Diverse potential of secretome from natural killer cells and monocyte-derived macrophages in activating stellate cells.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-ß1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Can an integrated intervention package including peer support increase the proportion of health facility births? A cluster randomised controlled trial in Northern Uganda.

OBJECTIVE: To assess the effect of an integrated intervention package compared with routine government health services on the frequency of health facility births. SETTING: Three subcounties of Lira district in Northern Uganda. DESIGN: A cluster randomised controlled trial where a total of 30 clusters were randomised in a ratio of 1:1 to intervention or standard of care. PARTICIPANTS: Pregnant women at ≥28 weeks of gestation. INTERVENTIONS: Participants in the intervention arm received an integrated intervention package of peer support, mobile phone messaging and birthing kits during pregnancy while those in the control arm received routine government health services ('standard of care'). PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was the proportion of women giving birth at a health facility in the intervention arm compared with the control arm. Secondary outcomes were perinatal and neonatal deaths. RESULTS: In 2018-2019, 995 pregnant women were included in 15 intervention clusters and 882 in 15 control clusters. The primary outcome was ascertained for all except one participant who died before childbirth. In the intervention arm, 754/994 participants (76%) gave birth at a health facility compared with 500/882 (57%) in the control arm. Participants in the intervention arm were 35% more likely to give birth at a health facility compared with participants in the control arm, (risk ratio 1.35 (95% CI 1.20 to 1.51)) and (risk difference 0.20 (95% CI 0.13 to 0.27)). Adjusting for baseline differences generated similar results. There was no difference in secondary outcomes (perinatal or neonatal mortality or number of postnatal visits) between arms. CONCLUSION: The intervention was successful in increasing the proportion of facility-based births but did not reduce perinatal or neonatal mortality. TRIAL REGISTRATION NUMBER: NCT02605369.

Changes in nutrient composition and diet quality in the first 6 months following bariatric surgery: An observational cohort study.

BACKGROUND: Bariatric surgery (BS) may result in inadequate nutrient intake and poor diet quality, which can lead to nutritional complications. The present study aimed to evaluate changes in macro- and micronutrient composition and diet quality in the first 6 months following BS. METHODS: One hundred seven participants undergoing BS (Roux-en-Y gastric bypass: n = 87, sleeve gastrectomy: n = 20) completed 3-day food records before and 6 months after surgery. Changes in energy, macronutrient (carbohydrates, protein, fat, dietary fibre) and micronutrient intake (folate, vitamin B12, vitamin D, calcium, iron) were evaluated. Diet quality was assessed by adherence to the Dutch food-based dietary guidelines. RESULTS: After BS, we observed a significant decrease in intake of energy and all macro- and micronutrients (p < 0.01 for all), except for calcium (-39.0 ± 404.6 mg; p = 0.32). Overall, nutrient composition slightly changed with an increase in the relative intake of protein (+1.1 ± 4.3 energy percentage [en%]; p = 0.01) and mono- and disaccharides (+4.2 ± 6.4 en%; p < 0.001) post-surgery. Consumption (median [Q1, Q3]) of vegetables (-50 [-120, 6] g day-1 ), wholegrain products (-38 [-81, -8] g day-1 ), liquid fats (-5 [-13, 2] g day-1 ), red meat (-3 [-30, 4] g day-1 ), processed meat (-32 [-55, 13] g day-1 ), sodium (-0.7 [-1.1, -0.2] g day-1 ) and unhealthy food choices (-2.4 [-5.0, 0.6] serves week-1 ) significantly decreased after BS (p < 0.01 for all). CONCLUSIONS: Our results demonstrate both favourable and unfavourable changes in macro- and micronutrient composition and diet quality in the first 6 months following BS. Insight into these changes can improve dietary counselling in this population. Future research into underlying causes, consequences and long-term changes in dietary intake is needed.

An optimized protocol for isolation of hepatic leukocytes retrieved from murine and NASH liver biopsies.

Immune dysregulation and inflammation by hepatic-resident leukocytes is considered a key step in disease progression of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis toward cirrhosis and hepatocellular carcinoma. Here, we provide a protocol for isolation and characterization of liver-resident immune cells from fine-needle biopsies obtained from a rodent model and humans. We describe steps for isolating leukocytes, cell sorting, and RNA extraction and sequencing. We then detail procedures for low-input mRNA sequencing analyses.

Factors influencing delayed discharge after day-surgery laparoscopic cholecystectomy: the DeDiLaCo study protocol.

OBJECTIVE: Laparoscopic cholecystectomy (LC) is the gold standard for most benign gallbladder diseases. Early discharge (<24 hours) has the same outcomes as longer (>24 hours) hospital stay. Nevertheless, the rate of delayed discharge >24 hours range from 4.6% to 37%. The primary endpoint of this Italian nationwide study is to analyze the prevalence of patients undergoing elective LC who experienced a delayed discharge >24 hours and identify potential limiting factors of early discharge. Results from these analyses will be used to select patients who can be safely discharged on the same day after surgery. Secondary endpoints will be to evaluate the patient's quality of life (QoL), assess the direct health costs associated with late discharge, and quantify the patient's involvement in the treatment process. PATIENTS AND METHODS: This prospective, observational study was conducted following a resident-led model and the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines. All patients were treated according to the local hospital protocol and received routine care as standard therapy. RESULTS: We expected to obtain the enrollment of at least 500 patients based on an assumed difference in discharge delay between the reference and the recruitable population of 6% and the identification of factors related to discharge failure within 24 h. Early discharge after LC leads to advantages both in terms of clinical outcomes and quality of life of the patient, and it is highly effective in terms of health costs and shortening the waiting list. However, clinical reality differs from the results of randomized studies by a complex series of non-objectionable real-world data influencing treatment plans. Therefore, we expected to identify independent predictors and factors of failure of early discharge. CONCLUSIONS: Clinical reality often differs from randomized trial results. In Italy, the vast majority of delayed discharges after LC may not be related to surgery and can be prevented both with logistical reorganization and with a readjustment of the trust reimbursement policies.

Myelin dysfunction drives amyloid-ß deposition in models of Alzheimer's disease.

The incidence of Alzheimer's disease (AD), the leading cause of dementia, increases rapidly with age, but why age constitutes the main risk factor is still poorly understood. Brain ageing affects oligodendrocytes and the structural integrity of myelin sheaths1, the latter of which is associated with secondary neuroinflammation2,3. As oligodendrocytes support axonal energy metabolism and neuronal health4-7, we hypothesized that loss of myelin integrity could be an upstream risk factor for neuronal amyloid-ß (Aß) deposition, the central neuropathological hallmark of AD. Here we identify genetic pathways of myelin dysfunction and demyelinating injuries as potent drivers of amyloid deposition in mouse models of AD. Mechanistically, myelin dysfunction causes the accumulation of the Aß-producing machinery within axonal swellings and increases the cleavage of cortical amyloid precursor protein. Suprisingly, AD mice with dysfunctional myelin lack plaque-corralling microglia despite an overall increase in their numbers. Bulk and single-cell transcriptomics of AD mouse models with myelin defects show that there is a concomitant induction of highly similar but distinct disease-associated microglia signatures specific to myelin damage and amyloid plaques, respectively. Despite successful induction, amyloid disease-associated microglia (DAM) that usually clear amyloid plaques are apparently distracted to nearby myelin damage. Our data suggest a working model whereby age-dependent structural defects of myelin promote Aß plaque formation directly and indirectly and are therefore an upstream AD risk factor. Improving oligodendrocyte health and myelin integrity could be a promising target to delay development and slow progression of AD.

Botulinum toxin infusion into the mesenteric artery has selective action on peristalsis in a rat model: experimental research.

OBJECTIVE: Botulinum toxin type A (BoNT/A) reversibly blocks neurotransmission at voluntary and autonomic cholinergic nerve terminals, inducing paralysis. The aim of this study was to block panenteric peristalsis in rats through BoNT/A administration into the superior mesenteric artery (SMA) and to understand whether the toxin's action is selectively restricted to the perfused territory. MATERIALS AND METHODS: Rats were infused through a 0.25-mm surgically inserted SMA catheter with different doses of BoNT/A (10 U, 20 U, 40 U BOTOX®, Allergan Inc.) or with saline for 24 h. Animals were free to move on an unrestricted diet. As a sign of bowel peristalsis impairment, body weight and oral/water intake were collected for 15 days. Statistical analysis was conducted with nonlinear mixed effects models to study the variation over time of the response variables. In three 40 U-treated rats, the selectivity of the intra-arterial delivered toxin action was studied by examining bowel and voluntary muscle samples and checking the presence of BoNT/A-cleaved SNAP-25 (the smoking gun of the toxin action) using the Immunofluorescence (IF) method through a specific antibody recognition. RESULTS: While control rats exhibited an increasing body weight, treated rats showed an initial dose-dependent weight reduction (p<0.001 control vs. treated) with recovery after Day 11 for 10 and 20 U-treated rats. Food and water intake over time showed significantly different half-saturation constants with rats treated with higher doses who reached half of the maximum achievable in a greater number of days (p<0.0001 control vs. treated rats). BoNT/A-cleaved SNAP-25 was identified in bowel wall NMJs and not in voluntary muscles, demonstrating the remarkable selectivity of arterially infused BoNT/A. CONCLUSIONS: Blockade of intestinal peristalsis, can be induced in rats by slow infusion of BoNT/A into the SMA. The effect is long-lasting, dose-dependent and selective. BoNT/A delivery into the SMA through a percutaneous catheter could prove clinically useful in the treatment of entero-atmospheric fistula by temporarily reducing fistula output.

Probiotic Lactobacillus rhamnosus EM1107 prevents hyperglycemia, alveolar bone loss, and inflammation in a rat model of diabetes and periodontitis.

BACKGROUND: This study evaluated the antihyperglycemic, anti-bone-resorptive, and anti-inflammatory efficacy of the probiotic Lactobacillus rhamnosus EM1107 in an experimental model of ligature-induced periodontitis in diabetic rats treated with metformin (Met). METHODS: A total of 114 male Wistar rats was randomly divided into six groups: (1) control, (2) experimental periodontitis (EP), (3) EP + diabetes mellitus (DM), (4) EP + probiotic (Prob), (5) EP + DM + Prob, and (6) EP + DM + Prob + Met. The animals received probiotic gavage during the 30 days of the experiment. DM was induced on the 14th day of the experiment with a single injection of streptozotocin into the penile vein, followed by ligature for EP induction and Met gavage on the 19th day and euthanasia on the 30th day. Heart blood, gingival and periodontal tissue, and hemimaxillae were collected. Biomolecular analysis, immunoenzymatic assays, histomorphology, and microtomographic analysis were performed. Data were statistically analyzed (p < 0.05). RESULTS: There was a significant reduction in interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the Prob groups (p < 0.05) as well as in blood glucose levels in the Prob and Met groups (p < 0.001). In addition, histomorphological analysis revealed that the Prob groups had a reduction in inflammatory infiltrate. Tartrate-resistant acid phosphatase (TRAP) and microtomographic analyses showed that the EP/DM/Prob/Met group had significantly lower linear and volumetric bone loss than those who had no treatment (p < 0.01). SOD and GPx immunostaining decreased in all groups receiving probiotics. CONCLUSION: The findings suggest the immunoinflammatory efficacy of the probiotic L. rhamnosus EM1107 administered either alone or in association with Met in type 1 DM associated with periodontitis.

Low-Dose rIL-15 Protects from Nephrotoxic Serum Nephritis via CD8+ T Cells.

Rapid progressive glomerulonephritis (GN) often leads to end-stage kidney disease, driving the need for renal replacement therapy and posing a global health burden. Low-dose cytokine-based immunotherapies provide a new strategy to treat GN. IL-15 is a strong candidate for the therapy of immune-mediated kidney disease since it has proven to be tubular-protective before. Therefore, we set out to test the potential of low-dose rIL-15 treatment in a mouse model of nephrotoxic serum nephritis (NTS), mimicking immune complex-driven GN in humans. A single low-dose treatment with rIL-15 ameliorated NTS, reflected by reduced albuminuria, less tissue scarring, fewer myeloid cells in the kidney, and improved tubular epithelial cell survival. In addition, CD8+ T cells, a primary target of IL-15, showed altered gene expression and function corresponding with less cytotoxicity mediated by rIL-15. With the use of transgenic knock-out mice, antibody depletion, and adoptive cell transfer studies, we here show that the beneficial effects of rIL-15 treatment in NTS depended on CD8+ T cells, suggesting a pivotal role for them in the underlying mechanism. Our findings add to existing evidence of the association of IL-15 with kidney health and imply a potential for low-dose rIL-15 immunotherapies in GN.

Nutritional Deficiencies 3 Years After Sleeve Gastrectomy Can Be Limited by a Specialized Multivitamin Supplement.

PURPOSE: Lifelong daily multivitamin supplementation is highly recommended after sleeve gastrectomy (SG). Based on previous research, a specialized multivitamin supplement (MVS) for SG patients was developed and optimized (WLS Optimum 1.0 and 2.0). This study presents its mid-term effectives and compares micronutrient status of SG patients using this specialized MVS to users of standard MVS (sMVS) and non-users of multivitamin supplementation during the first three years post-surgery. MATERIALS AND METHODS: Of the 226 participants that were included at baseline, yearly follow-up blood tests were completed by 193 participants (85%) at 12 months, 176 participants (78%) at 24 months, and 140 participants (62%) at 36 months of follow-up. At each time point, participants were divided into four groups: (1) Optimum 1.0, (2) Optimum 2.0, (3) sMVS, and (4) non-users. Serum concentrations (linear mixed-effects models) and the prevalence of micronutrient deficiencies (chi-square tests) during follow-up were compared between the groups. RESULTS: Users of specialized MVS (Optimum 1.0 and 2.0) had higher serum concentrations of hemoglobin, folic acid, and vitamin D compared to sMVS users and non-users during follow-up. Serum concentrations of vitamin B12 and (corrected) calcium were also higher in specialized MVS users than in non-users. Overall, fewer deficiencies for folic acid and vitamin D were observed in the Optimum groups. CONCLUSION: Although the perfect multivitamin supplement for all SG patients does not exist, WLS Optimum was more effective in sustaining normal serum concentrations than standard, over-the-counter supplementation. Non-users of MVS presented with most micronutrient deficiencies and will evidently develop poor nutritional status on the longer term.

Brain activities of streptozotocin-induced diabetic Wistar rats treated with gliclazide: Behavioural, biochemical and histomorphology studies.

Gliclazide (GLD), a sulphonylurea is efficacious in the treatment of diabetes type-2. However, there is limited information on its activity in the brain, especially in diabetics. This research investigated the brain activities of GLD following streptozotocin-induced diabetes in Wistar rats. Twenty five adult male Wistar rats (200-250 g) were grouped (n = 5) as: Control (distilled water, 5 mL/kg) and GLD (150 mg/kg) groups; and the diabetic groups, untreated streptozotocin (STZ, 35 mg/kg), and STZ (35 mg/kg) treated with GLD (150 mg/kg) for two and four weeks, and already on high fat diet. The animals' body weights and blood glucose levels were checked weekly. After the experimental duration, spontaneous alternation and novel object recognition tests were carried out and the animals sacrificed. Perfusion with phosphate buffered saline preceded brain excision for biochemical analyses, with halves fixed in 10% neutral buffered formalin for histology. Compared with the control, results showed (p < 0.05) declined spontaneous alternation and exploratory activities with no preference for familiar or novel objects, body weights loss, raised blood glucose, increased malondialdehyde with decreased superoxide dismutase concentrations, and no apparent adverse effect on hippocampal and prefrontal cortical Nissl substance in the untreated diabetic group. The adverse observations were attenuated in the GLD treated diabetic groups; although the spontaneous alternation in the four weeks GLD treated diabetic group improved (p < 0.05), exploration of objects increased (p < 0.05) without preference. The present results showed that treatment with GLD for two and four weeks mitigated STZ activities, even though there was less improvement in neurocognitive activities.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Introducing the brain erythropoietin circle to explain adaptive brain hardware upgrade and improved performance.

PREFACE: Executive functions, learning, attention, and processing speed are imperative facets of cognitive performance, affected in neuropsychiatric disorders. In clinical studies on different patient groups, recombinant human (rh) erythropoietin (EPO) lastingly improved higher cognition and reduced brain matter loss. Correspondingly, rhEPO treatment of young rodents or EPO receptor (EPOR) overexpression in pyramidal neurons caused remarkable and enduring cognitive improvement, together with enhanced hippocampal long-term potentiation. The 'brain hardware upgrade', underlying these observations, includes an EPO induced ~20% increase in pyramidal neurons and oligodendrocytes in cornu ammonis hippocampi in the absence of elevated DNA synthesis. In parallel, EPO reduces microglia numbers and dampens their activity and metabolism as prerequisites for undisturbed EPO-driven differentiation of pre-existing local neuronal precursors. These processes depend on neuronal and microglial EPOR. This novel mechanism of powerful postnatal neurogenesis, outside the classical neurogenic niches, and on-demand delivery of new cells, paralleled by dendritic spine increase, let us hypothesize a physiological procognitive role of hypoxia-induced endogenous EPO in brain, which we imitate by rhEPO treatment. Here we delineate the brain EPO circle as working model explaining adaptive 'brain hardware upgrade' and improved performance. In this fundamental regulatory circle, neuronal networks, challenged by motor-cognitive tasks, drift into transient 'functional hypoxia', thereby triggering neuronal EPO/EPOR expression.

A Quantitative Study of Particle Dispersion due to Respiratory Support Modalities in PC-12 Aircraft: Prehospital Patient Transport.

OBJECTIVE: It is unclear whether supplemental oxygen and noninvasive ventilation respiratory support devices increase the dispersion of potentially infectious bioaerosols in a pressurized air medical cabin. This study quantitatively compared particle dispersion from respiratory support modalities in an air medical cabin during flight. METHODS: Dispersion was measured in a fixed wing air ambulance during flight with a breathing medical mannequin simulator exhaling nebulized saline from the lower respiratory tract with the following respiratory support modalities: a nasal cannula with a surgical mask, high-flow nasal oxygen (HFNO) with a surgical mask, and noninvasive bilevel positive airway pressure (BiPAP) ventilation. RESULTS: Nasal cannula oxygen with a surgical mask was associated with the highest particle concentrations. In the absence of mask seal leaks, BiPAP was associated with 1 order of magnitude lower particle concentration compared with a nasal cannula with a surgical mask. Particle concentrations associated with HFNO with a surgical mask were lower than a nasal cannula with a surgical mask but higher than BiPAP. CONCLUSIONS: Particle dispersion associated with the use of BiPAP and HFNO with a surgical mask is lower than nasal cannula oxygen with a surgical mask. These findings may assist air medical organizations with operational decisions where little data exist about respiratory particle dispersion.

Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 ß-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem.

While Extended-Spectrum ß-Lactamases (ESBL) and AmpC ß-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding proteins, thereby inhibiting their activity. Further, it has been shown that Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC ß-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the Escherichia coli porins OmpC and OmpF, its chromosomal AmpC ß-lactamase, and the plasmid-mediated CMY-2 ß-lactamase. Bla CMY-2-like positive E. coli isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like ß-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant E. coli.

B Cell Composition Is Altered After Kidney Transplantation and Transitional B Cells Correlate With SARS-CoV-2 Vaccination Response.

BACKGROUND: The COVID-19 pandemic has major implications on kidney transplant recipients (KTRs) since they show increased mortality due to impaired immune responses to SARS-CoV-2 infection and a reduced efficacy of SARS-CoV-2 vaccination. Surprisingly, dialysis patients have shown superior seroconversion rates after vaccination compared to KTRs. Therefore, we investigated peripheral blood B cell (BC) composition before and after kidney transplantation (KT) and aimed to screen the BC compartment to explain impaired antibody generation. METHODS: A total of 105 patients were recruited, and multicolor flow cytometric phenotyping of peripheral venous blood BC subpopulations was performed before and 1 year after KT. Complete follow-up was available for 71 individuals. Anti-SARS-CoV-2 antibodies were collected retrospectively and were available for 40 subjects, who had received two doses of an mRNA-based vaccine (BNT162b2 or mRNA-1273). RESULTS: Overall, relative BC frequencies within lymphocytes decreased, and their absolute counts trended in the same direction 1 year after KT as compared to CKD G5 patients. Frequencies and absolute numbers of naïve BCs remained stable. Frequencies of double negative BCs, a heterogeneous subpopulation of antigen experienced BCs lacking CD27 expression, were increased after KT, yet their absolute counts were similar at both time points. Transitional BCs (TrBCs) and plasmablasts were significantly reduced after KT in absolute and relative terms. Memory BCs were affected differently since class-switched and IgM-only subsets decreased after KT, but unswitched and IgD-only memory BCs remained unchanged. CD86+ and CD5+ expression on BCs was downregulated after KT. Correlational analysis revealed that TrBCs were the only subset to correlate with titer levels after SARS-CoV-2 vaccination. Responders showed higher TrBCs, both absolute and relative, than non-responders. CONCLUSION: Together, after 1 year, KTRs showed persistent and profound compositional changes within the BC compartment. Low TrBCs, 1 year after KT, may account for the low serological response to SARS-CoV-2 vaccination in KTRs compared to dialysis patients. Our findings need confirmation in further studies as they may guide vaccination strategies.

NMDAR1 autoantibodies amplify behavioral phenotypes of genetic white matter inflammation: a mild encephalitis model with neuropsychiatric relevance.

Encephalitis has an estimated prevalence of ≤0.01%. Even with extensive diagnostic work-up, an infectious etiology is identified or suspected in <50% of cases, suggesting a role for etiologically unclear, noninfectious processes. Mild encephalitis runs frequently unnoticed, despite slight neuroinflammation detectable postmortem in many neuropsychiatric illnesses. A widely unexplored field in humans, though clearly documented in rodents, is genetic brain inflammation, particularly that associated with myelin abnormalities, inducing primary white matter encephalitis. We hypothesized that "autoimmune encephalitides" may result from any brain inflammation concurring with the presence of brain antigen-directed autoantibodies, e.g., against N-methyl-D-aspartate-receptor NR1 (NMDAR1-AB), which are not causal of, but may considerably shape the encephalitis phenotype. We therefore immunized young female Cnp-/- mice lacking the structural myelin protein 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) with a "cocktail" of NMDAR1 peptides. Cnp-/- mice exhibit early low-grade inflammation of white matter tracts and blood-brain barrier disruption. Our novel mental-time-travel test disclosed that Cnp-/- mice are compromised in what-where-when orientation, but this episodic memory readout was not further deteriorated by NMDAR1-AB. In contrast, comparing wild-type and Cnp-/- mice without/with NMDAR1-AB regarding hippocampal learning/memory and motor balance/coordination revealed distinct stair patterns of behavioral pathology. To elucidate a potential contribution of oligodendroglial NMDAR downregulation to NMDAR1-AB effects, we generated conditional NR1 knockout mice. These mice displayed normal Morris water maze and mental-time-travel, but beam balance performance was similar to immunized Cnp-/-. Immunohistochemistry confirmed neuroinflammation/neurodegeneration in Cnp-/- mice, yet without add-on effect of NMDAR1-AB. To conclude, genetic brain inflammation may explain an encephalitic component underlying autoimmune conditions.

Methylene Blue not Contraindicated in Treating Hemodynamic Instability in Pediatric and Neonate Patients.

The present review was carried out to describe publications on the use of methylene blue (MB) in pediatrics and neonatology, discussing dose, infusion rate, action characteristics, and possible benefits for a pediatric patient group. The research was performed on the data sources PubMed, BioMed Central, and Embase (updated on Aug 31, 2020) by two independent investigators. The selected articles included human studies that evaluated MB use in pediatric or neonatal patients with vasoplegia due to any cause, regardless of the applied methodology. The MB use and 0 to 18-years-old patients with vasodilatory shock were the adopted criteria. Exclusion criteria were the use of MB in patients without vasoplegia and patients ≥ 18-years-old. The primary endpoint was the increase in mean arterial pressure (MAP). Side effects and dose were also evaluated. Eleven studies were found, of which 10 were case reports, and 1 was a randomized clinical study. Only two of these studies were with neonatal patients (less than 28 days-old), reporting a small number of cases (1 and 6). All studies described the positive action of MB on MAP, allowing the decrease of vasoactive amines in several of them. No severe side effects or death related to the use of the medication were reported. The maximum dose used was 2 mg/kg, but there was no consensus on the infusion rate and drug administration timing. Finally, no theoretical or experimental basis sustains the decision to avoid MB in children claiming it can cause pulmonary hypertension. The same goes for the concern of a possible deleterious effect on inflammatory distress syndrome.