Difference In Absolute Number Research Articles

Baseline CD57 + and CD16 + NK-Cells Predict Treatment Outcome in Non-Transplant Eligible Newly Diagnosed Multiple Myeloma Patients Treated with Daratumumab-Ixazomib-Dexamethasone

Background Non-fit multiple myeloma (MM) patients have inferior survival, increased toxicity, and higher rates of treatment discontinuation compared with their fit peers. Effective and less toxic treatment strategies are therefore urgently needed for this vulnerable population. The HOVON-143 trial (EudraCT: 2016-002600-90) was specifically designed to assess the efficacy and safety of daratumumab-ixazomib-dexamethasone in non-transplant eligible (NTE), intermediate fit and frail newly diagnosed (ND)MM patients. Patients were treated with nine 28-day induction cycles comprising daratumumab 16 mg/kg intravenously (cycles 1-2: days 1, 8, 15, and 22; cycles 3-6: days 1 and 15; cycles 7-9: day 1), ixazomib 4 mg orally (days 1, 8, and 15) and dexamethasone orally (only on days that daratumumab was given; cycle 1-2: 20 mg; cycle 3-9: 10 mg), followed by 8-week maintenance cycles consisting of daratumumab (day 1) and ixazomib (days 1, 8, 15, 29, 36, 43) for two years or until disease progression. Aims Since daratumumab has well-known NK-cell-mediated effector mechanisms and T-cell-stimulating immunomodulatory effects, we monitored these cell subsets in bone marrow (BM) and peripheral blood (PB) samples obtained before and during treatment. We specifically aimed to better understand these cell subsets related to frailty status, their predictive value for treatment outcome, and their dynamics during treatment. Methods 126 NDMM patients, among which 64 intermediate fit and 62 frail, were monitored. BM was collected at study-entry (baseline) and at disease progression; PB was collected at baseline, before cycle 4, before maintenance, and at disease progression. Absolute numbers of total T-cells and NK-cells (in PB) or percentages of T-cell and NK-cell subsets (in PB and BM) were profiled by flow cytometry. Data were analyzed using FlowSOM and UMAP. Total cells and cell subsets at baseline were compared between intermediate fit and frail patients, between responders (≥ partial response) and non-responders (< partial response), and associated with progression-free survival (PFS) and overall survival (OS). Results At baseline, we found no difference in absolute numbers of total T-cells and NK-cells between intermediate fit and frail patients. Besides, NK-cell phenotype was similar between both groups. However, within the T-cell compartment frail patients had a decreased CD4 +:CD8 + T-cell ratio, reduced naïve CD4 + and CD8 + T cells, and increased PD1 + CD4 + T-cells compared with intermediate fit patients in both BM and PB, independent of age. Interestingly, differences in T-cell phenotype were more pronounced in female than in male patients. Response data were available for 124 patients, of which 92 (74%) responded to treatment. In both BM and PB, preliminary results showed no clear association between response and absolute numbers of total T-cells or certain T-cell phenotypes at baseline. However, responders had higher absolute numbers of total NK-cells and higher percentages of NK-cells positive for maturation marker CD57 or fc-gamma receptor CD16 than non-responders, while higher absolute numbers of total NK-cells and higher percentages of CD57 + NK-cells were also associated with superior PFS and OS (Figure 1). Conversely, higher proportions of baseline NK-cells positive for inhibitory markers TIGIT and PD1 were associated with non-response, while higher percentages of CD56 bright and PD1 + NK-cells were negatively associated with PFS and OS. As expected, absolute numbers of total NK-cells markedly declined during the treatment course. Interestingly, percentages of CD57 + and CD16 + NK-cells decreased during treatment and remained low at disease progression, while percentages of CD56 bright and NKG2A + NK-cells substantially increased compared with baseline. Conclusions Frail patients had a distinct T-cell profile compared with intermediate fit patients, while NK-cell profiles were similar. Treatment outcome of NTE-NDMM patients treated with daratumumab-ixazomib-dexamethasone was positively associated with higher absolute numbers of total NK-cells and higher percentages of CD57 + and CD16 + NK-cells at baseline, corresponding to a mature and cytotoxic phenotype capable of mediating potent antibody-dependent cellular cytotoxicity. This study highlights the importance of NK-cell phenotype for the effectivity of daratumumab-based combination therapies in NTE-NDMM.

Concordance between COVID-19 mortality statistics derived from clinical audit and death certificates in Östergötland county, Sweden

ObjectivesDespite early notions that correct attribution of deaths caused by SARS-CoV-2 infection is critical to the understanding of the COVID-19 pandemic, three years later, the accuracy of COVID-19 death counts is still contested. We aimed to compare official death statistics with cause-of-death assessments made in a clinical audit routine by experienced physicians having access to the full medical record. Study designHealth service quality evaluation. MethodsIn Östergötland county (pop. 465,000), Sweden, a clinical audit team assessed from the start of the pandemic the cause of death in individuals having deceased after testing positive for SARS-CoV-2. We estimated the concordance between official data on COVID-19 deaths and data from the clinical audit using correlations (r) between the cause-of-death categories and discrepancies between the absolute numbers of categorised deaths. ResultsThe concordance between the data sources was poor regarding whether COVID-19 was the underlying or a contributing cause of death. Grouping of the causes increased the correlations to acceptable strength. Also including deaths implicated by a positive SARS-CoV-2 test in the clinical categorisation of COVID-19 deaths reduced the difference in absolute number of deaths; with these modifications, the concordance was acceptable before the COVID-19 vaccination program was initiated (r = 0.97; symmetric mean absolute percentage error (SMAPE) = 19%), while a difference in the absolute numbers of deaths remained in the vaccination period (r = 0.94; SMAPE = 35%). ConclusionsThis study highlights that carefulness is warranted when COVID-19 death statistics are used in health service planning and resonates a need for further research on cause-of-death recording methodologies.

BLOOD PRESSURE IN ATRIAL FIBRILLATION AND IN SINUS RHYTHM DURING 24 HOUR AMBULATORY BLOOD PRESSURE MONITORING: DATA FROM THE TEMPLAR PROJECT

Objective: Hypertension is the most common risk factor for atrial fibrillation (AF), and accounts for a worse prognosis. Blood pressure (BP) control is accordingly essential in AF. However, the interpretation of BP levels is difficult in AF, since differences may be dependent on rhythm, particularly when using automated oscillometric devices. We used data from the TEMPLAR project to compare oscillometric ambulatory BP monitoring (ABPM) intra-individually in AF and sinus rhythm (SR). Design and method: Among the 4398 individuals in TEMPLAR > 65 years investigated with ABPM and Afib Microlife detector (rhythm indicated with each BP measurement), those with > 30% of readings indicating AF were selected (as previously recommended), and episodes > 30 mins for AF and SR were chosen. Systolic and diastolic BP, mean arterial pressure (MAP), pulse pressure (PP), averaged real variability (ARV), and its coefficient of variation (CV) were assessed for AF and SR, separated for 24 h, daytime, night-time, and for heart rate (HR) < = 90 and > = 90 bpm. Results: We included 430 individuals (mean age 78 ± 7 years, 57% females, 77% with hypertension and 72% on treatment, 4% with previous AF). Mean BP of all measurements included were 124.2/69.0, 126.3/71.1, and 119.5/64.0 mmHg, for 24 h, daytime, and night-time, respectively. There were no differences in MAP for AF vs SR (p = 0.47). Systolic BP tended to be lower in AF (24 h 123.6 ± 13.9 vs 124.7 ± 16.1 mmHg in SR, p = 0.05), and night-time diastolic BP was higher in AF (64.6 ± 10.9 vs 63.3 ± 10.4 mmHg in SR, p = 0.01). PP was lower in AF (24 h 54.6 ± 10.9 vs 55.7 ± 12.8 mmHg in SR, p = 0.002). Diastolic, but not systolic BP variability (ARV and CV) was higher in AF (24 h, p < 0.001). Results were similar with HR < = 90 and > = 90 bpm. Conclusions: Using an oscillometric ABPM device, our results suggest that MAP is similar and reliable in both AF and SR. Lower systolic and higher diastolic BP in AF affects PP, which together with a high variability might impact arterial structure. However, differences in absolute numbers are small, and do not imply different BP strategies and target values in AF.

Longitudinal Associations between Concurrent Changes in Phenotypic Frailty and Lower Urinary Tract Symptoms among Older Men

BackgroundLower urinary tract symptoms (LUTS) are associated with prevalent frailty and functional impairment, but longitudinal associations remain unexplored.ObjectivesTo assess the association of change in phenotypic frailty with concurrent worsening LUTS severity among older men without clinically significant LUTS at baseline.DesignMulticenter, prospective cohort study.SettingPopulation-based.ParticipantsParticipants included community-dwelling men age ≥65 years at enrollment in the Osteoporotic Fractures in Men study.MeasurementsData were collected at 4 visits over 7 years. Phenotypic frailty score (range: 0–5) was defined at each visit using adapted Fried criterion and men were categorized at baseline as robust (0), pre-frail (1–2), or frail (3–5). Within-person change in frailty was calculated at each visit as the absolute difference in number of criteria met compared to baseline. LUTS severity was defined using the American Urologic Association Symptom Index (AUASI; range: 0–35) and men with AUASI ≥8 at baseline were excluded. Linear mixed effects models were adjusted for demographics, health-behaviors, and comorbidities to quantify the association between within-person change in frailty and AUASI.ResultsAmong 3235 men included in analysis, 48% were robust, 45% were pre-frail, and 7% were frail. Whereas baseline frailty status was not associated with change in LUTS severity, within-person increases in frailty were associated with greater LUTS severity (quadratic P<0.001). Among robust men at baseline, mean predicted AUASI during follow-up was 4.2 (95% CI 3.9, 4.5) among those meeting 0 frailty criteria, 4.6 (95% CI 4.3, 4.9) among those meeting 1 criterion increasing nonlinearly to 11.2 (95% CI 9.8, 12.6) among those meeting 5 criteria.ConclusionsGreater phenotypic frailty was associated with nonlinear increases in LUTS severity in older men over time, independent of age and comorbidities. Results suggest LUTS and frailty share an underlying mechanism that is not targeted by existing LUTS interventions.

Risk of SARS-CoV-2 reinfection and COVID-19 hospitalisation in individuals with natural and hybrid immunity: a retrospective, total population cohort study in Sweden

BackgroundReal-world evidence supporting vaccination against COVID-19 in individuals who have recovered from a previous SARS-CoV-2 infection is sparse. We aimed to investigate the long-term protection from a previous infection (natural immunity) and whether natural immunity plus vaccination (hybrid immunity) was associated with additional protection.MethodsIn this retrospective cohort study, we formed three cohorts using Swedish nationwide registers managed by the Public Health Agency of Sweden, the National Board of Health and Welfare, and Statistics Sweden. Cohort 1 included unvaccinated individuals with natural immunity matched pairwise on birth year and sex to unvaccinated individuals without natural immunity at baseline. Cohort 2 and cohort 3 included individuals vaccinated with one dose (one-dose hybrid immunity) or two doses (two-dose hybrid immunity) of a COVID-19 vaccine, respectively, after a previous infection, matched pairwise on birth year and sex to individuals with natural immunity at baseline. Outcomes of this study were documented SARS-CoV-2 infection from March 20, 2020, until Oct 4, 2021, and inpatient hospitalisation with COVID-19 as main diagnosis from March 30, 2020, until Sept 5, 2021.FindingsCohort 1 was comprised of 2 039 106 individuals, cohort 2 of 962 318 individuals, and cohort 3 of 567 810 individuals. During a mean follow-up of 164 days (SD 100), 34 090 individuals with natural immunity in cohort 1 were registered as having had a SARS-CoV-2 reinfection compared with 99 168 infections in non-immune individuals; the numbers of hospitalisations were 3195 and 1976, respectively. After the first 3 months, natural immunity was associated with a 95% lower risk of SARS-CoV-2 infection (adjusted hazard ratio [aHR] 0·05 [95% CI 0·05–0·05] p<0·001) and an 87% (0·13 [0·11–0·16]; p<0·001) lower risk of COVID-19 hospitalisation for up to 20 months of follow-up. During a mean follow-up of 52 days (SD 38) in cohort 2, 639 individuals with one-dose hybrid immunity were registered with a SARS-CoV-2 reinfection, compared with 1662 individuals with natural immunity (numbers of hospitalisations were eight and 113, respectively). One-dose hybrid immunity was associated with a 58% lower risk of SARS-CoV-2 reinfection (aHR 0·42 [95% CI 0·38–0·47]; p<0·001) than natural immunity up to the first 2 months, with evidence of attenuation thereafter up to 9 months (p<0·001) of follow-up. During a mean follow-up of 66 days (SD 53) in cohort 3, 438 individuals with two-dose hybrid immunity were registered as having had a SARS-CoV-2 reinfection, compared with 808 individuals with natural immunity (numbers of hospitalisations were six and 40, respectively). Two-dose hybrid immunity was associated with a 66% lower risk of SARS-CoV-2 reinfection (aHR 0·34 [95% CI 0·31–0·39]; p<0·001) than natural immunity, with no significant attenuation up to 9 months (p=0·07). To prevent one reinfection in the natural immunity cohort during follow-up, 767 individuals needed to be vaccinated with two doses. Both one-dose (HR adjusted for age and baseline date 0·06 [95% CI 0·03–0·12]; p<0·001) and two-dose (HR adjusted for age and baseline date 0·10 [0·04–0·22]; p<0·001) hybrid immunity were associated with a lower risk of COVID-19 hospitalisation than natural immunity.InterpretationThe risk of SARS-CoV-2 reinfection and COVID-19 hospitalisation in individuals who have survived and recovered from a previous infection remained low for up to 20 months. Vaccination seemed to further decrease the risk of both outcomes for up to 9 months, although the differences in absolute numbers, especially in hospitalisations, were small. These findings suggest that if passports are used for societal restrictions, they should acknowledge either a previous infection or vaccination as proof of immunity, as opposed to vaccination only.FundingNone.

Interplay of infection and vaccination in long-term protection from COVID-19

Interplay of infection and vaccination in long-term protection from COVID-19

Mediating role of multimorbidity in inequality in mortality: a register study on the Danish population

BackgroundThe social inequality in mortality is due to differential incidence of several disorders and injury types, as well as differential survival. The resulting clustering and possible interaction in disadvantaged groups...

The Traf2 and Nck-Interacting Kinase Is a Conserved Regulator of Self-Renewal in Hematopoietic and Leukemia Stem Cells

The Traf2 and Nck-interacting kinase (TNIK) is a member of the germinal center kinase (GCK) family. We have recently found that signaling through the tumor necrosis factor superfamily receptor CD27 activates Wnt target genes through Traf2-TNIK-β-catenin signaling, promoting leukemia development and progression in acute and chronic myeloid leukemia (AML and CML).To explore how TNIK signaling regulates hematopoietic and leukemia stem cells (HSCs and LSCs), we generated a constitutive TNIK-/- knockout mouse.Under steady-state conditions, total bone marrow (BM) cellularity of naïve TNIK-/- mice was slightly reduced (mean BM count (x108): TNIK-/- = 1.42; TNIKwt/wt = 2.15, p < 0.0132). In contrast, no differences in absolute number of lineage negative (Lin-) cells, as well as the more primitive subset Lin-; Sca1+; c-kit+ (LSK) cells could be observed. Similarly, proportions of long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs) and multipotent progenitors (MPPs) did not differ between TNIK-/- and TNIKwt/wt mice. Functionally, naïve TNIK-/- and TNIKwt/wt HSCs displayed in vitro a comparable serial colony formation capacity in methylcellulose. Altogether these data suggest that TNIK does not affect the maintenance and self-renewal of HSCs under steady-state conditions.Next the impact of TNIK on hematopoietic recovery after genotoxic stress was analyzed in vivo. TNIK-/- and TNIKwt/wt mice were injected i.p. with 5-Fluorouracil (5-FU) (150mg/g bodyweight) and BM cellularity as well as HSC phenotype and function were assessed 9 days post injection. The total BM cellularity was significantly higher in TNIK-/- mice compared to controls (mean BM count (x106/ml): TNIK-/- =10.25; TNIKwt/wt =4.55, p< 0.0382). Furthermore, LSKs were significantly reduced in frequency in TNIK-/- mice (% LSK of whole BM: TNIK-/- = 2.93; TNIKwt/wt = 7.073, p < 0.0009) but not in absolute numbers. Interestingly, however, the total number LT-HSCs was significantly reduced in the BM of TNIK-/- mice (mean LT-HSC count (x103): TNIK-/- = 5.628; TNIKwt/wt = 16.725, p < 0.0101). These results were functionally confirmed in vivo by competitive BM transplantation (BMTx) of whole BM from 5-FU treated mice into lethally irradiated secondary recipients. Mice receiving BM from 5-FU-treated TNIK-/- mice showed a significantly reduced peripheral engraftment capacity 6 weeks post BMTx compared to controls (mean peripheral blood chimerism: TNIK-/- = 64.6%; TNIKwt/wt = 35.4%, p < 0.0001), suggesting that TNIK signaling mediates HSC maintenance during genotoxic stress conditions in vivo.In parallel the role of TNIK signaling was analyzed in myeloid LSCs. CML-like disease was induced in mice by transplanting pMSCV-BCR-ABL1-IRES-GFP transduced TNIK-/- or TNIKwt/wt LSKs into BL/6 mice. Even though CML developed similarly as indicated by measuring BCR-ABL1-GFP+ granulocytes in the peripheral blood, primary TNIK-/- CML mice survived significantly longer compared to TNIKwt/wt CML mice (Median survival (days): TNIK-/- = 33, TNIKwt/wt = 23.5, p < 0.0216) with 2 out of 7 mice surviving long-term. Next, we determined the effect of TNIK signaling on the functionality of FACS sorted Lin-; GFP+; Sca1+; c-kit+ LSC in vitro in serial colony forming assays and in vivo in secondary transplantations. Serial colony formation capacity of TNIK-/- CML LSCs was significantly reduced after re-plating in vitro (CFUs fold change (TNIK-/- vs TNIKwt/wt): 1st Plating = 0.89, P<0.5149, 2nd Plating = 0.42, 9 < 0.0002). In line with these findings, only 1 out of 9 mice secondarily transplanted with LSCs from primary TNIK-/- CML mice developed CML within 90 days, while all 9 recipients receiving TNIKwt/wt LSCs succumbed to the disease within 23 days after transplantation (p < 0.0001). These results suggest that TNIK signaling also promotes self-renewal of CML LSCs.To summarize, we have described for the first time TNIK signaling as a commonly conserved regulator in stress-induced hematopoiesis and CML, which promotes stem cell self-renewal and maintenance. As TNIK inhibitors are currently developed for the treatment of different kinds of solid tumors, TNIK may be an attractive therapeutic target for LSCs in myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Abstract 16654: Covid - 19 - Acute Myocardial Infarction and Heart Failure Mortality Trends in a Non-epicenter Region

Introduction: As the novel coronavirus gained acceleration, there are several reports of a decreasing number of hospital admissions for acute myocardial infarctions (AMI) worldwide. It is unclear if this is due to an actual decrease in their incidence or due to patients avoiding hospital visits during the pandemic. We examined the trends in admissions for AMI, acute decompensated heart failure (HF) and their associated mortality in an integrated health system (7 hospitals) in Pittsburgh (Pennsylvania), a non-epicenter region. Hypothesis: If patients are presenting late due to avoiding hospitals, AMI and HF would be associated with increased mortality rates. Methods: We collected data on the total number of admissions and deaths related to AMI (ST and non-ST elevation), HF using ICD-10 codes, during the period of January-April 2020. We compared this with data from the same period in 2018, 2019. Mortality rate was calculated as deaths per 100 admissions. Results: There were 505 AMI admissions in January-April 2020 and 37 died compared to 23 deaths amongst 645 admissions in the same period of 2018 and 2019 (averaged). Similarly, there were 1030 HF admissions in January-April 2020 and 46 died in comparison to 39 deaths amongst 1280 admissions in 2018 and 2019 (averaged). The absolute number of AMI and HF admissions was lower in 2020 (p&lt;0.05, p&lt;0.01). There was no difference in absolute number of deaths attributed to HF or AMI between 2020 and the preceding years (p=0.3). However, mortality rate was higher for HF in 2020 compared to prior 2 years. Conclusions: In this non-epicenter region, there was a significant decrease in admissions for AMI and HF in 2020 compared to 2018-2019 while the absolute number of deaths were similar. Similar mortality rates for AMI across the years suggests that patients were presenting appropriately and that the true incidence was likely low. However, higher mortality rate with HF may suggest a delayed presentation albeit without accounting for confounders.

Epicardial adipose tissue CD4+ T cells as novel drivers in the development of atrial fibrillation

Abstract Background Epicardial adipose tissue (EAT) is now a well-established independent risk factor for all forms of atrial fibrillation (AF). However, the cellular profile and immune mediators within EAT remain poorly defined. Purpose This study sought to define the immunological signature of EAT critical to its role in the development of both persistent AF and de novo post-operative AF (POAF). Methods Adult patients undergoing elective cardiac surgery with no prior AF history and those with pre-existing AF were recruited to undergo EAT sampling alongside subcutaneous adipose tissue (SAT) and pre-operative blood (adipose tissue and systemic controls respectively) sampling. The blood and tissue samples were immediately taken to the laboratory for immune cell isolation, flow cytometry and quantitative polymerase chain reaction (qPCR) analysis. Results A total of 71 patients were recruited including 9 patients with a prior history of AF. Of the 62 a priori sinus rhythm (SR) patients, 42 remained in SR post-operatively and 20 developed de novo POAF. No differences in absolute numbers of immune cells in the blood, EAT or SAT were detected on flow cytometry analysis between the three groups of patients. However, there was a significant increase (p&amp;lt;0.01) in EAT-resident CD4+ memory T cell populations in pre-existing AF patients and a trend towards significance (p=0.09) in POAF patients. qPCR analysis of immune mediator expression in EAT demonstrated pre-existing AF patients uniquely showed a significant reduction (p&amp;lt;0.005) in the pro-fibrotic mediator transforming growth factor-β (TGF-β) compared to the pre-existing AF patients (graph 1). Conclusions and implications Resident CD4+ memory T cell populations are locally and exclusively elevated in the EAT of pre-existing AF and POAF patients. This would suggest primed antigen-specific CD4+ T cells are present prior to the development of AF in patients who go on to develop de novo POAF. The reduced TGF-β levels observed in the EAT of AF patients indicates the EAT immune mediator profile differs to the underlying myocardial tissue, where increased TGF-β levels have previously been described in the persistent AF cohort. Therapies targeting this EAT-resident CD4+ T cell population would provide a novel approach to the management of the inflammatory components of AF genesis. Graph 1 Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Barts Charity, Abbott

Comparison of the Effects of Three Anticoagulants on Hematological Analytes in the Eastern Indigo Snake (Drymarchon couperi)

Studies on the effects of anticoagulants on blood samples in reptiles are limited. Eastern indigo snakes (Drymarchon couperi) have unique physiological features that are still not well understood. The objective of this study was to evaluate the effects of lithium heparin, tripotassium ethylendiaminetetraacetic acid (EDTA), trisodium citrate, and the use of no anticoagulant on various hematological analytes of the eastern indigo snake. Blood was collected from 10 female eastern indigo snakes and transferred in aliquots into three anticoagulants; it was also processed without anticoagulant. Packed cell volume (PCV), total solids (TS), plasma color, and data from blood film evaluations were compared. Significant differences were found for PCV (P < 0.0001) and TS (P < 0.0001). Both analytes were significantly lower in citrated samples, and heparinized samples had lower TS than EDTA-preserved and non-anticoagulated samples. Although there were no differences in absolute numbers and morphological quality among treatments, heparinized blood samples had a higher heterophil percentage (P = 0.039) compared to non-anticoagulated samples, and both heparinized and EDTA-preserved samples had lower azurophil percentages (P = 0.039) than non-anticoagulated samples. Hemolysis was absent in all samples. Heparinization produced a blue background hue on blood films that was absent in all other methods. This preliminary study demonstrates that heparinized, EDTA-preserved, and non-anticoagulated blood samples are equally superior for hematological evaluation over citrated samples because of its effect on the PCV. This finding has clinical application and it is advised to avoid citrate as an anticoagulant in eastern indigo snakes.

Clinical and Immune Features of Hospitalized Pediatric Patients With Coronavirus Disease 2019 (COVID-19) in Wuhan, China.

The epidemiologic and clinical characteristics of pediatric patients with coronavirus disease 2019 (COVID-19) have been reported, but information on immune features associated with disease severity is scarce. To delineate and compare the immunologic features of mild and moderate COVID-19 in pediatric patients. This single-center case series included 157 pediatric patients admitted to Wuhan Children's Hospital with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data were collected from January 25 to April 18, 2020. Documented SARS-CoV-2 infection. Clinical and immunologic characteristics were collected and analyzed. Outcomes were observed until April 18, 2020. Of the 157 pediatric patients with COVID-19, 60 (38.2%) had mild clinical type with pneumonia, 88 (56.1%) had moderate cases, 6 (3.8%) had severe cases, and 3 (1.9%) were critically ill. The 148 children with mild or moderate disease had a median (interquartile range [IQR]) age of 84 (18-123) months, and 88 (59.5%) were girls. The most common laboratory abnormalities were increased levels of alanine aminotransferase (ALT) (median [IQR], 16.0 [12.0-26.0] U/L), aspartate aminotransferase (AST) (median [IQR], 30.0 [23.0-41.8] U/L), creatine kinase MB (CK-MB) activity (median [IQR], 24.0 [18.0-34.0] U/L), and lactate dehydrogenase (LDH) (median [IQR], 243.0 [203.0-297.0] U/L), which are associated with liver and myocardial injury. Compared with mild cases, levels of inflammatory cytokines including interleukin 6, tumor necrosis factor α, and interferon γ were unchanged, whereas the level of immune suppressive interleukin 10 was markedly increased in moderate cases compared with mild cases (median [IQR], 3.96 [3.34-5.29] pg/mL vs 3.58 [3.10-4.36] pg/mL; P = .048). There was no statistically significant difference in absolute number of lymphocytes (including T cells and B cells) between mild and moderate cases, but moderate cases were associated with a decrease in neutrophil levels compared with mild cases (median [IQR], 2310/μL [1680/μL-3510/μL] vs 3120/μL [2040/μL-4170/μL]; P = .01). Immunoglobin G and the neutrophil to lymphocyte ratio were negatively associated with biochemical indices related to liver and myocardial injury (immunoglobulin G, ALT: r, -0.3579; AST: r, -0.5280; CK-MB activity: r, -0.4786; LDH: r, -0.4984; and neutrophil to lymphocyte ratio, ALT: r, -0.1893; AST: r, -0.3912; CK-MB activity: r, -0.3428; LDH: r, -0.3234), while counts of lymphocytes, CD4+ T cells, and interleukin 10 showed positive associations (lymphocytes, ALT: r, 0.2055; AST: r, 0.3615; CK-MB activity: r, 0.338; LDH: r, 0.3309; CD4+ T cells, AST: r, 0.4701; CK-MB activity: r, 0.4151; LDH: r, 0.4418; interleukin 10, ALT: r, 0.2595; AST: r, 0.3386; CK-MB activity: r, 0.3948; LDH: r, 0.3794). In this case series, systemic inflammation rarely occurred in pediatric patients with COVID-19, in contrast with the lymphopenia and aggravated inflammatory responses frequently observed in adults with COVID-19. Gaining a deeper understanding of the role of neutrophils, CD4+ T cells, and B cells in the pathogenesis of SARS-CoV-2 infection could be important for the clinical management of COVID-19.

The effect of pneumococcal immunization on total and antigen-specific B cells in patients with severe chronic kidney disease

BackgroundWhile the 23-valent pneumococcal polysaccharide vaccine (PPV23) is routinely used in Canada and some other countries to prevent pneumococcal infection in adults with chronic kidney disease (CKD), patients develop a suboptimal antibody response to PPV23 due to their immune dysfunction. The 13-valent pneumococcal conjugate vaccine (PCV13) has superior immunogenicity in some categories of immunocompromised adults; however, its effect on the immune response in CKD patients has only been addressed by two recent studies with conflicting results. The effect of PPV23 or PCV13 on B cells in these patients has not been previously studied. We studied the absolute numbers and proportions of B cells and subpopulations in two groups of adult patients with severe CKD pre- and 7 days post-immunization with PCV13: pneumococcal vaccine naïve and previously immunized with PPV23 (over one year ago).ResultsPPV23 immunized patients had significantly lower proportions and absolute numbers of class switched memory (CD19 + CD27 + IgM-), as well as lower absolute numbers of IgM memory (CD19 + CD27 + IgM+) and class switched B cells (CD19 + CD27-IgM-) compared to PPV23 naïve patients. Following PCV13 immunization, the differences in absolute numbers of B-cell subpopulations between groups remained significant. The PPV23 immunized group had higher proportions of CD5- B cells along with lower proportions and absolute numbers of CD5+ B cells compared to PPV23 naïve patients both pre- and post-immunization with PCV13. However, previous PPV23 immunization did not have a noticeable effect on the numbers of total IgG or serotype 6B and 14 specific antibody-secreting cells detected 7 days post-immunization with PCV13. Nevertheless, fold increase in anti-serotype 14 IgG concentrations 28 days post-PCV13 was greater in PPV23 naïve than in previously immunized patients.ConclusionsThe results suggest that immunization with PPV23 may result in long-term changes in B-cell subpopulations such as increased prevalence of CD5- B cells and decreased prevalence of class switched memory B cells in the peripheral blood. Because previous immunization with PPV23 in patients with CKD is associated with a significant decrease in the total class switched memory B cells in response to subsequent immunization with PCV13, this may reduce PCV13 immunogenicity in the setting of PPV23 followed by PCV13.Trial registrationRegistered February 24, 2015 at ClinicalTrials.gov (NCT 02370069).

Natural immunity against Haemophilus influenzae type a and B-cell subpopulations in adult patients with severe chronic kidney disease

Individuals suffering from severe chronic kidney disease (CKD) are immunocompromised and therefore highly susceptible to various infections including Haemophilus influenzae type a (Hia), an emerging pathogen in North American Indigenous populations. Immunocompromised Indigenous adults are considered a target for a new Hia vaccine under development. In an attempt to foresee their response to Hia immunization, we studied natural immunity against Hia and B-cell subpopulations in sixty patients with CKD residing in a geographic region with noticeable presence of Hia invasive disease. Serum bactericidal activity (SBA) against Hia, concentrations of IgG and IgM antibodies specific to Hia capsular polysaccharide, and B-cell subpopulations were studied in patients with CKD and 35 healthy controls of the same age. Of the patients with CKD, proportions and absolute numbers of B-cell subpopulations were determined for 28 patients. The patients had lower SBA titres compared to controls. Although no significant differences in anti-Hia IgG or IgM antibody concentrations between control and CKD groups were found, IgM antibody concentrations were higher in Indigenous than non-Indigenous patients. Patients with CKD had a higher proportion of B cells (CD19+), class switched memory B cells (CD19+CD27+IgM-) and a lower proportion of CD19+CD27-IgM- B cells compared to healthy controls. Non-Indigenous patients with CKD had significantly higher proportions of IgM memory B cells and CD19+CD27-IgM- B cells compared to Indigenous patients with no significant difference in absolute numbers. Because 72% of CKD patients had detectable SBA titres and 100% had detectable IgG and IgM antibodies it is possible that a portion of IgM memory B cells and class switched memory B cells are specific for Hia resulting from a natural exposure to the pathogen. The data suggest that a Hia-conjugate vaccine may be immunogenic in adult patients with CKD as it will potentially induce re-activation of immunological memory against Hia.

Dysregulated monocyte compartment in human obesity

Abstract Background Monocytes can be divided in classical monocytes (CM), intermediate monocytes (IM) and non-classical monocytes (NCM). CD56+ monocytes are part of the classical monocytes. Aim of the study was to assess the occurrence of the different monocyte subpopulations in human obesity. Methods 110 age-matched subjects were included in the study and classified according to the definition of the World Health Organization into lean (n=27), overweight (n=23) and obese (n=60). Monocyte subpopulations were analyzed by flow cytometry (antibody staining of CD14, CD16, CD56). Results Obese subject showed a higher BMI, fat mass, and waist circumference, deterioration of metabolic parameters, and the absolute amount of leukocytes and monocytes were increased. The calculated absolute numbers of CM, IM and CD56+ monocytes were significantly increased in obese subjects compared to lean and overweight. In contrast, the absolute numbers of NCM were equal between lean, overweight and obese subjects. Strong associations between the absolute numbers of CM, IM and CD56+ monocytes with the BMI, fat mass, and waist circumference were detectable in the whole cohort. Triglyceride, HDL-c values and HbA1c were also strongly correlated with numbers of CM, IM and CD56+ monocytes. Obese subjects with impaired glucose tolerance had increased numbers of CD56+ monocytes in comparison to obese subsects with normal glucose tolerance. Stratification of obese subjects in subjects with and without type 2 diabetes mellitus showed no difference in absolute numbers of CM but significant increased numbers of IM and NCM. Conclusion The amount of CM, IM and CD56+ monocytes are expanded in the blood of obesity subjects and contribute to monocytosis in obesity.

Donor T Cell Independent Mechanism May Contribute to Enhancing Steroid Refractory Gvhd in Murine Models

Steroids are the first line therapy for acute GVHD but 50% of patients develop steroid refractory GVHD (SR-GVHD). The development of therapies for SR-GVHD is limited by incomplete understanding of its pathophysiology. Here we generated SR-GVHD murine model by developing an MHC matched, multiple minor histocompatibility antigens (miHAs) mismatched BMT and determined whether donor T cells plays an important role in mediating SR-GVHD. To this end, we utilized C3H.swaB6 model. Recipient B6 animals were lethally irradiated and transplanted with splenic T cells along with BM cells from either syngeneic or allogeneicdonors. Dexamethasone (DEX) was administered from day +7 to day +21 at a dose of 0.1mg/kg. We observed three phenotypes in response to DEX: 1) steroid refractory (REF), characterized by progressive GVHD with severe peak GVHD clinical scores, progressive weight loss and early mortality; 2) stable GVHD (ST), characterized by moderate GVHD clinical scores, body weight loss, and minimal late mortality; 3) steroid responsive (RES) GVHD, characterized by near complete remission of GVHD clinical scores, weight loss, and no mortality. These criteria clearly separated treatment animals into three groups and resulted in significantly higher day +21 GVHD specific histopathological scores of GVHD target organs in REF relative to RES animals. We next sought to determine whether steroid refractoriness can be modeled at a later time-point, to account for any contribution from radiation toxicity. To achieve this, we utilized the same model except that DEX was administered from day +21 to +35. We again obtained similar overall survival and GVHD specific histopathological scores in target organs as in our early steroid treatment model. We next determined whether the steroid refractoriness correlated with changes in donor T cells. However, we found no difference in the proportion of donor activated, effector or memory T cells among groups. In addition, there was no difference in absolute number or percent of donor splenic T cells or their subsets including Tregs and exhausted T cells. Furthermore, there were no differences in serum inflammatory cytokines on day 7 or 14 of DEX treatment in the early and late steroid models, respectively. Because target tissue intrinsic mechanisms are critical for initiating GVHD, we tested whether gut homeostasis related proteins were altered in SR-GVHD and found that the expression of CD324 in gut epithelial cells is significantly increased in SR-GVHD. In conclusion, we established clinically relevant SR-GVHD murine models. Donor T cell characteristics were not significantly different between groups. However, CD324 expression in gut epithelial cells is dramatically increased in refractory animals. These data suggest that donor T cell independent mechanisms may also contribute to steroid refractoriness more so than was previously considered.

Donor T Cell Independent Mechanism Is Critical for Mediating Steroid Refractory Gvhd in Murine Models

Acute graft-versus-host disease (GVHD) is a major complication of allogeneic cell transplantation (Allo-HCT). Steroids have remained the first line therapy for acute GVHD. However, 30-50% of patients develop steroid refractory GVHD (SR-GVHD) which shows extremely poor prognosis. The development of therapies for SR-GVHD is limited by an incomplete understanding of its pathophysiology because of the lack of clinical animal models. Here we generated SR-GVHD murine models by developing a major histocompatibility (MHC) matched, multiple minor histocompatibility antigens (miHAs) mismatched as well as an MHC mismatched haploidentical murine bone marrow transplantation (BMT) models and determined whether donor T cells are crucial role in mediating SR-GVHD.Clinically, most allo-HCTs are human leukocyte antigen (HLA) matched. Therefore, we utilized the clinically relevant MHC matched multiple miHAs mismatched C3H.swàB6 model. Recipient B6 WT animalswere lethally irradiated and transplanted with splenic T cells along with bone marrow (BM) cells from either syngeneic B6 or allogeneic C3H.swdonors. Dexamethasone (DEX) was administered from day +7 to day +21 at a dose of 0.1mg/kg. We observed three phenotypes in response to DEX: 1) steroid refractory (REF), characterized by progressive GVHD with severe peak GVHD clinical scores, progressive weight loss and early mortality; 2) stable GVHD (ST), characterized by moderate GVHD clinical scores, moderate body weight loss, and minimal late mortality; 3) steroid responsive (RES) GVHD, characterized by near complete normalization of GVHD clinical scores, near complete normalization of weight loss, and no mortality. We were able to differentiate among these three phenotypes based on GVHD clinical score and body weight change on day +21 after allo-BMT which included14 days of DEX treatment. These criteria clearly separated steroid treatment animals into three groups based on mortality and resulted in significantly higher day +21 GVHD specific histopathological scores of GVHD target organs in REF relative to RES animals. To eliminate strain dependent factors, we used another BMT model, B6àB6D2F1 and found a similar differential response to steroid; albeit with a slightly different distribution amongst the groups and greater mortality in the ST and RES groups.We next sought to determine whether steroid refractoriness can be modeled at a later time-point, to account for any contribution from radiation toxicity. To achieve this, we utilized the same C3H.sw→B6 model except that DEX was administered from day +21 to +35. We again obtained similar overall survival and GVHD specific histopathological scores in target organs as in our early steroid treatment model.We next determined whether the steroid refractoriness correlated with changes in donor T cell, such as persistent activation, in REF animals. However, to our surprise, in both our early and late steroid treatment models, we found no difference in the proportion of donor activated, effector or memory T cells between the REF and RES groups. In addition, there was no difference in absolute number or percent of donor splenic T cells or their subsets including Tregsand exhausted T cells. Furthermore, there were no differences in serum inflammatory cytokines, donor intrahepatic lymphocytes, GI tract donor intraepithelial lymphocytes, or donor myeloid cells on day 7 or 14 of DEX treatment in the early and late steroid models, respectively.Because target tissue intrinsic mechanisms are critical for initiating GVHD, we tested whether gut homeostasis related proteins were altered in SR-GVHD and found that the expression of CD324 (E-cadherin) in gut epithelial cells is significantly increased in SR-GVHD.In conclusion, we established clinically relevant SR-GVHD murine models. To our surprise, donor T cell characteristics were not significantly different between groups treated with steroids. However, CD324 expression in gut epithelial cells is dramatically increased in refractory animals. These data suggest that donor T cell independent mechanisms may also contribute to steroid refractoriness more so than was previously considered. DisclosuresIshizawa:Chugai: Honoraria; Sanofi: Research Funding; Phizer: Research Funding; Celgene: Honoraria; Eisai: Honoraria; Janssen: Honoraria; Otsuka: Research Funding.

What is the true tuberculosis mortality burden? Differences in estimates by the World Health Organization and the Global Burden of Disease study

The World Health Organization (WHO) and the Global Burden of Disease (GBD) study at the Institute for Health Metrics and Evaluation (IHME) periodically provide global estimates of tuberculosis (TB) mortality. We compared the 2015 WHO and GBD TB mortality estimates and explored which factors might drive the differences. We extracted the number of estimated TB-attributable deaths, disaggregated by age, HIV status, sex and country from publicly available WHO and GBD datasets for the year 2015. We 'standardized' differences between sources by adjusting each country's difference in absolute number of deaths by the average number of deaths estimated by both sources. For 195 countries with estimates from both institutions, WHO estimated 1768482 deaths attributable to TB, whereas GBD estimated 1322916 deaths, a difference of 445566 deaths or 29% of the average of the two estimates. The countries with the largest absolute differences in deaths were Nigeria (216621), Bangladesh (49863) and Tanzania (38272). The standardized difference was not associated with HIV prevalence, prevalence of multidrug resistance or global region, but did show correlation with the case detection rate as estimated by WHO [r = -0.37, 95% confidence interval (CI): -049; -0.24] or, inversely, with case detection rate based on GBD data (r = 0.44, 95% CI: 0.31; 0.54). Countries with a recent national prevalence survey had higher standardized differences (higher estimates by WHO) than those without (P = 0.006). After exclusion of countries with recent prevalence surveys, the overall correlation between both estimates was r = 0.991. A few countries account for the large global discrepancy in TB mortality estimates. The differences are due to the methodological approaches used by WHO and GBD. The use and interpretation of prevalence survey data and case detection rates seem to play a role in the observed differences.

Proportional thresholding in resting-state fMRI functional connectivity networks and consequences for patient-control connectome studies: Issues and recommendations

Graph theoretical analysis has become an important tool in the examination of brain dysconnectivity in neurological and psychiatric brain disorders. A common analysis step in the construction of the functional graph or network involves “thresholding” of the connectivity matrix, selecting the set of edges that together form the graph on which network organization is evaluated. To avoid systematic differences in absolute number of edges, studies have argued against the use of an “absolute threshold” in case-control studies and have proposed the use of “proportional thresholding” instead, in which a pre-defined number of strongest connections are selected as network edges, ensuring equal network density across datasets. Here, we systematically studied the effect of proportional thresholding on the construction of functional matrices and subsequent graph analysis in patient-control functional connectome studies. In a few simple experiments we show that differences in overall strength of functional connectivity (FC) – as often observed between patients and controls – can have predictable consequences for between-group differences in network organization. In individual networks with lower overall FC the proportional thresholding algorithm has to select more edges based on lower correlations, which have (on average) a higher probability of being spurious, and thus introduces a higher degree of randomness in the resulting network. We show across both empirical and artificial patient-control datasets that lower levels of overall FC in either the patient or control group will most often lead to differences in network efficiency and clustering, suggesting that differences in FC across subjects will be artificially inflated or translated into differences in network organization. Based on the presented case-control findings we inform about the caveats of proportional thresholding in patient-control studies in which groups show a between-group difference in overall FC. We make recommendations on how to examine, report and to take into account overall FC effects in future patient-control functional connectome studies.

Histopathological analysis of infiltrating T cell subsets in acute T cell-mediated rejection in the kidney transplant.

AIMTo compare the differential immune T cell subset composition in patients with acute T cell-mediated rejection in the kidney transplant with subset composition in the absence of rejection, and to explore the association of their respective immune profiles with kidney transplant outcomes.METHODSA pilot cross-sectional histopathological analysis of the immune infiltrate was performed using immunohistochemistry in a cohort of 14 patients with acute T cell-mediated rejection in the kidney transplant and 7 kidney transplant patients with no rejection subjected to biopsy to investigate acute kidney transplant dysfunction. All patients were recruited consecutively from 2012 to 2014 at the Singapore General Hospital. Association of the immune infiltrates with kidney transplant outcomes at up to 54 mo of follow up was also explored prospectively.RESULTSIn comparison to the absence of rejection, acute T cell-mediated rejection in the kidney transplant was characterised by numerical dominance of cytotoxic T lymphocytes over Foxp3+ regulatory T cells, but did not reach statistical significance owing to the small sample size in our pilot study. There was no obvious difference in absolute numbers of infiltrating cytotoxic T lymphocytes, Foxp3+ regulatory T cells and Th17 cells between the two patient groups when quantified separately. Our exploratory analysis on associations of T cell subset quantifications with kidney transplant outcomes revealed that the degree of Th17 cell infiltration was significantly associated with shorter time to doubling of creatinine and shorter time to transplant loss.CONCLUSIONAlthough this was a small pilot study, results support our suspicion that in kidney transplant patients the immune balance in acute T cell-mediated rejection is tilted towards the pro-rejection forces and prompt larger and more sophisticated studies.