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1.
Transfusion ; 62(5): 948-953, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35470900

RESUMO

BACKGROUND: Alloimmunization can be a significant barrier to red blood cell (RBC) transfusion. While alloantigen matching protocols hold promise in reducing alloantibody formation, transfusion-dependent patients can still experience RBC alloimmunization and associated complications even when matching protocols are employed. As a result, complementary strategies capable of actively preventing alloantibody formation following alloantigen exposure are warranted. STUDY DESIGN AND METHODS: We examined whether pharmacological removal of macrophages using clodronate may provide an additional strategy to actively inhibit RBC alloimmunization using two preclinical models of RBC alloimmunization. To accomplish this, mice were treated with clodronate, followed by transfusion of RBCs expressing the HOD (HEL, OVA, and Duffy) or KEL antigens. On days 5 and 14 post transfusion, anti-HOD or anti-KEL IgM and IgG antibodies were evaluated. RESULTS: Low dose clodronate effectively eliminated key marginal zone macrophage populations from the marginal sinus. Prior treatment with clodronate, but not empty liposomes, also significantly inhibited IgM and IgG anti-HOD alloantibody formation following transfusion of HOD RBCs. Similar exposure to clodronate inhibited IgM and IgG antibody formation following KEL RBC transfusion. CONCLUSIONS: Clodronate can inhibit anti-HOD and anti-KEL antibody formation following RBC transfusion in preclinical models. These results suggest that clodronate may provide an alternative approach to actively inhibit or prevent the development of alloantibodies following RBC transfusion, although future studies will certainly be needed to fully explore this possibility.


Assuntos
Ácido Clodrônico , Isoantígenos , Animais , Ácido Clodrônico/farmacologia , Eritrócitos , Humanos , Imunoglobulina G , Imunoglobulina M , Isoanticorpos , Camundongos
2.
Transfusion ; 62(4): 770-782, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35274303

RESUMO

BACKGROUND: In hematologic and transfusion medicine research, measurement of red blood cell (RBC) in vivo kinetics must be safe and accurate. Recent reports indicate use of biotin-labeled RBC (BioRBC) to determine red cell survival (RCS) offers substantial advantages over 51 Cr and other labeling methods. Occasional induction of BioRBC antibodies has been reported. STUDY DESIGN AND METHODS: To investigate the causes and consequences of BioRBC immunization, we reexposed three previously immunized adults to BioRBC and evaluated the safety, antibody emergence, and RCS of BioRBC. RESULTS: BioRBC re-exposure caused an anamnestic increase of plasma BioRBC antibodies at 5-7 days; all were subclass IgG1 and neutralized by biotinylated albumin, thus indicating structural specificity for the biotin epitope. Concurrently, specific antibody binding to BioRBC was observed in each subject. As biotin label density increased, the proportion of BioRBC that bound increased antibody also increased; the latter was associated with proportional accelerated removal of BioRBC labeled at density 6 µg/mL. In contrast, only one of three subjects exhibited accelerated removal of BioRBC density 2 µg/mL. No adverse clinical or laboratory events were observed. Among three control subjects who did not develop BioRBC antibodies following initial BioRBC exposure, re-exposure induced neither antibody emergence nor accelerated BioRBC removal. DISCUSSION: We conclude re-exposure of immunized subjects to BioRBC can induce anamnestic antibody response that can cause an underestimation of RCS. To minimize chances of antibody induction and underestimation of RCS, we recommend an initial BioRBC exposure volume of ≤10 mL and label densities of ≤18 µg/mL.


Assuntos
Biotina , Eritrócitos , Adulto , Anticorpos/metabolismo , Biotina/química , Sobrevivência Celular , Contagem de Eritrócitos , Eritrócitos/metabolismo , Humanos
3.
Methods Mol Biol ; 2442: 533-548, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320544

RESUMO

Cellular turnover represents a fundamental aspect of immunological homeostasis. While many factors appear to regulate leukocyte removal during inflammatory resolution, recent studies suggest that members of the galectin family play a unique role in orchestrating this process. Unlike cellular removal through apoptotic cell death, several members of the galectin family induce surface expression of phosphatidylserine (PS), a phagocytic marker on cells undergoing apoptosis, in the absence of cell death. However, similar to PS on cells undergoing apoptosis, galectin-induced PS exposure sensitizes cells to phagocytic removal. As galectins appear to prepare cells for phagocytic removal without actually inducing apoptotic cell death, this process has recently been coined preaparesis. Given the unique characteristics of galectin-induced PS exposure in the context of preaparesis, we will examine unique considerations when evaluating the potential impact of different galectin family members on PS exposure and cell viability.


Assuntos
Apoptose , Galectinas , Leucócitos , Fagocitose , Fosfatidilserinas , Apoptose/imunologia , Galectinas/metabolismo , Células HL-60 , Humanos , Leucócitos/imunologia , Fosfatidilserinas/metabolismo
4.
Methods Mol Biol ; 2442: 549-564, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320545

RESUMO

Reactive oxygen species (ROS) have been extensively studied in biology in the past years. This class of molecules can be derived from endogenous sources (e.g., phagocytic cells as neutrophils, eosinophils, monocytes, macrophages, and organelles as mitochondria and peroxisomes) and participate in physiological and pathological conditions. The beneficial and harmful effects of ROS depend on redox regulation, which establishes the balance between their production and the activity of antioxidant systems to prevent oxidative stress in vivo. Neutrophils are the immune effectors most well depicted with an intense oxidative burst in response to tissue inflammation. Several proteins and members of the galectin family are involved in this fine modulation of ROS production by neutrophils. Interestingly, studies have indicated that Galectin-1 (Gal-1) can up- or downregulate ROS production by neutrophils even when exposed to N-formyl-Met-Leu-Phe (fMLP) or Phorbol Myristate Acetate (PMA), both of which are potent neutrophil stimulants that trigger high levels of ROS production. Similarly, Galectin-3 (Gal-3) induces ROS in neutrophils from a sterile or nonsterile inflammatory environment, possibly creating a negative loop that could control ROS production. Besides, superoxide production is also induced by Galectin-8 (Gal-8) and Galectin-9 (Gal-9) in neutrophils but in a different manner. We describe herein the luminol and lucigenin-dependent chemiluminescence technique by using a luminometer as a method of assessment to measure ROS production by human neutrophils isolated and exposed to purified human recombinant Gal-1. The protocol described herein could be applied for the investigation of the role of other galectins in the modulation of ROS production by neutrophils.


Assuntos
Galectinas , Neutrófilos , Espécies Reativas de Oxigênio , Galectinas/genética , Galectinas/metabolismo , Galectinas/farmacologia , Humanos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Acetato de Tetradecanoilforbol/farmacologia
5.
Methods Mol Biol ; 2442: 663-683, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320552

RESUMO

Galectin-1 is a small (14.5 kDa) multifunctional protein with cell-cell and cell-ECM adhesion due to interactions with the carbohydrate recognition domain (CRD). In two types of muscular dystrophies, this lectin protein has shown therapeutic properties, including positive regulation of skeletal muscle differentiation and regeneration. Both Duchenne and limb-girdle muscular dystrophy 2B (LGMD2B) are subtypes of muscular dystrophies characterized by deficient membrane repair, muscle weakness, and eventual loss of ambulation. This chapter explains confocal techniques such as laser injury, calcium imaging, and galectin-1 localization to examine the effects of galectin-1 on membrane repair in injured LGMD2B models.


Assuntos
Galectina 1 , Distrofia Muscular do Cíngulo dos Membros , Sarcolema , Galectina 1/metabolismo , Galectina 1/farmacologia , Galectina 1/uso terapêutico , Humanos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/tratamento farmacológico , Sarcolema/efeitos dos fármacos , Sarcolema/fisiologia
6.
Blood Adv ; 6(8): 2628-2645, 2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35286375

RESUMO

Advances in the development of novel treatment options for hemophilia A are prevalent. However, the anti-factor VIII (FVIII) neutralizing antibody (inhibitor) response to existing FVIII products remains a major treatment challenge. Although some novel products are designed to function in the presence of inhibitors, they do not specific address the immunogenicity risk or mechanistic causes of inhibitor development, which remain unclear. Furthermore, most preclinical studies supporting clinical gene therapy programs have reported immunogenicity signals in animal models, especially at higher vector doses and sometimes using multiple vector designs. In these settings, immunogenicity risk factor determination, comparative immunogenicity of competing vector designs, and the potential for obtaining meaningful prognostic data remain relatively unexplored. Additionally, there remains the opportunity to investigate clinical gene therapy as an alternative to standard immune tolerance induction therapy. The current study was designed to address these issues through longitudinal dose-response evaluation of 4 adeno-associated viral (AAV) vector candidates encoding 2 different FVIII transgenes in a murine model of hemophilia A. Plasma FVIII activity and anti-FVIII antibody data were used to generate a pharmacokinetic model that (1) identifies initial AAV-FVIII product expression kinetics as the dominant risk factor for inhibitor development, (2) predicts a therapeutic window where immune tolerance is achieved, and (3) demonstrates evidence of gene therapy-based immune tolerance induction. Although there are known limitations to the predictive value of preclinical immunogenicity testing, these studies can uncover or support the development of design principles that can guide the development of safe and effective genetic medicines.


Assuntos
Hemofilia A , Hemostáticos , Animais , Fator VIII/genética , Fator VIII/uso terapêutico , Terapia Genética , Hemofilia A/genética , Hemofilia A/terapia , Camundongos , Transgenes
7.
Methods Mol Biol ; 2442: 55-74, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320519

RESUMO

Galectins are lectins having the capacity to recognize ß-galactose-containing glycan structures and are widely distributed among various taxa. However, the exact physiological and biochemical functions mediated by galectins that necessitate their wide occurrence among diverse species have not yet been delineated in a precise manner. Purification of recombinant galectins in active form is a fundamental requirement to elucidate their biological function. In this chapter, we are describing methods to recombinantly express and purify galectins using three different methods of affinity purification, i.e., lactosyl-Sepharose chromatography for fungal galectin Coprinopsis cinerea galectin 2 (CGL2), nickel-chromatography for histidine-tagged human galectin-7, and glutathione-Sepharose chromatography for Glutathione S-transferase-tagged (GST-tagged) human galectin-7. Step-by-step instructions are provided for obtaining the above-mentioned recombinant galectins that retain carbohydrate-binding activity and are suitable for conducting biochemical experiments.


Assuntos
Galectina 2 , Galectinas , Carboidratos , Cromatografia de Afinidade , Galactose , Galectinas/química , Humanos
8.
Methods Mol Biol ; 2442: 75-87, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320520

RESUMO

Galectins can display unique sensitivity to oxidative changes that result in significant conformational alterations that prevent carbohydrate recognition. While a variety of approaches can be utilized to prevent galectin oxidation, several of these require inclusion of reducing agents that not only prevent galectins from undergoing oxidative inactivation but can also interfere with normal redox potentials required for fundamental cellular processes. To overcome the limitations associated with placing cells in an artificial reducing environment, cysteine residues on galectins can be directly alkylated with iodoacetamide to form a stable thioether adduct that is resistant to further modification. Iodoacetamide alkylated galectin remains stable over prolonged periods of time and retains the carbohydrate binding and biological activities of the protein. As a result, this approach allows examination of the biological roles of a stabilized form of galectin-1 without introducing the confounding variables that can occur when typical soluble reducing agents are employed.


Assuntos
Galectina 1 , Galectinas , Alquilação , Galectina 1/química , Galectina 1/metabolismo , Galectinas/metabolismo , Iodoacetamida , Espectrometria de Massas
9.
Methods Mol Biol ; 2442: 151-168, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320525

RESUMO

Glycan binding proteins (GBPs) possess the unique ability to regulate a wide variety of biological processes through interactions with highly modifiable cell surface glycans. While many studies demonstrate the impact of glycan modification on GBP recognition and activity, the relative contribution of subtle changes in glycan structure on GBP binding can be difficult to define. To overcome limitations in the analysis of GBP-glycan interactions, recent studies utilized glycan microarray platforms containing hundreds of structurally defined glycans. These studies not only provided important information regarding GBP-glycan interactions in general but have also resulted in significant insight into binding specificity and biological activity of the galectin family. We will describe the methods used when employing glycan microarray platforms to examine galectin-glycan binding specificity and function.


Assuntos
Galectinas , Polissacarídeos , Proteínas de Transporte/metabolismo , Galectinas/metabolismo , Análise em Microsséries/métodos , Polissacarídeos/química , Ligação Proteica
10.
Methods Mol Biol ; 2442: 187-203, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320527

RESUMO

We have utilized simple flow cytometric and fluorescence-based solid phase assays to study the interaction of glycan binding proteins (GBP) to cell surface glycoconjugates. These methods utilize commonly employed flow cytometry techniques and commercially available streptavidin-coated microplates to immobilize various biotinylated ligands, such as glycopeptides, oligosaccharides, and whole cells. Using this approach, fluorescently labeled GBPs, in particular, members of the galectin family, can be interrogated for potential interactions with cell surface carbohydrates, including elucidation of the potential impact of alterations in glycosylation on carbohydrate recognition. Using these approaches, we present examples of flow cytometric and fluorescence-based solid phase assays to study galectin-carbohydrate interactions.


Assuntos
Galectinas , Glicoconjugados , Carboidratos , Citometria de Fluxo , Galectinas/metabolismo , Polissacarídeos/metabolismo
11.
Methods Mol Biol ; 2442: 215-232, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320529

RESUMO

Glycosylation is one of the most common protein posttranslational modifications. Most human lymphocyte membrane receptors are modified by diverse glycan structures, and functional studies have indicated that a family of glycan-binding proteins, galectins, can significantly modulate lymphocyte development and function by interacting with these glycans. Several galectins have a varying degree of affinity for the N-acetyllactosamine (LacNAc) disaccharide, and some critical lymphocyte receptors can be modified by glycan structures carrying this motif. However, the site-specific glycan composition on primary lymphocyte membrane receptors in healthy individuals is largely limited. The main reason for the limitation is low abundance of available material from a single donor and compositional heterogeneity in glycan structures that can potentially modify a protein. Donor-dependent variability in N-glycan structures on CD16a isolated from primary NK cells of healthy human donors was recently reported. NK cell CD16a is glycosylated at five N-glycosylation sites, and two of the five sites are modified, almost exclusively, by N-glycans with multiple LacNAc repeats which can serve as ligands for endogenous galectins. Thus, the protocol described in this section can be utilized to identify galectin ligands at specific glycosylation sites of endogenous membrane receptor from circulating primary human lymphocytes.


Assuntos
Galectinas , Glicoproteínas de Membrana , Galectinas/metabolismo , Glicosilação , Humanos , Células Matadoras Naturais/metabolismo , Ligantes , Glicoproteínas de Membrana/metabolismo
12.
Methods Mol Biol ; 2442: 289-306, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320532

RESUMO

Galectins are multifunctional glycan-binding proteins present in various tissues that participate in multiple physiological and pathological processes and are considered as not only biomarkers of human diseases but also molecular targets for treating cancer and inflammatory illnesses in many organs. In the glycobiology field, it is crucial to determine the pattern of galectin expression and location in cells and tissues. Confocal microscopy is a powerful imaging technology that represents a unique approach to investigate the expression and location of biomolecules in various tissues and cells. The confocal microscope acquires images of the specimen through the reflected or fluorescent light from the objective's focal plane, using laser light focused on a small spot inside the tissue or cell. This technique provides high-resolution and high-contrast images without artifacts generated by conventional microscopy and enables reconstruction of virtual tridimensional images by acquiring multiple sections from several focal planes, which makes it possible to obtain the precise spatial location of any cellular structure or molecule. Furthermore, confocal microscopy is a non-invasive tissue imaging strategy used in clinical practices. We describe herein the immunofluorescence confocal method for examining galectins in frozen tissue sections and mammalian cell culture.


Assuntos
Galectinas , Testes Imunológicos , Animais , Técnicas de Cultura de Células , Imunofluorescência , Humanos , Mamíferos , Microscopia Confocal/métodos
13.
Methods Mol Biol ; 2442: 339-352, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320534

RESUMO

Molecular imaging (MI) is a non-invasive growing technology that allows the investigation of cellular and molecular processes in basic and clinical research and medicine. Luminescent proteins and radionuclides can be associated to target molecules providing high-definition and real-time image of whole body in few minutes or hours. Several MI studies have enabled the determination of molecular partners, in vivo tracking, and fate of compounds in different disorders. Considering that galectins are multifaceted proteins with great impact in many biological events, here we describe methods and strategies to generate labeled galectins for in vivo non-invasive imaging studies.


Assuntos
Galectinas , Imagem Molecular , Proteínas Luminescentes
14.
Methods Mol Biol ; 2442: 517-531, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320543

RESUMO

Over a century ago, Karl Landsteiner discovered that blood group antigens could predict the immunological outcome of red blood cell transfusion. While the discovery of ABO(H) blood group antigens revolutionized transfusion medicine, many questions remain regarding the development and regulation of naturally occurring anti-blood group antibody formation. Early studies suggested that blood group antibodies develop following stimulation by bacteria that express blood group antigens. While this may explain the development of anti-blood group antibodies in blood group-negative individuals, how blood group-positive individuals protect themselves against blood group-positive microbes remained unknown. Recent studies suggest that several members of the galectin family specifically target blood group-positive microbes, thereby providing innate immune protection against blood group antigen-positive microbes regardless of the blood group status of an individual. Importantly, subsequent studies suggest that this unique form of immunity may not be limited to blood group expressing microbes, but may reflect a more generalized form of innate immunity against molecular mimicry. As this form of antimicrobial activity represents a unique and unprecedented form of immunity, we will examine important considerations and methodological approaches that can be used when seeking to ascertain the potential antimicrobial activity of various members of the galectin family.


Assuntos
Antígenos de Grupos Sanguíneos , Galectinas , Galectinas/metabolismo , Humanos , Imunidade Inata , Mimetismo Molecular
16.
Methods Mol Biol ; 2442: 1-40, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320517

RESUMO

Galectins are a large family of carbohydrate binding proteins with members in nearly every lineage of multicellular life. Through tandem and en-mass genome duplications, over 15 known vertebrate galectins likely evolved from a single common ancestor extant in pre-chordate lineages. While galectins have divergently evolved numerous functions, some of which do not involve carbohydrate recognition, the vast majority of the galectins have retained the conserved ability to bind variably modified polylactosamine (polyLacNAc) residues on glycans that modify proteins and lipids on the surface of host cells and pathogens. In addition to their direct role in microbial killing, many proposed galectin functions in the immune system and cancer involve crosslinking glycosylated receptors and modifying signaling pathways or sensitivity to antigen from the outside in. However, a large body of work has uncovered intracellular galectin functions mediated by carbohydrate- and non-carbohydrate-dependent interactions. In the cytoplasm, galectins can tune intracellular kinase and G-protein-coupled signaling cascades important for nutrient sensing, cell cycle progression, and transformation. Particularly, but interconnected pathways, cytoplasmic galectins serve the innate immune system as sensors of endolysosomal damage, recruiting and assembling the components of autophagosomes during intracellular infection through carbohydrate-dependent and -independent activities. In the nucleus, galectins participate in pre-mRNA splicing perhaps through interactions with non-coding RNAs required for assembly of spliceosomes. Together, studies of galectin function paint a picture of a functionally dynamic protein family recruited during eons of evolution to regulate numerous essential cellular processes in the context of multicellular life.


Assuntos
Galectinas , Sistema Imunitário , Ciclo Celular , Galectinas/metabolismo , Glicosilação , Sistema Imunitário/metabolismo , Transdução de Sinais
17.
J Biol Chem ; 298(4): 101704, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35148986

RESUMO

While adaptive immunity recognizes a nearly infinite range of antigenic determinants, immune tolerance renders adaptive immunity vulnerable to microbes decorated in self-like antigens. Recent studies suggest that sugar-binding proteins galectin-4 and galectin-8 bind microbes expressing blood group antigens. However, the binding profile and potential antimicrobial activity of other galectins, particularly galectin-9 (Gal-9), has remained incompletely defined. Here, we demonstrate that while Gal-9 possesses strong binding preference for ABO(H) blood group antigens, each domain exhibits distinct binding patterns, with the C-terminal domain (Gal-9C) exhibiting higher binding to blood group B than the N-terminal domain (Gal-9N). Despite this binding preference, Gal-9 readily killed blood group B-positive Escherichia coli, whereas Gal-9N displayed higher killing activity against this microbe than Gal-9C. Utilization of microarrays populated with blood group O antigens from a diverse array of microbes revealed that Gal-9 can bind various microbial glycans, whereas Gal-9N and Gal-9C displayed distinct and overlapping binding preferences. Flow cytometric examination of intact microbes corroborated the microbial glycan microarray findings, demonstrating that Gal-9, Gal-9N, and Gal-9C also possess the capacity to recognize distinct strains of Providencia alcalifaciens and Klebsiella pneumoniae that express mammalian blood group-like antigens while failing to bind related strains that do not express mammalian-like glycans. In each case of microbial binding, Gal-9, Gal-9N, and Gal-9C induced microbial death. In contrast, while Gal-9, Gal-9N, and Gal-9C engaged red blood cells, each failed to induce hemolysis. These data suggest that Gal-9 recognition of distinct microbial strains may provide antimicrobial activity against molecular mimicry.


Assuntos
Anti-Infecciosos , Antígenos de Grupos Sanguíneos , Galectinas , Animais , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Galectinas/metabolismo , Mamíferos/metabolismo , Polissacarídeos/metabolismo
18.
medRxiv ; 2022 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-35132426

RESUMO

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

19.
Medicine (Baltimore) ; 101(2): e28489, 2022 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-35029197

RESUMO

OBJECTIVES: Investigate polymorphisms and expressions of human leukocyte antigen-G (HLA-G), galectin-1 (Gal-1), and interleukin-10 (IL-10) in people living with HIV (PLHIV) with and without comorbidities to help understanding the mechanisms involved in triggering these disorders in PLHIV and in their prognosis. DESIGN: Here we evaluated the potential correlation between the genetic polymorphism and/or protein levels of HLA-G, Gal-1, and IL-10 with and without comorbidities of PLHIV. METHODS: Two hundred HIV patients under antiretroviral treatment (83 with comorbidities and 117 without comorbidities) and 200 healthy individuals (controls) were genotyped, using PCR, for HLA-G 14-base pair polymorphism located at the 3' untranslated region in exon 8 insertion/insertion (Ins/Ins: low HLA-G expression) or deletion/deletion (Del/Del: high HLA-G expression). Soluble levels of HLA-G (sHLA-G), Gal-1, and IL-10 were quantified by enzyme-linked immunosorbet assay. RESULTS: HIV patients without comorbidities exhibited higher frequency of 14-base pair Del/Del genotype than HIV patients with comorbidities. As expected, HIV patients Ins/Ins with and without comorbidities produced less sHLA-G than controls. However, HIV patients Del/Del with comorbidities expressed sHLA-G more than controls and HIV patients Del/Del without comorbidities. Interestingly, patients that showed low levels sHLA-G, and presence of comorbidities, exhibited high Gal-1 serum levels. However, an increase in soluble levels of IL-10 in PLHIV was observed when compared to controls, especially in the PLHIV group without comorbidities suggesting, a protective role of IL-10 in the development of comorbidities. CONCLUSIONS: These data suggested that the high expression of sHLA-G and IL-10 or Gal-1 could be associated and could be associated with the development or not of comorbidities in PLHIV.


Assuntos
Galectina 1 , Infecções por HIV , Antígenos HLA-G , Interleucina-10 , Regiões 3' não Traduzidas , Estudos de Casos e Controles , Comorbidade , Galectina 1/genética , Genótipo , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Antígenos HLA-G/genética , Humanos , Interleucina-10/genética , Polimorfismo Genético
20.
J Immunol ; 208(4): 991-997, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35039331

RESUMO

RBC transfusion therapy is essential for the treatment of anemia. A serious complication of transfusion is the development of non-ABO alloantibodies to polymorphic RBC Ags; yet, mechanisms of alloantibody formation remain unclear. Storage of mouse RBCs before transfusion increases RBC immunogenicity through an unknown mechanism. We previously reported that sterile, stored mouse RBCs activate splenic dendritic cells (DCs), which are required for alloimmunization. Here we transfused mice with allogeneic RBCs to test whether stored RBCs activate pattern recognition receptors (PRRs) on recipient DCs to induce adaptive immunity. TLRs are a class of PRRs that regulate DC activation, which signal through two adapter molecules: MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation and migration, and the subsequent alloantibody response to transfused RBCs require MyD88 but not TRIF, suggesting that a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.


Assuntos
Imunidade Adaptativa , Eritrócitos/imunologia , Eritrócitos/metabolismo , Imunidade Inata , Fator 88 de Diferenciação Mieloide/metabolismo , Animais , Biomarcadores , Transfusão de Eritrócitos , Imunofluorescência , Humanos , Isoanticorpos/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética
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