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Platelets, anucleate blood cells derive from megakaryocytes, are involved in cardiovascular diseases and tumors. FcγRIIA, the only FcγR expressed on human platelets, is known for its role in immune-related diseases. A growing body of evidence reveals that platelet FcγRIIA is a potential target for the prevention and control of cardiovascular disease and cancer, and is an advantageous biomarker. In this review, we describe the structure and physiological function of platelet FcγRIIA, its regulatory role in cardiovascular disease and cancer, and its potential clinical application. • Platelets are a group of highly reactive and sensitive cells that play a crucial role in human disease. • FcγRIIA, the only FcγR expressed on human platelets, is abnormal expressed in cardiovascular diseases and cancers. • Platelet FcγRIIA is a good biomarker for the diagnosis and treatment of cardiovascular diseases and tumors. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19), the illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and spread very quickly across the world. Different responses to infections have been related to fragment crystallizable gamma-receptor II alpha (FcγRIIA) polymorphisms. The purpose of this investigation was to determine if FCγRIIA rs1801274 polymorphism was related to COVID-19 mortality among different variants of SARS-CoV-2. The FCγRIIA rs1801274 polymorphism was genotyped using the polymerase chain reaction-restriction fragment length polymorphism technique in 1,734 recovered and 1,450 deceased patients. Deceased patients had significantly higher minor allele frequency of the FCγRIIA rs1801274 G allele than in the recovered cases. The COVID-19 mortality was associated with FCγRIIA rs1801274 GG and AG genotypes in the Delta variant and with FCγRIIA rs1801274 GG genotypes in the Alpha and Omicron BA.5 variants. The reverse transcription-quantitative polymerase chain reaction Ct values revealed statistically significant differences between individuals with a G allele and those with an A allele. In conclusion, among the several SARS-CoV-2 variants, there may be a correlation between the mortality rate of COVID-19 and the G allele of FCγRIIA rs1801274. To confirm our findings, thorough research is still required. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19), which was caused by the coronavirus - severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was globally responsible for remarkable morbidity and mortality. Several highly effective vaccines for COVID-19 were developed and disseminated worldwide within an unprecedented timescale. Rare but dangerous clotting and thrombocytopenia events, and subsequent coagulation abnormalities, have been reported after massive vaccination against SARS-CoV-2. Soon after their global rollout, reports of a morbid clinical syndrome following vaccination with adenovirus-DNA-based vaccines appeared. In the spring of 2021, reports of a novel, rare and morbid clinical syndrome, with clinically devastating and fatal complication after vaccination with adenovirus-based coronavirus vaccines (Janssen/Johnson & Johnson and Astra-Zeneca vaccines) led to a brief suspension of their use by several countries. Those complications were associated with unusual cerebral and splanchnic venous thrombosis, and circulating autoantibodies directed against anti-platelet factor 4 (PF4), a protein secreted from platelets, leading to the designation: Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT). The reported VITT incidence remains very low and does not affect the overall benefit of immunization, however, if left untreated, VITT can be debilitating or even fatal. VITT resembled specific adverse drugs' reactions that also involved the production of autoantibodies and subsequent abnormal platelet activation through platelet FcγRIIa. These unusual but well-documented drug reactions were heparin-induced thrombocytopenia (HIT), streptokinase- (SK), and anisoylated plasminogen-streptokinase activator complex- (APSAC) associated with platelet-activating antibodies. There was considerable overlapping of clinical features between VITT, COVID-19 and these adverse drugs' reactions. We review the phenomenon of VITT against the backdrop of shared and common mechanisms that underlie HIT-, SK-, and APSAC-platelet FcγRIIa-dependent platelet activation. An understanding of VITT's pathogenesis may be achieved by comparing and contrasting VITT-, HIT-, SK- and APSAC-induced platelet activation mechanisms, their respective physiopathology and similarities. Discussing these conditions in parallel provides insight into complex immunological disorders and diseases associated with abnormal hemostasis and thrombosis in particular.

In recent years, it has been established that atherosclerosis is an autoimmune disease. However, little is currently known about the role of FcγRIIA in atherosclerosis. Herein, we sought to investigate the relationship between FcγRIIA genotypes and the effectiveness of different IgG subclasses in treating atherosclerosis. We constructed and produced different subtypes of IgG and Fc-engineered antibodies. In vitro, we observed the effect of different subtypes of IgG and Fc-engineered antibodies on the differentiation of CD14+ monocytes from patients or healthy individuals. In vivo, Apoe−/− mice were fed a high-fat diet (HFD) for 20 weeks and administered injections of different CVI-IgG subclasses or Fc-engineered antibodies. Flow cytometry was used to assess the polarization of monocytes and macrophages. Although CVI-IgG4 reduced the release of MCP-1 compared to the other subtypes, IgG4 did not yield an anti-inflammatory effect by induction of human monocyte and macrophage differentiation in vitro. Furthermore, genetic polymorphisms of FcγRIIA were not associated with different CVI-IgG subclasses during the treatment of atherosclerosis. In vivo, CVI-IgG1 decreased Ly6Chigh monocyte differentiation and promoted M2 macrophage polarization. We also found that the secretion of IL-10 was upregulated in the CVI-IgG1-treated group, whereas V11 and GAALIE exerted no significant effect. These findings highlight that IgG1 is the optimal subtype for treating atherosclerosis, and CVI-IgG1 can induce monocyte/macrophage polarization. Overall, these results have important implications for the development of therapeutic antibodies. [ABSTRACT FROM AUTHOR]

ABSTRACT Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)–related extrahepatic manifestation that is characterized by the abnormal presence of immune complexes (ICs). This may be due to the reduced uptake and clearance of ICs. The C-type lectin member 18A (CLEC18A) is a secretory protein that is expressed abundantly in hepatocytes. We previously observed that CLEC18A increased significantly in the phagocytes and sera of patients with HCV, particularly those with MC. Herein, we explored the biological functions of CLEC18A in the MC syndrome development of patients with HCV by using an in vitro cell-based assay with quantitative reverse transcription-PCR, immunoblotting, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. HCV infection or Toll-like receptor 3/7/8 activation could induce CLEC18A expression in Huh7.5 cells. Upregulated CLEC18A interacts with Rab5 and Rab7 and enhances type I/III interferon production to inhibit HCV replication in hepatocytes. However, overexpressed CLEC18A suppressed phagocytic activity in phagocytes. Significantly decreased levels of the Fc gamma receptor (FcγR) IIA were found in the neutrophils of HCV patients, particularly in those with MC (P

Fragment crystallizable gamma receptors (FcγRs) mediate various cellular responses with significant cardiovascular implications. They contribute to the anticancer activity of trastuzumab (TRZ), a recombinant humanized monoclonal antibody that interferes with human epidermal growth factor receptor 2 (HER2), thereby blocking its physiological function in cardiac cells. This is responsible for cardiac complications that hamper TRZ clinical application. In this study we investigated the involvement of FcγRs in the TRZ cardiotoxicity. We used a recombinant antigen-binding fragment (Fab) of TRZ (rFab-HER2) to examine whether the absence of the Fc region resulted in fewer cardiomyocyte toxicity while preserving TRZ's ability to inhibit HER2. When exposed to rFab-HER2, AC16 human adult ventricular cardiomyocytes were less vulnerable to damage and death, than to TRZ. Specifically, TRZ exhibited cytotoxicity at a lower concentration (150 µg/mL, corresponding to ~1 µM) compared to rFab-HER2 (250 µg/mL, corresponding to ~5 µM). Like TRZ, rFab-HER2 negatively modulated HER2 levels in cardiomyocyte (without inducing cytotoxic activity in BJ human fibroblast cells that either did not express or express very low levels of HER2) and inhibited the downstream ERK/AKT cascades. But rFab-HER2 did not alter cardiomyocyte mitochondrial dynamic balance, and affect apoptosis and inflammation, while it limited cytosolic and mitochondrial ROS indicators. On contrary, the Fc region (50-250 μg/mL) exerted direct cytotoxic action on cardiomyocytes (but not on human fibroblasts that lacked Fc receptors). TRZ (150 μg/mL) markedly upregulated the expression level of FcγRIIA (a FcγRs strongly involved in TRZ-induced antibody-dependent cellular toxicity) in cardiomyocytes, whereas the Fab fragment (150 μg/mL) had no effect. Our results demonstrate that Fc region plays an important pathogenic role in TRZ-induced cardiomyocyte toxicity. In addition, targeting FcγRIIA might contribute to the off-target effects of TRZ therapy., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

The immune system is comprised of two principal interconnected components called innate and adaptive immunity. While the innate immune system mounts a nonspecific response that provides protection against the spread of foreign pathogens, the adaptive immune system has developed to specifically recognize a given pathogen and lead to immunological memory. Platelets are small fragments produced from megakaryocytes in bone marrow and lungs. They circulate throughout the blood to monitor the integrity of the vasculature and to prevent bleeding. Given their large repertoire of immune receptors and inflammatory molecules, platelets and megakaryocytes can contribute to both innate and adaptive immunity. In adaptive immunity, platelets and megakaryocytes can process and present antigens to lymphocytes. Moreover, platelets, via FcγRIIA, rapidly respond to pathogens in an immune host when antibodies are present. This manuscript reviews the reported contributions of platelets and megakaryocytes with emphasis on antigen presentation and antibody response in adaptive immunity.

In recent years, it has been established that atherosclerosis is an autoimmune disease. However, little is currently known about the role of FcγRIIA in atherosclerosis. Herein, we sought to investigate the relationship between FcγRIIA genotypes and the effectiveness of different IgG subclasses in treating atherosclerosis. We constructed and produced different subtypes of IgG and Fc-engineered antibodies. In vitro, we observed the effect of different subtypes of IgG and Fc-engineered antibodies on the differentiation of CD14+ monocytes from patients or healthy individuals. In vivo, Apoe−/− mice were fed a high-fat diet (HFD) for 20 weeks and administered injections of different CVI-IgG subclasses or Fc-engineered antibodies. Flow cytometry was used to assess the polarization of monocytes and macrophages. Although CVI-IgG4 reduced the release of MCP-1 compared to the other subtypes, IgG4 did not yield an anti-inflammatory effect by induction of human monocyte and macrophage differentiation in vitro. Furthermore, genetic polymorphisms of FcγRIIA were not associated with different CVI-IgG subclasses during the treatment of atherosclerosis. In vivo, CVI-IgG1 decreased Ly6Chigh monocyte differentiation and promoted M2 macrophage polarization. We also found that the secretion of IL-10 was upregulated in the CVI-IgG1-treated group, whereas V11 and GAALIE exerted no significant effect. These findings highlight that IgG1 is the optimal subtype for treating atherosclerosis, and CVI-IgG1 can induce monocyte/macrophage polarization. Overall, these results have important implications for the development of therapeutic antibodies.

Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes. [ABSTRACT FROM AUTHOR]

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. [ABSTRACT FROM AUTHOR]

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

Abstract Background Anti‐platelet factor 4 (PF4) antibodies that activate platelets via FcγRIIA drive the pathophysiology of vaccine‐induced immune thrombocytopenia and thrombosis (VITT). Evolution of these antibodies and their ability to activate platelets after initial treatment remains unknown. Objectives To assess how clinical and platelet parameters, anti‐PF4 antibody levels, and patient serum reactivity changes during follow‐up after VITT presentation. Methods We describe cases of seven discharged VITT patients that were followed from diagnosis up to 280 days (range 199–280) after vaccination. We measured anti‐PF4 antibodies and PF4 levels in patient serum during follow‐up and tested the ability of patient serum to activate healthy donor platelets and patient platelets over time. Results Anti‐PF4 immunoglobulin G antibody levels are very high at diagnosis (0.9–2.6 OD) and remain relatively high (>1.0 OD) in all patients, except one treated with rituximab, at 7 months post vaccination. All patients were on direct oral anticoagulants throughout follow‐up and no patients had recurrent thrombosis. Patients’ platelets during follow‐up have normal FcγRIIA levels and responsiveness to platelet agonists. Patient diagnostic serum strongly activated control platelets, either alone or with PF4. Most follow‐up serum alone was weaker at stimulating donor and patient platelets. However, follow‐up serum beyond 150 days still strongly activated platelets with PF4 addition in three patients. Patient serum PF4 levels were lower than controls at diagnosis but returned within normal range by day 50. Conclusions Explanations for reduced platelet activation during follow‐up, despite similar total anti‐PF4 antibody levels, remains unclear. Clinical implications of persistent anti‐PF4 antibodies in VITT require further study.

Low-affinity Fcγ-receptors that recognize the Fc portion of immunoglobulin (Ig) molecules, usually being in antigen-bound state, thus representing a link between innate and adaptive immunity. They play a significant role in inflammatory and infectious diseases. Among them, a separate FcγRII family (CD32) is discerned, which is characterized by transmission of intracellular signal independently of the common γ-chain, they have one α-chain containing two extracellular immunoglobulin-like domains. FcγRII receptors are present in almost all cells of the innate immune system: monocytes and macrophages, neutrophils, eosinophils, dendritic cells, as well as on B-lymphocytes and platelets. They perform two main functions: target recognition, facilitation of phagocytosis and destruction of antibody-opsonized cells by monocytes/ macrophages (including pathogenic cells). In parallel, the phagocytes are activated via the cytokine synthesis stimulation. The FcγRIIA (CD32a) and FcγRIIC (CD32c) activating receptors, like as FcγRIIB (CD32b) inhibiting receptors are present among the members of the FcγRII family. The low-affinity FcγRII receptors bind to IgG, with immune complexes being their natural ligands. High levels of immune complexes are usually found in both chronic viral infections and autoimmune diseases. There are shown polymorphic variants of the CD32a gene, which can affect the receptor function, and, thereby, causing susceptibility for different infections, influence the development of autoimmune diseases and primary immunodeficiencies. Activation of the CD32a receptor induces the production of pro-inflammatory cytokines, including TNFα and interferons, that are involved into inflammation in systemic lupus erythematosus, Kawasaki disease, Graves’ disease and rheumatoid arthritis. It has been shown that antibacterial activity of platelets is carried out via the CD32a receptor. The study of CD32a expression in people The CD32a receptor is considered a biomarker of cells that are a reservoir of HIV infection. At the present time, however, many questions remain regarding the mechanisms of CD32a expression of on HIV-infected cells and the role of CD32a in the formation of an HIV reservoir and/or development of appropriate resistance. In addition to HIV infection, the significance of FcγR receptors is shown in other infectious diseases, for example, with influenza and dengue virus infections. Better understanding of the CD32a structure and function will help to assess its role in immunopathogenesis of different conditions. This review focuses on the role of CD32a in development of the normal immune response in normal state and various diseases.

Soluble forms of the low-affinity immunoglobulin receptor FcγRIIa (sFcγRIIa) lacking the cytoplasmic tail have been reported in plasma however the mechanism and functional consequences are unknown. This study aimed to evaluate mechanisms of FcγRIIa release compared to GPVI release from platelets, and examine whether genetic polymorphisms at positions 27 and 131 within FcγRIIa correlate with platelet FcγRIIa stability and function. Enzyme-linked immunosorbent assays (ELISAs) were used to measure plasma sFcγRIIa and sGPVI levels. FcγRIIa genotype at positions 27 and 131 was evaluated. sFcγRIIa levels were not significantly different between non-131HH and 131HH but were significantly lower in 27W than non-27W. Treatment of platelets with aggregated immunoglobulin (Ig) G induced release of FcγRIIa and GPVI, but only sGPVI release was statistically significant, required functional FcγRIIa, and was blocked by inhibitors of signaling pathways and metalloproteinases. This indicated that sFcγRIIa was not released from platelets by metalloproteolysis. sFcγRIIa levels were not correlated with sGPVI levels in healthy individuals however levels of sFcγRIIa and sGPVI in plasma from patients with rheumatoid arthritis (RA) were significantly elevated above levels found in healthy individuals. Elevated level of sFcγRIIa in RA patients may reflect active immune-based arthritis and be predictive of active inflammation.

Aim To analyze the impact of CD32a polymorphism rs1801274 on the occurrence of Kawasaki disease (KD) through the meta-analysis.Methods The correlation between CD32a polymorphism rs1801274 and the susceptibility to KD was appraised using summarized odds ratios (ORs) with their 95% confidence intervals (95% CIs). Besides, stratification analyses were further implemented on the basis of ethnicity and control source, respectively. Between-study heterogeneity was checked adopting chi-square-based Q test, with p

Fcγ-receptors (FcγRs) including FcγRII (CD32) gene family members are expressed on leukocytes, bind the crystallizable fragment (Fc) region of immunoglobulin G (IgG), and bridge humoral and cellular immunity. FcγRIIA and FcγRIIB have opposing roles, with the former responsible for activation and the latter for inhibition of immune cell signaling and effector functions. The extracellular domains of human and murine FcγRIIs share multiple conserved N- glycosylation sites. Understanding the role(s) of FcγRIIA and FcγRIIB glycosylation in autoimmune diseases is precluded by a lack of effective methods to study disease-associated changes in glycosylation. To address this barrier, we developed a method to assess site-specific glycosylation of human FcγRIIA and FcγRIIB, and the mouse ortholog of human FcγRIIB. Among the receptors, conserved glycosylation sites are compared, with the N144/145 site displaying predominantly complex glycans in recombinant FcγRIIs. Differences in sialylation between recombinant human FcγRIIA H/R134 (H/R131) variants at a nearby N145 N -glycosylation site are reported. Further, a potential human FcγRIIA O -glycosylation site, S179 (S212), is reported in recombinant FcγRIIA. The robust method to assess site-specific glycosylation of FcγRIIs reported here, can be utilized to study the potential role of FcγRII family glycosylation in disease. Data are available via ProteomeXchange with identifier PXD049429.

Bacterial infections are a major cause of morbidity and mortality in chronic lymphocytic leukemia (CLL), and infection risk increases in patients treated with the Bruton's tyrosine kinase (Btk) inhibitor, ibrutinib. Btk and related kinases (like Tec) are expressed in non-leukemic hematopoietic cells and can be targeted by ibrutinib. In platelets, ibrutinib therapy is associated with bleeding complications mostly due to off-target effects. But the ability of platelets to respond to bacteria in CLL, and the potential impact of ibrutinib on platelet innate immune functions remain unknown. FcγRIIA is a tyrosine kinase-dependent receptor critical for platelet activation in response to IgG-coated pathogens. Crosslinking of this receptor with monoclonal antibodies causes downstream activation of Btk and Tec in platelets, however, this has not been investigated in response to bacteria. We asked whether ibrutinib impacts on FcγRIIA-mediated activation of platelets derived from CLL patients and healthy donors after exposure to Staphylococcus aureus Newman and Escherichia coli RS218. Platelet aggregation, α-granule secretion and integrin αIIbβ3-dependent scavenging of bacteria were detected in CLL platelets but impaired in platelets from ibrutinib-treated patients and in healthy donor-derived platelets exposed to ibrutinib in vitro. While levels of surface FcγRIIA remained unaffected, CLL platelets had reduced expression of integrin αIIbβ3 and GPVI compared to controls regardless of therapy. In respect of intracellular signaling, bacteria induced Btk and Tec phosphorylation in both CLL and control platelets that was inhibited by ibrutinib. To address if Btk is essential for platelet activation in response to bacteria, platelets derived from X-linked agammaglobulinemia patients (lacking functional Btk) were exposed to S. aureus Newman and E. coli RS218, and FcγRIIA-dependent aggregation was observed. Our data suggest that ibrutinib impairment of FcγRIIA-mediated platelet activation by bacteria results from a combination of Btk and Tec inhibition, although off-target effects on additional kinases cannot be discarded. This is potentially relevant to control infection-risk in CLL patients and, thus, future studies should carefully evaluate the effects of CLL therapies, including Btk inhibitors with higher specificity for Btk, on platelet-mediated immune functions. [ABSTRACT FROM AUTHOR]

FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR; FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab')2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs. [ABSTRACT FROM AUTHOR]

Bacterial infections are a major cause of morbidity and mortality in chronic lymphocytic leukemia (CLL), and infection risk increases in patients treated with the Bruton’s tyrosine kinase (Btk) inhibitor, ibrutinib. Btk and related kinases (like Tec) are expressed in non-leukemic hematopoietic cells and can be targeted by ibrutinib. In platelets, ibrutinib therapy is associated with bleeding complications mostly due to off-target effects. But the ability of platelets to respond to bacteria in CLL, and the potential impact of ibrutinib on platelet innate immune functions remain unknown. FcγRIIA is a tyrosine kinase-dependent receptor critical for platelet activation in response to IgG-coated pathogens. Crosslinking of this receptor with monoclonal antibodies causes downstream activation of Btk and Tec in platelets, however, this has not been investigated in response to bacteria. We asked whether ibrutinib impacts on FcγRIIA-mediated activation of platelets derived from CLL patients and healthy donors after exposure to Staphylococcus aureus Newman and Escherichia coli RS218. Platelet aggregation, α-granule secretion and integrin αIIbβ3-dependent scavenging of bacteria were detected in CLL platelets but impaired in platelets from ibrutinib-treated patients and in healthy donor-derived platelets exposed to ibrutinib in vitro. While levels of surface FcγRIIA remained unaffected, CLL platelets had reduced expression of integrin αIIbβ3 and GPVI compared to controls regardless of therapy. In respect of intracellular signaling, bacteria induced Btk and Tec phosphorylation in both CLL and control platelets that was inhibited by ibrutinib. To address if Btk is essential for platelet activation in response to bacteria, platelets derived from X-linked agammaglobulinemia patients (lacking functional Btk) were exposed to S. aureus Newman and E. coli RS218, and FcγRIIA-dependent aggregation was observed. Our data suggest that ibrutinib impairment of FcγRIIA-mediated platelet activation by bacteria results from a combination of Btk and Tec inhibition, although off-target effects on additional kinases cannot be discarded. This is potentially relevant to control infection-risk in CLL patients and, thus, future studies should carefully evaluate the effects of CLL therapies, including Btk inhibitors with higher specificity for Btk, on platelet-mediated immune functions.

FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR; FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab’)2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs.

Thrombosis is a hallmark of the fatal fungal infection mucormycosis. Yet, the platelet activation pathway in response to mucormycetes is unknown. In this study we determined the platelet aggregation potential of Mucor circinelloides (M. circinelloides) NRRL3631, characterized the signaling pathway facilitating aggregation in response to fungal spores, and identified the influence of the spore developmental stage upon platelet aggregation potential. Using impedance and light-transmission aggregometry, we showed that M. circinelloides induced platelet aggregation in whole blood and in platelet-rich plasma, respectively. The formation of large spore-platelet aggregates was confirmed by light-sheet microscopy, which showed spores dispersed throughout the aggregate. Aggregation potential was dependent on the spore’s developmental stage, with the strongest platelet aggregation by spores in mid-germination. Inhibitor studies revealed platelet aggregation was mediated by the low affinity IgG receptor FcγRIIA and integrin αIIbβ3; Src and Syk tyrosine kinase signaling; and the secondary mediators TxA2 and ADP. Flow cytometry of antibody stained platelets showed that interaction with spores increased expression of platelet surface integrin αIIbβ3 and the platelet activation marker CD62P. Together, this is the first elucidation of the signaling pathways underlying thrombosis formation during a fungal infection, highlighting targets for therapeutic intervention.

The immune system is comprised of two principal interconnected components called innate and adaptive immunity. While the innate immune system mounts a nonspecific response that provides protection against the spread of foreign pathogens, the adaptive immune system has developed to specifically recognize a given pathogen and lead to immunological memory. Platelets are small fragments produced from megakaryocytes in bone marrow and lungs. They circulate throughout the blood to monitor the integrity of the vasculature and to prevent bleeding. Given their large repertoire of immune receptors and inflammatory molecules, platelets and megakaryocytes can contribute to both innate and adaptive immunity. In adaptive immunity, platelets and megakaryocytes can process and present antigens to lymphocytes. Moreover, platelets, via FcγRIIA, rapidly respond to pathogens in an immune host when antibodies are present. This manuscript reviews the reported contributions of platelets and megakaryocytes with emphasis on antigen presentation and antibody response in adaptive immunity. [ABSTRACT FROM AUTHOR]

Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes.

Melioidosis is a life-threatening infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei. An effective vaccine is needed, but data on protective immune responses in human melioidosis are lacking. We used ELISA and an antibody-dependent cellular phagocytosis assay to identify the major features of protective antibodies in patients with acute melioidosis in Thailand. We found that high levels of B. pseudomallei-specific IgG2 are associated with protection against death in a multivariable logistic regression analysis adjusting for age, diabetes, renal disease, and neutrophil count. Serum from melioidosis survivors enhanced bacteria uptake into human monocytes expressing FcγRIIa-H/R131, an intermediate-affinity IgG2-receptor, compared with serum from nonsurvivors. We did not find this enhancement when using monocytes carrying the low IgG2-affinity FcγRIIa-R131 allele. The findings indicate the importance of IgG2 in protection against death in human melioidosis, a crucial finding for antibody-based therapeutics and vaccine development. [ABSTRACT FROM AUTHOR]

To analyze the impact of CD32a polymorphism rs1801274 on the occurrence of Kawasaki disease (KD) through the meta-analysis. The correlation between CD32a polymorphism rs1801274 and the susceptibility to KD was appraised using summarized odds ratios (ORs) with their 95% confidence intervals (95% CIs). Besides, stratification analyses were further implemented on the basis of ethnicity and control source, respectively. Between-study heterogeneity was checked adopting chi-square-based Q test, with p <.05 as significant level. And results from Q test determined which model would be employed for OR calculation, fixed- or random-effects. Sensitivity analysis was accomplished to test the stability of final results. Potential publication bias among included studies was investigated using Begg's funnel plot and Egger's test. If publication bias was significant, its influence on overall estimates would be measured adopting the trim-and-fill method. CD32a polymorphism rs1801274 significantly increased KD risk in total analysis under the comparisons of AA vs. GG, AA + AG vs. GG, AA vs. GG + AG, A vs. G and AG vs. GG (OR = 2.69, 95% CI = 1.39–5.20; OR = 2.00, 95% CI = 1.23–3.26; OR = 1.90, 95% CI = 1.23–2.94; OR = 1.77, 95% CI = 1.34–2.34; OR = 1.53, 95% CI = 1.07–2.19). After stratification analysis by ethnicity, similar tendency was also observed in Caucasian and Asian subgroups under corresponding genetic models. And parallel results were replicated in population-based and other-source subgroups after stratified analysis by control source, under some contrasts. CD32a polymorphism rs1801274 has strong relation to KD onset, and the presence of its A allele could elevate the disease incidence. [ABSTRACT FROM AUTHOR]

Abstract The interaction of tumor cells with platelets is a prerequisite for successful hematogenous metastatic dissemination. Upon tumor cell arrival in the blood, tumor cells immediately activate platelets to form a permissive microenvironment. Platelets protect tumor cells from shear forces and assault of NK cells, recruit myeloid cells by secretion of chemokines, and mediate an arrest of the tumor cell platelet embolus at the vascular wall. Subsequently, platelet-derived growth factors confer a mesenchymal-like phenotype to tumor cells and open the capillary endothelium to expedite extravasation in distant organs. Finally, platelet-secreted growth factors stimulate tumor cell proliferation to micrometastatic foci. This review provides a synopsis on the current literature on platelet-mediated effects in cancer metastasis and particularly focuses on platelet adhesion receptors and their role in metastasis. Immunoreceptor tyrosine-based activation motif (ITAM) and hemi ITAM (hemITAM) comprising receptors, especially, glycoprotein VI (GPVI), FcγRIIa, and C-type lectin-like-2 receptor (CLEC-2) are turned in the spotlight since several new mechanisms and contributions to metastasis have been attributed to this family of platelet receptors in the last years.

High platelet reactivity leading to spontaneous platelet aggregation (SPA) is a hallmark of cardiovascular diseases; however, the mechanism underlying SPA remains obscure. Platelet aggregation in stirred hirudin-anticoagulated blood was measured by multiple electrode aggregometry (MEA) for 10 min. SPA started after a delay of 2–3 min. In our cohort of healthy blood donors (n = 118), nine donors (8%) with high SPA (>250 AU*min) were detected. Pre-incubation of blood with two different antibodies against the platelet Fc-receptor (anti-FcγRIIA, CD32a) significantly reduced high SPA by 86%. High but not normal SPA was dose-dependently and significantly reduced by blocking Fc of human IgG with a specific antibody. SPA was completely abrogated by blood pre-incubation with the reversible Btk-inhibitor (BTKi) fenebrutinib (50 nM), and 3 h after intake of the irreversible BTKi ibrutinib (280 mg) by healthy volunteers. Increased SPA was associated with higher platelet GPVI reactivity. Anti-platelet factor 4 (PF4)/polyanion IgG complexes were excluded as activators of the platelet Fc-receptor. Our results indicate that high SPA in blood is due to platelet FcγRIIA stimulation by unidentified IgG complexes and mediated by Btk activation. The relevance of our findings for SPA as possible risk factor of cardiovascular diseases and pathogenic factor contributing to certain autoimmune diseases is discussed.

Aim: Previous studies have discussed association of FcγRIIA‐R/H131 polymorphism and systemic lupus erythematosus (SLE), lupus nephritis (LN) risk. However, conclusions were inconsistent. Methods: A meta‐analysis was performed in this study with allelic contrast (allele R vs H), additive model (genotype RR vs HH), recessive model (genotype RR vs RH + HH), and dominant model (genotype RR + RH vs HH). Results: A total of 33 studies discussed the correlation between FcγRIIA‐R/H131 polymorphism and SLE, involving 5652 SLE patients and 6322 controls. Allele R was significantly related to SLE in the overall population (odds ratio [OR] = 1.238, P <.001), Asian (OR = 1.237, P <.001) and European population (OR = 1.212, P =.012). Additive, recessive and dominant models were correlating with SLE in the overall population (OR = 1.448, P <.001; OR = 1.303, P <.001; OR = 1.310, P <.001), Asian population (OR = 1.640, P =.001; OR = 1.437, P <.001; OR = 1.344, P =.005), respectively. In addition, 22 studies evaluated relation of FcγRIIA‐R/H131 polymorphism with LN, involving 2065 patients with LN, and 2023 patients without LN. Results showed that allele R and the other 3 models related to LN susceptibility in the overall population when discussing differences of polymorphism between patients with/without LN. We further compared differences of polymorphism between patients with LN and controls, showing that additive and recessive models related to LN risk in the overall population, Asian, European and North American populations. Conclusion: In summary, FcγRIIA‐R/H131 polymorphism is associated with SLE and LN. [ABSTRACT FROM AUTHOR]

Soluble forms of the low-affinity immunoglobulin receptor FcγRIIa (sFcγRIIa) lacking the cytoplasmic tail have been reported in plasma however the mechanism and functional consequences are unknown. This study aimed to evaluate mechanisms of FcγRIIa release compared to GPVI release from platelets, and examine whether genetic polymorphisms at positions 27 and 131 within FcγRIIa correlate with platelet FcγRIIa stability and function. Enzyme-linked immunosorbent assays (ELISAs) were used to measure plasma sFcγRIIa and sGPVI levels. FcγRIIa genotype at positions 27 and 131 was evaluated. sFcγRIIa levels were not significantly different between non-131HH and 131HH but were significantly lower in 27W than non-27W. Treatment of platelets with aggregated immunoglobulin (Ig) G induced release of FcγRIIa and GPVI, but only sGPVI release was statistically significant, required functional FcγRIIa, and was blocked by inhibitors of signaling pathways and metalloproteinases. This indicated that sFcγRIIa was not released from platelets by metalloproteolysis. sFcγRIIa levels were not correlated with sGPVI levels in healthy individuals however levels of sFcγRIIa and sGPVI in plasma from patients with rheumatoid arthritis (RA) were significantly elevated above levels found in healthy individuals. Elevated level of sFcγRIIa in RA patients may reflect active immune-based arthritis and be predictive of active inflammation. [ABSTRACT FROM AUTHOR]

Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy. [Display omitted] • Preferred transduction of cells expressing two surface markers by bispecific AAVs. • Incorporation of two DARPins without detectable structural changes in the capsid. • Increased uptake rates of bispecific versus monospecific AAVs. • New options for precise in vivo gene delivery to therapy-relevant cells. • Targeting of the HIV reservoir as potential application. [ABSTRACT FROM AUTHOR]

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn’s disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 109/L, respectively, to ≈13 days (both HR and RR) at 350 × 109/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.

Thrombosis is a hallmark of the fatal fungal infection mucormycosis. Yet, the platelet activation pathway in response to mucormycetes is unknown. In this study we determined the platelet aggregation potential of Mucor circinelloides (M. circinelloides) NRRL3631, characterized the signaling pathway facilitating aggregation in response to fungal spores, and identified the influence of the spore developmental stage upon platelet aggregation potential. Using impedance and light-transmission aggregometry, we showed that M. circinelloides induced platelet aggregation in whole blood and in platelet-rich plasma, respectively. The formation of large spore-platelet aggregates was confirmed by light-sheet microscopy, which showed spores dispersed throughout the aggregate. Aggregation potential was dependent on the spore's developmental stage, with the strongest platelet aggregation by spores in mid-germination. Inhibitor studies revealed platelet aggregation was mediated by the low affinity IgG receptor FcγRIIA and integrin αIIbβ3; Src and Syk tyrosine kinase signaling; and the secondary mediators TxA2 and ADP. Flow cytometry of antibody stained platelets showed that interaction with spores increased expression of platelet surface integrin αIIbβ3 and the platelet activation marker CD62P. Together, this is the first elucidation of the signaling pathways underlying thrombosis formation during a fungal infection, highlighting targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Safety and efficacy of therapeutic antibodies are often dependent on their interaction with Fc receptors for IgG (FcγRs). The Göttingen minipig represents a valuable species for biomedical research but its use in preclinical studies with therapeutic antibodies is hampered by the lack of knowledge about the porcine FcγRs. Genome analysis and sequencing now enabled the localization of the previously described FcγRIIIa in the orthologous location to human FCGR3A. In addition, we identified nearby the gene coding for the hitherto undescribed putative porcine FcγRIIa. The 1′241 bp long FCGR2A cDNA translates to a 274aa transmembrane protein containing an extracellular region with high similarity to human and cattle FcγRIIa. Like in cattle, the intracellular part does not contain an immunoreceptor tyrosine-based activation motif (ITAM) as in human FcγRIIa. Flow cytometry of the whole blood and single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) of Göttingen minipigs revealed the expression profile of all porcine FcγRs which is compared to human and mouse. The new FcγRIIa is mainly expressed on platelets making the minipig a good model to study IgG-mediated platelet activation and aggregation. In contrast to humans, minipig blood monocytes were found to express inhibitory FcγRIIb that could lead to the underestimation of FcγR-mediated effects of monocytes observed in minipig studies with therapeutic antibodies. [ABSTRACT FROM AUTHOR]

Objective: Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development.Methods: VIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates.Results: We generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies.Conclusions: VIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases. [ABSTRACT FROM AUTHOR]

Abstract Human Fc-gamma receptors (FcγRs) FcγRIIA and FcγRIIIA contain amino acid variants with both high and low affinities for IgG that modulate antibody-mediated effector functions. Recent HIV vaccine trials suggested that these FcγR variants can influence susceptibility to HIV infection, which prompted us to fully assess the role of FcγR variants on HIV acquisition, viral control, and disease progression in two longitudinal heterosexual transmission cohorts with HIV subtypes A and C as the major circulating viruses. For 836 participants, molecular genotyping resolved genetic variations encoding the FcγRIIA (131 H/R) and FcγRIIIA (158 V/F) single nucleotide polymorphisms. Kaplan-Meier curves, Cox proportional hazards models, and linear regression models did not reveal any clear or consistent FcγR association with time to HIV acquisition, viral load in early infection, or extent of CD4 + T-cell decline over time after infection. Overall, previous epidemiological findings on FcγR variants and vaccine efficacy are not readily applicable to heterosexual HIV transmission. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19), which was caused by the coronavirus - severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was globally responsible for remarkable morbidity and mortality. Several highly effective vaccines for COVID-19 were developed and disseminated worldwide within an unprecedented timescale. Rare but dangerous clotting and thrombocytopenia events, and subsequent coagulation abnormalities, have been reported after massive vaccination against SARS-CoV-2. Soon after their global rollout, reports of a morbid clinical syndrome following vaccination with adenovirus-DNA-based vaccines appeared. In the spring of 2021, reports of a novel, rare and morbid clinical syndrome, with clinically devastating and fatal complication after vaccination with adenovirus-based coronavirus vaccines (Janssen/Johnson & Johnson and Astra-Zeneca vaccines) led to a brief suspension of their use by several countries. Those complications were associated with unusual cerebral and splanchnic venous thrombosis, and circulating autoantibodies directed against anti-platelet factor 4 (PF4), a protein secreted from platelets, leading to the designation: Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT). The reported VITT incidence remains very low and does not affect the overall benefit of immunization, however, if left untreated, VITT can be debilitating or even fatal. VITT resembled specific adverse drugs' reactions that also involved the production of autoantibodies and subsequent abnormal platelet activation through platelet Fc γ RIIa. These unusual but well-documented drug reactions were heparin-induced thrombocytopenia (HIT), streptokinase- (SK), and anisoylated plasminogen-streptokinase activator complex- (APSAC) associated with platelet-activating antibodies. There was considerable overlapping of clinical features between VITT, COVID-19 and these adverse drugs' reactions. We review the phenomenon of VITT against the backdrop of shared and common mechanisms that underlie HIT-, SK-, and APSAC-platelet Fc γ RIIa-dependent platelet activation. An understanding of VITT's pathogenesis may be achieved by comparing and contrasting VITT-, HIT-, SK- and APSAC-induced platelet activation mechanisms, their respective physiopathology and similarities. Discussing these conditions in parallel provides insight into complex immunological disorders and diseases associated with abnormal hemostasis and thrombosis in particular., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Abdelouahed, Yateem and Fredericks.)

Intravenous Immunoglobulin (IVIg) is used to treat autoimmune or inflammatory diseases, but its mechanism of action is not completely understood. We asked whether IVIg can induce interleukin-10 (IL-10) and reduce pro-inflammatory cytokine production in human monocytes, and whether this response is reduced in monocytes from people with an Fcγ receptor IIA (FcγRIIA) gene variant, which is associated with increased risk of inflammatory diseases and poor response to antibody-based biological therapy. IVIg increased IL-10 production and reduced pro-inflammatory cytokine production in response to bacterial lipopolysaccharide (LPS), which required FcγRI and FcγRIIB and activation of MAPKs, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38. IL-10 production was lower and pro-inflammatory cytokine production was higher in monocytes from people with the FcγRIIA risk variant and the risk variant prevented IL-10 production in response to (IVIg+LPS). Finally, we show that IVIg did not induce MAPK activation in monocytes from people with the risk variant. Our results demonstrate that IVIg can skew human monocytes to an anti-inflammatory, IL-10-producing activation state, which is compromised in monocytes from people with the FcγRIIA risk variant. This research has profound implications for the use of IVIg because 25% of the population is homozygous for the FcγRIIA risk variant and its efficacy may be reduced in those individuals. In addition, this research may be useful to develop new therapeutic strategies to replace IVIg by cross-linking FcγRIs and FcγRIIBs to promote anti-inflammatory macrophage activation, independent of the FcγRIIA genotype. [ABSTRACT FROM AUTHOR]

Background: In chronic immune thrombocytopenic purpura (ITP), rituximab removes the harmful autoantibodies through antibody-dependent cellular cytotoxicity. The response to rituximab in ITP is variable; the effectiveness of rituximab is influenced by the process of activation of effector fragment C gamma receptors (FcγRs). Genetic factors may affect the response to rituximab. Objectives: The influence of FcγRIIa (H131R) and FcγRIIIa (V158F) gene polymorphisms on the response to rituximab in ITP. Methods: One hundred ITP patients were genotyped for FcγRIIa (H131R) and FcγRIIIa (V158F) gene polymorphisms using the polymerase chain reaction-restriction fragment length polymorphism assay. The response at the end of the third month was assessed by direct platelets count. Polymorphisms were analyzed in relation to the response. Results: The mean platelets count at end of weeks 1-4 of rituximab was statistically significantly higher in patients who achieved complete response (CR) than partial response or no response (P-value = .001). Although RR (44.4%) and HR (38.9%) genotypes were observed to be higher in patients who achieved CR compared with the wild (HH) genotype (16.7%), it was not statistically significantly different (P-value = .648). Conclusion: The higher platelet count achieved early is predictive for a better response to rituximab later. FCγRIIA polymorphisms did not significantly influence response to rituximab in ITP. [ABSTRACT FROM AUTHOR]

Background: Brucellosis is the major bacterial zoonoses of global importance caused by Brucella spps. FCγRIIA receptor plays a central role in phagocytosis of IgG2-opsonized bacteria. FCγRIIA exhibits allelic polymorphisms with different capacities for binding IgG2 and phagocytosis. Cells expressing Fc γ RIIa-H131, bind more efficiently to complexes of IgG2 than those expressing the Fc γ RII A -R131 variant. The purpose of this study was to evaluate the association of FCγRIIA polymorphisms with susceptibility to or severity of brucellosis. Materials and Methods: In this study, we evaluated FCγRIIA polymorphisms (R/R131, R/H131, H/H131) in 67 patients with brucellosis and 67 age, sex and geographical matched healthy volunteers. FCγRIIA genotyping was performed by using a sequence-specific primer polymerase chain reaction (SSP-PCR). Results: The comparison of the FCγRIIA genotypes distribution in patients with brucellosis and controls showed a higher frequency in FCγRIIA-R/R131 homozygosity in patients than controls (47.8% vs. 28.4%). Logistic regression analysis showed that there is a significant correlation between R/R131 genotype and brucellosis (OR=2.3, 95%CI=1.3-4.2, P=0.04). Although the frequency of the FCγRIIA-R/R131 was higher in patients with chronic brucellosis compared with acute brucellosis, we did not find any statistically significant differences (53.8% vs. 46.3%, P=0.65). Conclusion: The result of this study showed that the homozygous genotype of FCγRIIA-R/R131 in patients with brucellosis may be associated with susceptibility to brucellosis as a genetic risk factor.

Introduction: Fc gamma receptor (FcγR) IIa is considered the most widely distributed of the three classes of Fc receptors, and it expresses an allelic polymorphism. This type of polymorphism may modify the immune response and may be an important factor for some diseases. The aim of the study reported herein was to evaluate the association between the FcγRIIa polymorphism and susceptibility to both end-stage renal disease (ESRD) and acute kidney graft rejection (AR) in children who have undergone renal transplantation. Material and methods: The study evaluated 70 children who had undergone transplantation and 60 healthy subjects. AR was observed in 25 children. Results: FcgRIIa genotypes and alleles were significantly different between transplantation patients and the control group. The assessment for FcgR of the groups in which AR was present showed that there was only a risk of having an acute rejection in homozygous genotype RR. Conclusions: FcγRIIa RR genotype and allele frequency was increased in paediatric renal transplant recipients. The present findings showed that FcγRIIa genotype may be related to ESRD disease susceptibility, and FcγRIIa polymorphisms seemed to affect AR. [ABSTRACT FROM AUTHOR]

Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)-related extrahepatic manifestation that is characterized by the abnormal presence of immune complexes (ICs). This may be due to the reduced uptake and clearance of ICs. The C-type lectin member 18A (CLEC18A) is a secretory protein that is expressed abundantly in hepatocytes. We previously observed that CLEC18A increased significantly in the phagocytes and sera of patients with HCV, particularly those with MC. Herein, we explored the biological functions of CLEC18A in the MC syndrome development of patients with HCV by using an in vitro cell-based assay with quantitative reverse transcription-PCR, immunoblotting, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. HCV infection or Toll-like receptor 3/7/8 activation could induce CLEC18A expression in Huh7.5 cells. Upregulated CLEC18A interacts with Rab5 and Rab7 and enhances type I/III interferon production to inhibit HCV replication in hepatocytes. However, overexpressed CLEC18A suppressed phagocytic activity in phagocytes. Significantly decreased levels of the Fc gamma receptor (FcγR) IIA were found in the neutrophils of HCV patients, particularly in those with MC ( P  < 0.005). We demonstrated that CLEC18A could inhibit FcγRIIA expression in a dose-dependent manner through the production of NOX-2-dependent reactive oxygen species to impair the uptake of ICs. Additionally, CLEC18A suppresses the Rab7 expression that is induced by starvation. Overexpressed CLEC18A does not affect autophagosome formation but does reduce the recruitment of Rab7 to autophagosomes, thereby retarding the maturation of autophagosomes and affecting autophagosome-lysosome fusion. We offer a novel molecular machinery with which to understand the association of HCV infection with autoimmunity and propose that CLEC18A may act as a candidate biomarker for HCV-associated MC. IMPORTANCE During infection, the host immune system produces cellular factors to protect against pathogen invasion. However, when the immune response overreacts and there is dysregulated cytokine homeostasis, autoimmunity occurs following an infection. We identified a cellular factor that is involved in HCV-related extrahepatic manifestation, namely, CLEC18A, which is expressed abundantly in hepatocytes and phagocytes. It inhibits HCV replication in hepatocytes by interacting with Rab5/7 and enhancing type I/III IFN expression. However, overexpressed CLEC18A inhibited FcγRIIA expression in phagocytes to impair phagocytosis. Furthermore, the interaction between CLEC18A and Rab5/7 may reduce the recruitment of Rab7 to autophagosomes and thereby retard autophagosome maturation and cause immune complex accumulation. A decreasing trend in CLEC18A levels that was accompanied by reduced HCV RNA titers and diminished cryoglobulin was observed in the sera of HCV-MC patients after direct-acting antiviral therapy. CLEC18A may be used for the evaluation of anti-HCV therapeutic drug effects and could be a potential predisposing factor for the development of MC syndrome., Competing Interests: The authors declare no conflict of interest.

AbstractBackground and purpose: Brucellosis is a major bacterial zoonosis of global importance. Effective host defence against brucella depends on immunoglobulin G-mediated phagocytosis of the bacteria and it has been shown that the FC RIIA polymorphism (FCγ RIIA-R131 vs FCγ RIIA –H131) determines the capacity of immunoglobulin G-mediated phagocytosis via this receptor. The aim of this study was to evaluate the FCγRIIA polymorphism in patients with brucellosis and to reveal any relation between this polymorphism and disease progression.Materials and Methods: In this study we evaluated FCγRIIA polymorphisms (R/R131, R/H131, H/H131) in 67 patients with serologically proven brucellosis and 67 healthy volunteers matched for age, sex and geographical area. FCγRIIA polymorphism was determined using a polymerase chain reaction method (SSCP-PCR).Results: The frequency of FCγ RIIA-R/R131 genotype was higher in patients with brucellosis compared with controls (47.8% vs 28.4%). This genotype has a (OR=2.1, 95%CI=1.3-4.2, P=0.039) significant correlation with brucellosis. However, no significant difference was found between patients with brucellosis and controls (P=0.2)Although the frequency of FCγRIIA-R/R131 was higher in patients with brucellosis compared with controls, we did not find any statistically significant differences (53.8% vs46.3%, P=0.2). As a result, there was no significant association between FCγRIIA genotype and severity of brucellosis.Conclusion: Based on the results, the dominance of homozygous genotype of FCγRIIA-R/R131 in patients with bracellosis emphasize the importance of this predisposing genetic risk factor in contracting the disease.

Autoantigen development is poorly understood at the atomic level. Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by antibodies to an antigen composed of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs). In solution, PF4 exists as an equilibrium among monomers, dimers and tetramers. Structural studies of these interacting components helped delineate a multi-step process involved in the pathogenesis of HIT. First, heparin binds to the ‘closed’ end of the PF4 tetramer and stabilizes its conformation; exposing the ‘open’ end. Second, PF4 arrays along heparin/GAG chains, which approximate tetramers, form large antigenic complexes that enhance antibody avidity. Third, pathogenic HIT antibodies bind to the ‘open’ end of stabilized PF4 tetramers to form an IgG/PF4/heparin ternary immune complex and also to propagate the formation of ‘ultralarge immune complexes’ (ULCs) that contain multiple IgG antibodies. Fourth, ULCs signal through FcγRIIA receptors, activating platelets and monocytes directly and generating thrombin, which transactivates hematopoietic and endothelial cells. A non-pathogenic anti-PF4 antibody prevents tetramer formation, binding of pathogenic antibody, platelet activation and thrombosis, providing a new approach to manage HIT. An improved understanding of the pathogenesis of HIT may lead to novel diagnostics and therapeutics for this autoimmune disease. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) causes a spectrum of illness from asymptomatic infection, to a mild febrile illness, to occasional more severe complications including hemorrhage and shock. Dengue is endemic in the state of Morelos, Mexico. Two single nucleotide polymorphisms (SNPs), rs1801274 of FcγRIIa and rs4804803 of DC-SIGN, have been associated with protection from or susceptibility to severe dengue infection. Both of these polymorphisms are located in genes for receptors with important roles in dengue pathogenesis, and their relationship with the clinical presentation of dengue infection in Mexican populations is unknown. In this study, real-time PCR was used to characterize the distribution of rs1801274 and rs4804803 in subjects with asymptomatic dengue infection ( n = 145), uncomplicated dengue ( n = 67), and severe dengue ( n = 36) in Morelos. In contrast with previous studies, the histidine (A) variant of rs1801274 was associated with more mild infection: carrying the histidine allele (either homozygous or heterozygous) was associated with protection from symptomatic infection compared with asymptomatic (OR 0.51, p = 0.038). Histidine homozygotes were also less likely to present severe dengue (OR 0.34, p = 0.05). Logistic regression models confirm this association (OR 0.48, p = 0.04) and also indicate that the G allele of rs4804803 is associated with symptomatic dengue (OR 2.3, p = 0.08), after accounting for other biological factors including history of infection. This variant was rare in this study population, with a frequency of 5.4%. These findings reflect the complexity of influences on the development of severe dengue infection. The inclusion of asymptomatic infections and adjusted case definitions likely do not explain the entire disparity with previous findings. Interactions with other polymorphisms may explain why the association of rs1801274 is reversed in this population compared to others. This study demonstrates the importance of genetic association studies in multiple genetically distinct populations. [ABSTRACT FROM AUTHOR]