Investigation of crystalline lens overshooting: ex vivo experiment and optomechanical simulation results.

Introduction: Crystalline lens overshooting refers to a situation in which the lens momentarily shifts too much from its typical location immediately after stopping the rotational movement of the eye globe. This movement can be observed using an optical technique called Purkinje imaging. Methods: In this work, an experimental setup was designed to reproduce this effect ex vivo using a fresh porcine eye. The sample was rotated 90° around its centroid using a high-velocity rotation stage, and the Purkinje image sequences were recorded, allowing us to quantify the overshooting effect. The numerical part of the study consisted of developing a computational model of the eye, based on the finite element method, that allowed us to understand the biomechanical behavior of the different tissues in this dynamic scenario. A 2D fluid-structure interaction model of the porcine eye globe, considering both the solid parts and humors, was created to reproduce the experimental outcomes. Results: Outputs of the simulation were analyzed using an optical simulation software package to assess whether the mechanical model behaves optically like the real ex vivo eye. The simulation predicted the experimental results by carefully adjusting the mechanical properties of the zonular fibers and the damping factor. Conclusion: This study effectively demonstrates the importance of characterizing the dynamic mechanical properties of the eye tissues to properly comprehend and predict the overshooting effect.

An integrated model of the human cornea as a linear biaxial birefringent medium.

A novel model of human corneal birefringence is presented. The cornea is treated as a homogeneous biaxial linear birefringent medium in which the values of the binormal axes angle and organization of the main refractive indices vary continuously from the apex to the limbus. In its central part, the angle between binormal axes is 35°, and para centrally, it smoothly increases to 83.7°. The values of the main refractive indices (nx, ny, nz) change, as well as their order, from nx < nz < ny to nz < nx < ny. The transition between these two states was described with a normal distribution (µ = 0.45, σ = 0.1). The presented model corresponds with the experimental results presented in the literature. To our knowledge, it is the first model that presents the anisotropic properties' distributions of the entire cornea. The presented model facilitates a better understanding of the corneal birefringence phenomenon directly related to its lamellar structure.

Complexity of crystalline lens wobbling investigated by means of combined mechanical and optical simulations.

Crystalline lens wobbling is a phenomenon when the lens oscillates briefly from its normal position immediately after stopping the rotational movement of the eye globe. It can be observed by means of Purkinje imaging. The aim of this research is to present the data and computation workflow that involve both biomechanical and optical simulations that can mimic this effect, aimed to better understanding of lens wobbling. The methodology described in the study allows to visualize both the dynamic changes of the lens conformation within the eye and its optical effect in terms of Purkinje performance.

Opto-mechanical self-adjustment model of the human eye.

The eye has specific optical and biomechanical properties that jointly regulate the eye's quality of vision, shape, and elasticity. These two characteristics are interdependent and correlated. Contrary to most currently available computational models of the human eye that only focus on biomechanical or optical aspects, the current study explores the inter-relationships between biomechanics, structure, and optical properties. Possible combinations of mechanical properties, boundary conditions, and biometrics were specified to ensure the opto-mechanical (OM) integrity to compensate for physiological changes in intraocular pressure (IOP) without compromising image acuity. This study evaluated the quality of the vision by analyzing the minimum spot diameters formed on the retina and drew how the self-adjustment mechanism affects the eye globe shape by adopting a finite element (FE) model of the eyeball. The model was verified by a water drinking test with biometric measurement (OCT Revo NX, Optopol) and tonometry (Corvis ST, Oculus).

Selected Predictors of COVID-19 Mortality in the Hospitalised Patient Population in a Single-Centre Study in Poland.

Background: The correct analysis of COVID-19 predictors could substantially improve the clinical decision-making process and enable emergency department patients at higher mortality risk to be identified. Methods: We retrospectively explored the relationship between some demographic and clinical factors, such as age and sex, as well as the levels of ten selected factors, namely, CRP, D-dimer, ferritin, LDH, RDW-CV, RDW-SD, procalcitonin, blood oxygen saturation, lymphocytes, and leukocytes, and COVID-19 mortality risk in 150 adult patients diagnosed with COVID-19 at Provincial Specialist Hospital in Zgierz, Poland (this hospital was transformed, in March 2020, into a hospital admitting COVID-19 cases only). All blood samples for testing were collected in the emergency room before admission. The length of stay in the intensive care unit and length of hospitalisation were also analysed. Results: The only factor that was not significantly related to mortality was the length of stay in the intensive care unit. The odds of dying were significantly lower in males, patients with a longer hospital stay, patients with higher lymphocyte levels, and patients with higher blood oxygen saturation, while the chances of dying were significantly higher in older patients; patients with higher RDW-CV and RDW-SD levels; and patients with higher levels of leukocytes, CRP, ferritin, procalcitonin, LDH, and D-dimers. Conclusions: Six potential predictors of mortality were included in the final model: age, RDW-CV, procalcitonin, and D-dimers level; blood oxygen saturation; and length of hospitalisation. The results obtained from this study suggest that a final predictive model with high accuracy in mortality prediction (over 90%) was successfully built. The suggested model could be used for therapy prioritization.

UCART19, a first-in-class allogeneic anti-CD19 chimeric antigen receptor T-cell therapy for adults with relapsed or refractory B-cell acute lymphoblastic leukaemia (CALM): a phase 1, dose-escalation trial.

BACKGROUND: The prognosis for adults with relapsed or refractory B-cell acute lymphoblastic leukaemia remains poor. UCART19, an allogeneic genome-edited anti-CD19 chimeric antigen receptor (CAR) T-cell product derived from healthy donors and available for immediate clinical use, offers a potential therapeutic option for such patients. The CALM trial is a first-in-human study evaluating the safety and antileukaemic activity of UCART19 in adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. METHODS: This phase 1, open-label study was conducted at eight centres across France, the UK, the USA, and Japan. Adult patients aged 16-70 years with CD19-positive relapsed or refractory B-cell acute lymphoblastic leukaemia who had morphological relapse or a minimal residual disease level of at least 1 × 10-3 and had exhausted standard treatment options were enrolled in the study, which comprised a dose-escalation phase of up to three UCART19 doses followed by a safety expansion phase. Patients underwent lymphodepletion with fludarabine (30 mg/m2 per day intravenously for 3 days) and cyclophosphamide (500 mg/m2 per day intravenously for 3 days) with or without alemtuzumab (1 mg/kg or 40 mg or 60 mg over 5 days) and received UCART19 doses of 6 × 106, 6-8 × 107, or 1·8-2·4 × 108 total CAR T cells intravenously, followed by safety evaluation and disease response assessments. The primary endpoint was incidence and severity of adverse events. Secondary endpoints were the overall response rate, duration of response, relapse-free survival, progression-free survival, and overall survival. This trial is registered with ClinicalTrials.gov (NCT02746952) and is complete. FINDINGS: Between Aug 1, 2016, and June 30, 2020, 25 patients were enrolled in the study and treated with UCART19. Median duration of follow-up was 12·8 months (IQR 2·8-24·8). Median age was 37 years (IQR 28-45). 14 (56%) patients were male and 11 (44%) female. 17 (68%) patients were White, two (8%) Black, two (8%) Asian, and four (16%) from other racial or ethnic groups. Three patients developed dose-limiting toxicities (one at each dose level); one had grade 4 cytokine release syndrome and two had grade 4 prolonged cytopenias. Grade 3 or higher cytokine release syndrome was reported in six (24%) patients and grade 3 or higher neurological toxicity in one (4%) patient. Grade 3 or higher infections occurred in seven (28%) patients, and grade 4 prolonged cytopenia in four (16%) patients. Two (8%) patients developed grade 1 acute cutaneous graft-versus-host disease. 14 patients died, nine from progressive disease and five from infections or other complications, of which four were considered to be related to UCART19 or lymphodepletion, or both. After a median of follow-up of 12·8 months (IQR 2·8-24·8), overall response rate was 48% (95% CI 28-69; 12 of 25 patients), duration of response and median relapse-free survival were 7·4 months (95% CI 1·8 to not calculable), progression-free survival was 2·1 months (95% CI 1·2-2·8), and overall survival was 13·4 months (95% CI 4·8-23·0). INTERPRETATION: UCART19 had a manageable safety profile, and showed evidence of antileukaemic activity in heavily pretreated adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. This study shows that allogeneic off-the-shelf CAR T cells can be used safely to treat patients with relapsed B-cell acute lymphoblastic leukaemia. FUNDING: Servier.

Corneal Behavior during Tonometer Measurement during the Water Drinking Test in Eyes with XEN GelStent in Comparison to Non-Implanted Eyes.

Biomechanics of the cornea have significant influences on the non-contact measurement of the intraocular pressure. The corneal behaviour during tonometry is a fundamental factor in estimating its value. The aim of the study was to analyse the behaviour of the cornea during tonometric measurement with the forced change in intraocular pressure during the water drinking test. Ocular Response Analyser (Reichert) was used to the measurement. Besides four basic parameters connected with intraocular pressure (IOPg, IOPcc) and biomechanics (corneal hysteresis CH and corneal resistance factor (CRF), other parameters representing the behaviour of the cornea during a puff of air were analysed. There were 47 eyes included in the study, including 27 eyes with a XEN GelStent implanted and 20 without it. The eyes of people with monocular implementation were the reference group. The values of analysed parameters were compared before and after 10, 25, 40, and 55 min after drinking the water. The intraocular pressure increased by 2.4 mmHg (p < 0.05) for eyes with a XEN stent and 2.2 mmHg for eyes without a stent (p < 0.05) in the tenth minute after drinking of water. This change caused a decreasing of corneal hysteresis (p < 0.05) without significant changes in the corneal resistance factor (p > 0.05). Corneal hysteresis changed similarly in the reference group and the group with a XEN GelStent. The analysis of additional parameters showed a difference in the behaviour of the cornea in eyes with a XEN GelStent in comparison to the corneas of eyes without a stent. This was particularly visible in the analysis of the cornea's behaviour during the second applanation, when the cornea returns to its baseline state after deformation caused by air puff tonometry.

Allogeneic Anti-BCMA CAR T Cells Are Superior to Multiple Myeloma-derived CAR T Cells in Preclinical Studies and May Be Combined with Gamma Secretase Inhibitors.

Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however, all patients ultimately still progress from their disease. Lack of CAR T-cell persistence, impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile, fitness, and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts, higher CD4/CD8 ratio, and expanded naïve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production, patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells, decreased central memory phenotype, and increased checkpoint inhibitory markers compared with HD-derived products, which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly, HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion, allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. Significance: Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately, patients still relapse. In this study, we propose to use T cells from HD volunteers, which have a stronger T-cell fitness, higher cancer killing capacity, and are ready to be administered when needed.

Clinical Pharmacology and Determinants of Response to UCART19, an Allogeneic Anti-CD19 CAR-T Cell Product, in Adult B-cell Acute Lymphoblastic Leukemia.

Background: UCART19 is an "off-the-shelf" genome-edited anti-CD19 chimeric antigen receptor (CAR)-T cell product, manufactured from unrelated healthy donor cells. Methods: UCART19 was administered to 25 adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) in the CALM trial. All patients underwent lymphodepletion with fludarabine and cyclophosphamide ± alemtuzumab and received one of three ascending doses of UCART19. Given the allogeneic nature of UCART19, we analyzed the impact of lymphodepletion, HLA disparities, and host immune system reconstitution on its kinetics, along with other factors known to affect autologous CAR-T cell clinical pharmacology. Results: Responder patients (12/25) had higher UCART19 expansion (C max) and exposure (AUCTlast) than nonresponders (13/25), as measured by transgene levels in peripheral blood. The persistence of CAR+ T cells did not exceed 28 days in 10/25 patients and lasted beyond 42 days in 4/25. No significant correlation was found between UCART19 kinetics and administered cell dose, patient and product characteristics or HLA disparities. However, the number of prior lines of therapy and absence of alemtuzumab negatively impacted UCART19 expansion and persistence. Alemtuzumab exposure positively affected IL7 and UCART19 kinetics, while negatively correlating with host T lymphocyte AUC0-28. Conclusions: UCART19 expansion is a driver of response in adult patients with R/R B-ALL. These results shed light on the factors associated with UCART19 kinetics, which remain highly affected by the impact of alemtuzumab on IL7 and host-versus-graft rejection. Significance: First description of the clinical pharmacology of a genome-edited allogeneic anti-CD19 CAR-T cell product showing the crucial role of an alemtuzumab-based regimen in sustaining UCART19 expansion and persistence through increased IL7 availability and decreased host T lymphocyte population.

Innate-like Gene Expression of Lung-Resident Memory CD8+ T Cells during Experimental Human Influenza: A Clinical Study.

Rationale: Suboptimal vaccine immunogenicity and antigenic mismatch, compounded by poor uptake, means that influenza remains a major global disease. T cells recognizing peptides derived from conserved viral proteins could enhance vaccine-induced cross-strain protection. Objectives: To investigate the kinetics, phenotypes, and function of influenza virus-specific CD8+ resident memory T (Trm) cells in the lower airway and infer the molecular pathways associated with their response to infection in vivo. Methods: Healthy volunteers, aged 18-55, were inoculated intranasally with influenza A/California/4/09(H1N1). Blood, upper airway, and (in a subgroup) lower airway samples were obtained throughout infection. Symptoms were assessed by using self-reported diaries, and the nasal viral load was assessed by using quantitative PCR. T-cell responses were analyzed by using a three-color FluoroSpot assay, flow cytometry with MHC I-peptide tetramers, and RNA sequencing, with candidate markers being confirmed by using the immunohistochemistry results for endobronchial biopsy specimens. Measurements and Main Results: After challenge, 57% of participants became infected. Preexisting influenza-specific CD8+ T cells in blood correlated strongly with a reduced viral load, which peaked at Day 3. Influenza-specific CD8+ T cells in BAL fluid were highly enriched and predominantly expressed the Trm markers CD69 and CD103. Comparison between preinfection CD8+ T cells in BAL fluid and blood by using RNA sequencing revealed 3,928 differentially expressed genes, including all major Trm-cell markers. However, gene set enrichment analysis of BAL-fluid CD8+ T cells showed primarily innate cell-related pathways and, during infection, included upregulation of innate chemokines (Cxcl1, Cxcl10, and Cxcl16) that were also expressed by CD8+ cells in bronchial tissues. Conclusions: CD8+ Trm cells in the human lung display innate-like gene and protein expression that demonstrates blurred divisions between innate and adaptive immunity. Clinical study registered with www.clinicaltrials.gov (NCT02755948).

Detection of respiratory syncytial virus defective genomes in nasal secretions is associated with distinct clinical outcomes.

Respiratory syncytial virus (RSV) causes respiratory illness in children, immunosuppressed individuals and the elderly. However, the viral factors influencing the clinical outcome of RSV infections remain poorly defined. Defective viral genomes (DVGs) can suppress virus replication by competing for viral proteins and by stimulating antiviral immunity. We studied the association between detection of DVGs of the copy-back type and disease severity in three RSV A-confirmed cohorts. In hospitalized children, detection of DVGs in respiratory samples at or around the time of admission associated strongly with more severe disease, higher viral load and a stronger pro-inflammatory response. Interestingly, in experimentally infected adults, the presence of DVGs in respiratory secretions differentially associated with RSV disease severity depending on when DVGs were detected. Detection of DVGs early after infection associated with low viral loads and mild disease, whereas detection of DVGs late after infection, especially if DVGs were present for prolonged periods, associated with high viral loads and severe disease. Taken together, we demonstrate that the kinetics of DVG accumulation and duration could predict clinical outcome of RSV A infection in humans, and thus could be used as a prognostic tool to identify patients at risk of worse clinical disease.

How a dynamic optical system maintains image quality: Self-adjustment of the human eye.

The eyeball is continually subjected to forces that cause alterations to its shape and dimensions, as well as to its optical components. Forces that induce accommodation result in an intentional change in focus; others, such as the effect of intraocular pressure fluctuations, are more subtle. Although the mechanical properties of the eyeball and its components permit mediation of such subtle forces, the concomitant optical changes are not detected by the visual system. Optical self-adjustment is postulated as the mechanism that maintains image quality. The purpose of this study was to investigate how self-adjustment occurs by using an optical model of the eyeball and to test the requisite optical and biometric conditions.

Short-Time Changes of Intraocular Pressure and Biomechanics of the Anterior Segment of the Eye during Water Drinking Test in Patients with XEN GelStent.

PURPOSE: Little is known about short-term changes in intraocular pressure (IOP) following minimally invasive glaucoma surgeries, such as post-XEN GelStent implantation. Although the importance of corneal biomechanics in glaucoma diagnostics has been reported, little work has been conducted on postoperative description of changes when the structure of the anterior segment is altered. The aim of presented study was to evaluate the changes in the biomechanical parameters of the anterior segment of the post-XEN GelStent implantation eyes. PATIENTS AND METHODS: This investigator-initiated, open-label, prospective, single-center study recruited patients. Patients with primary open-angle glaucoma (POAG) after XEN GelStent implantation versus matched POAG controls (considered as control group/CG) treated pharmacologically were screened. Water loading was conducted using 10 mL of water per kilogram of body weight for ≤5 min. Goldmann applanation tonometry (GAT), corneal hysteresis (CH), and corneal resistance factor (CRF) were performed before water loading and after every 15 min up to 1 h. RESULTS: The water drinking test (WDT) was positive in 3.7% (1 out of 27) of patients in the post-XEN group compared with 22.7% (5 out of 22) of patients in the control group (CG; p < 0.05). Mean fluctuations in GAT during the WDT were higher in the CG group (3.6 ± 2.5 mmHg vs. 2.9 ± 1.3 mmHg, p < 0.001). CRF and CH changed significantly only in the post-XEN group. The mean peak of CH and CRF occurred at 15 and 30 min of the test in the post-XEN group (p = 0.001). CONCLUSION: WDT is important to assess the ability of compensation mechanisms to reduce fluctuations in IOP after water upload. The relationship between biomechanics of the anterior segment and glaucoma may have substantial impact on surgical outcome evaluation.

Genome-edited, donor-derived allogeneic anti-CD19 chimeric antigen receptor T cells in paediatric and adult B-cell acute lymphoblastic leukaemia: results of two phase 1 studies.

BACKGROUND: Genome-edited donor-derived allogeneic anti-CD19 chimeric antigen receptor (CAR) T cells offer a novel form of CAR-T-cell product that is available for immediate clinical use, thereby broadening access and applicability. UCART19 is one such product investigated in children and adults with relapsed or refractory B-cell acute lymphoblastic leukaemia. Two multicentre phase 1 studies aimed to investigate the feasibility, safety, and antileukaemic activity of UCART19 in children and adults with relapsed or refractory B-cell acute lymphoblastic leukaemia. METHODS: We enrolled paediatric or adult patients in two ongoing, multicentre, phase 1 clinical trials to evaluate the safety and antileukaemic activity of UCART19. All patients underwent lymphodepletion with fludarabine and cyclophosphamide with or without alemtuzumab, then children received UCART19 at 1·1-2·3 × 106 cells per kg and adults received UCART19 doses of 6 × 106 cells, 6-8 × 107 cells, or 1·8-2·4 × 108 cells in a dose-escalation study. The primary outcome measure was adverse events in the period between first infusion and data cutoff. These studies were registered at ClinicalTrials.gov, NCT02808442 and NCT02746952. FINDINGS: Between June 3, 2016, and Oct 23, 2018, seven children and 14 adults were enrolled in the two studies and received UCART19. Cytokine release syndrome was the most common adverse event and was observed in 19 patients (91%); three (14%) had grade 3-4 cytokine release syndrome. Other adverse events were grade 1 or 2 neurotoxicity in eight patients (38%), grade 1 acute skin graft-versus-host disease in two patients (10%), and grade 4 prolonged cytopenia in six patients (32%). Two treatment-related deaths occurred; one caused by neutropenic sepsis in a patient with concurrent cytokine release syndrome and one from pulmonary haemorrhage in a patient with persistent cytopenia. 14 (67%) of 21 patients had a complete response or complete response with incomplete haematological recovery 28 days after infusion. Patients not receiving alemtuzumab (n=4) showed no UCART19 expansion or antileukaemic activity. The median duration of response was 4·1 months with ten (71%) of 14 responders proceeding to a subsequent allogeneic stem-cell transplant. Progression-free survival at 6 months was 27%, and overall survival was 55%. INTERPRETATION: These two studies show, for the first time, the feasibility of using allogeneic, genome-edited CAR T cells to treat patients with aggressive leukaemia. UCART19 exhibited in-vivo expansion and antileukaemic activity with a manageable safety profile in heavily pretreated paediatric and adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. The results this study are an encouraging step forward for the field of allogeneic CAR T cells, and UCART19 offers the opportunity to treat patients with rapidly progressive disease and where autologous CAR-T-cell therapy is unavailable. FUNDING: Servier.

Neutrophilic inflammation in the respiratory mucosa predisposes to RSV infection.

The variable outcome of viral exposure is only partially explained by known factors. We administered respiratory syncytial virus (RSV) to 58 volunteers, of whom 57% became infected. Mucosal neutrophil activation before exposure was highly predictive of symptomatic RSV disease. This was associated with a rapid, presymptomatic decline in mucosal interleukin-17A (IL-17A) and other mediators. Conversely, those who resisted infection showed presymptomatic activation of IL-17- and tumor necrosis factor-related pathways. Vulnerability to infection was not associated with baseline microbiome but was reproduced in mice by preinfection chemokine-driven airway recruitment of neutrophils, which caused enhanced disease mediated by pulmonary CD8+ T cell infiltration. Thus, mucosal neutrophilic inflammation at the time of RSV exposure enhances susceptibility, revealing dynamic, time-dependent local immune responses before symptom onset and explaining the as-yet unpredictable outcomes of pathogen exposure.

Organ Donation in Intensive Care Units of Hospitals in Warsaw.

BACKGROUND: In recent years a systematic decrease in donation activity in Poland (15.4 vs 13.0) has been observed. A significant reduction has been noticed in Warsaw (36.2 vs 19.2) as well. METHODS: Data on deaths of patients admitted to intensive care units (ICUs) of Warsaw hospitals in from 2014 to 2018 were analyzed. Analysis was conducted in compliance with the Helsinki Congress and the Istanbul Declaration. Population of the city during this period averaged 1,753,480, although specialized capital hospitals service substantially bigger area than Warsaw alone. There are 18,500 to 18,600 deaths affecting this population every year. A total of 333 patients diagnosed as having brain death were included in analysis. RESULTS: In 42 cases (12.7%) data collection was given up because of lack of authorization. In all donors, death was diagnosed according to neurologic criteria. The most common causes were vascular brain diseases (64.1%) and craniocerebral trauma (21.4%). In 14.5% brain death occurred from other reasons. There are 20 hospitals with ICUs in Warsaw, which totals 318 intensive care beds. Program of identification of deceased organ donors was active in only 14 hospitals. A total of 243 potential donors (73%) were identified in the 3 most active hospitals. CONCLUSION: Analysis of ICU deaths of Warsaw hospitals showed a gradual decrease in the number of reported donors (from 75 in 2014 to 46 in 2018), although the number of all deaths did not decrease (the number of deaths in ICUs was on average 2.571/y).

Impact of Blood Loss and Intraoperative Blood Transfusion During Liver Transplantation on the Incidence of Early Biliary Complications and Mortality.

Biliary complications are one of the most serious and dangerous complications following liver transplantation. Factors that may determine their occurrence are still being assessed. The retrospective analysis of 239 consecutive liver transplantations (LT) performed between January 2013 and December 2018 was conducted in compliance with the Helsinki Congress and the Istanbul Declaration. We divided recipients into 2 groups depending on whether biliary complications occurred. The first (biliary complication [BC group]) consisted of patients who developed biliary complications (n = 41) and the second (nonbiliary complications [NBC group]) without them (n = 198). Demographic and statistical data analysis showed no differences between the groups in terms of age, Model for End-Stage Liver Disease with sodium serum concentration (MELD-Na) score, and average cold or warm ischemia time. In comparison, estimated intraoperative blood loss, 1341 mL in the NBC and 1399 mL in the BC, was not significantly different, as were the number of transfused red blood cells (RBC) units, which were respectively 1.7 and 2.1 (P = ns). The recipients' hemoglobin levels just before surgery were (11.5 g/dL vs 11.6 g/dL; P = ns) and after transplantation (9.8 g/dL vs 9.8 g/dL; P = ns). Eleven patients died within 30 days of transplantation. This group was characterized by a higher MELD-Na score (25 vs 17; P = .01), lower pretransplant hemoglobin level (10 g/dL vs 11.6 g/dL; P = .02), and the number of transfused RBC units (3.3 vs 1.7; P = .01). However, there was no correlation between intraoperative blood loss, the number of transfused RBC units, pre- and postoperative hemoglobin levels, and the incidence of biliary complications after LT. Lower pretransplant hemoglobin levels and a higher amount of intraoperatively transfused blood products were associated with a higher fatality rate after LT.

Epitope-specific airway-resident CD4+ T cell dynamics during experimental human RSV infection.

BACKGROUNDRespiratory syncytial virus (RSV) is an important cause of acute pulmonary disease and one of the last remaining major infections of childhood for which there is no vaccine. CD4+ T cells play a key role in antiviral immunity, but they have been little studied in the human lung.METHODSHealthy adult volunteers were inoculated i.n. with RSV A Memphis 37. CD4+ T cells in blood and the lower airway were analyzed by flow cytometry and immunohistochemistry. Bronchial soluble mediators were measured using quantitative PCR and MesoScale Discovery. Epitope mapping was performed by IFN-γ ELISpot screening, confirmed by in vitro MHC binding.RESULTSActivated CD4+ T cell frequencies in bronchoalveolar lavage correlated strongly with local C-X-C motif chemokine 10 levels. Thirty-nine epitopes were identified, predominantly toward the 3' end of the viral genome. Five novel MHC II tetramers were made using an immunodominant EFYQSTCSAVSKGYL (F-EFY) epitope restricted to HLA-DR4, -DR9, and -DR11 (combined allelic frequency: 15% in Europeans) and G-DDF restricted to HLA-DPA1*01:03/DPB1*02:01 and -DPA1*01:03/DPB1*04:01 (allelic frequency: 55%). Tetramer labeling revealed enrichment of resident memory CD4+ T (Trm) cells in the lower airway; these Trm cells displayed progressive differentiation, downregulation of costimulatory molecules, and elevated CXCR3 expression as infection evolved.CONCLUSIONSHuman infection challenge provides a unique opportunity to study the breadth of specificity and dynamics of RSV-specific T-cell responses in the target organ, allowing the precise investigation of Trm recognizing novel viral antigens over time. The new tools that we describe enable precise tracking of RSV-specific CD4+ cells, potentially accelerating the development of effective vaccines.TRIAL REGISTRATIONClinicalTrials.gov NCT02755948.FUNDINGMedical Research Council, Wellcome Trust, National Institute for Health Research.

Monitoring Allogeneic CAR-T Cells Using Flow Cytometry.

Chimeric antigen receptor T cell (CAR-T) therapies have now entered mainstream clinical practice with two approved autologous CAR-T products targeting CD19 and numerous other products in early and late phase clinical trials. This has led to a demand for highly sensitive, specific, and easily reproducible methods to monitor CAR-T cells in patients. Here we describe a flow cytometry based protocol for detection of allogeneic CAR-T cells and for monitoring their phenotype and numbers in blood and bone marrow of patients following CAR-T treatment.