RESUMO
Patients with relapsed T cell acute lymphoblastic leukemia (T-ALL) have limited therapeutic options and poor prognosis. The finding of efficient strategies against this refractory neoplasm is a medical priority. Superantigens (SAgs) are viral and bacterial proteins that bind to major histocompatibility complex class II molecules as unprocessed proteins and subsequently interact with a high number of T cells expressing particular T cell receptor Vß chains. Although on mature T cells, SAgs usually trigger massive cell proliferation producing deleterious effects on the organism, in contrast, on immature T cells, they may trigger their death by apoptosis. On this basis, it was hypothesized that SAgs could also induce apoptosis in neoplastic T cells that are usually immature cells that probably conserve their particular Vß chains. In this work, we investigated the effect of the SAg Staphylococcus aureus enterotoxin E (SEE) (that specifically interacts with cells that express Vß8 chain), on human Jurkat T- leukemia line, that expresses Vß8 in its T receptor and it is a model of the highly aggressive recurrent T-ALL. Our results demonstrated that SEE could induce apoptosis in Jurkat cells in vitro. The induction of apoptosis was specific, correlated to the down regulation of surface Vß8 TCR expression and was triggered, at least in part, through the Fas/FasL extrinsic pathway. The apoptotic effect induced by SEE on Jurkat cells was therapeutically relevant. In effect, upon transplantation of Jurkat cells in the highly immunodeficient NSG mice, SEE treatment reduced dramatically tumor growth, decreased the infiltration of neoplastic cells in the bloodstream, spleen and lymph nodes and, most importantly, increased significantly the survival of mice. Taken together, these results raise the possibility that this strategy can be, in the future, a useful option for the treatment of recurrent T-ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Superantígenos , Humanos , Camundongos , Animais , Enterotoxinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Apoptose , Receptores de Antígenos de Linfócitos TRESUMO
Gastropod Molluscs rely exclusively on the innate immune system to protect from pathogens, defending their embryos through maternally transferred effectors. In this regard, Pomacea snail eggs, in addition to immune defenses, have evolved the perivitellin-2 or PV2 combining two immune proteins into a neurotoxin: a lectin and a pore-forming protein from the Membrane Attack Complex/Perforin (MACPF) family. This binary structure resembles AB-toxins, a group of toxins otherwise restricted to bacteria and plants. Many of these are enterotoxins, leading us to explore this activity in PV2. Enterotoxins found in bacteria and plants act mainly as pore-forming toxins and toxic lectins, respectively. In animals, although both pore-forming proteins and lectins are ubiquitous, no enterotoxins have been reported. Considering that Pomacea snail eggs ingestion induce morpho-physiological changes in the intestinal mucosa of rodents and is cytotoxic to intestinal cells in culture, we seek for the factor causing these effects and identified PmPV2 from Pomacea maculata eggs. We characterized the enterotoxic activity of PmPV2 through in vitro and in vivo assays. We determined that it withstands the gastrointestinal environment and resisted a wide pH range and enzymatic proteolysis. After binding to Caco-2 cells it promoted changes in surface morphology and an increase in membrane roughness. It was also cytotoxic to both epithelial and immune cells from the digestive system of mammals. It induced enterocyte death by a lytic mechanism and disrupted enterocyte monolayers in a dose-dependent manner. Further, after oral administration to mice PmPV2 attached to enterocytes and induced large dose-dependent morphological changes on their small intestine mucosa, reducing the absorptive surface. Additionally, PmPV2 was detected in the Peyer's patches where it activated lymphoid follicles and triggered apoptosis. We also provide evidence that the toxin can traverse the intestinal barrier and induce oral adaptive immunity with evidence of circulating antibody response. As a whole, these results indicate that PmPV2 is a true enterotoxin, a role that has never been reported to lectins or perforin in animals. This extends by convergent evolution the presence of plant- and bacteria-like enterotoxins to animals, thus expanding the diversity of functions of MACPF proteins in nature.
Assuntos
Enterotoxinas/farmacologia , Imunidade Inata/imunologia , Mucosa Intestinal/efeitos dos fármacos , Venenos de Moluscos/farmacologia , Caramujos/imunologia , Animais , Complexo de Ataque à Membrana do Sistema Complemento , Camundongos , Óvulo/imunologia , Óvulo/metabolismo , Perforina/metabolismoRESUMO
Staphylococcus aureus colonizes the skin of atopic dermatitis (AD) individuals, but the impact of its enterotoxins on the chronic activation of CD4+ T cells demands further analysis. We aimed to analyze the CD4+ T cell anergy profile and their phenotypic and functional features through differential expression of cellular activation markers, cytokine production and response to staphylococcal enterotoxin A (SEA). A panel of 84 genes relevant to T cell anergy was assessed by PCR array in FACS-sorted CD4+ T cells, and the most prominent genes were validated by RT-qPCR. We evaluated frequencies of circulating CD4+ T cells secreting single or multiple (polyfunctional) cytokines (IL-17A, IL-22, TNF, IFN-γ, and MIP-1ß) and expression of activation marker CD38 in response to SEA stimulation by flow cytometry. Our main findings indicated upregulation of anergy-related genes (EGR2 and IL13) promoted by SEA in AD patients, associated to a compromised polyfunctional response particularly in CD4+CD38+ T cells in response to antigen stimulation. The pathogenic role of staphylococcal enterotoxins in adult AD can be explained by their ability to downmodulate the activated effector T cell response, altering gene expression profile such as EGR2 induction, and may contribute to negative regulation of polyfunctional CD4+ T cells in these patients.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Enterotoxinas/farmacologia , Transcriptoma/efeitos dos fármacos , Adulto , Feminino , Humanos , Masculino , Adulto JovemRESUMO
ABSTRACT Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50 kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.
Assuntos
Animais , Masculino , Ratos , Sobrevivência Celular/efeitos dos fármacos , Plesiomonas/metabolismo , Vesículas Citoplasmáticas , Fatores de Virulência , Rios/microbiologia , Enterotoxinas/farmacologia , Células Vero , Testes de Neutralização , Microscopia Eletrônica de Varredura , Chlorocebus aethiops , Plesiomonas/patogenicidade , Plesiomonas/ultraestrutura , Dose Letal MedianaRESUMO
Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Vesículas Citoplasmáticas , Enterotoxinas/farmacologia , Plesiomonas/metabolismo , Rios/microbiologia , Fatores de Virulência , Animais , Chlorocebus aethiops , Dose Letal Mediana , Masculino , Microscopia Eletrônica de Varredura , Testes de Neutralização , Plesiomonas/patogenicidade , Plesiomonas/ultraestrutura , Coelhos , Células VeroRESUMO
Macrophage migration inhibitory factor (MIF) mediates immunity against Toxoplasma gondii infection by inducing inflammatory cytokines required to control the parasite replication. However, the role of this inflammatory mediator in the cell-mediated immune response against this infection is still poorly understood. Here, we used T. gondii-infected WT and Mif (-/-) mice to analyze the role of MIF in the maturation of CD11b(+) and CD8α (+) dendritic cells (DCs). We found that MIF promotes maturation of CD11b(+) but not CD8α (+) DCs, by inducing IL-12p70 production and CD86 expression. Infected Mif (-/-) mice showed significantly lower numbers of TNF and inducible nitric oxide synthase- (iNOS-) producing DCs (TipDCs) compared to infected WT mice. The adoptive transfer of Ly6C(high) monocytes into infected WT or Mif (-/-) mice demonstrated that MIF participates in the differentiation of Ly6C(high) monocytes into TipDCs. In addition, infected Mif (-/-) mice display a lower percentage of IFN-γ-producing natural killer (NK) cells compared to WT mice, which is associated with reducing numbers of TipDCs in Mif (-/-) mice. Furthermore, administration of recombinant MIF (rMIF) into T. gondii-infected Mif (-/-) mice restored the numbers of TipDCs and reversed the susceptible phenotype of Mif (-/-) mice. Collectively, these results demonstrate an important role for MIF inducing cell-mediated immunity to T. gondii infection.
Assuntos
Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Monócitos/metabolismo , Toxoplasmose/metabolismo , Animais , Enterotoxinas/farmacologia , Feminino , Galactosamina/farmacologia , Imunidade Celular/imunologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Oxirredutases Intramoleculares/genética , Lipopolissacarídeos/farmacologia , Fatores Inibidores da Migração de Macrófagos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/efeitos dos fármacos , Neutrófilos/microbiologia , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/patogenicidade , Toxoplasmose/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 µmol/L STa (30 minutes), 25 µmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 µmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 µmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 µmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.
Assuntos
AMP Cíclico/metabolismo , Enterotoxinas/farmacologia , Células Epiteliais/efeitos dos fármacos , Escherichia coli , Temperatura Alta , Intestinos/citologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Adulto , Linhagem Celular Tumoral , GMP Cíclico/metabolismo , Estabilidade de Medicamentos , Enterotoxinas/química , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Prótons , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/ß-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA for 24 h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of ß-catenin and Western blotting for ß-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the ß-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/ß-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3ß inhibitor), or constitutively active ß-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA is important for TcdA antiproliferative effects.
Assuntos
Toxinas Bacterianas/farmacologia , Enterotoxinas/farmacologia , Células Epiteliais/metabolismo , Mucosa Intestinal/citologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
The disturbed cytokine-chemokine network could play an important role in the onset of diseases with inflammatory processes such as chronic idiopathic urticaria (CIU). Our main objectives were to evaluate the relation between proinflammatory chemokine serum levels from CIU patients and their response to autologous skin test (ASST) and basophil histamine release (BHR). We also aimed to assess the chemokine secretion by peripheral blood mononuclear cells (PBMC) upon polyclonal stimulus and to evaluate chemokine C-C ligand 2/C-X-C chemokine 8 (CCL2/CXCL8) and Toll-like receptor-4 (TLR-4) expression in monocytes. We observed significantly higher serum levels of the CXCL8, CXCL9, CXCL10 and CCL2 in CIU patients compared to the healthy group, regardless of the BHR or ASST response. The basal secretion of CCL2 by PBMC or induced by Staphylococcus aureus enterotoxin A (SEA) was higher in CIU patients than in the control group, as well as for CXCL8 and CCL5 secretions upon phytohaemagglutinin stimulation. Also, up-regulation of CCL2 and CXCL8 mRNA expression was found in monocytes of patients upon SEA stimulation. The findings showed a high responsiveness of monocytes through CCL2/CXCL8 expression, contributing to the creation of a proinflammatory environment in CIU.
Assuntos
Quimiocina CCL2/biossíntese , Interleucina-8/biossíntese , Leucócitos Mononucleares/metabolismo , Monócitos/metabolismo , Urticária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Teste de Degranulação de Basófilos , Quimiocina CCL2/genética , Quimiocina CCL2/fisiologia , Quimiocinas/sangue , Doença Crônica , Enterotoxinas/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Interleucina-8/genética , Interleucina-8/fisiologia , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Fito-Hemaglutininas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Testes Cutâneos , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/genética , Regulação para Cima/efeitos dos fármacos , Urticária/sangue , Urticária/imunologia , Adulto JovemRESUMO
Introducción: La poliposis nasal (PN) se presenta frecuentemente asociada a asma bronquial (AB). La enterotoxina estafilocócica B (SEB) jugaría un papel en su patogenia. No se ha estudiado si el perfil de citoquinas inducido por SEB en linfocitos T (LT) de pacientes con PNyAB difiere del de controles sanos. Objetivo: Comparar el perfil de citoquinas de LT de sangre periférica de pacientes con PN-AByde controles, estimulados con SEB o concanavalina A (ConA). Material y método: Células mononucleares de sangre periférica de 9 pacientes con PN-AB y de 6 controles se estimularon con SEB o ConA. El porcentaje LT CD4+ productores de interferón (IFN)-y, interleuquina (IL) IL-4, IL-5, IL-17 e IL-21 se determinó mediante citometrfa de flujo. Resultados: El grupo PN-AB presentó un menor porcentaje de LT productores de IL-5 que los controles al estimularse con SEB y con ConA. No hubo diferencia en las otras citoquinas estudiadas. Discusión: Nuestros resultados en sangre periférica difieren de lo descrito en tejido de pólipos nasales. Conclusión: Se sugiere que la respuesta inflamatoria de la PN se originaría localmente ya que los LT de sangre de pacientes con PN-AB no muestran una polarización hacia perfiles proinflamatorios con los estímulos utilizados.
Introduction: Nasal poliposis (NP) is frequently associated with bronchial asthma (BA) and its pathogenesis is still unknown. Staphylococcal enterotoxin B (SEB) has been implicated in the development of NP, however if the SEB-induced cytoklne profile of peripheral blood T lymphocytes (TL) of PN-BA patients differs from that of normal controls has not been studied. Aim: To compare the cytoklne profile of CD4+ TL from NP-BA and controls stimulated with SEB or concanavalin A (ConA). Material and method: Peripheral blood mononuclear cells from 9 NP-BA patients and from 6 controls were stimulated with SEB or ConA. The percentage of interferon (IFN)-y, interleukin {II) 11-4,11-5,11-17, and 11-21 producing TL was analyzed by flow cytometry Results: The percentage of SEB and ConA stimulated CD4+ IL-5-producing TLs was lower in the NP-BA group compared to the control group. There were no differences in the other cytokine-producing populations. Discussion: Unlike what is described in nasal polyp tissue, our findings show a diminished production of IL-5 by peripheral TL from the NP-AB group. Conclusion: A local sinonasal origin of the chronic inflammation is suggested since peripheral blood TL of NP-BA patients do not show a pro-inflammatory polarization with the tested stimuli.