Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T-cell interactions.

COVID-19 can cause severe neurological symptoms, but the underlying pathophysiological mechanisms are unclear. Here, we interrogated the brain stems and olfactory bulbs in postmortem patients who had COVID-19 using imaging mass cytometry to understand the local immune response at a spatially resolved, high-dimensional, single-cell level and compared their immune map to non-COVID respiratory failure, multiple sclerosis, and control patients. We observed substantial immune activation in the central nervous system with pronounced neuropathology (astrocytosis, axonal damage, and blood-brain-barrier leakage) and detected viral antigen in ACE2-receptor-positive cells enriched in the vascular compartment. Microglial nodules and the perivascular compartment represented COVID-19-specific, microanatomic-immune niches with context-specific cellular interactions enriched for activated CD8+ T cells. Altered brain T-cell-microglial interactions were linked to clinical measures of systemic inflammation and disturbed hemostasis. This study identifies profound neuroinflammation with activation of innate and adaptive immune cells as correlates of COVID-19 neuropathology, with implications for potential therapeutic strategies.

Exhaustion of CD39-Expressing CD8+ T Cells in Crohn's Disease Is Linked to Clinical Outcome.

BACKGROUND & AIMS: Exhaustion of CD8 T cells has been suggested to inform different clinical outcomes in Crohn's disease, but detailed analyses are lacking. This study aimed to identify the role of exhaustion on a single-cell level and identify relevant CD8 T cell populations in Crohn's disease. METHODS: Blood and intestinal tissue from 58 patients with Crohn's disease (active disease or remission) were assessed for CD8 T cell expression of exhaustion markers and their cytokine profile by highly multiplexed flow and mass cytometry. Key disease-associated subsets were sorted and analyzed by RNA sequencing. CD39 inhibition assays were performed in vitro. RESULTS: Activated CD39+ and CD39+PD-1+ CD8 T cell subsets expressing multiple exhaustion markers were enriched at low frequency in active Crohn's disease. Their cytokine production capacity was inversely linked to the Harvey-Bradshaw Index. Subset-level protein and transcriptome profiling revealed co-existence of effector and exhaustion programs in CD39+ and CD39+ PD-1+CD8 T cells, with CD39+ cells likely originating from the intestine. CD39 enzymatic activity controlled T cell cytokine production. Importantly, transcriptional exhaustion signatures were enriched in remission in CD39-expressing subsets with up-regulation of TOX. Subset-level transcriptomics revealed a CD39-related gene module that is associated with the clinical course. CONCLUSIONS: These data showed a role for the exhaustion of peripheral CD39-expressing CD8 T cell subsets in Crohn's disease. Their low frequency illustrated the utility of single-cell cytometry methods for identification of relevant immune populations. Importantly, the link of their exhaustion status to the clinical activity and their specific gene signatures have implications for exhaustion-based personalized medicine approaches.

Nonlinear Effects in Asymmetric Catalysis by Design: Concept, Synthesis, and Applications.

Asymmetric synthesis constitutes a key technology for the preparation of enantiomerically pure compounds as well as for the selective control of individual stereocenters in the synthesis of complex compounds. It is thus of extraordinary importance for the synthesis of chiral drugs, dietary supplements, flavors, and fragrances, as well as novel materials with tunable and reconfigurable chiroptical properties or the assembly of complex natural products. Typically, enantiomerically pure catalysts are used for this purpose. To prepare enantiomerically pure ligands or organocatalysts, one can make use of the natural chiral pool. Ligands and organocatalysts with an atropisomeric biphenyl and binaphthyl system have become popular, as they are configurationally stable and contain a C2-symmetric skeleton, which has been found to be particularly privileged. For catalysts with opposite configurations, both product enantiomers can be obtained. Configurationally flexible biphenyl systems initially appeared to be unsuitable for this purpose, as they racemize after successful enantiomer separation and thus are neither storable nor afford a reproducible enantioselectivity. However, there are strategies that exploit the dynamics of such ligands to stereoconvergently enrich one of the catalyst enantiomers. This can be achieved, for example, by coordinating an enantiomerically pure additive to a ligand-metal complex, which results in deracemization of the configurationally flexible biphenyl system, thereby enriching the thermodynamically preferred diastereomer. In this Account, we present our strategy to design stereochemically flexible catalysts that combine the properties of supramolecular recognition, stereoconvergent alignment, and catalysis. Such systems are capable to recognize the chirality of the target product, leading to an increase in enantioselectivity during asymmetric catalysis. We have systematically developed and investigated these smart catalyst systems and have found ways to specifically design and synthesize them for various applications. In addition to (i) reaction product-induced chiral amplification, we have developed systems with (ii) intermolecular and (iii) intramolecular recognition, and successfully applied them in asymmetric catalysis. Our results pave the way for new applications such as temperature-controlled enantioselectivity, controlled inversion of enantioselectivity with the same chirality of the recognition unit, generation of positive nonlinear effects, and targeted design of autocatalytic systems through dynamic formation of transient catalysts. Understanding such systems is of enormous importance for catalytic processes leading to symmetry breaking and amplification of small imbalances of enantiomers and offer a possible explanation of homochirality of biological systems. In addition, we are learning how to target supramolecular interactions to enhance enantioselectivities in asymmetric catalysis through secondary double stereocontrol. Configurationally flexible catalysts will enable future resource-efficient development of asymmetric syntheses, as enantioselectivities can be fully switched by stereoselective alignment of the stereochemically flexible ligand core on demand.

Synthesis and stereodynamics of intramolecular hemiacetals in biaryl aldehyde-alcohols.

Soai's asymmetric autocatalysis represents a highly remarkable example for spontaneous symmetry breaking and enantioselective amplification in the enantioselective alkylation of pyrimidine-5-carbaldehydes to the corresponding chiral pyrimidine alcohols. Recently, zinc hemiacetalate complexes, formed from pyrimidine-5-carbaldehydes and the chiral product alcohol, were identified by in situ high-resolution mass spectrometric measurements as highly active transient asymmetric catalysts in this autocatalytic transformation. To study the formation of such hemiacetals and their stereodynamic properties, we focused on the synthesis of coumarin homolog biaryl systems with carbaldehyde and alcohol substituents. Such systems are able to form hemiacetals by intramolecular cyclization. An interesting feature of the substituted biaryl backbone is that tropos and atropos systems can be obtained, enabling or disabling the intramolecular cyclization to hemiacetals. Biaryl structures with various functional groups were synthesized, and the equilibrium and stereodynamics between the closed and open structures were investigated by dynamic enantioselective HPLC (DHPLC). The enantiomerization barriers ΔGǂ and activation parameters ΔHǂ and ΔSǂ were determined from temperature dependent kinetic measurements.

T-cell exhaustion and residency dynamics inform clinical outcomes in hepatocellular carcinoma.

BACKGROUND & AIMS: Despite recent translation of immunotherapies into clinical practice, the immunobiology of hepatocellular carcinoma (HCC), in particular the role and clinical relevance of exhausted and liver-resident T cells remain unclear. We therefore dissected the landscape of exhausted and resident T cell responses in the peripheral blood and tumor microenvironment of patients with HCC. METHODS: Lymphocytes were isolated from the blood, tumor and tumor-surrounding liver tissue of patients with HCC (n = 40, n = 10 treated with anti-PD-1 therapy). Phenotype, function and response to anti-PD-1 were analyzed by mass and flow cytometry ex vivo and in vitro, tissue residence was further assessed by immunohistochemistry and imaging mass cytometry. Gene signatures were analyzed in silico. RESULTS: We identified significant enrichment of heterogeneous populations of exhausted CD8+ T cells (TEX) in the tumor microenvironment. Strong enrichment of severely exhausted CD8 T cells expressing multiple immune checkpoints in addition to PD-1 was linked to poor progression-free and overall survival. In contrast, PD-1 was also expressed on a subset of more functional and metabolically active CD103+ tissue-resident memory T cells (TRM) that expressed few additional immune checkpoints and were associated with better survival. TEX enrichment was independent of BCLC stage, alpha-fetoprotein levels or age as a variable for progression-free survival in our cohort. These findings were in line with in silico gene signature analysis of HCC tumor transcriptomes from The Cancer Genome Atlas. A higher baseline TRM/TEX ratio was associated with disease control in anti-PD-1-treated patients. CONCLUSION: Our data provide information on the role of peripheral and intratumoral TEX-TRM dynamics in determining outcomes in patients with HCC. The dynamics between exhausted and liver-resident T cells have implications for immune-based diagnostics, rational patient selection and monitoring during HCC immunotherapies. LAY SUMMARY: The role of the immune response in hepatocellular carcinoma (HCC) remains unclear. T cells can mediate protection against tumor cells but are frequently dysfunctional and exhausted in cancer. We found that patients with a predominance of exhausted CD8+ T cells (TEX) had poor survival compared to patients with a predominance of tissue-resident memory T cells (TRM). This correlated with the molecular profile, metabolic and functional status of these cell populations. The enrichment of TEX was independently associated with prognosis in addition to disease stage, age and tumor markers. A high TRM proportion was also associated with better outcomes following checkpoint therapy. Thus, these T-cell populations are novel biomarkers with relevance in HCC.

Alkyl backbone variations in common ß-diketiminate ligands and applications to N-heterocyclic silylene chemistry.

We report the extension of the common ß-diketimine proligand class, RArnacnacH (HC(RCNAr)2H), where R is an alkyl group such as Et or iPr, plus Ph, and Ar is a sterically demanding aryl substituent such as Dip = 2,6-diispropylphenyl, Dep = 2,6-diethylphenyl, Mes = 2,4,6-trimethylphenyl or mesityl, Xyl = 2,6-dimethylphenyl, via one-pot condensation procedures. When a condensation reaction is carried out using the chemical dehydrating agent PPSE (polyphosphoric acid trimethylsilylester), ß-diketiminate phosphorus(V) products such as (iPrMesnacnac)PO2 can also be obtained, which can be converted to the respective proligand iPrMesnacnacH via alkaline hydrolysis. The RArnacnacH proligands can be converted to their alkali metal complexes with common methods and we have found that deprotonation of iPrDipnacnacH is significantly more sluggish than that of related ß-diketimines with smaller backbone alkyl groups. The basicity of the RArnacnac- anions can play a role in the success of their salt metathesis chemistry and we have prepared and structurally characterised the EtDipnacnac-derived silicon(II) compounds (EtDipnacnac)SiBr and (EtDipnacnac')Si, where EtDipnacnac' is the deprotonated variant MeCHC(NDip)CHC(NDip)Et.

Norovirus-Specific CD8+ T Cell Responses in Human Blood and Tissues.

BACKGROUND & AIMS: Noroviruses (NoVs) are the leading cause of acute gastroenteritis worldwide and are associated with significant morbidity and mortality. Moreover, an asymptomatic carrier state can persist following acute infection, promoting NoV spread and evolution. Thus, defining immune correlates of NoV protection and persistence is needed to guide the development of future vaccines and limit viral spread. Whereas antibody responses following NoV infection or vaccination have been studied extensively, cellular immunity has received less attention. Data from the mouse NoV model suggest that T cells are critical for preventing persistence and achieving viral clearance, but little is known about NoV-specific T-cell immunity in humans, particularly at mucosal sites. METHODS: We screened peripheral blood mononuclear cells from 3 volunteers with an overlapping NoV peptide library. We then used HLA-peptide tetramers to track virus-specific CD8+ T cells in peripheral, lymphoid, and intestinal tissues. Tetramer+ cells were further characterized using markers for cellular trafficking, exhaustion, cytotoxicity, and proliferation. RESULTS: We defined 7 HLA-restricted immunodominant class I epitopes that were highly conserved across pandemic strains from genogroup II.4. NoV-specific CD8+ T cells with central, effector, or tissue-resident memory phenotypes were present at all sites and were especially abundant in the intestinal lamina propria. The properties and differentiation states of tetramer+ cells varied across donors and epitopes. CONCLUSIONS: Our findings are an important step toward defining the breadth, distribution, and properties of human NoV T-cell immunity. Moreover, the molecular tools we have developed can be used to evaluate future vaccines and engineer novel cellular therapeutics.

[Autoimmune mediated cholestatic liver diseases]. / Autoimmune cholestatische Lebererkrankungen.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are rare, autoimmune mediated cholestatic liver diseases. Other auto-immune diseases are often associated with PBC and PSC, and inflammatory bowel disease is present in the majority of PSC patients. In the course of disease, chronic inflammation in the liver leads to fibrotic restructuring and ultimately cirrhosis. The diagnosis of PBC is confirmed serologically and PSC is diagnosed via cholangiography, whereas MRCP is preferred over ERCP. For PBC, the first line therapy is ursodeoxycholic acid (UDCA). Prognosis is strongly dependent on the response to UDCA. The only approved second line therapy is obeticholic acid (OCA). Alternatively, Budesonide or Fibrates are often used off-label. In the management of PSC, prevention and adequate treatment of bacterial cholangitis play a major role. For both PBC and PSC novel treatments are currently being tested in clinical trials. Disease management should address compromising symptoms like pruritus and sicca as well as complications due to maldigestion and concomitant autoimmune diseases. The only curative treatment available is liver transplantation and should be considered at a MELD score of 15.

Impact of viral multiplex real-time PCR on management of respiratory tract infection: a retrospective cohort study.

BACKGROUND: Significance and clinical utility of multiple virus detection by multiplex real-time polymerase chain reaction (rtPCR) in respiratory tract infection remain unclear. METHODS: This retrospective cohort study analyzed how virus detection affected clinical management. During a 27-month period, clinical and laboratory information was collected from all children and adults in two Swiss tertiary centres whose respiratory samples were tested for respiratory viruses with a 16-plex rtPCR test. RESULTS: Pathogens were identified in 140 of 254 patients (55%); of those patients, there was ≥1 virus in 91 (65%), ≥ 1 bacterium in 53 (38%), and ≥1 virus and bacterium in 11 (8%). Of 80 patients with viral infection, 59 (74%) received antibiotics. Virus detection was associated with discontinuation of antibiotics in 2 of 20 adults (10%) and 6 of 14 children (43%). Overall 12 adults (34%) and 18 children (67%) were managed correctly without antibiotics after virus detection (p = 0.01). When taking biomarkers, radiologic presentations, and antibiotic pre-treatment into account, the impact of rtPCR and appropriateness of therapy for clinically viral infections increased to 100% in children and 62% in adults. CONCLUSIONS: A substantial reduction of unnecessary antibiotic prescriptions seems possible. Appropriate application of rtPCR results in respiratory tract infections should be encouraged.

Differential impact of zinc deficiency on phagocytosis, oxidative burst, and production of pro-inflammatory cytokines by human monocytes.

Zinc deficiency has a fundamental influence on the immune defense, with multiple effects on different immune cells, resulting in a major impairment of human health. Monocytes and macrophages are among the immune cells that are most fundamentally affected by zinc, but the impact of zinc on these cells is still far from being completely understood. Therefore, this study investigates the influence of zinc deficiency on monocytes of healthy human donors. Peripheral blood mononuclear cells, which include monocytes, were cultured under zinc deficient conditions for 3 days. This was achieved by two different methods: by application of the membrane permeable chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) or by removal of zinc from the culture medium using a CHELEX 100 resin. Subsequently, monocyte functions were analyzed in response to Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. Zinc depletion had differential effects. On the one hand, elimination of bacterial pathogens by phagocytosis and oxidative burst was elevated. On the other hand, the production of the inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 was reduced. This suggests that monocytes shift from intercellular communication to basic innate defensive functions in response to zinc deficiency. These results were obtained regardless of the method by which zinc deficiency was achieved. However, CHELEX-treated medium strongly augmented cytokine production, independently from its capability for zinc removal. This side-effect severely limits the use of CHELEX for investigating the effects of zinc deficiency on innate immunity.

The silent epidemic of falls from buildings: analysis of risk factors.

This study wanted to search for potential risk factors associated with falls from windows and balconies in order to eventually improve prevention. All children under the age of 16 years suffering from head injuries/multiple trauma due to falls from windows or balconies treated over the last 7 years at the intensive care unit (ICU) of the University Children's Hospital Zürich were analysed retrospectively (group A). Fifty patients out of all children suffering from head injuries/multiple trauma due to other types of accidents in the same period were selected at random as controls (group B). Out of a total of 241 children with head injury and/or multiple trauma, 31 (13%) fell out of a building. Twenty-seven of these victims (87%) fell from the third floor or lower. Twenty-one of the falls (68%) occurred at home. Fifteen children (49%) climbed on a piece of furniture before falling. In almost 20% of the accidents dangerous balcony or house constructions led to the fall. Parents did not witness the fall, except for three cases (10%) with direct parental involvement (one mother jumped out with her child, two mothers threw their child out of the window). Two children (6%) attempted suicide. Children aged 0-5 years were predominantly represented (84%), and all six children who died were in this age group. There were significantly more patients with foreign nationalities and lower socio-professional categories in group A than in group B. In both groups, the accidents concerned the youngest child of the family in approximately 50% and happened mostly during summer evenings. There were no significant differences in injured systems and in injury severity between the two groups. This study identified young age, an immigrant family setting, low socio-professional category of the parents, dangerous house constructions, inappropriate furniture placement, and summertime evenings as risk factors for serious building falls in children. This information may foster focused prevention.