURPOSE: Malnutrition is highly prevalent in head and neck cancer (HNC) patients, with weight loss being one of the major nutritional indicators. The objective of this study was to investigate the impact of weight loss on treatment interruptions and unplanned hospital admissions in HNC patients undergoing radiotherapy (RT) with or without chemotherapy. METHODS: In this retrospective cohort study, consecutive HNC patients who started RT between January 2011 and December 2019 were included. Data from a total of 1086 subjects with 747 (68.8%) nasopharyngeal carcinomas (NPCs) and 31.2% (N=339) non-NPC patients were analysed. Body weight (BW) was measured before, during, and after RT treatment. Factors associated with ≥10% weight loss, treatment interruption, and unplanned admissions were analysed using multivariate logistic regression. RESULTS: The prevalence of ≥10% weight loss was 26.8% (N=288), with 32.7% (N=243) in NPC and 13.5% (N=45) in non-NPC patients. The prevalence of RT delay in patients with ≥10% vs. <10% weight loss was 6.2% vs. 7.0% (p=0.668) in NPC patients and 42.2% vs. 50.5% (p=0.300) in non-NPC patients. The prevalence of unplanned admissions in patients with ≥10% vs. <10% weight loss was 51.9% vs. 25.3% (p<0.001) in NPC patients and 68.9% vs. 27.0% (p<0.001) in non-NPC patients. CONCLUSION: In our study, ≥10% weight loss was found to be associated with a higher rate of unplanned admissions but not with RT delay or chemotherapy interruption. CLINICAL IMPLICATIONS: With the knowledge of the impact of weight loss on hospital admissions and the characteristics of patients with weight loss, nutritional intervention can be effectively focused on the stratification of patients for intensive nutritional support to reduce weight loss.

Ninety-Day Stroke or Transient Ischemic Attack Recurrence in Patients Prescribed Anticoagulation in the Emergency Department With Atrial Fibrillation and a New Transient Ischemic Attack or Minor Stroke.

Background For patients with atrial fibrillation seen in the emergency department (ED) following a transient ischemic attack (TIA) or minor stroke, the impact of initiating oral anticoagulation immediately rather than deferring the decision to outpatient follow-up is unknown. Methods and Results We conducted a planned secondary data analysis of a prospective cohort of 11 507 adults in 13 Canadian EDs between 2006 and 2018. Patients were eligible if they were aged 18 years or older, with a final diagnosis of TIA or minor stroke with previously documented or newly diagnosed atrial fibrillation. The primary outcome was subsequent stroke, recurrent TIA, or all-cause mortality within 90 days of the index TIA diagnosis. Secondary outcomes included stroke, recurrent TIA, or death and rates of major bleeding. Of 11 507 subjects with TIA/minor stroke, atrial fibrillation was identified in 11.2% (1286, mean age, 77.3 [SD 11.1] years, 52.4% male). Over half (699; 54.4%) were already taking anticoagulation, 89 (6.9%) were newly prescribed anticoagulation in the ED. By 90 days, 4.0% of the atrial fibrillation cohort had experienced a subsequent stroke, 6.5% subsequent TIA, and 2.6% died. Results of a multivariable logistic regression indicate no association between prescribed anticoagulation in the ED and these 90-day outcomes (composite odds ratio, 1.37 [95% CI, 0.74-2.52]). Major bleeding was found in 5 patients, none of whom were in the ED-initiated anticoagulation group. Conclusions Initiating oral anticoagulation in the ED following new TIA was not associated with lower recurrence rates of neurovascular events or all-cause mortality in patients with atrial fibrillation.

Transforming Growth Factor ß Signaling Promotes HIV-1 Infection in Activated and Resting Memory CD4+ T Cells.

Understanding the facilitator of HIV-1 infection and subsequent latency establishment may aid the discovery of potential therapeutic targets. Here, we report the elevation of plasma transforming growth factor ß (TGF-ß) during acute HIV-1 infection among men who have sex with men (MSM). Using a serum-free in vitro system, we further delineated the role of TGF-ß signaling in mediating HIV-1 infection of activated and resting memory CD4+ T cells. TGF-ß could upregulate both the frequency and expression of the HIV-1 coreceptor CCR5, thereby augmenting CCR5-tropic viral infection of resting and activated memory CD4+ T cells via Smad3 activation. The production of live HIV-1JR-FL upon infection and reactivation was increased in TGF-ß-treated resting memory CD4+ T cells without increasing CD4 expression or inducing T cell activation. The expression of CCR7, a central memory T cell marker that serves as a chemokine receptor to facilitate T cell trafficking into lymphoid organs, was also elevated on TGF-ß-treated resting and activated memory CD4+ T cells. Moreover, the expression of CXCR3, a chemokine receptor recently reported to facilitate CCR5-tropic HIV-1 infection, was increased on resting and activated memory CD4+ T cells upon TGF-ß treatment. These findings were coherent with the observation that ex vivo CCR5 and CXCR3 expression on total resting and resting memory CD4+ T cells in combination antiretroviral therapy (cART)-naive and cART-treated patients were higher than in healthy individuals. Overall, the study demonstrated that TGF-ß upregulation induced by acute HIV-1 infection might promote latency reservoir establishment by increasing infected resting memory CD4+ T cells and lymphoid organ homing of infected central memory CD4+ T cells. Therefore, TGF-ß blockade may serve as a potential supplementary regimen for HIV-1 functional cure by reducing viral latency. IMPORTANCE Incomplete eradication of HIV-1 latency reservoirs remains the major hurdle in achieving a complete HIV/AIDS cure. Dissecting the facilitator of latency reservoir establishment may aid the discovery of druggable targets for HIV-1 cure. This study showed that the T cell immunomodulatory cytokine TGF-ß was upregulated during the acute phase of infection. Using an in vitro serum-free system, we specifically delineated that TGF-ß promoted HIV-1 infection of both resting and activated memory CD4+ T cells via the induction of host CCR5 coreceptor. Moreover, TGF-ß-upregulated CCR7 or CXCR3 might promote HIV-1 latent infection by facilitating lymphoid homing or IP-10-mediated viral entry and DNA integration, respectively. Infected resting and central memory CD4+ T cells are important latency reservoirs. Increased infection of these cells mediated by TGF-ß will promote latency reservoir establishment during early infection. This study, therefore, highlighted the potential use of TGF-ß blockade as a supplementary regimen with cART in acute patients to reduce viral latency.

Prospective Validation of Computed Tomography to Identify Patients at High Risk for Stroke After Transient Ischemic Attack or Minor Stroke.

BACKGROUND: Computed tomography (CT) findings of acute and chronic ischemia are associated with subsequent stroke risk in patients with transient ischemic attack. We sought to validate these associations in a large prospective cohort of patients with transient ischemic attack or minor stroke. METHODS: This prospective cohort study enrolled emergency department patients from 13 hospitals with transient ischemic attack who had CT imaging. Primary outcome was stroke within 90 days. Secondary outcomes were stroke within 2 or 7 days. CT findings were abstracted from radiology reports and classified for the presence of acute ischemia, chronic ischemia, or microangiopathy. Multivariable logistic regression was used to test associations with primary and secondary end points. RESULTS: From 8670 prospectively enrolled patients between May 2010 and May 2017, 8382 had a CT within 24 hours. From this total population, 4547 (54%) patients had evidence of acute ischemia, chronic ischemia, or microangiopathy on CT, of whom 175 had a subsequent stroke within 90 days (3.8% subsequent stroke rate; adjusted odds ratio [aOR], 2.33 [95% CI, 1.62-3.36]). This was in comparison to those with CT imaging without ischemia. Findings associated with an increased risk of stroke at 90 days were isolated acute ischemia (6.0%; aOR, 2.42 [95% CI, 1.03-5.66]), acute ischemia with microangiopathy (10.7%; aOR, 3.34 [95% CI, 1.57-7.14]), chronic ischemia with microangiopathy (5.2%; aOR, 1.83 [95% CI, 1.34-2.50]), and acute ischemia with chronic ischemia and microangiopathy (10.9%; aOR, 3.49 [95% CI, 1.54-7.91]). Acute ischemia with chronic ischemia and microangiopathy were most strongly associated with subsequent stroke within 2 days (aOR, 4.36 [95% CI, 1.31-14.54]) and 7 days (aOR, 4.50 [95% CI, 1.73-11.69]). CONCLUSIONS: In patients with transient ischemic attack or minor stroke, CT evidence of acute ischemia with chronic ischemia or microangiopathy significantly increases the risk of subsequent stroke within 90 days of index visit. The combination of all 3 findings results in the greatest early risk.

A Dynamic Programming Algorithm for Finding an Optimal Sequence of Informative Measurements.

An informative measurement is the most efficient way to gain information about an unknown state. We present a first-principles derivation of a general-purpose dynamic programming algorithm that returns an optimal sequence of informative measurements by sequentially maximizing the entropy of possible measurement outcomes. This algorithm can be used by an autonomous agent or robot to decide where best to measure next, planning a path corresponding to an optimal sequence of informative measurements. The algorithm is applicable to states and controls that are either continuous or discrete, and agent dynamics that is either stochastic or deterministic; including Markov decision processes and Gaussian processes. Recent results from the fields of approximate dynamic programming and reinforcement learning, including on-line approximations such as rollout and Monte Carlo tree search, allow the measurement task to be solved in real time. The resulting solutions include non-myopic paths and measurement sequences that can generally outperform, sometimes substantially, commonly used greedy approaches. This is demonstrated for a global search task, where on-line planning for a sequence of local searches is found to reduce the number of measurements in the search by approximately half. A variant of the algorithm is derived for Gaussian processes for active sensing.

Risk of stroke is low after transient ischemic attack presentation with isolated dizziness.

OBJECTIVE: Stroke presenting as dizziness is a diagnostic challenge in frontline settings, given the multitude of benign conditions that present similarly. The risk of stroke after episodic dizziness is unknown, leading to divergent guidance on optimal workup and management. Prior TIA risk scores have shown a history of dizziness is a negative predictor of subsequent stroke. Our objective was to assess the subsequent stroke risk within 90 days following emergency department assessment (ED) for isolated dizziness diagnosed as TIA during the index visit. METHODS: We conducted prospective, multicenter cohort studies at 13 Canadian EDs over 11 years. We enrolled patients diagnosed with TIA and compared patients with isolated dizziness to those with other neurological deficits. Our primary outcome was subsequent stroke within 90 days. Secondary outcomes were subsequent stroke within 2, 7, and 30 days, respectively, as well as subsequent TIA within 90 days. RESULTS: Only 4/483 (0.8%) patients with isolated dizziness had a stroke within 90 days compared to 320/11024 (2.9%) of those with any focal neurological sign or symptom (RR 0.29, 95% CI 0.11-0.76). Over the first 90 days, the two groups differ significantly in their probability of stroke (p = 0.007). Subsequent TIA was also significantly less common in the isolated dizziness group (1.7% vs. 5.6%, p = 0.001) with a relative risk of 0.30 (95% CI 0.15-0.60). CONCLUSION: The risk of subsequent stroke following ED presentation for TIA is low when the presenting symptoms are isolated dizziness.

RéSUMé: OBJECTIF: Les accidents vasculaires cérébraux (AVC) se présentant sous forme de vertiges constituent un défi diagnostique en première ligne, étant donné la multitude d'affections bénignes qui se présentent de la même manière. Le risque d'accident vasculaire cérébral (AVC) après des vertiges épisodiques est inconnu, ce qui donne lieu à des conseils divergents sur le bilan et la prise en charge optimaux. Des scores de risque d'AIT antérieurs ont montré que des antécédents de vertiges sont un facteur prédictif négatif d'accident vasculaire cérébral ultérieur. Notre objectif était d'évaluer le risque ultérieur d'accident vasculaire cérébral (AVC) dans les 90 jours suivant l'évaluation aux urgences d'un étourdissement isolé diagnostiqué comme un AIT lors de la visite de référence. MéTHODES: Nous avons mené des études de cohorte prospectives multicentriques dans 13 services d'urgence canadiens pendant 11 ans. Nous avons recruté des patients ayant reçu un diagnostic d'AIT et avons comparé les patients présentant des vertiges isolés à ceux présentant d'autres déficits neurologiques. Nous avons inscrit des patients ayant reçu un diagnostic d'AIT et comparé des patients ayant des étourdissements isolés à ceux présentant d'autres déficits neurologiques. Notre résultat primaire était l'AVC subséquent dans les 90 jours. Les résultats secondaires étaient l'AVC subséquent dans les 2, 7 et 30 jours, respectivement, ainsi que l'AIT subséquent dans les 90 jours. RéSULTATS: Seuls 4/483 (0,8 %) des patients présentant des vertiges isolés ont eu un AVC dans les 90 jours, contre 320/11 024 (2,9 %) de ceux présentant un signe ou symptôme neurologique focal (RR 0,29, IC 95 % 0,11-0,76). Au cours des 90 premiers jours, les deux groupes diffèrent significativement en termes de probabilité d'AVC (p = 0,007). L'AIT ultérieur était également significativement moins fréquent dans le groupe des vertiges isolés (1,7 % contre 5,6 %, p = 0,001) avec un risque relatif de 0,30 (IC 95 % 0,15-0,60). CONCLUSIONS: Le risque d'AVC ultérieur après une présentation aux urgences pour un AIT est faible lorsque les symptômes présentés sont des étourdissements isolés.

Hyperinflammatory Syndrome, Natural Killer Cell Function, and Genetic Polymorphisms in the Pathogenesis of Severe Dengue.

BACKGROUND: Severe dengue, characterized by shock and organ dysfunction, is driven by an excessive host immune response. We investigated the role of hyperinflammation in dengue pathogenesis. METHODS: Patients recruited into an observational study were divided into 3 plasma leak severity grades. Hyperinflammatory biomarkers were measured at 4 time points. Frequencies, activation, and cytotoxic potential of natural killer (NK) cells were analyzed by flow cytometry. RNA was extracted from sorted CD56+ NK cells and libraries were prepared using SMART-Seq and sequenced using HiSeq3000 (Illumina). RESULTS: Sixty-nine patients were included (grade 0, 42 patients; grade 1, 19 patients; grade 2, 8 patients). Patients with grade 2 leakage had higher biomarkers than grade 0, including higher peak ferritin levels (83.3% vs 45.2%) and H-scores (median, 148.5 vs 105.5). NK cells from grade 2 patients exhibited decreased expression of perforin and granzyme B and activation markers. RNA sequencing revealed 3 single-nucleotide polymorphisms in NK cell functional genes associated with more severe leakage-NK cell lectin-like receptor K1 gene (KLRK1) and perforin 1 (PRF1). CONCLUSIONS: Features of hyperinflammation are associated with dengue severity, including higher biomarkers, impaired NK cell function, and polymorphisms in NK cell cytolytic function genes (KLRK1 and PRF1). Trials of immunomodulatory therapy in these patients is now warranted.

External Validation of a Nomogram to Predict Survival and Benefit of Concurrent Chemoradiation for Stage II Nasopharyngeal Carcinoma.

A nomogram was recently published by Sun et al. to predict overall survival (OS) and the additional benefit of concurrent chemoradiation (CCRT) vs. radiotherapy (RT) alone, in stage II NPC treated with conventional RT. We aimed to assess the predictors of OS and to externally validate the nomogram in the IMRT era. We analyzed stage II NPC patients treated with definitive RT alone or CCRT between 2001 and 2011 under the territory-wide Hong Kong NPC Study Group 1301 study. Clinical parameters were studied using the Cox proportional hazards model to estimate OS. The nomogram by Sun et al. was applied with 1000 times bootstrap resampling to calculate the concordance index, and we compared the nomogram predicted and observed 5-year OS. There were 482 patients included. The 5-year OS was 89.0%. In the multivariable analysis, an age > 45 years was the only significant predictor of OS (HR, 1.98; 95%CI, 1.15-3.44). Other clinical parameters were insignificant, including the use of CCRT (HR, 0.99; 95%CI, 0.62-1.58). The nomogram yielded a concordance index of 0.55 (95% CI, 0.49-0.62) which lacked clinically meaningful discriminative power. The nomogram proposed by Sun et al. should be interpreted with caution when applied to stage II NPC patients in the IMRT era. The benefit of CCRT remained controversial.

Predictors of neurologists confirming or overturning emergency physicians' diagnosis of TIA or stroke.

BACKGROUND: Transient ischemic attack (TIA) and non-disabling stroke are common emergency department (ED) presentations. Currently, there are no prospective multicenter studies determining predictors of neurologists confirming a diagnosis of cerebral ischemia in patients discharged with a diagnosis of TIA or stroke. The objectives were to (1) calculate the concordance between emergency physicians and neurologists for the outcome of diagnosing TIA or stroke, and (2) identify characteristics associated with neurologists diagnosing a stroke mimic. METHODS: This was a planned sub-study of a prospective cohort study at 14 Canadian EDs enrolling patients diagnosed with TIA or non-disabling stroke from 2006 to 2017. Logistic regression was used to identify factors associated with neurologists' diagnosis of cerebral ischemia. Our primary outcome was the composite outcome of cerebral ischemia (TIA or non-disabling stroke) based on the neurologists' assessment. RESULTS: The diagnosis of cerebral ischemia was confirmed by neurologists in 5794 patients (55.4%). The most common identified stroke mimics were migraine (18%), peripheral vertigo (7%), syncope (4%), and seizure (3%). Over a third of patients (38.4%) ultimately had an undetermined aetiology for their symptoms. The strongest predictors of cerebral ischemia confirmation were infarct on CT (OR 1.83, 95% CI 1.65-2.02), advanced age (OR comparing 75th-25th percentiles 1.67, 1.55-1.80), language disturbance (OR 1.92, 1.75-2.10), and smoking (OR 1.67, 1.46-1.91). The strongest predictors of stroke mimics were syncope (OR 0.59, 0.48-0.72), vertigo (OR 0.52, 0.45-0.59), bilateral symptoms (OR 0.60, 0.50-0.72), and confusion (OR 0.50, 0.44-0.57). CONCLUSION: Physicians should have a high index of suspicion of cerebral ischemia in patients with advanced age, smoking history, language disturbance, or infarcts on CT. Physicians should discriminate in which patients to pursue stroke investigations on when deemed at minimal risk of cerebral ischemia, including those with isolated vertigo, syncope, or bilateral symptoms.

RéSUMé: CONTEXTE: L'accident ischémique transitoire (AIT) et l'accident vasculaire cérébral (AVC) non invalidant sont des présentations courantes dans les services d'urgence. Actuellement, il n'existe pas d'études prospectives multicentriques déterminant les facteurs prédictifs de la confirmation par les neurologues d'un diagnostic d'ischémie cérébrale chez les patients sortis de l'hôpital avec un diagnostic d'AIT ou d'AVC. Les objectifs étaient de (1) calculer la concordance entre les urgentistes et les neurologues pour le résultat du diagnostic de l'AIT ou de l'AVC, et (2) identifier les caractéristiques associées au diagnostic par les neurologues d'une imitation d'AVC. MéTHODES: Il s'agissait d'une sous-étude planifiée d'une étude de cohorte prospective dans 14 services d'urgence canadiens recrutant des patients diagnostiqués avec un AIT ou un AVC non invalidant de 2006 à 2017. Une régression logistique a été utilisée pour identifier les facteurs associés au diagnostic d'ischémie cérébrale par les neurologues. Notre résultat principal était le résultat composite de l'ischémie cérébrale (AIT ou accident vasculaire cérébral non invalidant) selon l'évaluation des neurologues. RéSULTATS: Le diagnostic d'ischémie cérébrale a été confirmé par des neurologues chez 5 794 patients (55,4 %). Les imitateurs d'AVC identifiés les plus courants étaient la migraine (18 %), le vertige périphérique (7 %), la syncope (4 %) et les convulsions (3 %). Plus d'un tiers des patients (38,4 %) avaient finalement une étiologie indéterminée pour leurs symptômes. Les prédicteurs les plus forts de la confirmation de l'ischémie cérébrale étaient l'infarctus au scanner (OR 1.83, IC 95 % 1.65­2.02), l'âge avancé (OR comparant les 75e et 25e percentiles 1.67, 1.55­1.80), les troubles du langage (OR 1.92, 1.75­2.10) et le tabagisme (OR 1.67, 1.46­1.91). Les prédicteurs les plus forts d'imitateurs d'AVC étaient la syncope (OR 0.59, 0.48­0.72), le vertige (OR 0.52, 0.45­0.59), les symptômes bilatéraux (OR 0.60, 0.50­0.72) et la confusion (OR 0.50, 0.44­0.57). CONCLUSION: Les médecins devraient avoir un indice élevé de suspicion d'ischémie cérébrale chez les patients ayant un âge avancé, des antécédents de tabagisme, des troubles du langage ou des infarctus au scanner. Les médecins doivent distinguer les patients sur lesquels poursuivre des investigations sur un AVC lorsqu'ils sont jugés à risque minimal d'ischémie cérébrale, y compris ceux présentant des vertiges isolés, une syncope ou des symptômes bilatéraux.

Sustained viremia suppression by SHIVSF162P3CN-recalled effector-memory CD8+ T cells after PD1-based vaccination.

HIV-1 functional cure requires sustained viral suppression without antiretroviral therapy. While effector-memory CD8+ T lymphocytes are essential for viremia control, few vaccines elicit such cellular immunity that could be potently recalled upon viral infection. Here, we investigated a program death-1 (PD1)-based vaccine by fusion of simian immunodeficiency virus capsid antigen to soluble PD1. Homologous vaccinations suppressed setpoint viremia to undetectable levels in vaccinated macaques following a high-dose intravenous challenge by the pathogenic SHIVSF162P3CN. Poly-functional effector-memory CD8+ T cells were not only induced after vaccination, but were also recalled upon viral challenge for viremia control as determined by CD8 depletion. Vaccine-induced effector memory CD8+ subsets displayed high cytotoxicity-related genes by single-cell analysis. Vaccinees with sustained viremia suppression for over two years responded to boost vaccination without viral rebound. These results demonstrated that PD1-based vaccine-induced effector-memory CD8+ T cells were recalled by AIDS virus infection, providing a potential immunotherapy for functional cure.

Prospective validation of Canadian TIA Score and comparison with ABCD2 and ABCD2i for subsequent stroke risk after transient ischaemic attack: multicentre prospective cohort study.

OBJECTIVE: To validate the previously derived Canadian TIA Score to stratify subsequent stroke risk in a new cohort of emergency department patients with transient ischaemic attack. DESIGN: Prospective cohort study. SETTING: 13 Canadian emergency departments over five years. PARTICIPANTS: 7607 consecutively enrolled adult patients attending the emergency department with transient ischaemic attack or minor stroke. MAIN OUTCOME MEASURES: The primary outcome was subsequent stroke or carotid endarterectomy/carotid artery stenting within seven days. The secondary outcome was subsequent stroke within seven days (with or without carotid endarterectomy/carotid artery stenting). Telephone follow-up used the validated Questionnaire for Verifying Stroke Free Status at seven and 90 days. All outcomes were adjudicated by panels of three stroke experts, blinded to the index emergency department visit. RESULTS: Of the 7607 patients, 108 (1.4%) had a subsequent stroke within seven days, 83 (1.1%) had carotid endarterectomy/carotid artery stenting within seven days, and nine had both. The Canadian TIA Score stratified the risk of stroke, carotid endarterectomy/carotid artery stenting, or both within seven days as low (risk ≤0.5%; interval likelihood ratio 0.20, 95% confidence interval 0.09 to 0.44), medium (risk 2.3%; interval likelihood ratio 0.94, 0.85 to 1.04), and high (risk 5.9% interval likelihood ratio 2.56, 2.02 to 3.25) more accurately (area under the curve 0.70, 95% confidence interval 0.66 to 0.73) than did the ABCD2 (0.60, 0.55 to 0.64) or ABCD2i (0.64, 0.59 to 0.68). Results were similar for subsequent stroke regardless of carotid endarterectomy/carotid artery stenting within seven days. CONCLUSION: The Canadian TIA Score stratifies patients' seven day risk for stroke, with or without carotid endarterectomy/carotid artery stenting, and is now ready for clinical use. Incorporating this validated risk estimate into management plans should improve early decision making at the index emergency visit regarding benefits of hospital admission, timing of investigations, and prioritisation of specialist referral.

The use of ketamine in the management of refractory cancer pain in a palliative care unit.

BACKGROUND: Ketamine has been used as an adjuvant to opioid therapy for the management of refractory cancer pain but the current evidence is insufficient to draw any conclusions regarding its efficacy. We aimed to assess the response to ketamine in patients with refractory cancer pain treated in an oncology palliative care unit. METHODS: Patients with refractory cancer pain despite opioid dose escalation were selected for a trial of parenteral ketamine infusion according to a local protocol. The medical records of those patients treated between January 2004 and December 2018 were retrospectively reviewed. The primary endpoint of the study was a favorable response to ketamine, defined as a reduction in regular opioid dose with no increase in pain intensity or a reduction in pain intensity by ≥2 points on the numerical rating scale (NRS) with a stable regular opioid dose. The secondary endpoint was adverse events associated with ketamine. RESULTS: Among the 70 patients, mean pain score on NRS improved from 7.0 to 4.0 after ketamine (P<0.001). Forty-nine patients had a reduction of pain score by ≥2 points on NRS, 33 had ≥50% reduction in pain intensity. The median decrease in regular opioid dose was 25.5%, and the mean difference was -133.2 mg (P=0.002). A favorable response to ketamine was observed in 52 patients (74.3%). The use of more than one coanalgesic (odds ratio 3.451; 95% CI: 1.087-10.960; P=0.036) was associated with a favorable response to ketamine on multivariate analysis. Adverse events were mostly mild, with the commonest being drowsiness (45.7%), hypertension (34.3%) and nightmares (25.7%). Only five and three patients required temporary suspension and early termination of ketamine infusion respectively. CONCLUSIONS: These data demonstrated the efficacy and safety of ketamine in a population of patients with refractory cancer pain. The use of more than one coanalgesic was associated with a favorable response to ketamine. Further large and multicentered studies are warranted to confirm these data.

Patterns and infection outcomes of bacterial colonization in patients with indwelling abdominal drains for malignant ascites.

BACKGROUND: Indwelling abdominal drains for intermittent drainage is an effective treatment for refractory malignant ascites, bacterial colonization and subsequent drain-related infection is however a common concern. This study aimed to investigate the patterns of bacterial colonization and the subsequent infection outcomes in patients with indwelling abdominal drains. METHODS: All consecutive advanced cancer patients with newly inserted indwelling abdominal drains and who were under the service of the ascites clinic of our institution for intermittent drainage between January 2011 and March 2018 were screened for study eligibility. Patients with positive surveillance ascitic fluid culture without immediate drain-related infection were included in the final analysis. Clinical information during the drainage period was prospectively collected using standardized clinical assessment forms. These assessment forms and other medical records were retrospectively reviewed. RESULTS: Sixty nine patients developed bacterial colonization without immediate infection during the study period. The most common cancer diagnosis was hepatocellular carcinoma (HCC), which comprise 30.4% of the population. Central venous catheters (CVCs) were inserted in 76.8% of patients and pigtail drains in 23.2% as the indwelling abdominal drain. The median duration from drain insertion to the development of bacterial colonization was 18.0 days. Staphylococci, Diphtheroid bacilliand Enterococci were the most common types of bacteria isolated during colonization. Thirty patients (43.5%) developed drain-related infection subsequently and the median time from bacterial colonization to development of infection was 14.5 days. The incidence rate of drain-related infection after bacterial colonization was 1.78 per 100-catheter days and the 1-month infection-free survival was 54.4%. Five patients (7.2%) developed peritonitis and 4 of them died from the infection episode. Decrease in body mass index (BMI) (P=0.03), having 3 or more episodes of drainage in the ascites clinic before bacterial colonization (P=0.03), presence of Escherichia coli (P=0.04) and Bacillus species (P=0.04) in surveillance ascitic fluid culture were significantly correlating with infection outcomes in univariate analyses. HCC as cancer diagnosis (OR 8.85, 95% CI: 1.86-42.07, P=0.006) and decrease in body weight (OR 1.20, 95% CI: 1.02-1.42, P=0.03) were significant factors that correlated with infection outcomes in multivariate analysis. CONCLUSIONS: Bacterial colonization and subsequent progression into drain-related infection are common in patients on indwelling abdominal drains for malignant ascites. Staphylococci is the most common type of bacteria causing both colonization and subsequent drain-related infection. HCC and decrease in body weight are significant factors that correlate with infection outcomes after bacterial colonization.

NK cells are activated and primed for skin-homing during acute dengue virus infection in humans.

Despite animal models showing that natural killer (NK) cells are important players in the early defense against many viral infections, the NK cell response is poorly understood in humans. Here we analyze the phenotype, temporal dynamics, regulation and trafficking of NK cells in a patient cohort with acute dengue virus infection. NK cells are robustly activated and proliferate during the first week after symptom debut. Increased IL-18 levels in plasma and in induced skin blisters of DENV-infected patients, as well as concomitant signaling downstream of the IL-18R, suggests an IL-18-dependent mechanism in driving the proliferative NK cell response. Responding NK cells have a less mature phenotype and a distinct chemokine-receptor imprint indicative of skin-homing. A corresponding NK cell subset can be localized to skin early during acute infection. These data provide evidence of an IL-18-driven NK cell proliferation and priming for skin-homing during an acute viral infection in humans.

Vedolizumab-mediated integrin α4ß7 blockade does not control HIV-1SF162 rebound after combination antiretroviral therapy interruption in humanized mice.

OBJECTIVE: The combined combination antiretroviral therapy (cART) and anti-α4ß7 RM-Act-1 antibody therapy allows macaques to durably control simian immunodeficiency virus (SIV) rebound after withdrawal of the interventions. Here, we aimed to investigate whether vedolizumab (VDZ), a clinical-grade humanized anti-α4ß7 antibody, would have similar effects in controlling live HIV-1 infection in humanized mice. DESIGN AND METHODS: The integrin α4ß7 blockade by VDZ was evaluated on human primary memory CD4+ T (MEMT) cells and retinoic acid-induced gut-homing α4ß7+MEMT cells (α4ß7+MEMT) in vitro. The antiretroviral activity of VDZ was determined using pseudotyped R5-tropic HIV-1SF162, which displays binding activity to α4ß7. The preventive and immunotherapeutic efficacies of VDZ were further investigated in humanized mice using the live HIV-1SF162 strain compared with RM-Act-1. RESULTS: VDZ effectively and dose-dependently blocked the binding of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1), the native ligand of α4ß7, to α4ß7+MEMT cells. HIV-1SF162 not only displayed binding capacity to α4ß7-expressing cells, but also showed an increased infectivity in retinoic acid-induced α4ß7+MEMT cells pretreated with VDZ. Moreover, VDZ failed to prevent live HIV-1SF162 infection and did not reduce the peak viral load when administrated prior to viral challenge in humanized mice. Lastly, in immunotherapeutic settings, the withdrawal of combined cART with either VDZ or RM-Act-1 resulted in an uncontrolled HIV-1SF162 rebound in humanized mice, whereas the α4ß7 molecules remained significantly blocked on circulating CD4+ T cells. CONCLUSION: VDZ neither prevents nor controls HIV-1SF162 infection both in vitro and in humanized mice. Our findings have significant implications to the clinical application of VDZ in HIV-1 preventive and immunotherapeutic interventions.

Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection.

Newly emerging viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle Eastern respiratory syndrome CoVs (MERS-CoV), and H7N9, cause fatal acute lung injury (ALI) by driving hypercytokinemia and aggressive inflammation through mechanisms that remain elusive. In SARS-CoV/macaque models, we determined that anti-spike IgG (S-IgG), in productively infected lungs, causes severe ALI by skewing inflammation-resolving response. Alveolar macrophages underwent functional polarization in acutely infected macaques, demonstrating simultaneously both proinflammatory and wound-healing characteristics. The presence of S-IgG prior to viral clearance, however, abrogated wound-healing responses and promoted MCP1 and IL-8 production and proinflammatory monocyte/macrophage recruitment and accumulation. Critically, patients who eventually died of SARS (hereafter referred to as deceased patients) displayed similarly accumulated pulmonary proinflammatory, absence of wound-healing macrophages, and faster neutralizing antibody responses. Their sera enhanced SARS-CoV-induced MCP1 and IL-8 production by human monocyte-derived wound-healing macrophages, whereas blockade of FcγR reduced such effects. Our findings reveal a mechanism responsible for virus-mediated ALI, define a pathological consequence of viral specific antibody response, and provide a potential target for treatment of SARS-CoV or other virus-mediated lung injury.

Virotherapy-recruited PMN-MDSC infiltration of mesothelioma blocks antitumor CTL by IL-10-mediated dendritic cell suppression.

Antitumor cytotoxic T lymphocytes (CTLs) are essential for immune surveillance, yet the blockade of eliciting such CTLs during oncolytic virotherapy remains incompletely understood. Here, we show that oncolysis of mesothelioma by modified vaccinia Tiantan (MVTT) induces damage-associated molecular patterns exposure. Although MVTT leads to regression of established mesothelioma dose-dependently, antitumor CTLs are rarely induced. Mechanistically, MVTT virotherapy generates C-X-C chemokines that recruit CXCR2-expressing polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into tumor microenvironment, where they suppress dendritic cells (DCs) by producing IL-10 and halt CTL responses. During the virotherapy, however, depletion of PMN-MDSCs but not of monocytic (M)-MDSCs results in the induction of potent antitumor CTLs that not only eradicate established mesothelioma but also prevent the second tumor challenge. Our findings suggest that vaccinia virotherapy may combine strategies that prevent the chemotactic recruitment of PMN-MDSCs, block their suppression on DCs or deplete PMN-MDSCs in order to induce potent CTLs for tumor eradication.