Biallelic human SHARPIN loss of function induces autoinflammation and immunodeficiency.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.

Antigen receptor signaling and cell death resistance controls intestinal humoral response zonation.

Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous binding to multiple, diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount broadly reactive IgA to the gut microbiome remains elusive. Here, we have shown that IgA B cell receptor (BCR) is required for B cell fitness during the germinal center (GC) reaction in Peyer's patches (PPs) and for generation of gut-homing plasma cells (PCs). We demonstrate that IgA BCR drove heightened intracellular signaling in mouse and human B cells, and as a consequence, IgA+ B cells received stronger positive selection cues. Mechanistically, IgA BCR signaling offset Fas-mediated death, possibly rescuing low-affinity B cells to promote a broad humoral response to commensals. Our findings reveal an additional mechanism linking BCR signaling, B cell fate, and antibody production location, which have implications for how intestinal antigen recognition shapes humoral immunity.

Single-cell delineation of lineage and genetic identity in the mouse brain.

During neurogenesis, mitotic progenitor cells lining the ventricles of the embryonic mouse brain undergo their final rounds of cell division, giving rise to a wide spectrum of postmitotic neurons and glia1,2. The link between developmental lineage and cell-type diversity remains an open question. Here we used massively parallel tagging of progenitors to track clonal relationships and transcriptomic signatures during mouse forebrain development. We quantified clonal divergence and convergence across all major cell classes postnatally, and found diverse types of GABAergic neuron that share a common lineage. Divergence of GABAergic clones occurred during embryogenesis upon cell-cycle exit, suggesting that differentiation into subtypes is initiated as a lineage-dependent process at the progenitor cell level.

HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.

Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.

Guidelines for mouse and human DC generation.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

CD40 Drives Central Nervous System Autoimmune Disease by Inducing Complementary Effector Programs via B Cells and Dendritic Cells.

Costimulatory CD40 plays an essential role in autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis (MS). However, how CD40 drives autoimmune disease pathogenesis is not well defined. Here, we used a conditional knockout approach to determine how CD40 orchestrates a CNS autoimmune disease induced by recombinant human myelin oligodendrocyte glycoprotein (rhMOG). We found that deletion of CD40 in either dendritic cells (DCs) or B cells profoundly reduced EAE disease pathogenesis. Mechanistically, CD40 expression on DCs was required for priming pathogenic Th cells in peripheral draining lymph nodes and promoting their appearance in the CNS. By contrast, B cell CD40 was essential for class-switched MOG-specific Ab production, which played a crucial role in disease pathogenesis. In fact, passive transfer of MOG-immune serum or IgG into mice lacking CD40 on B cells but not DCs reconstituted autoimmune disease, which was associated with inundation of the spinal cord parenchyma by Ig and complement. These data demonstrate that CD40 supports distinct effector programs in B cells and DCs that converge to drive a CNS autoimmune disease and identify targets for intervention.

Developmental diversification of cortical inhibitory interneurons.

Diverse subsets of cortical interneurons have vital roles in higher-order brain functions. To investigate how this diversity is generated, here we used single-cell RNA sequencing to profile the transcriptomes of mouse cells collected along a developmental time course. Heterogeneity within mitotic progenitors in the ganglionic eminences is driven by a highly conserved maturation trajectory, alongside eminence-specific transcription factor expression that seeds the emergence of later diversity. Upon becoming postmitotic, progenitors diverge and differentiate into transcriptionally distinct states, including an interneuron precursor state. By integrating datasets across developmental time points, we identified shared sources of transcriptomic heterogeneity between adult interneurons and their precursors, and uncovered the embryonic emergence of cardinal interneuron subtypes. Our analysis revealed that the transcription factor Mef2c, which is linked to various neuropsychiatric and neurodevelopmental disorders, delineates early precursors of parvalbumin-expressing neurons, and is essential for their development. These findings shed new light on the molecular diversification of early inhibitory precursors, and identify gene modules that may influence the specification of human interneuron subtypes.

Cross-reactive antibody response to Monkeypox virus surface proteins in a small proportion of individuals with and without Chinese smallpox vaccination history.

BACKGROUND: After the eradication of smallpox in China in 1979, vaccination with the vaccinia virus (VACV) Tiantan strain for the general population was stopped in 1980. As the monkeypox virus (MPXV) is rapidly spreading in the world, we would like to investigate whether the individuals with historic VACV Tiantan strain vaccination, even after more than 40 years, could still provide ELISA reactivity and neutralizing protection; and whether the unvaccinated individuals have no antibody reactivity against MPXV at all. RESULTS: We established serologic ELISA to measure the serum anti-MPXV titer by using immunodominant MPXV surface proteins, A35R, B6R, A29L, and M1R. A small proportion of individuals (born before 1980) with historic VACV Tiantan strain vaccination exhibited serum ELISA cross-reactivity against these MPXV surface proteins. Consistently, these donors also showed ELISA seropositivity and serum neutralization against VACV Tiantan strain. However, surprisingly, some unvaccinated young adults (born after 1980) also showed potent serum ELISA activity against MPXV proteins, possibly due to their past infection by some self-limiting Orthopoxvirus (OPXV). CONCLUSIONS: We report the serum ELISA cross-reactivity against MPXV surface protein in a small proportion of individuals both with and without VACV Tiantan strain vaccination history. Combined with our serum neutralization assay against VACV and the recent literature about mice vaccinated with VACV Tiantan strain, our study confirmed the anti-MPXV cross-reactivity and cross-neutralization of smallpox vaccine using VACV Tiantan strain. Therefore, it is necessary to restart the smallpox vaccination program in high risk populations.

A combinatorial code of transcription factors specifies subtypes of visual motion-sensing neurons in Drosophila.

Direction-selective T4/T5 neurons exist in four subtypes, each tuned to visual motion along one of the four cardinal directions. Along with their directional tuning, neurons of each T4/T5 subtype orient their dendrites and project their axons in a subtype-specific manner. Directional tuning, thus, appears strictly linked to morphology in T4/T5 neurons. How the four T4/T5 subtypes acquire their distinct morphologies during development remains largely unknown. Here, we investigated when and how the dendrites of the four T4/T5 subtypes acquire their specific orientations, and profiled the transcriptomes of all T4/T5 neurons during this process. This revealed a simple and stable combinatorial code of transcription factors defining the four T4/T5 subtypes during their development. Changing the combination of transcription factors of specific T4/T5 subtypes resulted in predictable and complete conversions of subtype-specific properties, i.e. dendrite orientation and matching axon projection pattern. Therefore, a combinatorial code of transcription factors coordinates the development of dendrite and axon morphologies to generate anatomical specializations that differentiate subtypes of T4/T5 motion-sensing neurons.

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.

Characterization of cross-reactive monoclonal antibodies against SARS-CoV-1 and SARS-CoV-2: Implication for rational design and development of pan-sarbecovirus vaccines and neutralizing antibodies.

Emergence of various circulating SARS-CoV-2 variants of concern (VOCs) promotes the identification of pan-sarbecovirus vaccines and broadly neutralizing antibodies (bNAbs). Here, to characterize monoclonal antibodies cross-reactive against both SARS-CoV-1 and SARS-CoV-2 and to search the criterion for bNAbs against all emerging SARS-CoV-2, we isolated several SARS-CoV-1-cross-reactive monoclonal antibodies (mAbs) from a wildtype SARS-CoV-2 convalescent donor. These antibodies showed broad binding capacity and cross-neutralizing potency against various SARS-CoV-2 VOCs, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta), but failed to efficiently neutralize Omicron variant and its sublineages. Structural analysis revealed how Omicron sublineages, but not other VOCs, efficiently evade an antibody family cross-reactive against SARS-CoV-1 through their escape mutations. Further evaluation of a series of SARS-CoV-1/2-cross-reactive bNAbs showed a negative correlation between the neutralizing activities against SARS-CoV-1 and SARS-CoV-2 Omicron variant. Together, these results suggest the necessity of using cross-neutralization against SARS-CoV-1 and SARS-CoV-2 Omicron as criteria for rational design and development of potent pan-sarbecovirus vaccines and bNAbs.

An apoptosis-dependent checkpoint for autoimmunity in memory B and plasma cells.

B lymphocytes acquire self-reactivity as an unavoidable byproduct of antibody gene diversification in the bone marrow and in germinal centers (GCs). Autoreactive B cells emerging from the bone marrow are silenced in a series of well-defined checkpoints, but less is known about how self-reactivity that develops by somatic mutation in GCs is controlled. Here, we report the existence of an apoptosis-dependent tolerance checkpoint in post-GC B cells. Whereas defective GC B cell apoptosis has no measurable effect on autoantibody development, disruption of post-GC apoptosis results in accumulation of autoreactive memory B cells and plasma cells, antinuclear antibody production, and autoimmunity. The data presented shed light on mechanisms that regulate immune tolerance and the development of autoantibodies.

Water in bacterial biofilms: pores and channels, storage and transport functions.

Bacterial biofilms occur in many natural and industrial environments. Besides bacteria, biofilms comprise over 70 wt% water. Water in biofilms occurs as bound- or free-water. Bound-water is adsorbed to bacterial surfaces or biofilm (matrix) structures and possesses different Infra-red and Nuclear-Magnetic-Resonance signatures than free-water. Bound-water is different from intra-cellularly confined-water or water confined within biofilm structures and bacteria are actively involved in building water-filled structures by bacterial swimmers, dispersion or lytic self-sacrifice. Water-filled structures can be transient due to blocking, resulting from bacterial growth, compression or additional matrix formation and are generally referred to as "channels and pores." Channels and pores can be distinguished based on mechanism of formation, function and dimension. Channels allow transport of nutrients, waste-products, signalling molecules and antibiotics through a biofilm provided the cargo does not adsorb to channel walls and channels have a large length/width ratio. Pores serve a storage function for nutrients and dilute waste-products or antimicrobials and thus should have a length/width ratio close to unity. The understanding provided here on the role of water in biofilms, can be employed to artificially engineer by-pass channels or additional pores in industrial and environmental biofilms to increase production yields or enhance antimicrobial penetration in infectious biofilms.

Effect of Organic Solvents on the Structure and Activity of a Minimal Lipase.

Lipases are ubiquitously used in chemo-enzymatic synthesis and industrial applications. Nevertheless, the modulation of the activity of lipases by organic solvents still is not fully understood at the molecular level. We systematically investigated the activity and structure of lipase A from Bacillus subtilis in binary water-organic solvent mixtures of dimethyl sulfoxide (DMSO), acetonitrile (ACN), and isopropyl alcohol (IPA) using activity assays, fluorescence spectroscopy, molecular dynamics (MD) simulations, and FRET/MD analysis. The enzymatic activity strongly depended on the type and amount of organic solvent in the reaction media. Whereas IPA and ACN reduced the activity of the enzyme, small concentrations of DMSO led to lipase activation via an uncompetitive mechanism. DMSO molecules did not directly interfere with the binding of the substrate in the active site, contrary to what is known for other solvents and enzymes. We propose that the His156-Asp133 interaction, the binding of organic molecules to the active site, and the water accessibility of the substrate are key factors modulating the catalytic activity. Furthermore, we rationalized the role of solvent descriptors on the regulation of enzymatic activity in mixtures with low concentrations of the organic molecule, with prospective implications for the optimization of biocatalytic processes via solvent tuning.

THE NOVEL USE OF HIGH-FLOW POLYIMIDE CANNULAS TO IMPROVE SILICONE OIL INJECTABILITY IN VITREORETINAL SURGERY.

PURPOSE: The injection of high-viscosity silicone oil lengthens injection time. New polyimide cannulas offer a greater inner diameter than conventional metal cannulas at the same gauge. We compared the injection time for polyimide and metal cannulas at 23 G for a variety of silicone oils including a 12,500-mPas prototype oil. METHODS: In this laboratory study, injection time was measured three times per cannula and per oil. Warming the oil before injection to up to 42°C was also evaluated. Finally, the feasibility of polyimide cannulas was tested in vitrectomized porcine eyes. RESULTS: The 23-G polyimide cannula mostly decreased injection times. The time to inject 5 mL of Siluron Xtra and Siluron 5000 decreased by 6:02 (76.9%) minutes (483 vs. 121 seconds) and 12:01 (74.7%) minutes (973 vs. 252 seconds), respectively. Although the 23-G metal cannula failed to inject 12,500 mPas oil, 5 mL was injected in 10:21 minutes using the polyimide cannula. Prewarming Siluron 5000 to 42°C lowered the injection time by 9.0% and by 12.1% when using the metal or polyimide cannula, respectively. CONCLUSION: Polyimide cannulas allow a clinically relevant decrease in injection time. They may not only shorten surgery time but could also ease the use of next-generation ultra-high-viscosity silicone oils. Prewarming silicone oil leads to decreased injection times.

Syntheses of Polypeptides and Their Biomedical Application for Anti-Tumor Drug Delivery.

Polypeptides have attracted considerable attention in recent decades due to their inherent biodegradability and biocompatibility. This mini-review focuses on various ways to synthesize polypeptides, as well as on their biomedical applications as anti-tumor drug carriers over the past five years. Various approaches to preparing polypeptides are summarized, including solid phase peptide synthesis, recombinant DNA techniques, and the polymerization of activated amino acid monomers. More details on the polymerization of specifically activated amino acid monomers, such as amino acid N-carboxyanhydrides (NCAs), amino acid N-thiocarboxyanhydrides (NTAs), and N-phenoxycarbonyl amino acids (NPCs), are introduced. Some stimuli-responsive polypeptide-based drug delivery systems that can undergo different transitions, including stability, surface, and size transition, to realize a better anti-tumor effect, are elaborated upon. Finally, the challenges and opportunities in this field are briefly discussed.

Singlet-Oxygen Generation by Peroxidases and Peroxygenases for Chemoenzymatic Synthesis.

Singlet oxygen is a reactive oxygen species undesired in living cells but a rare and valuable reagent in chemical synthesis. We present a fluorescence spectroscopic analysis of the singlet-oxygen formation activity of commercial peroxidases and novel peroxygenases. Singlet-oxygen sensor green (SOSG) is used as fluorogenic singlet oxygen trap. Establishing a kinetic model for the reaction cascade to the fluorescent SOSG endoperoxide permits a kinetic analysis of enzymatic singlet-oxygen formation. All peroxidases and peroxygenases show singlet-oxygen formation. No singlet oxygen activity could be found for any catalase under investigation. Substrate inhibition is observed for all reactive enzymes. The commercial dye-decolorizing peroxidase industrially used for dairy bleaching shows the highest singlet-oxygen activity and the lowest inhibition. This enzyme was immobilized on a textile carrier and successfully applied for a chemical synthesis. Here, ascaridole was synthesized via enzymatically produced singlet oxygen.

Reasons for explantation of phakic intraocular lenses and associated perioperative complications: cross-sectional explant registry analysis.

BACKGROUND: We discuss the safety, since their introduction, of phakic intraocular lenses (pIOLs) to correct refractive errors in healthy eyes. We investigated the reasons for pIOL explantation and the associated perioperative complications. METHODS: This retrospective, cross-sectional study included 69 pIOLs, explanted at a single tertiary center between July 2005 and March 2020: 34 angle-supported (G1), 28 iris-fixated (G2) and seven posterior chamber (G3) pIOLs. Case data including the reason for explantation was taken from the patient records. Intra- and postoperative complications were evaluated for an association with the pIOL. RESULTS: The mean duration in the eye was 10.4 (0.2-28) years. Cataractogenesis and subsequent surgery that required pIOL explantation was the reason in 42% of all cases. In 22%, cataract in combination with endothelial damage prompted explantation, with 26, 18 and 14% for G1, G2 and G3 respectively. The second most common reasons were corneal damage alone in the angle-supported group (26%), IOL subluxation in the iris-fixated group (18%), and photopic disturbance in the posterior chamber group (29%). In 68% of all explantations, the surgical course was unremarkable, while in the remaining cases perioperative complications were associated with the lens in 45.7%. CONCLUSION: Overall, the need for cataract surgery was the most common reason for pIOL explantation. Corneal complications were more frequent in the angle-supported pIOLs and their removal was associated with higher rates of complication compared to the other groups.

Capsules from synthetic diblock-peptides as potential artificial oxygen carriers.

The design of an encapsulation system consisting of a synthetic peptide which is fully biodegradable into non-toxic constituents. This system should be capable of encapsulating perfluorinated hydrocarbons and should be a promising basis for oxygen carriers to be used as artificial blood replacement. A diblock-peptide is synthesised following a phosgene-free method and characterised by 1H-NMR. Subsequently, this diblock-peptide is self-assembled with perfluorodecalin (PFD) to form PFD-filled capsules as potential artificial oxygen carriers allowing for rapid oxygen uptake and release. The diblock-peptide Bu-PAsp10-PPhe10 is successfully synthesised and used to encapsulate PFD. The capsules have a spherical shape with an average diameter of 360 nm in stable aqueous dispersion. NMR measurements prove their physical capability for reversible uptake and release of oxygen. The resulting capsules are expected to be fully biodegradable and possibly could act as oxygen carriers for artificial blood replacement.

Functional Characterisation of ClpP Mutations Conferring Resistance to Acyldepsipeptide Antibiotics in Firmicutes.

Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10-6 . All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well-resolved N-terminal domains in the apo structure allow the pore-gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process.