RESUMO
OBJECTIVE: To evaluate the presence of cross-reactivity by anti-severe acute respiratory syndrome coronavirus 2 antibodies induced by the Pfizer-BioNTech vaccine against Trypanosoma cruzi proteins in a screening test. METHODS: Forty-three serum samples were obtained from personnel at the Hospital General Naval de Alta Especialidad in Mexico City who received one or two doses of the vaccine and were tested for T. cruzi infection using four tests: two 'in house' enzyme-linked immunosorbent assays (ELISAs), a commercial ELISA diagnostic kit and an immunoblot test. RESULTS: IgG antibodies against the T. cruzi proteins were present in the serum of unvaccinated subjects and subjects who had received one or two doses of the vaccine. The positivity of the samples against T. cruzi was ruled out by means of a Western Blot assay, where all samples were negative for T. cruzi. CONCLUSION: The data suggest that people convalescing from coronavirus disease 2019 and those who received the Pfizer-BioNTech vaccine exhibit cross-reactive antibodies against T. cruzi antigens in ELISA assays.
Assuntos
COVID-19 , Doença de Chagas , Trypanosoma cruzi , Vacinas , Humanos , Doença de Chagas/prevenção & controle , Doença de Chagas/diagnóstico , Western Blotting , Ensaio de Imunoadsorção Enzimática , Anticorpos AntiprotozoáriosRESUMO
Helicobacter pylori is a Gram-negative bacterium found on the luminal surface of the gastric mucosa in at least 50% of the world's human population. The protective effect of breastfeeding against H. pylori infection has been extensively reported; however, the mechanisms behind this protection remain poorly understood. Human IgA from colostrum has reactivity against H. pylori antigens. Despite that IgA1 and IgA2 display structural and functional differences, their reactivity against H. pylori had not been previously determined. We attested titers and reactivity of human colostrum-IgA subclasses by ELISA, immunoblot, and flow cytometry. Colostrum samples from healthy mothers had higher titers of IgA; and IgA1 mostly recognized H. pylori antigens. Moreover, we found a correlation between IgA1 reactivity and their neutralizing effect determined by inhibition of cytoskeletal changes in AGS cells infected with H. pylori. In conclusion, colostrum-IgA reduces H. pylori infection of epithelial gastric cells, suggesting an important role in preventing the bacteria establishment during the first months of life. As a whole, these results suggest that IgA1 from human colostrum provides protection that may help in the development of the mucosal immune system of newborn children.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Colostro/imunologia , Helicobacter pylori/imunologia , Imunoglobulina A Secretora/imunologia , Citoesqueleto , Células Epiteliais , Feminino , Mucosa Gástrica/imunologia , Infecções por Helicobacter/imunologia , Humanos , GravidezRESUMO
CD38 is a transmembrane glycoprotein expressed by T-cells. It has been reported that patients with systemic lupus erythematosus (SLE) showed increased CD38+CD25+ T-cells correlating with immune activation and clinical signs. Contrariwise, CD38 deficiency in murine models has shown enhanced autoimmunity development. Recent studies have suggested that CD38+ regulatory T-cells are more suppressive than CD38- regulatory T-cells. Thus, we have suggested that CD38 overexpression in SLE patients could play a role in regulating immune activation cells instead of enhancing it. This study found a correlation between CD38 with FoxP3 expression and immunosuppressive molecules (CD69, IL-10, CTLA-4, and PD-1) in T-cells from lupus-prone mice (B6.MRL-Faslpr/J). Additionally, B6.MRL-Faslpr/J mice showed a decreased proportion of CD38+ Treg cells regarding wild-type mice (WT). Furthermore, Regulatory T-Cells (Treg cells) from CD38-/- mice showed impairment in expressing immunosuppressive molecules and proliferation after stimulation through the T-cell receptor (TCR). Finally, we demonstrated an increased ratio of IFN-γ/IL-10 secretion in CD38-/- splenocytes stimulated with anti-CD3 compared with the WT. Altogether, our data suggest that CD38 represents an element in maintaining activated and proliferative Treg cells. Consequently, CD38 could have a crucial role in immune tolerance, preventing SLE development through Treg cells.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , Fatores de Transcrição Forkhead/imunologia , Imunossupressores/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T Reguladores/imunologia , ADP-Ribosil Ciclase 1/genética , Animais , Autoimunidade , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Tolerância Imunológica , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
A novel cell population denominated IFN-γ-producing killer dendritic cells (IKDCs) have been recently described. These cells are lymphocytes lacking B- or T- receptors, but they can be identified by the presence of B220+ CD38+ CD49b+ and low CD11c, among other cell surface markers. The main characteristics of IKDCs are the production of IFN-γ and the ability to spontaneously kill tumor cells. We found that this population increases in B6.MLR-Faslpr /J mice. Interestingly, IKDCs increase with age and are more abundant in mice older than 6 months onward. To analyze whether these cells have any role in the induction of the lupus-like phenotype in the B6.MLR-Faslpr /J mice, IKDCs were purified and transferred into 6-month-old B6.MRL-Faslpr /J mice, then the presence of anti-nuclear antibodies (ANAS) and anti-dsDNA antibodies were analyzed 2 and 4 months after the transfer. The results showed a reduction in the levels of these autoantibodies and increased survival of these mice, indicating that these cells may have a regulatory function. In vitro assays demonstrated that IKDCs reduced the proliferation of both autoreactive B and T cells, suggesting that these may be the mechanisms used by these cells to ameliorate the lupus-like phenotype in the B6.MRL-Faslpr /J mice.
Assuntos
Células Dendríticas/imunologia , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Proliferação de Células/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Linfócitos T/imunologiaRESUMO
CD38 is a 45,000 molecular weight transmembrane protein that is expressed in immature and mature lymphocytes. However, the expression and function of CD38 during B-cell differentiation in mice is poorly understood. Here, we report that CD38 is expressed from the earliest stages of B-cell development. Pre-pro-B, pro-B, pre-B and immature B cells from murine bone marrow all stained positive for CD38. Interestingly, CD38 expression increases with B-cell maturation. To assess the role of CD38 during B-cell maturation, CD38-deficient mice were analysed. CD38(-/-) mice showed a significant increase in both the frequency of B-lineage cells and the absolute numbers of pre-pro-B cells in bone marrow; however, no other differences were observed at later stages. CD38 cross-linking in Ba/F3 cells promoted apoptosis and marked extracellular signal-regulated kinase (ERK) phosphorylation, and these effects were reduced by treatment with the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and similar effects were observed in B-cell precursors from bone marrow. These data demonstrate that B-cell precursors in mouse bone marrow express functional CD38 and implicate the early ligation of CD38 in the ERK-associated regulation of the B-lineage differentiation pathway.
Assuntos
ADP-Ribosil Ciclase 1/genética , Células da Medula Óssea/citologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Células Precursoras de Linfócitos B/citologia , ADP-Ribosil Ciclase 1/biossíntese , Animais , Apoptose/imunologia , Células da Medula Óssea/imunologia , Diferenciação Celular/imunologia , Linhagem Celular , Linhagem da Célula/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Flavonoides/farmacologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/imunologiaRESUMO
Introduction: Lipopolysaccharide-responsive and beige-like anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PKA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although Lrba-/- mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells. Aim: To compare and evaluate spleen-derived B cell responses to BCR crosslinking in C57BL6 Lrba-/- and Lrba+/+ mice. Materials and methods: Spleen-derived B cells were obtained from 8 to 12-week-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinking was assessed by the F(ab')2 anti-µ chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot. Results: Lrba-/- B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinking in the Lrba-/- mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBα, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinking in Lrba-/ - B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected. Discussion: These results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter.
Assuntos
Linfócitos B , NF-kappa B , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Lipopolissacarídeos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
Introduction: Lrba is a cytoplasmic protein involved in vesicular trafficking. Lrba-deficient (Lrba-/-) mice exhibit substantially higher levels of IgA in both serum and feces than wild-type (WT) mice. Transforming growth factor ß1 (TGFß1) and its receptors (TGFßR I and II) is essential for differentiating IgA+ B cells. Furthermore, increased IgA production suggests a potential connection between Lrba and the TGFßR signaling pathway in IgA production. However, the specific function of Lrba in B cell biology remains unknown. Aim: Given the increased IgA levels in Lrba-/- mice, the goal in this work was to explore the lymph organs where the switch to IgA occurs, and if TGFßR function is affected. Methods: Non-immunized Lrba-/- mice were compared with Lrba+/+ mice. IgA levels in the serum and feces, as well as during peripheral B cell development, were determined. IgA+ B cells and plasma cells were assessed in the small intestine and secondary lymphoid organs, such as the spleen, mesenteric lymph nodes, and Peyer's patches. The TGFßR signaling pathway was evaluated by determining the expression of TGFßR on B cells. Additionally, SMAD2 phosphorylation was measured under basal conditions and in response to recombinant TGFß. Finally, confocal microscopy was performed to investigate a possible interaction between Lrba and TGFßR in B cells. Results: Lrba-/- mice exhibited significantly higher levels of circulating IgA, IgA+ B, and plasma cells than in peripheral lymphoid organs those in WT mice. TGFßR expression on the membrane of B cells was similar in both Lrba-/- and Lrba+/+ mice. However, intracellular TGFßR expression was reduced in Lrba-/- mice. SMAD2 phosphorylation showed increased levels under basal conditions; stimulation with recombinant TGFß elicited a poorer response than in that in Lrba+/+ B cells. Finally, we found that Lrba colocalizes with TGFßR in B cells. Conclusion: Lrba is essential in controlling TGFßR signaling, subsequently regulating SMAD2 phosphorylation on B cells. This mechanism may explain the increased differentiation of IgA+ B cells and production of IgA-producing plasma cells.
Assuntos
Linfócitos B , Diferenciação Celular , Imunoglobulina A , Transdução de Sinais , Animais , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/imunologia , Imunoglobulina A/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteína Smad2/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The suppressor effect of T regulatory lymphocytes in co-cultures with T effector cells obtained by magnetic columns from healthy donors and activated by CD3/CD28 was measured by a proliferation assay using BrdU incorporation and an ELISA test. Tritiated thymidine incorporation was used as a reference since it is the gold standard for proliferation assays. Both methods were used simultaneously in the same samples in order to compare them. Correlation between them was statistically significant (p < 0.001). The purification using magnetic columns was very efficient since CD4âºCD25⺠cells were also FOXP3⺠therefore; they were identified as suppressor T cells. The use of BrdU incorporation in suppression assays is an excellent method that avoids the use of radioactive contaminating materials.
Assuntos
Bioensaio , Bromodesoxiuridina/farmacologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Proliferação de Células , Células Cultivadas , Humanos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Timidina/farmacologia , TrítioRESUMO
The type II transmembrane glycoprotein CD38 has recently been implicated in regulating metabolism and the pathogenesis of multiple conditions, including aging, inflammation and cancer. CD38 is overexpressed in several tumor cells and microenvironment tumoral cells, associated to migration, angiogenesis, cell invasion and progression of the disease. Thus, CD38 has been used as a progression marker for different cancer types as well as in immunotherapy. This review focuses on describing the involvement of CD38 in various non-hematopoietic cancers.
Assuntos
Imunoterapia , Neoplasias , ADP-Ribosil Ciclase 1/metabolismo , Biomarcadores , Humanos , Fatores Imunológicos , Neoplasias/terapia , Microambiente TumoralRESUMO
The current pandemic generated by SARS-CoV-2 has led to mass vaccination with different biologics that have shown wide variations among human populations according to the origin and formulation of the vaccine. Studies evaluating the response in individuals with a natural infection before vaccination have been limited to antibody titer analysis and evaluating a few humoral and cellular response markers, showing a more rapid and intense humoral response than individuals without prior infection. However, the basis of these differences has not been explored in depth. In the present work, we analyzed a group of pro and anti-inflammatory cytokines, antibody titers, and cell populations in peripheral blood of individuals with previous SARS-CoV-2 infection using BNT162b2 biologic. Our results suggest that higher antibody concentration in individuals with an earlier disease could be generated by higher production of plasma cells to the detriment of the presence of memory B cells in the bloodstream, which could be related to the high baseline expression of cytokines (IL-6 and IL-10) before vaccination.
Assuntos
COVID-19 , Vacinas Virais , Vacina BNT162 , COVID-19/prevenção & controle , Humanos , Interleucina-10 , Interleucina-6 , Receptores CCR7 , SARS-CoV-2 , VacinaçãoRESUMO
BACKGROUND: Mongolian gerbils that are experimentally infected with Helicobacter pylori develop a chronic inflammation that is similar to natural infections in humans. The aim of this study was to compare the antigens of H. pylori cagPAI+ and cagPAI- strains that are expressed during Meriones unguiculatus colonization. MATERIALS AND METHODS: We identified H. pylori cagPAI+ and cagPAI- strain antigens via Western blotting of samples from Mongolian gerbils that were subjected to unique, mixed, and sequential bacterial infections. RESULTS: The antigens from the J99/CG3 (cagPAI+) strain had a lower molecular weight than the antigens from the 251F/CG3 (cagPAI-) strain. There were fewer identified antigens in the single unique infections compared with the mixed and sequential infections. The number of recognized antigens that had a frequency of recognition >60% was higher for the simultaneous and sequential infection groups compared with the single infection group. A 57-kDa antigen was present in >60% of the samples and four of the five experimental groups. Antigens specific to each bacterial strain were identified; the 190- and 158-kDa antigens appear to be specific for cagPAI-, and the 70-kDa antigen appears to be specific for cagPAI+. CONCLUSIONS: In this study, we identified antigens that are common and specific to the H. pylori cagPAI+ and cagPAI- strains.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Imunoglobulina G/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Western Blotting , Modelos Animais de Doenças , Ilhas Genômicas , Gerbillinae , Infecções por Helicobacter/microbiologia , Helicobacter pylori/química , Helicobacter pylori/fisiologia , Humanos , Imunoglobulina G/sangue , Masculino , Peso Molecular , CoelhosRESUMO
Cell spreading and phagocytosis are notably regulated by small GTPases and GAP proteins. TBC1D10C is a dual inhibitory protein with GAP activity. In immune cells, TBC1D10C is one of the elements regulating lymphocyte activation. However, its specific role in macrophages remains unknown. Here, we show that TBC1D10C engages in functions dependent on the cytoskeleton and plasma membrane reorganization. Using ex vivo and in vitro assays, we found that elimination and overexpression of TBC1D10C modified the cytoskeletal architecture of macrophages by decreasing and increasing the spreading ability of these cells, respectively. In addition, TBC1D10C overexpression contributed to higher phagocytic activity against Burkholderia cenocepacia and to increased cell membrane tension. Furthermore, by performing in vitro and in silico analyses, we identified 27 TBC1D10C-interacting proteins, some of which were functionally classified as protein complexes involved in cytoskeletal dynamics. Interestingly, we identified one unreported TBC1D10C-intrinsically disordered region (IDR) with biological potential at the cytoskeleton level. Our results demonstrate that TBC1D10C shapes macrophage activity by inducing reorganization of the cytoskeleton-plasma membrane in cell spreading and phagocytosis. We anticipate our results will be the basis for further studies focused on TBC1D10C. For example, the specific molecular mechanism in Burkholderia cenocepacia phagocytosis and functional analysis of TBC1D10C-IDR are needed to further understand its role in health and disease.
Assuntos
Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiologia , Fagocitose/fisiologia , Animais , Burkholderia cenocepacia/patogenicidade , Membrana Celular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Naringenin is flavonoid mainly found in citrus fruits which has shown several biological properties. In this work, we evaluated the therapeutic potential of the flavonoid Naringenin. Five-month-old B6.MRL-Faslpr/J lupus-prone mice were administered daily orally with Naringenin for seven months. We showed that Naringenin treatment at 50 or 100 mg/kg inhibited the splenomegaly and decreased the levels of anti-nuclear and anti-dsDNA autoantibodies. Furthermore, a reduction in serum concentration of TNF-α, IFN-γ and IL-6 was observed in the mice provided with Naringenin. Interestingly, serum levels of IL-10 increased. Naringenin decreased the frequency and absolute numbers of splenic effector memory T cells. Additionally, in order to be able to evaluate whether Naringenin prevented kidney damage, twelve-week-old MRL/MpJ-Faslpr/J mice, an accelerated lupus model, were orally administered with Naringenin at 100 mg/kg for six weeks. Surprisingly, Naringenin treatment prevented kidney damage and reduced the development of fibrosis similar to cyclophosphamide group. Moreover, Naringenin treatment increased the percentage of regulatory T cells in this aggressive model of lupus. Together, these results suggest a potential ability of Naringenin to reduce the autoimmunity in lupus-prone mice by modulation of T-cell subsets and cytokines profile that mitigate the development of important lupus clinical manifestations.
Assuntos
Citocinas/imunologia , Flavanonas/farmacologia , Memória Imunológica/efeitos dos fármacos , Nefrite Lúpica/tratamento farmacológico , Linfócitos T Reguladores/imunologia , Animais , Modelos Animais de Doenças , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Masculino , Camundongos , Linfócitos T Reguladores/patologiaRESUMO
BACKGROUND: Colostrum is the primary source of maternal immunoglobulin A (IgA) for the newborn. IgA participates in protection and regulation mechanisms of the immune response at the neonate's mucosa. Several studies have evaluated infectious diseases and vaccine protocols effects during pregnancy on maternal milk IgA levels, with the aim to understand lactation protecting effect on newborn. However, most of their results demonstrated that there were no differences in the total IgA levels. In humans, IgA has two subclasses (IgA1 and IgA2), they have an anatomical distribution among mucosal compartments, their levels vary after antigen stimulation and are also seen to describe differential affinities in colostrum. Although there are differences between IgA subclasses in several compartments, these studies have excluded specific colostrum IgA1 and IgA2 determination. METHODS: We analyzed data from 900 women in Mexico City. With Pearson correlation, we compared the number of infectious episodes during their pregnancy that was associated with mucosal compartments (skin, respiratory and gastrointestinal tracts) and colostrum IgA subclasses. RESULTS: We show a correlation between increased colostrum IgA1 levels and the number of infectious episodes at respiratory tract and the skin. In contrast, infections at the gastrointestinal tract correlated with increased IgA2 amounts. CONCLUSIONS: Infections present during pregnancy at certain mucosal site increase specific IgA subclasses levels in human colostrum. These results will help in understanding infections and immunizations effects on maternal IgA at the mammary gland, and their impact on the development and protection of the newborn.
RESUMO
Dengue viruses (DENVs; serotypes 1-4) are members of the flavivirus family. The envelope protein (E) of DENV has been defined as the principal antigenic target in terms of protection and diagnosis. Antibodies that can reliably detect the E surface glycoprotein are necessary for describing and mapping new DENV epitopes as well as for developing more reliable and inexpensive diagnostic assays. In this study, we describe the production and characterization of a monoclonal antibody (mAb) against a recombinant DENV-2 E protein that recognizes a sequential antigen in both native and recombinant form located in domain II of the E protein of all four DENV serotypes. We confirmed that this mAb, C21, recognizes a sequence located in the fusion peptide. In addition, C21 does not have neutralizing activity against DENV-2 in an in vitro system. Furthermore, the C21 mAb is an ideal candidate for the development of research reagents for studying DENV biology because it cross-reacts with the four dengue serotypes.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/análise , Aedes , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Reações Cruzadas , Vírus da Dengue/classificação , Vírus da Dengue/genética , Mapeamento de Epitopos , Epitopos/genética , Escherichia coli/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Tipagem Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
PfTBP is a transcriptional factor required by all three types of RNA polymerases in eukaryotic cells. In order to obtain a specific monoclonal antibody (MAb) against PfTBP, a DNA fragment of 684 base pairs (bp) that contained the complete PfTBP gene was amplified by polymerase chain reaction (PCR) and inserted into the pGEX prokaryotic expression vector. The recombinant protein (GST-PfTBP) was expressed in Escherichia coli, purified, and used as antigen to immunize mice. MAbs against PfTBP were obtained and hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Western blotting and immunofluorescence assays showed that MAb Pf.r1 recognized the PfTBP protein in nuclear extracts from Plasmodium falciparum as well as a native protein in the nuclei of this parasite. This MAb will be a helpful tool for the identification of the TBP associated factors (TAFs), which are apparently highly divergent with other eukaryotes. This information could help to identify new candidate gene products to develop novel drugs or vaccines.
Assuntos
Anticorpos Monoclonais , Anticorpos Antiprotozoários , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Proteína de Ligação a TATA-Box/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antiprotozoários/isolamento & purificação , Antígenos de Protozoários/genética , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , DNA de Protozoário/genética , Expressão Gênica , Genes de Protozoários , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismoRESUMO
Cross-linking of CD44 in vitro promotes chemokinesis and actin-based dendrite formation in T and B cells. However, the mechanisms by which the adhesion molecule CD44 induces cytoskeleton activation in lymphocytes are still poorly understood. In this study, we have investigated whether myosin isoforms are involved in CD44-dependent dendrite formation in activated B cells. Pharmacological inhibition of myosin with 2,3-butanedione monoxime strongly affected spreading and dendrite formation, suggesting that these cellular motors may participate in these phenomena. Furthermore, immunofluorescence analysis showed differences in subcellular localization of class I and class II myosin during B cell spreading. In response to CD44 cross-linking, myosin-1c was polarized to lamellipodia, where F-actin was high. In contrast, the distribution of cytosplasmic nonmuscle class II myosin was not altered. Expressions of myosin-1c and II were also demonstrated in B cells by Western blot. Although the inhibition of PLCgamma, PI3K and MEK-1 activation affected the spreading and dendrite formation in activated B cells, only PLCgamma and MEK-1 inhibition correlated with absence of myosin-1c polarization. Additionally, myosin-1c polarization was observed upon cross-linking of other surface molecules, suggesting a common mechanism for B cell spreading. This work shows that class I and class II myosin are expressed in B cells, are differentially distributed, and may participate in the morphological changes of these cells.
Assuntos
Linfócitos B/fisiologia , Movimento Celular/fisiologia , Extensões da Superfície Celular/química , Miosina Tipo II/análise , Miosina Tipo I/análise , Actinas/análise , Animais , Linfócitos B/química , Movimento Celular/efeitos dos fármacos , Extensões da Superfície Celular/efeitos dos fármacos , Diacetil/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores de Hialuronatos/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Miosina Tipo I/fisiologia , Miosina Tipo II/fisiologia , Baço/citologiaRESUMO
Cross-linking of CD38 on hematopoietic cells induces activation, proliferation and differentiation of mature T and B cells and mediates apoptosis of myeloid and lymphoid progenitor cells. In addition to acting as a signaling receptor, CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites, including cyclic adenosine diphosphate ribose (cADPR). It has been previously postulated that the calcium-mobilizing metabolites produced by CD38 may regulate its receptor-based activities. To test this hypothesis, we examined whether the enzyme activity of CD38 controls the apoptosis of an anti-CD38-stimulated leukemic B cell. We show that anti-CD38-induced apoptosis of Ba/F3 cells, a murine pro-B cell line, is not affected by blocking the calcium-mobilizing activity of cADPR or by inhibiting intracellular or extracellular calcium mobilization. In addition, we demonstrate that blocking CD38 enzyme activity with 2'-deoxy-2'-fluoro-nicotinamide arabinoside adenine dinucleotide has no effect on apoptosis and that Ba/F3 cells expressing catalytically inactive mutant forms of CD38 still undergo apoptosis upon CD38 cross-linking. Instead, we find that anti-CD38-induced apoptosis is dependent on tyrosine kinase and caspase activation, and that this process appears to be potentiated by the presence of membrane microdomains. Thus, the receptor-mediated functions of CD38 can be separated from its enzyme activity in a murine leukemic cell line, suggesting that CD38 plays multiple, but independent, biologic roles.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase/imunologia , Apoptose/imunologia , Linfócitos B/imunologia , Sinalização do Cálcio/imunologia , ADP-Ribosil Ciclase/antagonistas & inibidores , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/enzimologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , ADP-Ribose Cíclica/imunologia , ADP-Ribose Cíclica/metabolismo , Inibidores Enzimáticos/farmacologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Capeamento Imunológico/efeitos dos fármacos , Capeamento Imunológico/imunologia , Leucemia de Células B/imunologia , Leucemia de Células B/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/imunologia , Camundongos , NAD/análogos & derivados , NAD/farmacologiaRESUMO
We have obtained a specific monoclonal antibody (MAb) against the Plasmodium falciparum histone acetyl transferase (PfGcn5), a transcriptional factor that possesses HAT activity directed to the amino terminal of histone H3. To prepare this antibody, a 968-base pair (bp) DNA fragment of PfGcn5 gene corresponding to C-terminal domain was obtained by RT-PCR and cloned into prokaryotic expression vector pGEX-4T3. MAb against PfGcn5 was obtained with hybridoma technique and ELISA screening using either purified GSTPfGcn5 protein or purified GST protein alone as a control. One MAb, named Pf.r2, was able to identify the PfGcn5 protein in nuclear extract from P. falciparum and immunofluorescence assays. This MAb will be a helpful tool to perform a variety of assays to identify the other components of PfGcn5 complexes.