RESUMEN

Hematoxylin and eosin staining is widely used for routine histopathological analysis under light microscopic examination to determine alterations of tissue architecture and cellular components in animal studies. Aside from hematoxylin/eosin staining, periodic acid Schiff (PAS) staining is used to detect polysaccharides and carbohydrate-rich macromolecules, and is essential in immunological fields for evaluation of glomerular lesions of kidneys in autoimmune animals. Since erythrocytes are not stained by PAS, this stain is also helpful for identifying changes in immune cells in the red pulp of the spleen, which is filled with erythrocytes. This article describes a protocol to detect Mott cells, bizarre plasma cells containing immunoglobulin inclusion bodies (Russell bodies) in the cytoplasm. The protocol can be used for formalin-fixed, paraffin-embedded tissue sections, frozen tissue sections, tissue-touch preparations, blood films, and cytocentrifuged cell smears. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Detection of Mott cells by PAS staining in formalin-fixed, paraffin-embedded tissue sections Basic Protocol 2: Detection of Mott cells by PAS staining in frozen tissue sections, touch preparations, blood films, and cytocentrifuged cell smears.

Asunto(s)

Células Plasmáticas , Coloración y Etiquetado , Coloración y Etiquetado/métodos , Células Plasmáticas/inmunología , Células Plasmáticas/patología , Humanos , Reacción del Ácido Peryódico de Schiff , Animales , Cuerpos de Inclusión , Inmunoglobulinas/análisis , Inmunoglobulinas/inmunología , Adhesión en Parafina

RESUMEN

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboard titration test was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications.

Asunto(s)

Anticuerpos Antivirales , Pollos , Ensayo de Inmunoadsorción Enzimática , Fiebre Aftosa , Inmunoglobulinas , Enfermedades de las Aves de Corral , Animales , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulinas/inmunología , Inmunoglobulinas/análisis , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/prevención & control , Fiebre Aftosa/virología , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/virología , Virus de la Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/aislamiento & purificación , Sensibilidad y Especificidad , Yema de Huevo/inmunología

RESUMEN

In nephrotic syndrome, the podocyte filtration structures are damaged in a process called foot process effacement. This is mediated by the actin cytoskeleton; however, which actins are involved and how they interact with other filtration components, like the basement membrane, remains poorly understood. Here, we used the well-established Drosophila pericardial nephrocyte-the equivalent of podocytes in flies-knockdown models (RNAi) to study the interplay of the actin cytoskeleton (Act5C, Act57B, Act42A, and Act87E), alpha- and beta-integrin (basement membrane), and the slit diaphragm (Sns and Pyd). Knockdown of an actin gene led to variations of formation of actin stress fibers, the internalization of Sns, and a disrupted slit diaphragm cortical pattern. Notably, deficiency of Act5C, which resulted in complete absence of nephrocytes, could be partially mitigated by overexpressing Act42A or Act87E, suggesting at least partial functional redundancy. Integrin localized near the actin cytoskeleton as well as slit diaphragm components, but when the nephrocyte cytoskeleton or slit diaphragm was disrupted, this switched to colocalization, both at the surface and internalized in aggregates. Altogether, the data show that the interdependence of the slit diaphragm, actin cytoskeleton, and integrins is key to the structure and function of the Drosophila nephrocyte.

Asunto(s)

Citoesqueleto de Actina , Proteínas de Drosophila , Integrinas , Podocitos , Animales , Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Integrinas/metabolismo , Podocitos/metabolismo , Drosophila melanogaster/metabolismo , Drosophila/metabolismo , Actinas/metabolismo , Inmunoglobulinas

RESUMEN

BACKGROUND: HHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) represents a recently identified member of the B7 immune checkpoint family, characterized by limited expression in normal tissues but notable overexpression in various cancer types. Nevertheless, the precise function and interaction with immune cells remain poorly understood, particularly in laryngeal squamous cell carcinoma (LSCC). This investigation endeavored to elucidate the biological significance of HHLA2 within the tumor microenvironment of human LSCC tissues and delineate the clinical relevance and functional roles of HHLA2 in LSCC pathogenesis. METHODS: Through multiplexed immunohistochemistry analyses conducted on tissue microarrays sourced from LSCC patients (n = 72), the analysis was executed to assess the expression levels of HHLA2, density and spatial patterns of CD68+HLA-DR+CD163- (M1 macrophages), CTLA-4+CD4+FoxP3+ (CTLA-4+Treg cells), CTLA-4+CD4+FoxP3- (CTLA-4+Tcon cells), exhausted CD8+T cells, and terminally exhausted CD8+T cells in LSCC tissues. Survival analysis was conducted to evaluate the prognostic significance of HHLA2 and these immune checkpoints or immune cell populations, employing COX regression analysis to identify independent prognostic factors. RESULTS: Kaplan-Meier (K-M) survival curves revealed a significant association between HHLA2 expression and overall survival (OS) in LSCC. Elevated levels of HHLA2 were linked to reduced patient survival, indicating its potential as a prognostic marker (HR: 3.230, 95%CI 0.9205-11.34, P = 0.0067). Notably, increased infiltration of CD68+ cells (total macrophages), STING+CD68+HLA-DR+CD163- (STING+M1 macrophages), CTLA-4+CD4+FoxP3+, CTLA-4+CD4+FoxP3-, PD-1+LAG-3+CD8+T cells, and PD-1+LAG-3+TIM-3+CD8+T cells strongly linked to poorer survival outcomes (P < 0.05). A discernible trend was observed between the levels of these immune cell populations, STING+CD68+ (STING+ total macrophages), CD68+HLA-DR+CD163-, STING+CD68+CD163+HLA-DR- (STING+M2 macrophages), PD-1+LAG-3-CD8+T cells, PD-1+TIM-3+CD8+T cells, and PD-1+LAG-3+TIM-3-CD8+T cells and prognosis. Importantly, multivariate COX analysis identified HHLA2 as an independent predictive factor for OS in LSCC patients (HR = 3.86, 95% CI 1.08-13.80, P = 0.038). This underscored the potential of HHLA2 as a critical marker for predicting patient outcomes in LSCC. CONCLUSIONS: HHLA2 emerged as a detrimental prognostic biomarker for assessing OS in LSCC patients. Relative to other immune checkpoints, HHLA2 exhibited heightened predictive efficacy for the prognosis of LSCC patients.

Asunto(s)

Biomarcadores de Tumor , Neoplasias Laríngeas , Linfocitos Infiltrantes de Tumor , Microambiente Tumoral , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Inmunoglobulinas , Neoplasias Laríngeas/inmunología , Neoplasias Laríngeas/mortalidad , Neoplasias Laríngeas/patología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Pronóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/patología

RESUMEN

The Asian seabass (Lates calcarifer) faces significant disease threats, which are exacerbated by intensive farming practices and environmental changes. Therefore, understanding its immune system is crucial. The current study presents a comprehensive analysis of immune-related genes in Asian seabass peripheral blood leukocytes (PBLs) using Iso-seq technology, identifying 16 key pathways associated with 7857 immune-related genes, comprising 634 unique immune-related genes. The research marks the first comprehensive report on the entire immunoglobulin repertoire in Asian seabass, revealing specific characteristics of immunoglobulin heavy chain constant region transcripts, including IgM (Cµ, ighm), IgT (Cτ, ight), and IgD (Cδ, ighd). The study confirms the presence of membrane-bound form, ighmmb, ightmb, ighdmb of IgM, IgT and IgD and secreted form, ighmsc and ightsc of IgM and IgT, respectively, with similar structural patterns and conserved features in amino acids across immunoglobulin molecules, including cysteine residues crucial for structural integrity observed in other teleost species. In response to bacterial infections by Flavobacterium covae (formerly F. columnare genomovar II) and Streptococcus iniae, both secreted and membrane-bound forms of IgM (ighmmb and ighmsc) and IgT (ightmb and ightsc) show significant expression, indicating their roles in systemic and mucosal immunity. The expression of membrane-bound form IgD gene, ighdmb, predominantly exhibits targeted upregulation in PBLs, suggesting a regulatory role in B cell-mediated immunity. The findings underscore the dynamic and tissue-specific expression of immunoglobulin repertoires, ighmmb, ighmsc, ightmb, ightsc and ighdmb in Asian seabass, indicating a sophisticated immune response to bacterial pathogens. These findings have practical implications for fish aquaculture, and disease control strategies, serving as a valuable resource for advancing research in Asian seabass immunology.

Asunto(s)

Enfermedades de los Peces , Proteínas de Peces , Infecciones por Flavobacteriaceae , Flavobacterium , Inmunoglobulina D , Inmunoglobulina M , Inmunoglobulinas , Infecciones Estreptocócicas , Streptococcus iniae , Animales , Enfermedades de los Peces/inmunología , Flavobacterium/fisiología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Inmunoglobulina M/inmunología , Inmunoglobulina M/genética , Infecciones por Flavobacteriaceae/inmunología , Infecciones por Flavobacteriaceae/veterinaria , Infecciones por Flavobacteriaceae/genética , Inmunoglobulina D/genética , Inmunoglobulina D/inmunología , Inmunoglobulina D/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Streptococcus iniae/fisiología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Inmunidad Innata/genética , Lubina/inmunología , Lubina/genética

RESUMEN

Primary cytomegalovirus infection during pregnancy has a high risk of vertical transmission, with severe fetal sequelae mainly associated with first-trimester infections. We conducted a retrospective analysis of 200 IU/kg cytomegalovirus-specific hyperimmune globulin (HIG), used in first-trimester maternal primary infections for congenital infection prevention. The primary outcome was vertical transmission, defined as neonatal viruria or positive amniocentesis if pregnancy was discontinued. HIG, initially administered monthly and since 2019 biweekly, was discontinued in negative amniocentesis cases. Women declining amniocentesis and positive amniocentesis cases with normal sonography were offered monthly HIG until delivery as a treatment strategy. The total transmission rate was 29.9% (32/107; 10 pregnancy terminations with positive amniocentesis, 18 completed pregnancies with positive amniocentesis and 4 declining amniocentesis). Maternal viremia was the only factor associated with fetal transmission (OR 4.62, 95% CI 1.55-13.74). The transmission rate was not significantly different whether HIG was started during the first or second trimester (28.2% vs. 33.3%; p = 0.58), or between monthly and biweekly subgroups (25.7% vs. 37.8%, p = 0.193). Pre-treatment maternal viremia could inform decisions as a predictor of congenital infection.

Asunto(s)

Infecciones por Citomegalovirus , Transmisión Vertical de Enfermedad Infecciosa , Complicaciones Infecciosas del Embarazo , Primer Trimestre del Embarazo , Humanos , Femenino , Embarazo , Infecciones por Citomegalovirus/prevención & control , Infecciones por Citomegalovirus/transmisión , Infecciones por Citomegalovirus/inmunología , Estudios Retrospectivos , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Adulto , Complicaciones Infecciosas del Embarazo/prevención & control , Complicaciones Infecciosas del Embarazo/virología , Inmunoglobulinas/administración & dosificación , Inmunoglobulinas/uso terapéutico , Prevención Secundaria/métodos , Recién Nacido , Citomegalovirus/inmunología , Estudios de Cohortes , Amniocentesis , Viremia , Inmunoglobulinas Intravenosas

RESUMEN

Background: NAFLD is thought to affect approximately one-fourth of the world's population. Therefore, we evaluated the role of serum complement and immunoglobulins in the NAFLD pathogenesis. Patients and methods: 200 participants were used in this study, divided into two groups; Group I: 100 NAFLD patients and Group II: 100 healthy volunteers. The diagnosis of NAFLD is based on non-invasive methods, following the EASL guideline 2022. IgG, IgM, IgA, C3, and C4 assays were performed on all participants. Results: When the immunological profiles of patients with NAFLD and healthy controls were compared, it was found that the mean IgA in NAFLD patients was (4.20±5.07), whereas the mean IgA in healthy controls was (2.22±1.05) (P=0.000). Additionally, a significant increase in IgG was found in NAFLD patients (17.08±3.87) compared with healthy controls (11.59±3.34), with a P value of (p<0.001). complement C3 and complement C4 levels significantly increased in nonalcoholic fatty liver disease patients (1.28± 0.61 and 0.40 ± 0.19, respectively), compared to healthy controls (0.90 ±0.27 and 0.30 ±0.12, respectively), with a significant P value (p<0.001 for each). Conclusions: Elevated IgA, IgG, C3 and C4 exist in patients with NAFLD and could be associated with fatty liver development and progression of hepatic fibrosis in patients with NAFLD.

Asunto(s)

Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/inmunología , Enfermedad del Hígado Graso no Alcohólico/sangre , Masculino , Femenino , Adulto , Persona de Mediana Edad , Estudios de Casos y Controles , Complemento C3/análisis , Complemento C4/análisis , Complemento C4/metabolismo , Inmunoglobulina A/sangre , Inmunoglobulina A/inmunología , Inmunoglobulinas/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/análisis

RESUMEN

Facilitated subcutaneous immunoglobulin (fSCIG) 10% is an immunoglobulin replacement therapy that utilizes recombinant human hyaluronidase (rHuPH20) to enhance immunoglobulin dispersion and absorption, allowing for longer treatment intervals similar to intravenous immunoglobulin (up to once monthly). fSCIG 10% is indicated in the USA for treating adults and children aged ≥ 2 years with primary immunodeficiency diseases (PIDs). This prospective, non-interventional, open-label, multicenter, post-authorization safety study (NCT02593188) was conducted in the USA from November 2015 to October 2021 to assess the long-term safety of fSCIG 10% in routine clinical practice. Patients with PIDs aged ≥ 16 years who were prescribed and/or had started fSCIG 10% treatment were enrolled. In total, 253 patients were enrolled and included (full analysis set). Participants received fSCIG 10% treatment for a median (interquartile range) of 10.0 (3.5-11.8) months, with the majority of infusions administered every 4 weeks (54.4% [1197/2201 infusions]) and at home (62.6% [1395/2230 infusions]). Overall, 98.5% of infusions were administered without rate reduction, interruption, or discontinuation due to adverse events (AEs). Treatment-related, non-serious AEs were experienced by 52 patients (20.6%, 284 events). Two patients (0.8%) each experienced one treatment-related serious AE (aseptic meningitis and deep vein thrombosis). Development of antibodies against rHuPH20 was uncommon; 14/196 patients (7.1%) tested positive for binding antibodies (titer ≥ 1:160) with no neutralizing antibodies detected. There was no relationship between anti-rHuPH20 antibody positivity and the occurrence of treatment-related serious or non-serious AEs. Long-term, repeated self-administration of fSCIG 10% was well tolerated in US clinical practice by patients with PIDs.

Asunto(s)

Hialuronoglucosaminidasa , Humanos , Masculino , Femenino , Estados Unidos , Adulto , Adolescente , Estudios Prospectivos , Hialuronoglucosaminidasa/uso terapéutico , Hialuronoglucosaminidasa/administración & dosificación , Enfermedades de Inmunodeficiencia Primaria/tratamiento farmacológico , Persona de Mediana Edad , Infusiones Subcutáneas , Niño , Adulto Joven , Inmunoglobulinas/administración & dosificación , Inmunoglobulinas/efectos adversos , Inmunoglobulinas/uso terapéutico , Inyecciones Subcutáneas , Resultado del Tratamiento , Anciano , Preescolar , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Síndromes de Inmunodeficiencia/terapia

RESUMEN

Three hyperimmune egg-based formulations rich in immunoglobulin Y (IgY) were orally administered (daily, for up to 90 days) to C57BL/6 mice that were not microbially challenged. The serum levels of 32 cytokines were quantified every 30 days. Histopathology, hematology, and serum biochemistry investigations were also performed. As a sign of increased immune activity, lymphohistiocytic infiltrates were detected in the digestive tract and the liver after 30, 60, and 90 days of treatment. These infiltrates were also present in the lungs after 30 and 60 days, but not at 90 days. Blood analysis indicated systemic inflammation after 30 days of treatment: increases in pro-inflammatory cytokines, glycemia, total serum proteins, ALT, and ALP. After 60 and 90 days of treatment, the analyzed blood parameters showed mixed signs of both increased and decreased inflammation. The increased cytokines, which varied with formulation and time of exposure, indicated a combination of mostly Th17- and Th2-type immune responses. As the mice were healthy and housed in standardized sanitary conditions, and were not microbially challenged, the data were consistent with an interaction of IgY with the gut-associated lymphoid tissue as the main mechanism of action. This interaction generated a local immune response, which subsequently induced a systemic response.

Asunto(s)

Citocinas , Inmunidad Mucosa , Inmunoglobulinas , Ratones Endogámicos C57BL , Células Th17 , Células Th2 , Animales , Inmunoglobulinas/metabolismo , Administración Oral , Citocinas/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Ratones , Inmunidad Mucosa/efectos de los fármacos , Femenino , Huevos

RESUMEN

OBJECTIVE: The study aimed to characterize serum immunoglobulin (Ig) concentrations and their relationship with clinical and paraclinical features in patients with COPD group E in the stable stage. Additionally, the study focused on evaluating the relationship between serum Ig levels and the risk of exacerbations over the next 12 months, thereby clarifying the role of serum Ig deficiency in affecting the future risk for these patients. METHODS: A prospective observational study assessed IgG, IgA, IgM, and IgE levels in 67 COPD patients and 30 healthy controls at Military Hospital 103 from October 2017 to August 2020. Primary outcomes included Ig isotype levels in COPD patients, with secondary outcomes exploring differences compared to controls and associations with clinical variables. RESULTS: COPD patients showed significantly lower IgG concentrations and higher IgA levels than controls. IgM and IgE levels did not differ significantly. Subgroup analysis revealed notable decreases in IgG1 and IgG3 concentrations, with 10.4% of patients exhibiting reduced IgG levels and 0.3% diagnosed with common variable immunodeficiency. No significant associations were found between Ig levels and exacerbation risk or clinical variables. CONCLUSIONS: Serum IgG and IgM concentrations were significantly reduced in COPD patients compared to normal individuals, with IgG1 and IgG3 concentrations notably low. Serum IgA levels were significantly higher in COPD patients compared with normal controls. However, no significant association was found between Ig concentrations, particularly serum IgG deficiency and its subclasses, with the frequency and risk of exacerbations during 12 months of longitudinal follow-up. Caution is warranted in the use of immunoglobulin therapy in the treatment of COPD patients. TRIAL REGISTRATION: An independent ethics committee approved the study (Ethics Committee of Military Hospital 103 (No. 57/2014/VMMU-IRB), which was performed in accordance with the Declaration of Helsinki, Guidelines for Good Clinical Practice.

Asunto(s)

Enfermedad Pulmonar Obstructiva Crónica , Humanos , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/terapia , Masculino , Femenino , Estudios Prospectivos , Anciano , Persona de Mediana Edad , Inmunoglobulina G/sangre , Estudios de Casos y Controles , Inmunoglobulina A/sangre , Progresión de la Enfermedad , Inmunoglobulinas/sangre , Inmunoglobulina M/sangre

RESUMEN

Mink are susceptible to viruses such as SARS-CoV-2, H1N1 and H9N2, so they are considered a potential animal model for studying human viral infections. Therefore, it is important to study the immune system of mink. Immunoglobulin (Ig) is an important component of humoral immunity and plays an important role in the body's immune defense. In this study, we described the gene loci structure of mink Ig germline by genome comparison, and analysed the mechanism of expression diversity of mink antibody library by 5'RACE and next-generation sequencing (NGS). The results were as follows: the IgH, Igκ and Igλ loci of mink were located on chromosome 13, chromosome 8 and chromosome 3, respectively, and they had 25, 36 and 7 V genes, 3, 5 and 7 J genes and 10 DH genes, respectively. Mink Ig heavy chain preferred the IGHV1, IGHD2 and IGHJ4 subgroups, κ chain mainly use the IGKV1, IGKJ1 and IGHL4 subgroups, and λ chain mainly use the IGLV3 and IGLJ3 subgroups. Linkage diversity analysis revealed that N nucleotide insertion was the main factor affecting the linkage diversity of mink Igs. On the mutation types of mink Ig Somatic Hypermutation (SHM), the high mutation types of heavy chain were mainly G > A, C > T, T > C, A > G, C > A, G > T, A > C, and T > G; the high mutation types of κ chain were G > A and T > C; and the high mutation types of λ chain were G > A and A > G. The objective of this study was to analyse the loci structure and expression diversity of Ig in mink. The results contribute to our comprehension of Ig expression patterns in mink and were valuable for advancing knowledge in mink immunogenetics, exploring the evolution of adaptive immune systems across different species, and conducting comparative genomics research.

Asunto(s)

Visón , Animales , Visón/genética , Visón/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunidad Humoral/genética , COVID-19/inmunología , COVID-19/virología , Inmunoglobulinas/genética , Humanos , Mutación/genética , Cadenas Pesadas de Inmunoglobulina/genética , SARS-CoV-2/inmunología , Sitios Genéticos

RESUMEN

ABSTRACT: The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, it is not known whether "niche anchoring" of LSC affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C (Junctional Adhesion Molecule-C) expressed by hematopoietic stem cells (HSCs) and LSCs in an inducible mixed-lineage leukemia (iMLL)-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long-term to short-term HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSCs isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to activation protein-1 (AP-1) and tumor necrosis factor α (TNF-α)/NF-κB pathways. Human orthologs of dysregulated genes allowed to identify a score that was distinct from, and complementary to, the LSC-17 score. Substratification of patients with AML using LSC-17 and AP-1/TNF-α genes signature defined 4 groups with median survival ranging from <1 year to a median of "not reached" after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF-α gene signature as a proxy of LSC anchoring in bone marrow niches, which improves the prognostic value of the LSC-17 score. This trial was registered at www.ClinicalTrials.gov as #NCT02320656.

Asunto(s)

Moléculas de Adhesión Celular , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda , Células Madre Neoplásicas , Animales , Humanos , Ratones , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/genética , Modelos Animales de Enfermedad , Eliminación de Gen , Células Madre Hematopoyéticas/metabolismo , Inmunoglobulinas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Estudios Longitudinales

RESUMEN

Influenza virus contributes substantially to the global human and animal disease burden. To protect individuals against disease, strategies are needed to minimize the time an individual is at risk of developing disease symptoms. Passive immunization using avian IgY antibodies can protect individuals against a variety of pathogens, including influenza virus. Yet the effect of IgY administration on generation of protective immunity is largely unknown. To address the effect of passive immunization on the host immune response development, adult or aged, male and female C57BL/6NCrl mice received chicken IgY anti-H5N1, normal IgY or PBS intranasally four hours before, and 20 hours after intranasal infection with H1N1 influenza A virus (PR8). The mice receiving cross-reactive IgY anti-H5N1 were protected from disease and developed influenza virus-specific memory T cells similar to control-treated mice. When re-challenged with PR8 35 days post primary infection IgY anti-H5N1-treated mice were fully protected. Moreover, when challenged with heterologous H3N2 influenza A virus (X-31) or with PR8 three months post infection the mice were protected against severe disease and death, albeit a slight transient weight loss was noted. The results show that passive immunization with IgY anti-H5N1 is safe and protects mice against disease induced by influenza virus without inhibiting development of protective immunity after virus exposure. This indicate that passive immunization can be used as prophylactic therapy in combination with immunization to prevent disease.

Asunto(s)

Anticuerpos Antivirales , Inmunización Pasiva , Inmunoglobulinas , Subtipo H5N1 del Virus de la Influenza A , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae , Animales , Subtipo H5N1 del Virus de la Influenza A/inmunología , Inmunización Pasiva/métodos , Inmunoglobulinas/inmunología , Inmunoglobulinas/administración & dosificación , Femenino , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Masculino , Ratones , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Pollos/inmunología , Memoria Inmunológica , Reacciones Cruzadas/inmunología

RESUMEN

BACKGROUND: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod's immune response on a molecular level. RESULTS: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes. CONCLUSIONS: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Asunto(s)

Gadus morhua , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Gadus morhua/genética , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulinas/genética , Sitios Genéticos , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética

RESUMEN

It is widely acknowledged that immunoglobulins (Igs) are produced solely by B-lineage cells. The Ig gene is created by the rearrangement of a group of gene segments [variable (V), diversity (D), and joining (J) segments rearrangement, or V(D)J recombination], which results in the vast diversity of B cell-derived Ig responsible for recognising various antigens. Ig subsequently undergoes somatic hypermutation (SHM) and class switch recombination (CSR) after exposure to antigens, thus converting the low-affinity IgM to IgG, IgA, or IgE antibodies. IgM and IgD are primarily expressed in naïve B cells that have not been exposed to antigens, they do not undergo somatic hypermutation; hence, their variable region sequences remain the same as those in the germline. In contrast, IgG, IgA, and IgE are expressed in antigen-stimulated memory B cells or plasma cells, and thus, they often possess high-frequency mutations in their variable region sequences. Since the discovery that Ig can be produced by non-B cells, Qiu's group has investigated and compared the genetic characteristics of B cell-derived Ig and non-B cell-derived Ig. These findings demonstrated that non-B cell-derived Ig shares certain similarities with B cell-derived Ig in that the sequence of its constant region is identical to that of B cell-derived Ig, and its variable region is also strictly dependent on the rearrangement of V, D, and J gene segments. Moreover, akin to B cell-derived Ig, the V regions of IgM and IgD are rarely mutated, while IgG, IgA, and IgE produced by cancer cells are frequently mutated. However, the non-B cell-derived Ig V region sequence displays unique characteristics. (1) Unlike the vast diversity of B cell-derived Igs, non-B cell-derived Igs exhibit restricted diversity; cells from the same lineage always select the same V(D)J recombination patterns; (2) Both mRNA and proteins of RAG1/RAG2 recombinase have been detected in Ig positive cancer cell lines and normal tissues. But Ig recombination could also be found in RAG1-/- and RAG2-/- mice, suggesting that they are not necessary for the rearrangement of non-B cell-derived Igs. These features of non-B cell-derived Igs suggest a potentially undiscovered mechanism of V(D)J recombination, ligation, and SHM in non-B cells, which necessitates further investigation with advanced technology in molecular biology.

Asunto(s)

Linfocitos B , Genes de Inmunoglobulinas , Animales , Humanos , Ratones , Linfocitos B/inmunología , Genes de Inmunoglobulinas/genética , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Hipermutación Somática de Inmunoglobulina/genética

RESUMEN

INTRODUCTION: Shigellosis is a gastrointestinal disease causes high morbidity and mortality worldwide, however, there is no anti-Shigella vaccine. The use of antibiotics in shigellosis treatment exacerbates antibiotic resistance. Antibodies, particularly egg yolk antibody (IgY), offer a promising approach to address this challenge. This study aimed to investigate the prophylactic effect of IgY produced against a recombinant chimeric protein containing the immunogens IpaD, IpaB, StxB, and VirG from Shigella. METHODS: The chimeric protein, comprising IpaD, IpaB, StxB, and VirG, was expressed in E. coli BL21 and purified using the Ni-NTA column. Following immunization of chickens, IgY was extracted from egg yolk using the PEG-6000 method and analyzed through SDS-PAGE and ELISA techniques. Subsequently, the prophylactic efficacy of IgY was assessed by challenging of mice with 10 LD50 of S. dysenteriae and administering different concentrations of IgY (1.25, 2.5, 5, and 10 mg/kg) under various time conditions. RESULTS: The recombinant protein, weighing 82 kDa, was purified and confirmed by western blotting. The IgY concentration was determined as 9.5 mg/ml of egg yolk and the purity of the extracted IgY was over 90 %. The results of the ELISA showed that at least 19 ng of pure antibody identified recombinant protein and reacts with it. The challenge test employing IgY and Shigella demonstrated a direct correlation between the survival rate and antibody concentration, with increased concentrations leading to decreased mortality rates. Treatment of mice with 10 mg/kg IgY leads to 80 % survival of the mice against 10 LD50 S. dysenteriae. CONCLUSION: Our findings suggest that IgY may offer therapeutic potential in treating Shigella infections and combating antibiotic resistance.

Asunto(s)

Pollos , Disentería Bacilar , Yema de Huevo , Inmunoglobulinas , Animales , Inmunoglobulinas/inmunología , Ratones , Yema de Huevo/inmunología , Disentería Bacilar/prevención & control , Disentería Bacilar/inmunología , Shigella/inmunología , Proteínas Bacterianas/inmunología , Proteínas Recombinantes/inmunología , Femenino , Anticuerpos Antibacterianos/inmunología , Ratones Endogámicos BALB C , Antígenos Bacterianos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología

RESUMEN

Functional roles of SIGLEC15 in hepatocellular carcinoma (HCC) were not clear, which was recently found to be an immune inhibitor with similar structure of inhibitory B7 family members. SIGLEC15 expression in HCC was explored in public databases and further examined by PCR analysis. SIGLEC15 and PD-L1 expression patterns were examined in HCC samples through immunohistochemistry. SIGLEC15 expression was knocked-down or over-expressed in HCC cell lines, and CCK8 tests were used to examine cell proliferative ability in vitro. Influences of SIGLEC15 expression on tumor growth were examined in immune deficient and immunocompetent mice respectively. Co-culture system of HCC cell lines and Jurkat cells, flow cytometry analysis of tumor infiltrated immune cells and further sequencing analyses were performed to investigate how SIGLEC15 could affect T cells in vitro and in vivo. We found SIGLEC15 was increased in HCC tumor tissues and was negatively correlated with PD-L1 in HCC samples. In vitro and in vivo models demonstrated inhibition of SIGLEC15 did not directly influence tumor proliferation. However, SIGLEC15 could promoted HCC immune evasion in immune competent mouse models. Knock-out of Siglec15 could inhibit tumor growth and reinvigorate CD8+ T cell cytotoxicity. Anti-SIGLEC15 treatment could effectively inhibit tumor growth in mouse models with or without mononuclear phagocyte deletion. Bulk and single-cell RNA sequencing data of treated mouse tumors demonstrated SIGLEC15 could interfere CD8+ T cell viability and induce cell apoptosis. In all, SIGLEC15 was negatively correlated with PD-L1 in HCC and mainly promote HCC immune evasion through inhibition of CD8+ T cell viability and cytotoxicity.

Asunto(s)

Apoptosis , Antígeno B7-H1 , Linfocitos T CD8-positivos , Carcinoma Hepatocelular , Inmunoglobulinas , Neoplasias Hepáticas , Proteínas de la Membrana , Animales , Femenino , Humanos , Masculino , Ratones , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/inmunología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Proliferación Celular , Evasión Inmune , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Escape del Tumor/genética

RESUMEN

BACKGROUND: CD83 are closely related to the pathogenesis of immune thrombocytopenia (ITP), but the exact mechanism remains unclear. AIM: To explore the relationship between CD83 and CD4+ T cell subsets and clarify the role of CD83 in the pathogenesis of ITP. METHODS: RT-qPCR and Flow cytometry were used to illustrate CD83 expression. The downregulation and overexpression of DC-CD83 were co-cultured with CD4+ T cells to detect cell proliferation, co-cultured supernatant cytokines and Tregs expression. RESULTS: The results indicate that the ITP patients showed higher expression of CD83 than the healthy controls. The proliferation of CD4+ T cells was inhibited by downregulation of DCs-CD83 but promoted by overexpression of DCs-CD83. siRNA-CD83 inhibited proinflammatory IFN-γ and IL-17 secretion while raising TGF-ß, IL-10 concentrations. Overexpression of DCs-CD83 promoted Tregs expression. CONCLUSION: The Th1/Th2 and Th17/Tregs polarization were reversed via interfering DCs with siRNA-CD83. CD83 plays an important role in ITP pathogenesis, suggesting novel treatment for ITP patients.

Asunto(s)

Antígenos CD , Antígeno CD83 , Inmunoglobulinas , Glicoproteínas de Membrana , Púrpura Trombocitopénica Idiopática , Humanos , Púrpura Trombocitopénica Idiopática/inmunología , Púrpura Trombocitopénica Idiopática/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Antígenos CD/metabolismo , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Femenino , Masculino , Adulto , Persona de Mediana Edad , Citocinas/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo

RESUMEN

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Asunto(s)

Farmacorresistencia Bacteriana , Plásmidos , Animales , Plásmidos/genética , Ratones , Farmacorresistencia Bacteriana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Antibacterianos/farmacología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/farmacología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Femenino , Salmonella/genética , Salmonella/inmunología , Salmonella/efectos de los fármacos , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Ratones Endogámicos BALB C

RESUMEN

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319-519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD-lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD-lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Pollos , Inmunoglobulinas , SARS-CoV-2 , Animales , SARS-CoV-2/inmunología , Inmunoglobulinas/inmunología , Pollos/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Conejos , COVID-19/inmunología , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Humanos , Pruebas de Neutralización