Early postnatal high-dose fat-soluble enteral vitamin A supplementation for moderate or severe bronchopulmonary dysplasia or death in extremely low birthweight infants (NeoVitaA): a multicentre, randomised, parallel-group, double-blind, placebo-controlled, investigator-initiated phase 3 trial.

BACKGROUND: Vitamin A plays a key role in lung development, but there is no consensus regarding the optimal vitamin A dose and administration route in extremely low birthweight (ELBW) infants. We aimed to assess whether early postnatal additional high-dose fat-soluble enteral vitamin A supplementation versus placebo would lower the rate of moderate or severe bronchopulmonary dysplasia or death in ELBW infants receiving recommended basic enteral vitamin A supplementation. METHODS: This prospective, multicentre, randomised, parallel-group, double-blind, placebo-controlled, investigator-initiated phase 3 trial conducted at 29 neonatal intensive care units in Austria and Germany assessed early high-dose enteral vitamin A supplementation (5000 international units [IU]/kg per day) or placebo (peanut oil) for 28 days in ELBW infants. Eligible infants had a birthweight of more than 400 g and less than 1000 g; gestational age at birth of 32+0 weeks postmenstrual age or younger; and the need for mechanical ventilation, non-invasive respiratory support, or supplemental oxygen within the first 72 h of postnatal age after admission to the neonatal intensive care unit. Participants were randomly assigned by block randomisation with variable block sizes (two and four). All participants received basic vitamin A supplementation (1000 IU/kg per day). The composite primary endpoint was moderate or severe bronchopulmonary dysplasia or death at 36 weeks postmenstrual age, analysed in the intention-to-treat population. This trial was registered with EudraCT, 2013-001998-24. FINDINGS: Between March 2, 2015, and Feb 27, 2022, 3066 infants were screened for eligibility at the participating centres. 915 infants were included and randomly assigned to the high-dose vitamin A group (n=449) or the control group (n=466). Mean gestational age was 26·5 weeks (SD 2·0) and mean birthweight was 765 g (162). Moderate or severe bronchopulmonary dysplasia or death occurred in 171 (38%) of 449 infants in the high-dose vitamin A group versus 178 (38%) of 466 infants in the control group (adjusted odds ratio 0·99, 95% CI 0·73-1·55). The number of participants with at least one adverse event was similar between groups (256 [57%] of 449 in the high-dose vitamin A group and 281 [60%] of 466 in the control group). Serum retinol concentrations at baseline, at the end of intervention, and at 36 weeks postmenstrual age were similar in the two groups. INTERPRETATION: Early postnatal high-dose fat-soluble enteral vitamin A supplementation in ELBW infants was safe, but did not change the rate of moderate or severe bronchopulmonary dysplasia or death and did not substantially increase serum retinol concentrations. FUNDING: Deutsche Forschungsgemeinschaft and European Clinical Research Infrastructures Network (ECRIN).

Detailed statistical analysis plan for ALBINO: effect of Allopurinol in addition to hypothermia for hypoxic-ischemic Brain Injury on Neurocognitive Outcome - a blinded randomized placebo-controlled parallel group multicenter trial for superiority (phase III).

BACKGROUND: Despite therapeutic hypothermia (TH) and neonatal intensive care, 45-50% of children affected by moderate-to-severe neonatal hypoxic-ischemic encephalopathy (HIE) die or suffer from long-term neurodevelopmental impairment. Additional neuroprotective therapies are sought, besides TH, to further improve the outcome of affected infants. Allopurinol - a xanthine oxidase inhibitor - reduced the production of oxygen radicals and subsequent brain damage in pre-clinical and preliminary human studies of cerebral ischemia and reperfusion, if administered before or early after the insult. This ALBINO trial aims to evaluate the efficacy and safety of allopurinol administered immediately after birth to (near-)term infants with early signs of HIE. METHODS/DESIGN: The ALBINO trial is an investigator-initiated, randomized, placebo-controlled, double-blinded, multi-national parallel group comparison for superiority investigating the effect of allopurinol in (near-)term infants with neonatal HIE. Primary endpoint is long-term outcome determined as survival with neurodevelopmental impairment versus death versus non-impaired survival at 2 years. RESULTS: The primary analysis with three mutually exclusive responses (healthy, death, composite outcome for impairment) will be on the intention-to-treat (ITT) population by a generalized logits model according to Bishop, Fienberg, Holland (Bishop YF, Discrete Multivariate Analysis: Therory and Practice, 1975) and ."will be stratified for the two treatment groups. DISCUSSION: The statistical analysis for the ALBINO study was defined in detail in the study protocol and implemented in this statistical analysis plan published prior to any data analysis. This is in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. TRIAL REGISTRATION: ClinicalTrials.gov NCT03162653. Registered on 22 May 2017.

MICA Procedure vs Open Chevron Osteotomy for Hallux Valgus Correction: A Prospective Cohort Study.

Background: Currently, more than 150 surgical techniques have been described for the treatment of hallux valgus. The abundance of techniques indicates that there is no technique that has been designated as a gold standard. In recent years, a particular interest in the use of minimally invasive techniques has grown. The aim of this study was to prospectively compare clinical, radiologic, and postoperative outcomes between the MICA technique and open chevron technique over a 1-year follow-up period. Methods: Between January 2016 and August 2020, data were prospectively collected from consecutive patients preoperatively and at 6 weeks, 3 months, and 12 months following minimally invasive chevron and Akin (MICA) or open chevron osteotomies. Radiographic outcomes were measured using weightbearing radiographs preoperatively and at 3 and 12 months postoperatively. Clinical outcomes were measured using the American Orthopaedic Foot & Ankle Society (AOFAS), Manchester-Oxford Foot Questionnaire (MOXFQ), VAS (visual analog scale), Foot Function Index (FFI), Foot and Ankle Outcome Score (FAOS), and Euro-QoL-5D (EQ5D) questionnaires. Results: Of the 68 patients, 42 patients (62%) underwent a MICA surgery and 26 patients (38%) underwent open chevron osteotomy. Both groups showed significant improvement in HVA, IMA, and DMAA at the 1-year follow-up. Our findings show that both clinical and radiologic outcomes of the MICA technique are comparable to the conventional open technique. No significant differences were found in clinical outcomes (VAS, AOFAS, MOXFQ, FFI, and FAOS), complication rate, and operative times. Conclusion: These results show that MICA is a safe alternative for chevron osteotomy. The clinical and radiologic outcomes of these 2 techniques by 12 months are comparable. Level of Evidence: Level II, prospective cohort study.

Immune signatures of checkpoint inhibitor-induced autoimmunity-A focus on neurotoxicity.

BACKGROUND: Neurologic immune-related adverse events (irAE-n) are rare but severe toxicities of immune checkpoint inhibitor (ICI) treatment. To overcome diagnostic and therapeutic challenges, a better mechanistic understanding of irAE-n is paramount. METHODS: In this observational cohort study, we collected serum and peripheral blood samples from 34 consecutive cancer patients with irAE-n (during acute illness) and 49 cancer control patients without irAE-n (pre- and on-ICI treatment, n = 44 without high-grade irAEs, n = 5 with high-grade nonneurologic irAEs). Patients received either anti-programmed cell death protein (PD)-1 or anti-PD ligand-1 monotherapy or anti-PD-1/anti-cytotoxic T-lymphocyte-associated protein-4 combination therapy. Most common cancers were melanoma, lung cancer, and hepatocellular carcinoma. Peripheral blood immune profiling was performed using 48-marker single-cell mass cytometry and a multiplex cytokine assay. RESULTS: During acute illness, patients with irAE-n presented higher frequencies of cluster of differentiation (CD)8+ effector memory type (EM-)1 and central memory (CM) T cells compared to controls without irAEs. Multiorgan immunotoxicities (neurologic + nonneurologic) were associated with higher CD8+ EM1 T cell counts. While there were no B cell changes in the overall cohort, we detected a marked decrease of IgD- CD11c+ CD21low and IgD- CD24+ CD21high B cells in a subgroup of patients with autoantibody-positive irAE-n. We further identified signatures indicative of enhanced chemotaxis and inflammation in irAE-n patients and discovered C-X-C motif chemokine ligand (CXCL)10 as a promising marker to diagnose high-grade immunotoxicities such as irAE-n. CONCLUSIONS: We demonstrate profound and partly subgroup-specific immune cell dysregulation in irAE-n patients, which may guide future biomarker development and targeted treatment approaches.

Perioperative pain management for cleft palate surgery: a systematic review and procedure-specific postoperative pain management (PROSPECT) recommendations.

BACKGROUND/IMPORTANCE: Cleft palate surgery is associated with significant postoperative pain. Effective pain control can decrease stress and agitation in children undergoing cleft palate surgery and improve surgical outcomes. However, limited evidence often results in inadequate pain control after cleft palate surgery. OBJECTIVES: The aim of this review was to evaluate the available evidence and to develop recommendations for optimal pain management after cleft palate surgery using procedure-specific postoperative pain management (PROSPECT) methodology. EVIDENCE REVIEW: MEDLINE, Embase, and Cochrane Databases were searched for randomized controlled trials and systematic reviews assessing pain in children undergoing cleft palate repair published in English language from July 2002, through August 2023. FINDINGS: Of 1048 identified studies, 19 randomized controlled trials and 4 systematic reviews met the inclusion criteria. Interventions that improved postoperative pain, and are recommended, include suprazygomatic maxillary nerve block or palatal nerve block (if maxillary nerve block cannot be performed). Addition of dexmedetomidine to local anesthetic for suprazygomatic maxillary nerve block or, alternatively, as intravenous administration perioperatively is recommended. These interventions should be combined with a basic analgesic regimen including acetaminophen and nonsteroidal anti-inflammatory drugs. Of note, pre-incisional local anesthetic infiltration and dexamethasone were administered as a routine in several studies, however, because of limited procedure-specific evidence their contribution to pain relief after cleft palate surgery remains unknown. CONCLUSION: The present review identified an evidence-based analgesic regimen for cleft palate surgery in pediatric patients. PROSPERO REGISTRATION NUMBER: CRD42022364788.

Choline and Betaine Levels in Plasma Mirror Choline Intake in Very Preterm Infants.

Choline is essential for cell membrane formation and methyl transfer reactions, impacting parenchymal and neurological development. It is therefore enriched via placental transfer, and fetal plasma concentrations are high. In spite of the greater needs of very low birth weight infants (VLBWI), choline content of breast milk after preterm delivery is lower (median (p25-75): 158 mg/L (61-360 mg/L) compared to term delivery (258 mg/L (142-343 mg/L)). Even preterm formula or fortified breast milk currently provide insufficient choline to achieve physiological plasma concentrations. This secondary analysis of a randomized controlled trial comparing growth of VLBWI with different levels of enteral protein supply aimed to investigate whether increased enteral choline intake results in increased plasma choline, betaine and phosphatidylcholine concentrations. We measured total choline content of breast milk from 33 mothers of 34 VLBWI. Enteral choline intake from administered breast milk, formula and fortifier was related to the respective plasma choline, betaine and phosphatidylcholine concentrations. Plasma choline and betaine levels in VLBWI correlated directly with enteral choline intake, but administered choline was insufficient to achieve physiological (fetus-like) concentrations. Hence, optimizing maternal choline status, and the choline content of milk and fortifiers, is suggested to increase plasma concentrations of choline, ameliorate the choline deficit and improve growth and long-term development of VLBWI.

Allogeneic stem cell transplant for multiple myeloma & myelofibrosis with split-dose busulfan, fludarabine & cyclophosphamide.

Allogeneic stem cell transplant can have high morbidity and mortality in patients with myelofibrosis (MF) and multiple myeloma (MM). This phase 2 study used a novel myeloablative regimen of split-dose busulfan, fludarabine, and then post-transplant cyclophosphamide. Four patients with MF and 2 with MM were enrolled. At 1 year, non-relapse mortality was 33.3%, and overall survival was 50%. Incidence of acute and chronic GVHD was 33.3% and 16.7%, respectively. Those surviving beyond 1 year (MF = 1, MM = 2) had durable remissions with a median follow-up of 42 months. This small study demonstrates relative safety & favorable key outcomes using this novel approach.

SARS-CoV-2 specific plasma cells acquire long-lived phenotypes in human bone marrow.

BACKGROUND: SARS-CoV-2 specific antibody-secreting plasma cells (PC) mediating specific humoral immunity have been identified in the human bone marrow (BM) after COVID-19 or vaccination against SARS-CoV-2. However, it remained unclear whether or not they acquire phenotypes of human memory plasma cells. METHODS: SARS-CoV-2-specific human bone marrow plasma cells (BMPC) were characterised by tetramer-based, antigen-specific flow cytometry and FluoroSpot assay. FINDINGS: SARS-CoV-2 spike-S1-specific PC were detectable in all tested BM samples of previously vaccinated individuals, representing 0.22% of total BMPC. The majority of SARS-CoV-2-specific BMPC expressed IgG and their specificity for the spike S1 protein indicated emergence from a systemic vaccination response. Of note, one-fifth of SARS-CoV-2-specific BMPC showed the phenotype of memory plasma cells, i.e., downregulated CD19 and present or absent CD45 expression. INTERPRETATION: Our data indicate the establishment of phenotypically diverse SARS-CoV-2-specific PC in the human BM after basic mRNA immunization, including the formation of memory phenotypes. These results suggest the induction of durable humoral immunity after basic mRNA vaccination against SARS-CoV-2. FUNDING: The study was supported by funding by the DFG grants TRR130 TP24, ME 3644/8-1, and the Berlin Senate. SR received funding from DFGSFB-1444 C01 Central Service Project.

Peripheral Blood Immune Cell Composition After Autologous MSC Infusion in Kidney Transplantation Recipients.

Tacrolimus is the backbone of immunosuppressive agents to prevent transplant rejection. Paradoxically, tacrolimus is nephrotoxic, causing irreversible tubulointerstitial damage. Therefore, infusion of mesenchymal stromal cells (MSC) 6 and 7 weeks post-transplantation was assessed to facilitate withdrawal of tacrolimus in the randomized phase II TRITON trial. Here, we performed detailed analysis of the peripheral blood immune composition using mass cytometry to assess potential effects of MSC therapy on the immune system. We developed two metal-conjugated antibody panels containing 40 antibodies each. PBMC samples from 21 MSC-treated patients and 13 controls, obtained pre-transplant and at 24 and 52 weeks post-transplantation, were analyzed. In the MSC group at 24 weeks, 17 CD4+ T cell clusters were increased of which 14 Th2-like clusters and three Th1/Th2-like clusters, as well as CD4+FoxP3+ Tregs. Additionally, five B cell clusters were increased, representing either class switched memory B cells or proliferating B cells. At 52 weeks, CCR7+CD38+ mature B cells were decreased. Finally, eight Tc1 (effector) memory cytotoxic T cell clusters were increased. Our work provides a comprehensive account of the peripheral blood immune cell composition in kidney transplant recipients after MSC therapy and tacrolimus withdrawal. These results may help improving therapeutic strategies using MSCs with the aim to reduce the use of calcineurin inhibitors. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02057965.

Spatially Resolved Multi-Omics Single-Cell Analyses Inform Mechanisms of Immune Dysfunction in Pancreatic Cancer.

BACKGROUND & AIMS: As pancreatic ductal adenocarcinoma (PDAC) continues to be recalcitrant to therapeutic interventions, including poor response to immunotherapy, albeit effective in other solid malignancies, a more nuanced understanding of the immune microenvironment in PDAC is urgently needed. We aimed to unveil a detailed view of the immune micromilieu in PDAC using a spatially resolved multimodal single-cell approach. METHODS: We applied single-cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry, and mass cytometry to profile the immune compartment in treatment-naïve PDAC tumors and matched adjacent normal pancreatic tissue, as well as in the systemic circulation. We determined prognostic associations of immune signatures and performed a meta-analysis of the immune microenvironment in PDAC and lung adenocarcinoma on single-cell level. RESULTS: We provided a spatially resolved fine map of the immune landscape in PDAC. We substantiated the exhausted phenotype of CD8 T cells and immunosuppressive features of myeloid cells, and highlighted immune subsets with potentially underappreciated roles in PDAC that diverged from immune populations within adjacent normal areas, particularly CD4 T cell subsets and natural killer T cells that are terminally exhausted and acquire a regulatory phenotype. Differential analysis of immune phenotypes in PDAC and lung adenocarcinoma revealed the presence of extraordinarily immunosuppressive subtypes in PDAC, along with a distinctive immune checkpoint composition. CONCLUSIONS: Our study sheds light on the multilayered immune dysfunction in PDAC and presents a holistic view of the immune landscape in PDAC and lung adenocarcinoma, providing a comprehensive resource for functional studies and the exploration of therapeutically actionable targets in PDAC.

[Systematic risk management for a planned gas shutdown in the high-care facility of a university hospital]. / Systematisches Risikomanagement für eine geplante Gasabschaltung im High-Care-Bereich eines Universitätsklinikums.

BACKGROUND: In the course of building extension works at Dresden University Hospital, it was necessary to shut down the central medical gas supply in a building with 3 intensive care wards with 22 beds, an operating theater tract with 5 operating rooms and 6 normal wards each with 28 beds during ongoing services. Thus, for the construction phase there was a need to establish an interim decentralized gas supply with zero failure tolerance for the affected functional units . METHODS: Following established procedures for possible risk and failure analysis, a project group was set up by the hospital's emergency and disaster management officer to develop a project plan, a needs assessment and a communication plan. RESULTS: A variety of risk factors were systematically identified for which appropriate countermeasures needed to be designed. The needs assessment over 4 h based on physiological parameters for the maximum available 22 ventilator beds resulted in 26,000 l of oxygen and 26,000 l of compressed air. A total of 7 supply points were each equipped with two 50l cylinders for both oxygen and compressed air, with a total availability of 175,000 l of each of the 2 gases. Another eight cylinders each were held in reserve. The project was carried out on a Saturday without an elective surgery program, so that the operating rooms concerned could be closed. The timing was chosen so that double staffing of intensive care personnel was available during the afternoon shift change. In advance, as many of the patients on mechanical ventilation as possible were transferred within the hospital; however, nine of the mechanically ventilated patients had to remain. The technical intervention in the gas supply lasted only 2 h without affecting the patient's condition. During the 2­h interim supply, 16,500 l of compressed air and 8000 l of oxygen were consumed on the high-care wards. The calculated hourly consumption per ventilated patient was 917 l of air (15 l/min) and 444 l of oxygen (7 l/min). The quantity framework based on empirical values from intensive care medicine was significantly lower. This was more than compensated for by the 10-fold stocking of gas and the predictably lower number of ventilated patients than the maximum occupancy used as a basis. CONCLUSION: For technical interventions in high-risk areas, careful planning and execution in an effective team is required. Established procedures of project management and risk assessment help to avoid errors.

Different choline supplement metabolism in adults using deuterium labelling.

BACKGROUND: Choline deficiency leads to pathologies particularly of the liver, brain and lung. Adequate supply is important for preterm infants and patients with cystic fibrosis. We analysed the assimilation of four different enterally administered deuterium-labelled (D9-) choline supplements in adults. METHODS: Prospective randomised cross-over study (11/2020-1/2022) in six healthy men, receiving four single doses of 2.7 mg/kg D9-choline equivalent each in the form of D9-choline chloride, D9-phosphorylcholine, D9-alpha-glycerophosphocholine (D9-GPC) or D9-1-palmitoyl-2-oleoyl-glycero-3-phosphoryl-choline (D9-POPC), in randomised order 6 weeks apart. Plasma was obtained at baseline (t = - 0.1 h) and at 0.5 h to 7d after intake. Concentrations of D9-choline and its D9-labelled metabolites were analysed by tandem mass spectrometry. Results are shown as median and interquartile range. RESULTS: Maximum D9-choline and D9-betaine concentrations were reached latest after D9-POPC administration versus other components. D9-POPC and D9-phosphorylcholine resulted in lower D9-trimethylamine (D9-TMAO) formation. The AUCs (0-7d) of plasma D9-PC concentration showed highest values after administration of D9-POPC. D9-POPC appeared in plasma after fatty acid remodelling, predominantly as D9-1-palmitoyl-2-linoleyl-PC (D9-PLPC), confirming cleavage to 1-palmitoyl-lyso-D9-PC and re-acylation with linoleic acid as the most prominent alimentary unsaturated fatty acid. CONCLUSION: There was a delayed increase in plasma D9-choline and D9-betaine after D9-POPC administration, with no differences in AUC over time. D9-POPC resulted in a higher AUC of D9-PC and virtually absent D9-TMAO levels. D9-POPC is remodelled according to enterocytic fatty acid availability. D9-POPC seems best suited as choline supplement to increase plasma PC concentrations, with PC as a carrier of choline and targeted fatty acid supply as required by organs. This study was registered at Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020498, 22.01.2020. STUDY REGISTRATION: This study was registered at Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020498.

Choline supplementation for preterm infants: metabolism of four Deuterium-labeled choline compounds.

BACKGROUND: Supply of choline is not guaranteed in current preterm infant nutrition. Choline serves in parenchyma formation by membrane phosphatidylcholine (PC), plasma transport of poly-unsaturated fatty acids (PUFA) via PC, and methylation processes via betaine. PUFA-PC concentrations are high in brain, liver and lung, and deficiency may result in developmental disorders. We compared different deuterated (D9-) choline components for kinetics of D9-choline, D9-betaine and D9-PC. METHODS: Prospective study (1/2021-12/2021) in 32 enterally fed preterm infants (28 0/7-32 0/7 weeks gestation). Patients were randomized to receive enterally a single dose of 2.7 mg/kg D9-choline-equivalent as D9-choline chloride, D9-phosphoryl-choline, D9-glycerophosphorylcholine (D9-GPC) or D9-1-palmitoyl-2-oleoyl-PC(D9-POPC), followed by blood sampling at 1 + 24 h or 12 + 60 h after administration. Plasma concentrations were analyzed by tandem mass spectrometry. Results are expressed as median (25th/75th percentile). RESULTS: At 1 h, plasma D9-choline was 1.8 (0.9/2.2) µmol/L, 1.3 (0.9/1.5) µmol/L and 1.2 (0.7/1.4) µmol/L for D9-choline chloride, D9-GPC and D9-phosphoryl-choline, respectively. D9-POPC did not result in plasma D9-choline. Plasma D9-betaine was maximal at 12 h, with lowest concentrations after D9-POPC. Maximum plasma D9-PC values at 12 h were the highest after D9-POPC (14.4 (9.1/18.9) µmol/L), compared to the other components (D9-choline chloride: 8.1 [5.6/9.9] µmol/L; D9-GPC: 8.4 (6.2/10.3) µmol/L; D9-phosphoryl-choline: 9.8 (8.6/14.5) µmol/L). Predominance of D9-PC comprising linoleic, rather than oleic acid, indicated fatty-acyl remodeling of administered D9-POPC prior to systemic delivery. CONCLUSION: D9-Choline chloride, D9-GPC and D9-phosphoryl-choline equally increased plasma D9-choline and D9-betaine. D9-POPC shifted metabolism from D9-betaine to D9-PC. Combined supplementation of GPC and (PO) PC may be best suited to optimize choline supply in preterm infants. Due to fatty acid remodeling of (PO) PC during its assimilation, PUFA co-supplementation with (PO) PC may increase PUFA-delivery to critical organs. This study was registered (22.01.2020) at the Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020502. STUDY REGISTRATION: This study was registered at the Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020502.

Resident memory CD4+ T lymphocytes mobilize from bone marrow to contribute to a systemic secondary immune reaction.

Resident memory T lymphocytes (TRM ) of epithelial tissues and the Bm protect their host tissue. To what extent these cells are mobilized and contribute to systemic immune reactions is less clear. Here, we show that in secondary immune reactions to the measles-mumps-rubella (MMR) vaccine, CD4+ TRM are mobilized into the blood within 16 to 48 h after immunization in humans. This mobilization of TRM is cognate: TRM recognizing other antigens are not mobilized, unless they cross-react with the vaccine. We also demonstrate through methylome analyses that TRM are mobilized from the Bm. These mobilized cells make significant contribution to the systemic immune reaction, as evidenced by their T-cell receptor Vß clonotypes represented among the newly generated circulating memory T-cells, 14 days after vaccination. Thus, TRM of the Bm confer not only local, but also systemic immune memory.

The volume-regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling.

The volume-regulated anion channel (VRAC) is formed by LRRC8 proteins and is responsible for the regulatory volume decrease (RVD) after hypotonic cell swelling. Besides chloride, VRAC transports other molecules, for example, immunomodulatory cyclic dinucleotides (CDNs) including 2'3'cGAMP. Here, we identify LRRC8C as a critical component of VRAC in T cells, where its deletion abolishes VRAC currents and RVD. T cells of Lrrc8c-/- mice have increased cell cycle progression, proliferation, survival, Ca2+ influx and cytokine production-a phenotype associated with downmodulation of p53 signaling. Mechanistically, LRRC8C mediates the transport of 2'3'cGAMP in T cells, resulting in STING and p53 activation. Inhibition of STING recapitulates the phenotype of LRRC8C-deficient T cells, whereas overexpression of p53 inhibits their enhanced T cell function. Lrrc8c-/- mice have exacerbated T cell-dependent immune responses, including immunity to influenza A virus infection and experimental autoimmune encephalomyelitis. Our results identify cGAMP uptake through LRRC8C and STING-p53 signaling as a new inhibitory signaling pathway in T cells and adaptive immunity.

Complement activation induces excessive T cell cytotoxicity in severe COVID-19.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Differential metabolism of choline supplements in adult volunteers.

BACKGROUND: Adequate intake of choline is essential for growth and homeostasis, but its supply does often not meet requirements. Choline deficiency decreases phosphatidylcholine (PC) and betaine synthesis, resulting in organ pathology, especially of liver, lung, and brain. This is of particular clinical importance in preterm infants and cystic fibrosis patients. We compared four different choline supplements for their impact on plasma concentration and kinetics of choline, betaine as a methyl donor and trimethylamine oxide (TMAO) as a marker of bacterial degradation prior to absorption. METHODS: Prospective randomized cross-over study (1/2020-4/2020) in six healthy adult men. Participants received a single dose of 550 mg/d choline equivalent in the form of choline chloride, choline bitartrate, α-glycerophosphocholine (GPC), and egg-PC in randomized sequence at least 1 week apart. Blood was taken from t = - 0.1-6 h after supplement intake. Choline, betaine, TMAO, and total PC concentrations were analyzed by tandem mass spectrometry. Results are shown as medians and interquartile range. RESULTS: There was no difference in the AUC of choline plasma concentrations after intake of the different supplements. Individual plasma kinetics of choline and betaine differed and concentrations peaked latest for PC (at ≈3 h). All supplements similarly increased plasma betaine. All water-soluble supplements rapidly increased TMAO, whereas egg-PC did not. CONCLUSION: All supplements tested rapidly increased choline and betaine levels to a similar extent, with egg-PC showing the latest peak. Assuming that TMAO may have undesirable effects, egg-PC might be best suited for choline supplementation in adults. STUDY REGISTRATION: This study was registered at "Deutsches Register Klinischer Studien" (DRKS) (German Register for Clinical Studies), 17.01.2020, DRKS00020454.

Pharmacokinetic/Pharmacodynamic Modelling of Allopurinol, its Active Metabolite Oxypurinol, and Biomarkers Hypoxanthine, Xanthine and Uric Acid in Hypoxic-Ischemic Encephalopathy Neonates.

BACKGROUND: Allopurinol, an xanthine oxidase (XO) inhibitor, is a promising intervention that may provide neuroprotection for neonates with hypoxic-ischemic encephalopathy (HIE). Currently, a double-blind, placebo-controlled study (ALBINO, NCT03162653) is investigating the neuroprotective effect of allopurinol in HIE neonates. OBJECTIVE: The aim of the current study was to establish the pharmacokinetics (PK) of allopurinol and oxypurinol, and the pharmacodynamics (PD) of both compounds on hypoxanthine, xanthine, and uric acid in HIE neonates. The dosage used and the effect of allopurinol in this population, either or not undergoing therapeutic hypothermia (TH), were evaluated. METHODS: Forty-six neonates from the ALBINO study and two historical clinical studies were included. All doses were administered on the first day of life. In the ALBINO study (n = 20), neonates received a first dose of allopurinol 20 mg/kg, and, in the case of TH (n = 13), a second dose of allopurinol 10 mg/kg. In the historical cohorts (n = 26), neonates (all without TH) received two doses of allopurinol 20 mg/kg in total. Allopurinol and oxypurinol population PK, and their effects on inhibiting conversions of hypoxanthine and xanthine to uric acid, were assessed using nonlinear mixed-effects modelling. RESULTS: Allopurinol and oxypurinol PK were described by two sequential one-compartment models with an autoinhibition effect on allopurinol metabolism by oxypurinol. For allopurinol, clearance (CL) was 0.83 L/h (95% confidence interval [CI] 0.62-1.09) and volume of distribution (Vd) was 2.43 L (95% CI 2.25-2.63). For metabolite oxypurinol, CL and Vd relative to a formation fraction (fm) were 0.26 L/h (95% CI 0.23-0.3) and 11 L (95% CI 9.9-12.2), respectively. No difference in allopurinol and oxypurinol CL was found between TH and non-TH patients. The effect of allopurinol and oxypurinol on XO inhibition was described by a turnover model of hypoxanthine with sequential metabolites xanthine and uric acid. The combined allopurinol and oxypurinol concentration at the half-maximal XO inhibition was 0.36 mg/L (95% CI 0.31-0.42). CONCLUSION: The PK and PD of allopurinol, oxypurinol, hypoxanthine, xanthine, and uric acid in neonates with HIE were described. The dosing regimen applied in the ALBINO trial leads to the targeted XO inhibition in neonates treated with or without TH.

Either IL-7 activation of JAK-STAT or BEZ inhibition of PI3K-AKT-mTOR pathways dominates the single-cell phosphosignature of ex vivo treated pediatric T-cell acute lymphoblastic leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While TALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALL is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here we used single-cell mass cytometry to study signal transduction pathways such as JAK-STAT, PI3K-AKT-mTOR and MEK-ERK in 16 diagnostic and five relapsed T-ALL primary samples, and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKT-mTOR and MEK-ERK pathways and increased expression of proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy.