Brianna Dalesandro

Int J Mol Med. 2022 Apr;49(4):42. doi: 10.3892/ijmm.2022.5097. Epub 2022 Feb 4.

ABSTRACT

Anti‑CD19 chimeric antigen receptor (CAR)‑T cell therapy against refractory B‑cell malignancies shows excellent therapeutic effects. However, there are some obstacles to be overcome in this treatment. Since current CAR‑T cells target a single cell‑surface protein on tumor cells, the CAR‑T cells also attack normal cells expressing the protein. This is one of the major adverse effects of this therapy. To improve target‑cell‑specificity of this therapy, we established a novel CAR system, in which T‑cell activation was controlled by expression patterns of proteins on target cells. Our novel CAR‑T cells had two distinct CARs consisting of a 'Signal‑CAR', recognizing a protein on tumor cells, and a 'Scissors‑CAR', recognizing another protein on normal cells. The signal‑CAR had a peptide sequence which was cleaved by the Scissors‑CAR, and functional domains for cellular activation. The Scissors‑CAR had a protease domain that cleaved its recognition peptide sequence in the Signal‑CAR. When tumor cells expressed only the protein recognized by the Signal‑CAR, the tumor cells were attacked. By contrast, normal cells expressing both the proteins induced inactivation of the Signal‑CAR through cleavage of the recognition site when getting in contact with the CAR‑T cells. To establish this system, we invented a Scissors‑CAR that was dominantly localized on cell membranes and was activated only when the CAR‑T cells were in contact with the normal cells. Using a T‑cell line, Jurkat, and two proteins, CD19 and HER2, as target proteins, we showed that the anti‑CD19‑Signal‑CAR was cleaved by the anti‑HER2‑Scissors‑CAR when the CAR‑T cells were co‑cultivated with cells expressing both the proteins, CD19 and HER2. Furthermore, we demonstrated that primary CAR‑T cells expressing both the CARs showed attenuated cytotoxicity againsT cells with both the target proteins. Our novel system would improve safety of the CAR‑T cell therapy, leading to expansion of treatable diseases by this immunotherapy.

PMID:35119085 | DOI:10.3892/ijmm.2022.5097

Front Immunol. 2022 Jan 19;12:825825. doi: 10.3389/fimmu.2021.825825. eCollection 2021.

ABSTRACT

The resistance of Lactobacillus plantarum to vancomycin depends on its peptidoglycan composition. Vancomycin has poor binding affinity with peptidoglycan precursors ending in D-alanyl-D-lactate (D-Ala-D-Lac) but binds strongly to peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala), resulting in resistance and sensitivity, respectively. The ligase Ddl, which generates D-Ala-D-Lac or D-Ala-D-Ala incorporated into the peptidoglycan precursor chain, is responsible for this specificity. To study the effect of peptidoglycan precursors on immunity, we constructed several strains of L. plantarum expressing the ddl gene of Lactococcus lactis to change their peptidoglycan precursors. The change in the termini of the peptidoglycan precursors was determined by the sensitivity of the strains to vancomycin. The overexpression of ddl increased the susceptibility of the strains to vancomycin. We further explored the regulation of the macrophage inflammatory response pathway by the wild-type and constructed strains, and found that these strains induced the MyD88-dependent TRAF6/MAPK pathway, and the increase in D-Ala L. plantarum peptidoglycan precursors increased the secretion of the inflammatory factors IL-6, IL-1β and TNF-α. These results indicate that D-Ala-ended peptidoglycan precursors play a central role in the variable immunomodulatory ability of L. plantarum.

PMID:35126378 | PMC:PMC8807649 | DOI:10.3389/fimmu.2021.825825

Curr Med Chem. 2022 Feb 3. doi: 10.2174/0929867329666220203115122. Online ahead of print.

ABSTRACT

BACKGROUND: Peptidoglycan is an essential component of the cell wall in all bacteria. In particular, the cell walls of Gram-positive bacteria are composed mostly of a thick layer of peptidoglycan. Its accessibility has important implications for their sensing in whole bacterial detection methodologies. Indeed, there is an urgent demand for rapid tests which can identify whole bacteria, e.g., directly at the point of care. <p.

OBJECTIVE: The aim of this work is to explore the suitability of RipA, a key cell division protein of M. tuberculosis, for whole cell biosensing of Gram-positive bacteria.

METHODS: We here conducted Molecular Dynamics (MD) studies aimed at the understanding of the structural and dynamic features of active RipA and at the design of a suitable bioreceptor. Based on these studies, we engineered a RipA variant for covalent oriented immobilisation on golden surfaces and able to bind peptidoglycan, albeit without degrading it. Surface Plasmon Resonance (SPR) was employed to check the ability of functionalized golden chips to recognize whole bacteria.

RESULTS: MD analyses elucidated the structural details of the active form of RipA and suggested that this enzyme, once inactivated, presents a rigid and well-exposed peptidoglycan recognition cleft. We engineered RipA for proper oriented immobilisation on golden chips for SPR studies. Results show that, once chemically coupled to a golden chip, the developed RipA-based bioreceptor is able to detect B. subtilis, used as a model in a concentration-dependent mode. <p.

CONCLUSION: Results highlight the potential of the engineered molecule to be integrated in the development of early warning biosensors for Gram-positive contamination in clinical diagnosis or in food-borne infections. </p>.

PMID:35125077 | DOI:10.2174/0929867329666220203115122

Iran Biomed J. 2021 Nov 1;25(6):390-8. doi: 10.52547/ibj.25.6.390.

ABSTRACT

BACKGROUND: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS.

METHODS: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours).

RESULTS: : Increased efficiency was achieved by EnBase® compared to Luria–Bertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 μg/mL.

CONCLUSION: : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

PMID:34641643 | PMC:PMC8744694 | DOI:10.52547/ibj.25.6.390

Mol Biol Cell. 2022 May 15;33(6):ar57. doi: 10.1091/mbc.E21-10-0526. Epub 2022 Feb 2.

ABSTRACT

Lysosomal degradative compartments hydrolyze macromolecules to generate basic building blocks that fuel metabolic pathways in our cells. They also remove misfolded proteins and control size, function, and number of cytoplasmic organelles via constitutive and regulated autophagy. These catabolic processes attract interest because their defective functioning is linked to human disease and their molecular components are promising pharmacologic targets. The capacity to quantitatively assess them is highly sought-after. Here we present a tandem-fluorescent reporter consisting of a HaloTag-GFP chimera appended at the C- or at the N-terminus of select polypeptides to monitor protein and organelle delivery to the lysosomal compartment. The Halo-GFP changes color on fluorescent pulse with cell-permeable HaloTag ligands and, again, on delivery to acidic, degradative lysosomal compartments, where the fluorescent ligand-associated HaloTag is relatively stable, whereas the GFP portion is not, as testified by loss of the green fluorescence and generation of a protease-resistant, fluorescent HaloTag fragment. The Halo-GFP tandem fluorescent reporter presented in our study allows quantitative and, crucially, time-resolved analyses of protein and organelle transport to the lysosomal compartment by high resolution confocal laser scanning microscopy, antibody-free electrophoretic techniques and flow cytometry.

PMID:35108065 | PMC:PMC9265146 | DOI:10.1091/mbc.E21-10-0526

Nat Commun. 2022 Feb 1;13(1):590. doi: 10.1038/s41467-022-28257-0.

ABSTRACT

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

PMID:35105886 | PMC:PMC8807736 | DOI:10.1038/s41467-022-28257-0

ACS Biomater Sci Eng. 2022 Feb 1. doi: 10.1021/acsbiomaterials.1c01532. Online ahead of print.

ABSTRACT

Chimeric antigen receptor (CAR)-engineered adoptive cell therapy marks a revolution in cancer treatment based on the highly successful responses to CAR T cell therapy in the treatment of blood cancers. Due to the versatile structure of CARs, this technology can be easily adapted to other immune cell types, including macrophages and NKs, and applied in the treatment of many other cancers. However, high costs and fatal adverse effects represent significant concerns for future development. In vitro transcribed (IVT) mRNA therapeutics, which possess a high safety profile and straightforward production methods, could provide a useful alternative for CAR cell construction. However, the low stability and transfection efficiency of IVT-mRNA in immune cells limit further applications. In this work, we successfully engineered CAR macrophages (CAR-Ms) and CAR T cells with CAR mRNA using lipid nanoparticles (LNPs). Both the LNP formulations and mRNA modifications were optimized for in vitro mRNA transfection. More importantly, the CAR macrophages and CAR T cells both demonstrated significant cytotoxic effects on B lymphoma in vitro, underscoring the great potential of mRNA-engineered adoptive cell therapy.

PMID:35104103 | DOI:10.1021/acsbiomaterials.1c01532

Chemistry. 2022 Feb 1. doi: 10.1002/chem.202200146. Online ahead of print.

ABSTRACT

Monoclonal antibodies (mAbs) are one of the most rapidly growing drug classes used for the treatment of cancer, infectious and autoimmune diseases. Complement-dependent cytotoxicity (CDC) is one of the effector functions for antibodies to deplete target cells. We report here an efficient chemoenzymatic synthesis of structurally well-defined conjugates of a monoclonal antibody with a rhamnose- and an αGal trisaccharide-cluster to recruit natural anti-rhamnose and anti-αGal antibodies, respectively, to enhance the CDC-dependent targeted cell killing. The synthesis was achieved by using a modular antibody Fc-glycan remodeling method that includes site-specific chemoenzymatic Fc-glycan functionalization and subsequent click conjugation of synthetic rhamnose- and αGal trisaccharide-cluster to provide the respective homogeneous antibody conjugates. Cell-based assays indicated that the antibody-rhamnose cluster conjugates could mediate potent CDC activity for targeted cancer cell killing and showed much more potent efficacy than the antibody-αGal trisaccharide cluster conjugates for CDC effects.

PMID:35106843 | DOI:10.1002/chem.202200146

ACS Infect Dis. 2022 Jan 31. doi: 10.1021/acsinfecdis.1c00296. Online ahead of print.

ABSTRACT

The paradigm of antivirulence therapy dictates that bacterial pathogens are specifically disarmed but not killed by neutralizing their virulence factors. Clearance of the invading pathogen by the immune system is promoted. As compared to antibiotics, the pathogen-selective antivirulence drugs hold promise to minimize collateral damage to the beneficial microbiome. Also, selective pressure for resistance is expected to be lower because bacterial viability is not directly affected. Antivirulence drugs are being developed for stand-alone prophylactic and therapeutic treatments but also for combinatorial use with antibiotics. This Review focuses on drug modalities that target bacterial exotoxins after the secretion or release-upon-lysis. Exotoxins have a significant and sometimes the primary role as the disease-causing virulence factor, and thereby they are attractive targets for drug development. We describe the key pre-clinical and clinical trial data that have led to the approval of currently used exotoxin-targeted drugs, namely the monoclonal antibodies bezlotoxumab (toxin B/TcdB, Clostridioides difficile), raxibacumab (anthrax toxin, Bacillus anthracis), and obiltoxaximab (anthrax toxin, Bacillus anthracis), but also to challenges with some of the promising leads. We also highlight the recent developments in pre-clinical research sector to develop exotoxin-targeted drug modalities, i.e., monoclonal antibodies, antibody fragments, antibody mimetics, receptor analogs, neutralizing scaffolds, dominant-negative mutants, and small molecules. We describe how these exotoxin-targeted drug modalities work with high-resolution structural knowledge and highlight their advantages and disadvantages as antibiotic alternatives.

PMID:35099182 | DOI:10.1021/acsinfecdis.1c00296

Elife. 2022 Jan 24;11:e73178. doi: 10.7554/eLife.73178. Online ahead of print.

ABSTRACT

The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditionally LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.

PMID:35073258 | DOI:10.7554/eLife.73178

PLoS Pathog. 2022 Jan 25;18(1):e1010241. doi: 10.1371/journal.ppat.1010241. eCollection 2022 Jan.

ABSTRACT

Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan purified from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropeptides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)-meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This peptidoglycan has a reduced glycan chain average length and ~30% increase in the L,D-crosslink, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D-transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L,D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhimurium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remarkably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-containing muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D-alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.

PMID:35077524 | PMC:PMC8815878 | DOI:10.1371/journal.ppat.1010241

Appl Microbiol Biotechnol. 2022 Feb;106(3):981-993. doi: 10.1007/s00253-022-11795-4. Epub 2022 Jan 25.

ABSTRACT

Peptidoglycan (PGN) is a unique component in the cytoderm of prokaryotes which can be recognized by different pathogen-associated molecular patterns (PAMPs) in eukaryotes, followed by a cascade of immune responses via different pathways. This review outlined the basic structure of PGN, its immunologic functions. The immunomodulation pathways mediated by PGN were elaborated. PGN induces specific immunity through stimulating different cytokine release and Th1/Th2-dominated immune responses during humoral/cellular immune response. The nonspecific immunity activation by PGN involves immunomodulation by different pattern recognition receptors (PRRs) including PGN recognition proteins (PGRPs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), Toll-like receptors (TLRs), and C-type lectin receptors (CLRs). The sources and classification of PGRPs were summarized. In view of the stimulating activities of PGN and its monomers, the potential application of PGN as vaccine or adjuvant was prospected. This review provides systematic information on PGN functionalities from the point of immunoregulation, which might be useful in the deep exploitation of PGN.Key points. The immunological functions of PGN were illustrated. Cellular and humoral immunomodulation by PGN were outlined. The use of PGN as vaccine or adjuvant was prospected.

PMID:35076738 | DOI:10.1007/s00253-022-11795-4

Clin Exp Med. 2022 Jan 25. doi: 10.1007/s10238-022-00794-4. Online ahead of print.

ABSTRACT

For over 40 years, the gold standard treatment for non-muscular invasive bladder cancer (NMIBC) has been repeated administration of Mycobacterium bovis bacille Calmette-Guerin (BCG). Upon administration, BCG initiates a cascade of immunological events that lead to the recruitment of immune cells to the bladder that eliminates NMIBC cells in a multi-mechanistic, yet incompletely defined manner. Despite its effectiveness, live BCG immunotherapy is often impacted by limited supply and availability and can cause rare but serious side effects. Bacterial extracellular vesicles (EV) are nanoparticles secreted by live bacteria. EVs are composed of multiple surface proteins, sugars, and lipid that can elicit cellular responses and host recognition similar to live bacteria. In this study, we sought to evaluate the cellular responses of epithelial bladder cancer cells (BCC) to BCG EVs and live BCG. We compared the effect of each treatment on BCC cytokine production, cellular viability and apoptosis. Our data suggest that BCG EVs are as effective as live BCG in eliciting cytokine responses and halting cancer cell growth by, in part, inducing apoptosis. These results indicate that BCG EVs warrant investigation as an alternative to live BCG for NMIBC immunotherapy.

PMID:35076789 | DOI:10.1007/s10238-022-00794-4

Sci Rep. 2022 Jan 14;12(1):721. doi: 10.1038/s41598-021-04627-4.

ABSTRACT

Bacteriophages are highly abundant molecular machines that have evolved proteins to target the surface of host bacterial cells. Given the ubiquity of lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria, we reasoned that targeting proteins from bacteriophages could be leveraged to target the surface of Gram-negative pathogens for biotechnological applications. To this end, a short tail fiber (GP12) from the T4 bacteriophage, which infects Escherichia coli (E. coli), was isolated and tested for the ability to adhere to whole bacterial cells. We found that, surprisingly, GP12 effectively bound the surface of Pseudomonas aeruginosa cells despite the established preferred host of T4 for E. coli. In efforts to elucidate why this binding pattern was observed, it was determined that the absence of the O-antigen region of LPS on E. coli improved cell surface tagging. This indicated that O-antigens play a significant role in controlling cell adhesion by T4. Probing GP12 and LPS interactions further using deletions of the enzymes involved in the biosynthetic pathway of LPS revealed the inner core oligosaccharide as a possible main target of GP12. Finally, we demonstrated the potential utility of GP12 for biomedical applications by showing that GP12-modified agarose beads resulted in the depletion of pathogenic bacteria from solution.

PMID:35031652 | DOI:10.1038/s41598-021-04627-4

Small. 2022 Jan 25:e2104763. doi: 10.1002/smll.202104763. Online ahead of print.

ABSTRACT

The survival strategies of infectious organisms have inspired many therapeutics over the years. Indeed the advent of oncolytic viruses (OVs) exploits the uncontrolled replication of cancer cells for production of their progeny resulting in a cancer-targeting treatment that leaves healthy cells unharmed. Their success against inaccessible tumors however, is highly variable due to inadequate tumor targeting following systemic administration. Coassembling herpes simplex virus (HSV1716) with biocompatible magnetic nanoparticles derived from magnetotactic bacteria enables tumor targeting from circulation with magnetic guidance, protects the virus against neutralizing antibodies and thereby enhances viral replication within tumors. This approach additionally enhances the intratumoral recruitment of activated immune cells, promotes antitumor immunity and immune cell death, thereby inducing tumor shrinkage and increasing survival in a syngeneic mouse model of breast cancer by 50%. Exploiting the properties of such a nanocarrier, rather than tropism of the virus, for active tumor targeting offers an exciting, novel approach for enhancing the bioavailability and treatment efficacy of tumor immunotherapies for disseminated neoplasms.

PMID:35076148 | DOI:10.1002/smll.202104763

Elife. 2022 Jan 24;11:e73178. doi: 10.7554/eLife.73178.

ABSTRACT

The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditional LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and, contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.

PMID:35073258 | PMC:PMC8820737 | DOI:10.7554/eLife.73178

Proc Natl Acad Sci U S A. 2022 Jan 25;119(4):e2114478119. doi: 10.1073/pnas.2114478119.

ABSTRACT

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA-IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.

PMID:35058363 | PMC:PMC8795526 | DOI:10.1073/pnas.2114478119

Front Pharmacol. 2022 Jan 4;12:814113. doi: 10.3389/fphar.2021.814113. eCollection 2021.

ABSTRACT

Leukotriene synthesis in neutrophils is critical for host survival during infection. In particular, leukotriene B₄ (LTB₄) is a powerful neutrophil chemoattractant that plays a crucial role in neutrophil swarming. In this work, we demonstrated that preincubation of human neutrophils with Salmonella typhimurium strongly stimulated LTB₄ production induced by the bacterial chemoattractant, peptide N-formyl-L-methionyl-L-leucyl-l-phenylalanine (fMLP), while the reverse sequence of additions was ineffective. Preincubation with bacterial lipopolysaccharide or yeast polysaccharide zymosan particles gives weaker effect on fMLP-induced LTB₄ production. Activation of 5-lipoxygenase (5-LOX), a key enzyme in leukotrienes biosynthesis, depends on rise of cytosolic concentration of Ca²⁺ and on translocation of the enzyme to the nuclear membrane. Both processes were stimulated by S. typhimurium. With an increase in the bacteria:neutrophil ratio, the transformation of LTB₄ to ω-OH-LTB₄ was suppressed, which further supported increased concentration of LTB₄. These data indicate that in neutrophils gathered around bacterial clusters, LTB₄ production is stimulated and at the same time its transformation is suppressed, which promotes neutrophil swarming and elimination of pathogens simultaneously.

PMID:35058789 | PMC:PMC8764451 | DOI:10.3389/fphar.2021.814113

Mol Immunol. 2022 Jan;141:287-296. doi: 10.1016/j.molimm.2021.12.006. Epub 2021 Dec 10.

ABSTRACT

As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.

PMID:34915268 | PMC:PMC8660258 | DOI:10.1016/j.molimm.2021.12.006

Mater Horiz. 2022 Jan 17. doi: 10.1039/d1mh01813b. Online ahead of print.

ABSTRACT

Bacterial infection has become a global concern owing to the significant morbidity and mortality. Although the phagocytosis of bacteria by immune cells acts as the front line to protect human body from invading pathogens, the relatively slow encounter and insufficient capture of bacteria by immune cells often lead to an inefficient clearance of pathogens. Herein, a supramolecular artificial receptor-modified macrophage (SAR-Macrophage) was developed to enhance the recognition and latch of bacteria in the systemic circulation, mediated via strong and multipoint host-guest interactions between the artificial receptors (cucurbit[7]uril) on the macrophage and the guest ligands (adamantane) selectively anchored on Escherichia coli (E. coli). As a result, the SAR-Macrophage could significantly accelerate the recognition of E. coli, catch and internalize more pathogens, which subsequently induced the M1 polarization of macrophages to generate ROS and effectively kill the intracellular bacteria. Therefore, the SAR-Macrophage represents a simple, yet powerful anti-bacterial approach.

PMID:35037009 | DOI:10.1039/d1mh01813b