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Frontiers in Bioscience (Landmark... May 2024Recent studies highlighted the presence of anti-α-Gal antibodies in patients implanted with commercial bioprosthetic heart valves (BHVs). BHVs expose residual α-Gal...
BACKGROUND
Recent studies highlighted the presence of anti-α-Gal antibodies in patients implanted with commercial bioprosthetic heart valves (BHVs). BHVs expose residual α-Gal xenoantigen and their recognition by the circulating anti-Gal antibodies leads to opsonization of the device's tissue component with the consequent triggering of a deterioration pathway that culminates with calcification. Small animal models such as mice and rats have been broadly involved in the testing of biomaterials by subcutaneous implantation, especially for the effectiveness of BHVs anti-calcific treatments. However, since models employed for this purpose express α-Gal antigen, the implantation of BHVs' leaflets does not elicit a proper immunological response, so the calcification propensity may be dramatically underestimated.
METHODS
An α-Gal knockout (KO) mouse model has been created, using the CRISP/Cas9 approach, and adopted to assess the calcification potential of commercial BHVs leaflets through the surgical implantation in the back subcutis area. Calcium quantification was performed by inductively coupled plasma analysis; immune response against the BHVs leaflets and α-Gal silencing was evaluated through immunological assays.
RESULTS
Two months after the implantation of commercial BHV leaflets, the anti-Gal antibody titers in KO mice doubled when compared with those found in wild-type (WT) ones. Leaflets explanted from KO mice, after one month, showed a four-time increased calcium deposition concerning the ones explanted from WT. The degree of silencing of α-Gal varied, depending on the specific organ that was assessed. In any case, the animal model was suitable for evaluating implanted tissue responses.
CONCLUSIONS
Such mouse model proved to be an accurate tool for the study of the calcific propensity of commercial BHVs leaflets than those hitherto used. Given its reliability, it could also be successfully used to study even other diseases in which the possible involvement of α-Gal has been observed.
Topics: Animals; Heart Valve Prosthesis; Calcinosis; Mice, Knockout; Bioprosthesis; Mice; Disease Models, Animal; Mice, Inbred C57BL; Male
PubMed: 38812319
DOI: 10.31083/j.fbl2905181 -
EBioMedicine May 2024Natural killer (NK) cells are important innate immunity players and have unique abilities to recognize and eliminate cancer cells, particularly in settings of...
BACKGROUND
Natural killer (NK) cells are important innate immunity players and have unique abilities to recognize and eliminate cancer cells, particularly in settings of antibody-opsonization and antibody-dependant cellular cytotoxicity (ADCC). However, NK cell-based responses in bladder cancers to therapeutic antibodies are typically immunosuppressed, and these immunosuppressive mechanisms are largely unknown.
METHODS
Single cell RNA sequencing (scRNA-seq) and high-dimensional flow cytometry were used to investigate the phenotype of tumour-infiltrating NK cells in patients with bladder cancer. Further, in vitro, and in vivo models of this disease were used to validate these findings.
FINDINGS
NK cells within bladder tumours displayed reduced expression of FcγRIIIa/CD16, the critical Fc receptor involved in ADCC-mediated cytotoxicity, on both transcriptional and protein levels. Transcriptional signatures of transforming growth factor (TGF)-β-signalling, a pleiotropic cytokine known for its immunosuppressive and tissue residency-inducing effects, were upregulated in tumour-infiltrating NK cells. TGF-β mediated CD16 downregulation on NK cells, was further validated in vitro, which was accompanied by a transition into a tissue residency phenotype. This CD16 downregulation was also abrogated by TGF-βR signalling inhibition, which could also restore the ADCC ability of NK cells subject to TGF-β effects. In a humanized mouse model of bladder cancer, mice treated with a TGF-β inhibitor exhibited increased ADCC activity compared to mice treated only with antibodies.
INTERPRETATION
This study highlights how TGF-β-rich bladder cancers inhibit NK cell-mediated ADCC by downregulating CD16. TGF-β inhibition represents new avenues to reverse immunosuppression and enhance the tumoricidal capacity of NK cells in bladder cancer.
FUNDING
The Guimaraes Laboratory is funded by a US Department of Defense-Breast Cancer Research Program-Breakthrough Award Level 1 (#BC200025), a grant (#2019485) awarded through the Medical Research Future Fund (MRFF, with the support of the Queensland Children's Hospital Foundation, Microba Life Sciences, Richie's Rainbow Foundation, Translational Research Institute (TRI) and UQ), and a grant (#RSS_2023_085) funded by a Metro South Health Research Support Scheme. J.K.M.W. is funded by a UQ Research Training Program PhD Scholarship and N.O. is funded by a NHMRC Postgraduate Scholarship (#2021932).
PubMed: 38810560
DOI: 10.1016/j.ebiom.2024.105176 -
Journal, Genetic Engineering &... Jun 2024Malaria has remained a major health concern for decades among people living in tropical and sub-tropical countries. Plasmodium falciparum is one of the critical species...
BACKGROUND
Malaria has remained a major health concern for decades among people living in tropical and sub-tropical countries. Plasmodium falciparum is one of the critical species that cause severe malaria and is responsible for major mortality. Moreover, the parasite has generated resistance against all WHO recommended drugs and therapies. Therefore, there is an urgent need for preventive measures in the form of reliable vaccines to achieve the target of a malaria-free world. Surface proteins are the preferable choice for subunit vaccine development because they are rapidly detected and engaged by host immune cells and vaccination-induced antibodies. Additionally, abundant surface or membrane proteins may contribute to the opsonization of pathogens by vaccine-induced antibodies.
RESULTS
In our study, we have listed all those surface proteins from the literature that could be functionally important and essential for infection and immune evasion of the malaria parasite. Eight Plasmodium surface and membrane proteins from the pre-erythrocyte and erythrocyte stages were shortlisted. Thirty-seven epitopes (B-cell, CTL, and HTL epitopes) from these proteins were predicted using immune-informatic tools and joined with suitable peptide linkers to design a vaccine construct. A TLR-4 agonist peptide adjuvant was added at the N-terminus of the multi-epitope series, followed by the PADRE sequence and EAAAK linker. The TLR-4 receptor was docked with the construct's anticipated model structure. The complex of vaccine and TLR-4, with the lowest energy -1514, was found to be stable under simulated physiological settings.
CONCLUSION
This study has provided a novel multi-epitope construct that may be exploited further for the development of an efficient vaccine for malaria.
PubMed: 38797552
DOI: 10.1016/j.jgeb.2024.100377 -
Veterinary Sciences Apr 2024Human patients with type 1 diabetes mellitus (T1DM) are susceptible to several long-term complications that are related to glycemic control and immune dysregulation....
Human patients with type 1 diabetes mellitus (T1DM) are susceptible to several long-term complications that are related to glycemic control and immune dysregulation. Immune function remains relatively unexplored in dogs with naturally occurring diabetes mellitus (NODM). Calcitriol improves various aspects of immune function in a variety of species, but its effect in diabetic dogs remains unexplored. Therefore, the objectives of this study were to (i) evaluate immune function in dogs with NODM and determine if differences exist based on the level of clinical control and (ii) assess the immunomodulatory effects of calcitriol. Twenty diabetic dogs (clinically controlled, n = ten, not controlled, n = ten) and 20 non-diabetic, healthy control dogs were included in this prospective, case-control study. Whole blood was incubated with calcitriol (10 M) or negative control, after which the samples were divided for phagocytosis and leukocyte cytokine response experiments. The phagocytosis of opsonized () was evaluated with flow cytometry. The samples for leukocyte cytokine response evaluations were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or phosphate buffer solution (PBS; negative control), and tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, and IL-10 were measured in supernatant using a canine-specific multiplex bead-based assay. The leukocytes from diabetic dogs produced higher concentrations of IL-10 ( = 0.01), IL-6 ( < 0.0001), and IL-8 ( < 0.0001) than the control dogs while controlling for the intervention and stimulant. Calcitriol decreased the supernatant concentrations of TNF-α ( < 0.001) and IL-8 ( = 0.04) with concomitant increases in IL-6 ( = 0.005). Diabetic dogs had a lower percentage of leukocytes undergoing phagocytosis ( < 0.0001) but a higher number of bacteria phagocytized per cell ( = 0.001) when compared to the control dogs. Calcitriol had no effect on phagocytic capacity. Lastly, the status of clinical control in diabetic dogs did not yield differences in immune function. These results support that dogs with NODM exhibit immune dysregulation and warrant additional investigation.
PubMed: 38787165
DOI: 10.3390/vetsci11050193 -
Frontiers in Oncology 2024Targeted therapy has become crucial to modern translational science, offering a remedy to conventional drug delivery challenges. Conventional drug delivery systems... (Review)
Review
Targeted therapy has become crucial to modern translational science, offering a remedy to conventional drug delivery challenges. Conventional drug delivery systems encountered challenges related to solubility, prolonged release, and inadequate drug penetration at the target region, such as a tumor. Several formulations, such as liposomes, polymers, and dendrimers, have been successful in advancing to clinical trials with the goal of improving the drug's pharmacokinetics and biodistribution. Various stealth coatings, including hydrophilic polymers such as PEG, chitosan, and polyacrylamides, can form a protective layer over nanoparticles, preventing aggregation, opsonization, and immune system detection. As a result, they are classified under the Generally Recognized as Safe (GRAS) category. Serum, a biological sample, has a complex composition. Non-specific adsorption of chemicals onto an electrode can lead to fouling, impacting the sensitivity and accuracy of focused diagnostics and therapies. Various anti-fouling materials and procedures have been developed to minimize the impact of fouling on specific diagnoses and therapies, leading to significant advancements in recent decades. This study provides a detailed analysis of current methodologies using surface modifications that leverage the antifouling properties of polymers, peptides, proteins, and cell membranes for advanced targeted diagnostics and therapy in cancer treatment. In conclusion, we examine the significant obstacles encountered by present technologies and the possible avenues for future study and development.
PubMed: 38779096
DOI: 10.3389/fonc.2024.1391293 -
Pathogens & Immunity 2024The effective elimination of encapsulated bacteria like type a (Hia) relies on immune mechanisms such as complement-mediated opsonophagocytosis by neutrophils in...
BACKGROUND
The effective elimination of encapsulated bacteria like type a (Hia) relies on immune mechanisms such as complement-mediated opsonophagocytosis by neutrophils in coordination with opsonization by anti-capsular antibodies. This study evaluated if Hia could activate the immune response through neutrophils and if these responses differed between encapsulated versus unencapsulated or invasive versus non-invasive strains.
METHODS
HL-60-derived neutrophil-like cells (dHL-60), differentiated with 1.25% dimethyl sulfoxide over 9 days, were used in an opsonophagocytosis assay and infection model to measure Hia's susceptibility to killing and dHL-60 surface molecule expression, respectively. The impact of strain-specific features on the immune response was investigated using clinical isolates of a dominant North American sequence type (ST)-23, including Hia 11-139 (encapsulated, invasive), 14-61 (encapsulated, non-invasive), 13-0074 (unencapsulated, invasive), as well as a representative ST-4 isolate (Hia 13-240, encapsulated, invasive), and a nontypeable strain (NTHi 375, unencapsulated, non-invasive).
RESULTS
Unencapsulated and non-invasive Hi strains were more susceptible to killing by the innate immune response while the ST-23 invasive strain, Hia 11-139 required serum antibodies for destruction. Flow cytometry analysis showed increased expression of co-stimulatory molecule ICAM-1 and Fc receptors (CD89, CD64) but decreased expression of the Fc receptor CD16, revealing potential mechanisms of neutrophil-mediated defense against Hia that extend to both non-invasive and invasive strains.
CONCLUSIONS
Hia clinical isolates with diverse pathogenicity illustrated contrasting susceptibility to killing by immune mechanisms while maintaining the same capacity to activate neutrophil-like cells, further underscoring the need for additional studies on Hia's pathogenesis.
PubMed: 38774126
DOI: 10.20411/pai.v9i1.659 -
BioRxiv : the Preprint Server For... Apr 2024Reducing malaria transmission has been a major pillar of control programmes and is considered crucial for achieving malaria elimination. Gametocytes, the transmissible...
Reducing malaria transmission has been a major pillar of control programmes and is considered crucial for achieving malaria elimination. Gametocytes, the transmissible forms of the parasite, arise during the blood stage of the parasite and develop through 5 morphologically distinct stages. Immature gametocytes (stage I-IV) sequester and develop in the extravascular niche of the bone marrow and possibly spleen. Only mature stage V gametocytes re-enter peripheral circulation to be taken up by mosquitoes for successful onward transmission. We have recently shown that immature, but not mature gametocytes are targets of host immune responses and identified putative target surface antigens. We hypothesize that these antigens play a role in gametocyte sequestration and contribute to acquired transmission-reducing immunity. Here we demonstrate that surface antigen expression, serum reactivity by human IgG, and opsonic phagocytosis by macrophages all show similar dynamics during gametocyte maturation, i.e., on in immature and off in mature gametocytes. Moreover, the switch in surface reactivity coincides with reversal in phosphatidylserine (PS) surface exposure, a marker for red blood cell age and clearance. PS is exposed on the surface of immature gametocytes (as well as in late asexual stages) but is removed from the surface in later gametocyte stages (IV-V). Using parasite reverse genetics and drug perturbations, we confirm that parasite protein export into the host cell and phospholipid scramblase activity are required for the observed surface modifications in asexual and sexual stages. These findings suggest that the dynamic surface remodelling allows (i) immature gametocyte sequestration in bone marrow and (ii) mature gametocyte release into peripheral circulation and immune evasion, therefore contributing to mature gametocyte survival and onward transmission to mosquitoes. Importantly, blocking scramblase activity during gametocyte maturation results in efficient clearance of mature gametocytes, revealing a potential path for transmission blocking interventions. Our studies have important implications for our understanding of parasite biology and form a starting point for novel intervention strategies to simultaneously reduce parasite burden and transmission.
PubMed: 38746342
DOI: 10.1101/2024.04.30.591837 -
Redox Biology May 2024Monocyte-derived dendritic cells (MDDCs) are key players in the defense against fungal infection because of their outstanding capacity for non-opsonic phagocytosis and...
Monocyte-derived dendritic cells (MDDCs) are key players in the defense against fungal infection because of their outstanding capacity for non-opsonic phagocytosis and phenotypic plasticity. Accordingly, MDDCs rewire metabolism to meet the energetic demands for microbial killing and biomass synthesis required to restore homeostasis. It has been commonplace considering the metabolic reprogramming a mimicry of the Warburg effect observed in tumor cells. However, this may be an oversimplification since the offshoots of glycolysis and the tricarboxylic acid (TCA) cycle are connected in central carbon metabolism. Zymosan, the external wall of Saccharomyces cerevisiae, contains β-glucan and α-mannan chains that engage the C-type lectin receptors dectin-1/2 and Toll-like receptors. This makes it an optimal fungal surrogate for experimental research. Using real-time bioenergetic assays and [U-C]glucose labeling, central hubs connected to cytokine expression were identified. The pentose phosphate pathway (PPP) exhibited a more relevant capacity to yield ribose-5-phosphate than reducing equivalents of NADPH, as judged from the high levels of isotopologues showing C-labeling in the ribose moiety and the limited contribution of the oxidative arm of the PPP to the production of ROS by NADPH oxidases (NOX). The finding of C-label in the purine ring and in glutathione unveiled the contribution of serine-derived glycine to purine ring and glutathione synthesis. Serine synthesis also supported the TCA cycle. Zymosan exhausted NAD and ATP, consistent with intracellular consumption and/or extracellular export. Poly-ADP-ribosylated proteins detected in the nuclear fractions of MDDCs did not show major changes upon zymosan stimulation, which suggests its dependence on constitutive Fe(II)/2-oxoglutarate-dependent demethylation of 5-methylcytosine by TET translocases and/or demethylation of histone H3 lysine 27 by JMJD demethylases rather than on NOX activities. These results disclose a unique pattern of central carbon metabolism following fungal challenge, characterized by the leverage of glycolysis offshoots and an extensive recycling of NAD and poly(ADP-ribose).
PubMed: 38744190
DOI: 10.1016/j.redox.2024.103187 -
Nature Communications Apr 2024Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an...
Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3's Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGh. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh subclass in two distant biological systems provides new insights into antibody function.
Topics: Animals; Humans; Mice; Antibodies, Bacterial; Antibodies, Monoclonal; Antibodies, Viral; COVID-19; Immunoglobulin Fc Fragments; Immunoglobulin G; Mice, Inbred BALB C; Phagocytosis; Protein Engineering; SARS-CoV-2; Streptococcal Infections; Streptococcus pyogenes
PubMed: 38678029
DOI: 10.1038/s41467-024-47928-8 -
Veterinary Immunology and... Jun 2024Polymorphonuclear cells (PMN) provide a rapid response to infection and tissue damage and stress can modify these critical innate immune defences. The study of...
Polymorphonuclear cells (PMN) provide a rapid response to infection and tissue damage and stress can modify these critical innate immune defences. The study of adrenergic receptor (AR) expression and function in bovine PMNs is limited but both neutrophils and eosinophils express numerous AR genes but differ significantly in their expression of individual AR genes. A flow cytometric technique was developed to differentiate between bovine neutrophils and eosinophils so both neutrophil and eosinophil responses to adrenergic agonists could be analysed. Neutrophils and eosinophils displayed significantly different changes in CD11b, L-selectin, and CD44 expression when activated by bovine serum opsonized zymosan and recombinant bovine interferon gamma. The responses of activated and resting neutrophils and eosinophils were then compared following stimulation with endogenous adrenergic agonists, epinephrine (E) norepinephrine (NE), and synthetic agonists targeting α1-, α2-, or β-ARs. Both resting and activated neutrophils and eosinophils displayed differences in iROS, CD44, and L-selectin expression following stimulation with E and NE. Resting neutrophils displayed pro-inflammatory responses to both E and NE, while resting eosinophils displayed a pro-inflammatory response to only NE. No single synthetic adrenergic agonist fully recapitulated responses observed with either E or NE and responses to adrenergic agonists were dose-dependent. In conclusion, bovine eosinophils and neutrophils responded to multiple adrenergic agonists by altering expression of proteins involved in immune surveillance and pro-inflammatory responses. Significant differences in neutrophil and eosinophil responses to adrenergic agonists are consistent with their differences in AR gene expression. This highlights the importance of analysing separately these two PMN subpopulations when investigating the effects of either endogenous or synthetic AR agonists.
Topics: Animals; Cattle; Neutrophils; Eosinophils; L-Selectin; Norepinephrine; Epinephrine; Adrenergic Agonists; Hyaluronan Receptors; Flow Cytometry; CD11b Antigen; Neutrophil Activation; Receptors, Adrenergic
PubMed: 38669937
DOI: 10.1016/j.vetimm.2024.110758