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Janus NanoRods (JNR) are anisotropic and non-symmetrical colloids with two faces of different chemical composition. They are difficult to prepare because of their nanometric dimensions and strong anisotropy. Recently, a versatile strategy was developed, allowing the formation of JNR relying on the self-assembly in aqueous medium of two polymers end-functionalized with non-symmetrical and complementary hydrogen bonding stickers. However, the supramolecular JNR prepared following this strategy are out-of-equilibrium (frozen) and therefore their characteristics depend on the self-assembly process. The present study elucidates the formation mechanism of the JNR and the parameters of the self-assembly process influencing their characteristics. The polymers are initially dissolved as unimers in DMSO. Dropwise addition of water triggers the rapid assembly of more and more unimers into long nanocylinders that are unable to grow anymore once formed. Consequently, increasing the dropwise addition rate of water hardly impacts the process, whereas lowering the initial polymer concentration in DMSO reduces both the length and proportion of nanocylinders. Increasing temperature during water addition weakens hydrogen bonds, triggering the formation of a mixture of spheres and nanocylinders. Many supramolecular polymer assemblies are frozen in solution and these findings should help understanding how to control their characteristics, allowing to adapt them to a target application., (© 2024 The Author(s). Macromolecular Rapid Communications published by Wiley‐VCH GmbH.)

Neuropilin-2 (NRP2) is a cell surface receptor that plays key roles in lymphangiogenesis, but also in pathophysiological conditions such as cancer and inflammation. NRP2 targeting by efzofitimod, a novel immunomodulatory molecule, is currently being tested for the treatment of pulmonary sarcoidosis. To date, no anti-NRP2 antibodies are available for companion diagnostics. Here we describe the development and characterization of a novel NRP2 antibody. Using a variety of research techniques, that is, enzyme-linked immunoassay, Western blot, biolayer interferometry, and immunohistochemistry, we demonstrate that our antibody detects all major NRP2 isoforms and does not cross-react with NRP1. Using this antibody, we show high NRP2 expression in granulomas from sarcoidosis patient skin and lung biopsies. Our novel anti-NRP2 antibody could prove to be a useful clinical tool for sarcoidosis and other indications where NRP2 has been implicated. Clinical Trial Registration: clinicaltrials.gov NCT05415137.

Efzofitimod is a first-in-class biologic based on a naturally occurring splice variant of histidyl-tRNA synthetase (HARS) that downregulates immune responses via selective modulation of neuropilin-2 (NRP2). Preclinical data found high expression of NRP2 in sarcoidosis granulomas. Treatment with efzofitimod reduced the granulomatous inflammation induced by P. acnes in an animal model of sarcoidosis. A dose escalating trial of efzofitimod in sarcoidosis with chronic symptomatic pulmonary disease found that treatment with efzofitimod was associated with improved quality of life with a trend towards reduced glucocorticoid use and stable to improved pulmonary function. These studies have led to a large Phase 3 trial of efzofitimod in symptomatic pulmonary sarcoidosis.

Zwitterionic ring-opening polymerization (ZROP) of sarcosine-derived N- thiocarboxyanhydrides (Me-NNTAs) can be induced by using 1,1,3,3-tetramethylguanidine (TMG) initiators in CH 2 Cl 2 at 25 °C, rapidly producing well-defined polysarcosine polymers with controlled molecular weights ( M n = 1.9-37 kg/mol) and narrow molecular weight distributions ( Đ = 1.01-1.12). The reaction exhibits characteristics of a living polymerization, evidenced by pseudo-first-order polymerization kinetics, the linear increase of polymer molecular weight ( M n ) with conversion, and the successful chain extension experiments. The polymerization is proposed to proceed via propagating macro-zwitterions bearing a cationic 1,1,3,3-tetramethylguanidinium and an anionic thiocarbamate chain end. The TMG not only initiates the polymerization but also serves to stabilize the thiocarbamate chain end where the monomer addition occurs. Because of the enhanced hydrolytic stability of Me-NNTA, the polymerization can be conducted without the rigorous exclusion of moisture, further enhancing the appeal of the method to access well-defined polysarcosine., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background: Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infections in infants. There are still no vaccines or specific antiviral therapies against RSV, mainly due to the inadequate understanding of RSV pathogenesis. Recent data suggest a role for gut microbiota community structure in determining RSV disease severity. Our objective was to determine the gut microbial profile associated with severe RSV patients, which could be used to help identify at-risk patients and develop therapeutically protective microbial assemblages that may stimulate immuno-protection., Results: We enrolled 95 infants from Le Bonheur during the 2014 to 2016 RSV season. Of these, 37 were well-babies and 58 were hospitalized with RSV. Of the RSV infected babies, 53 remained in the pediatric ward (moderate) and 5 were moved to the pediatric intensive care unit at a later date (severe). Stool samples were collected within 72 h of admission; and the composition of gut microbiota was evaluated via 16S sequencing of fecal DNA. There was a significant enrichment in S24_7, Clostridiales, Odoribacteraceae, Lactobacillaceae, and Actinomyces in RSV (moderate and severe) vs. controls. Patients with severe RSV disease had slightly lower alpha diversity (richness and evenness of the bacterial community) of the gut microbiota compared to patients with moderate RSV and healthy controls. Beta diversity (overall microbial composition) was significantly different between all RSV patients (moderate and severe) compared to controls and had significant microbial composition separating all three groups (control, moderate RSV, and severe RSV)., Conclusions: Collectively, these data demonstrate that a unique gut microbial profile is associated with RSV disease and with severe RSV disease with admission to the pediatric intensive care unit. More mechanistic experiments are needed to determine whether the differences observed in gut microbiota are the cause or consequences of severe RSV disease.

Rationale: Studies of the immune responses at the site of respiratory syncytial virus (RSV) infection are sparse despite nearly five decades of research into understanding RSV disease. Objectives: To investigate the role of mucosal innate immune responses to RSV and respiratory viral load in infants hospitalized with the natural disease. Methods: Cytokines, viral load, and type 2 innate lymphoid cell (ILC2) levels in nasal aspirates, collected within 24 hours of enrollment, from infants hospitalized with RSV infection were quantified. Measurements and Main Results: RSV severity in infants was categorized based on admission to the general ward (moderate) or the pediatric ICU (severe). Evaluable subjects included 30 patients with severe and 63 patients with moderate disease (median age, 74 d; range, 9-297 d). ILC2s were found in the nasal aspirates of patients with severe disease (0.051% of total respiratory CD45 + cells) to a significantly greater extent than in patients with moderate disease (0.018%, P  = 0.004). Levels of IL-4, IL-13, IL-33, and IL-1β were significantly higher in nasal aspirates of patients with severe disease compared with those of patients with moderate disease. Factors associated with disease severity were gestational age (odds ratio, 0.49; 95% confidence interval, 0.29-0.82; P  = 0.007) and IL-4 (odds ratio, 9.67; 95% confidence interval, 2.45-38.15; P  = 0.001). Conclusions: This study shows, for the first time, that elevated levels of ILC2s is associated with infant RSV severity. The findings highlight the dominance of type-2 responses to RSV infection in infants and suggest an important role of ILC2 in shaping the immune response early during RSV infection.

Pneumonia due to methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of morbidity and mortality in infants particularly following lower respiratory tract viral infections such as Respiratory Syncytial Virus (RSV). However, the mechanisms by which co-infection of infants by MRSA and RSV cause increased lung pathology are unknown. Because the infant immune system is qualitatively and quantitatively different from adults we developed a model of infant MRSA pneumonia which will allow us to investigate the effects of RSV co-infection on disease severity. We infected neonatal and adult mice with increasing doses of MRSA and demonstrate that neonatal mice have delayed kinetics in clearing the bacteria in comparison to adult mice. There were differences in recruitment of immune cells into the lung following infection. Adult mice exhibited an increase in neutrophil recruitment that coincided with reduced bacterial titers followed by an increase in macrophages. Neonatal mice, however, exhibited an early increase in neutrophils that did not persist despite continued presence of the bacteria. Unlike the adult mice, neonatal mice failed to exhibit an increase in macrophages. Neonates exhibited a decrease in phagocytosis of MRSA suggesting that the decrease in clearance was partially due to deficient phagocytosis of the bacteria. Both neonates and adults responded with an increase in pro-inflammatory cytokines following infection. However, in contrast to the adult mice, neonates did not express constitutive levels of the anti-microbial peptide Reg3γ in the lung. Infection of neonates did not stimulate expression of the co-stimulatory molecule CD86 by dendritic cells and neonates exhibited a diminished T cell response compared to adult mice. Overall, we have developed a neonatal model of MRSA pneumonia that displays a similar delay in bacterial clearance as is observed in the neonatal intensive care unit and will be useful for performing co-infection studies., Competing Interests: The authors have declared that no competing interests exist.

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.

The infant immune response to respiratory syncytial virus (RSV) remains incompletely understood. Here we review the use of a neonatal mouse model of RSV infection to mimic severe infection in human infants. We describe numerous age-specific responses, organized by cell type, observed in RSV-infected neonatal mice and draw comparisons (when possible) to human infants., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The eukaryotic green algae, Chlamydomonas reinhardtii has been shown to be capable of producing a variety of recombinant proteins, but the true potential of this platform remains largely unexplored. To assess the potential of algae for the production of novel recombinant proteins, we generated a series of chimeric proteins containing a single chain antibody (scFv) targeting the B-cell surface antigen CD22, genetically fused to the eukaryotic ribosome inactivating protein, gelonin, from Gelonium multiflorm. These unique molecules, termed immunotoxins, are encoded as a single gene that produces an antibody--toxin chimeric protein capable of delivering a cytotoxic molecule to targeted B-cells. We show that the addition of an Fc domain of a human IgG1 to these fusion proteins results in the production of assembled dimeric immunotoxins, containing two cell binding scFvs and two gelonin molecules. Additionally, we demonstrate that these algal expressed proteins are capable of binding and reducing the viability of B-cell lymphomas, while treatment of T-cells, that lack the CD22 antigen, had no impact on cell viability. Since other protein expression platforms are incapable of folding and accumulating these complex immunotoxins as soluble and enzymatically active proteins, our studies document a novel and efficient method for immunotoxin production., (© 2013 Wiley Periodicals, Inc.)

Toll-like receptor 4 (TLR4), which signals through the adapter molecules myeloid differentiation factor 88 (MyD88) and toll/interleukin 1 receptor domain-containing adapter inducing IFN-β (TRIF), is required for protection against Gram-negative bacteria. TRIF is known to be important in TLR3-mediated antiviral signaling, but the role of TRIF signaling against Gram-negative enteropathogens is currently unknown. We show that TRIF signaling is indispensable for establishing innate protective immunity against Gram-negative Yersinia enterocolitica. Infection of wild-type mice rapidly induced both IFN-β and IFN-γ in the mesenteric lymph nodes. In contrast, TRIF-deficient mice were defective in these IFN responses and showed impaired phagocytosis in regional macrophages, resulting in greater bacterial dissemination and mortality. TRIF signaling may be universally important for protection against Gram-negative pathogens, as TRIF-deficient macrophages were also impaired in killing both Salmonella and Escherichia coli in vitro. The mechanism of TRIF-mediated protective immunity appears to be orchestrated by macrophage-induced IFN-β and NK cell production of IFN-γ. Sequential induction of IFN-β and IFN-γ leads to amplification of macrophage bactericidal activity sufficient to eliminate the invading pathogens at the intestinal interface. Our results demonstrate a previously unknown role of TRIF in host resistance to Gram-negative enteropathogens, which may lead to effective strategies for combating enteric infections.

Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-x(L)/Bcl-2 dependence. Surprisingly, analysis of Mcl-1-dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-x(L) in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.

Protein-based therapeutics are the fastest growing sector of drug development, mainly because of the high sensitivity and specificity of these molecules. Their high specificity leads to few side effects and excellent success rates in drug development. However, the inherent complexity of these molecules restricts their synthesis to living cells, making recombinant proteins expensive to produce. In addition to therapeutic uses, recombinant proteins also have a variety of industrial applications and are important research reagents. Eukaryotic algae offer the potential to produce high yields of recombinant proteins more rapidly and at much lower cost than traditional cell culture. Additionally, transgenic algae can be grown in complete containment, reducing any risk of environmental contamination. This system might also be used for the oral delivery of therapeutic proteins, as green algae are edible and do not contain endotoxins or human viral or prion contaminants.