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Bullous Pemphigoid is the most common auto-immune bullous skin disease. It is characterized by the production of auto-antibodies directed against 2 proteins of the hemi-desmosome (BP180 and BP230). We assessed the efficacy and mechanisms of action of rituximab, an anti-CD20 monoclonal antibody, in 17 patients with severe and relapsing type of bullous pemphigoid. The phenotype, cytokine gene expression, and rearrangement of BP180-specific B-cell receptor genes were performed over 2 years following treatment. At the end of the study, 5 patients had died, 3 had withdrawn from the study, and 9 patients were in complete remission. The one- and two-year relapse rates were 44.1% (95% Confidence Interval (CI): 21.0-76.0%) and 66.5%, (95% CI: 38.4-91.4%), respectively. Phenotypic analyses confirmed dramatic B-cell depletion, which lasted for 9 to 12 months. The ELISA values of serum anti-BP180 antibodies and the frequency of BP180-specific circulating B cells decreased dramatically following treatment, which paralleled the improvement of skin lesions. During B-cell reconstitution, a polyclonal IgM repertoire appeared and a shift in the rearrangement of the B-cell receptor genes of BP180-specific circulating B cells was observed. Concurrently, we observed a decrease of IL-15, IL-6 and TNFα expressing BP180-specific B cells, and the emergence of IL-10 and IL-1RA-expressing BP180-specific IgM+ B cells in patients in complete remission off therapy, suggesting the functional plasticity of BP180-specific auto-immune B cells after rituximab treatment.
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Autoantígenos/metabolismo , Linfócitos B/metabolismo , Citocinas/metabolismo , Colágenos não Fibrilares/metabolismo , Penfigoide Bolhoso/patologia , Idoso , Idoso de 80 Anos ou mais , Autoanticorpos/sangue , Autoantígenos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Interleucina-10/metabolismo , Interleucina-15/metabolismo , Interleucina-6/metabolismo , Masculino , Colágenos não Fibrilares/imunologia , Penfigoide Bolhoso/tratamento farmacológico , Penfigoide Bolhoso/imunologia , Fenótipo , Recidiva , Rituximab/uso terapêutico , Colágeno Tipo XVIIRESUMO
Newborns are highly susceptible to pathogenic infections with significant worldwide morbidity possibly due to an immature immune system. Recently, we reported that Propionibacterium strain, P. UF1, isolated from the gut microbiota of preterm infants, induced the differentiation of bacteria-specific Th17 cells. Here, we demonstrate that P. UF1 significantly increased the number of protective Th17 cells and maintained IL-10+ regulatory T cells (Tregs) in newborn mice. In addition, P. UF1 protected mice from intestinal Listeria monocytogenes (L. m) infection. P. UF1 also functionally sustained the gut microbiota and induced critical B vitamin metabolites implicated in the regulation of T cell immunity during L. m intestinal infection. Transcriptomic analysis of P. UF1-induced Th17 cells revealed genes involved in the differentiation and regulation of these cells. These results illustrate the potency of P. UF1 in the enhancement of neonatal host defense against intestinal pathogen infection.
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Microbioma Gastrointestinal/imunologia , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Propionibacterium/fisiologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Proliferação de Células , Tolerância Imunológica , Imunidade Inata , Imunomodulação , Interleucina-10/metabolismo , Interleucina-17/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vitaminas/metabolismoRESUMO
Regulation of Th17 and Th1 cell responses against intracellular pathogens, including Listeria monocytogenes (L. m), is critical to limit inflammation-induced tissue damage. We recently demonstrated the ability of P. UF1 bacterium derived from the intestinal bacterial commensals of preterm infants fed human breast milk to significantly mitigate pathogen-induced inflammation limiting colonic tissue damage. Here we further elucidated the potential of P. UF1 to also regulate innate and T cells, particularly Th17 and Th1 cells, against systemic L. m infection. Data demonstrate that P. UF1 not only robustly regulated protective Th17 and Th1 cells, but also sustained regulatory T cells (Treg cells) resulting in accelerated L. m clearance. Together, regulation of pathogenic inflammation by a novel probiotic bacterium such as P. UF1 may illuminate a new strategy to specifically control Th17-Th1 cells via IL-10+ Treg cells to limit systemic tissue damage induced by intracellular pathogens, including L. m.
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Antibiose , Listeria monocytogenes/imunologia , Listeriose/prevenção & controle , Probióticos/administração & dosagem , Propionibacterium/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Fezes/microbiologia , Microbioma Gastrointestinal/fisiologia , Listeriose/imunologia , Listeriose/microbiologia , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Propionibacterium/genética , Linfócitos T Reguladores/imunologia , Células Th1 , Células Th17RESUMO
Clostridium botulinum readily persists in the soil and secretes life-threatening botulinum neurotoxins (BoNTs) that are categorized into serotypes A to H, of which, serotype A (BoNT/A) is the most commonly occurring in nature. An efficacious vaccine with high longevity against BoNT intoxication is urgent. Herein, we developed a dual-route vaccine administered over four consecutive weeks by mucosal and parenteral routes, consisting of the heavy chain (Hc) of BoNT/A targeting dendritic cell peptide (DCpep) expressed by Lactobacillus acidophilus as a secretory immunogenic protein. The administered dual-route vaccine elicited robust and long-lasting memory B cell responses comprising germinal center (GC) B cells and follicular T cells (Tfh) that fully protected mice from lethal oral BoNT/A fatal intoxication. Additionally, passively transferring neutralizing antibodies against BoNT/A into naïve mice induced robust protection against BoNT/A lethal intoxication. Together, a targeted vaccine employing local and systemic administrative routes may represent a novel formulation eliciting protective B cell responses with remarkable longevity against threatening biologic agents such as BoNTs.
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Anticorpos Neutralizantes/imunologia , Vacinas Bacterianas/imunologia , Toxinas Botulínicas Tipo A/imunologia , Neurotoxinas/imunologia , Vacinação/métodos , Administração através da Mucosa , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/administração & dosagem , Linfócitos B/imunologia , Vacinas Bacterianas/administração & dosagem , Botulismo/prevenção & controle , Clostridium botulinum/imunologia , Células Dendríticas/química , Células Dendríticas/imunologia , Vias de Administração de Medicamentos , Imunização Passiva , Memória Imunológica , Lactobacillus acidophilus/química , Camundongos , Peptídeos/administração & dosagem , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , SorogrupoRESUMO
Noroviruses are the leading cause of food-borne gastroenteritis outbreaks and childhood diarrhoea globally, estimated to be responsible for 200,000 deaths in children each year 1-4 . Thus, reducing norovirus-associated disease is a critical priority. Development of vaccines and therapeutics has been hindered by the limited understanding of basic norovirus pathogenesis and cell tropism. While macrophages, dendritic cells, B cells and stem-cell-derived enteroids can all support infection of certain noroviruses in vitro 5-7 , efforts to define in vivo norovirus cell tropism have generated conflicting results. Some studies detected infected intestinal immune cells 8-12 , other studies detected epithelial cells 13 , and still others detected immune and epithelial cells 14-16 . Major limitations of these studies are that they were performed on tissue sections from immunocompromised or germ-free hosts, chronically infected hosts where the timing of infection was unknown, or following non-biologically relevant inoculation routes. Here, we report that the dominant cellular targets of a murine norovirus inoculated orally into immunocompetent mice are macrophages, dendritic cells, B cells and T cells in the gut-associated lymphoid tissue. Importantly, we also demonstrate that a norovirus can infect T cells, a previously unrecognized target, in vitro. These findings represent the most extensive analyses to date of in vivo norovirus cell tropism in orally inoculated, immunocompetent hosts at the peak of acute infection and thus they significantly advance our basic understanding of norovirus pathogenesis.
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Infecções por Caliciviridae/imunologia , Duodeno/imunologia , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/imunologia , Norovirus/imunologia , Norovirus/patogenicidade , Animais , Linfócitos B/imunologia , Infecções por Caliciviridae/patologia , Infecções por Caliciviridae/virologia , Linhagem Celular , Sobrevivência Celular , Células Dendríticas/imunologia , Modelos Animais de Doenças , Duodeno/patologia , Duodeno/virologia , Feminino , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Intestinos/patologia , Intestinos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Linfócitos T/virologiaRESUMO
Consumption of human breast milk (HBM) attenuates the incidence of necrotizing enterocolitis (NEC), which remains a leading and intractable cause of mortality in preterm infants. Here, we report that this diminution correlates with alterations in the gut microbiota, particularly enrichment of Propionibacterium species. Transfaunation of microbiota from HBM-fed preterm infants or a newly identified and cultured Propionibacterium strain, P. UF1, to germfree mice conferred protection against pathogen infection and correlated with profound increases in intestinal Th17 cells. The induction of Th17 cells was dependent on bacterial dihydrolipoamide acetyltransferase (DlaT), a major protein expressed on the P. UF1 surface layer (S-layer). Binding of P. UF1 to its cognate receptor, SIGNR1, on dendritic cells resulted in the regulation of intestinal phagocytes. Importantly, transfer of P. UF1 profoundly mitigated induced NEC-like injury in neonatal mice. Together, these results mechanistically elucidate the protective effects of HBM and P. UF1-induced immunoregulation, which safeguard against proinflammatory diseases, including NEC.
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Propionibacterium/imunologia , Células Th17/fisiologia , Animais , Proteínas de Bactérias/fisiologia , Diferenciação Celular , Colo/imunologia , Colo/microbiologia , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/fisiologia , Feminino , Microbioma Gastrointestinal , Genoma Bacteriano , Humanos , Imunomodulação , Recém-Nascido , Recém-Nascido Prematuro , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Propionibacterium/enzimologia , Propionibacterium/genética , Análise de Sequência de DNARESUMO
Interleukin (IL)-18 is an important effector of innate and adaptive immunity, but its expression must also be tightly regulated because it can potentiate lethal systemic inflammation and death. Healthy and septic human neonates demonstrate elevated serum concentrations of IL-18 compared with adults. Thus, we determined the contribution of IL-18 to lethality and its mechanism in a murine model of neonatal sepsis. We find that IL-18-null neonatal mice are highly protected from polymicrobial sepsis, whereas replenishing IL-18 increased lethality to sepsis or endotoxemia. Increased lethality depended on IL-1 receptor 1 (IL-1R1) signaling but not adaptive immunity. In genome-wide analyses of blood mRNA from septic human neonates, expression of the IL-17 receptor emerged as a critical regulatory node. Indeed, IL-18 administration in sepsis increased IL-17A production by murine intestinal γδT cells as well as Ly6G(+) myeloid cells, and blocking IL-17A reduced IL-18-potentiated mortality to both neonatal sepsis and endotoxemia. We conclude that IL-17A is a previously unrecognized effector of IL-18-mediated injury in neonatal sepsis and that disruption of the deleterious and tissue-destructive IL-18/IL-1/IL-17A axis represents a novel therapeutic approach to improve outcomes for human neonates with sepsis.
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Interleucina-17/antagonistas & inibidores , Interleucina-17/imunologia , Interleucina-18/imunologia , Sepse Neonatal/imunologia , Sepse Neonatal/terapia , Taxa de Sobrevida , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/uso terapêutico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Sepse Neonatal/patologia , Resultado do TratamentoRESUMO
Intestinal immunity is subject to complex and fine-tuned regulation dictated by interactions of the resident microbial community and their gene products with host innate cells. Deterioration of this delicate process may result in devastating autoinflammatory diseases, including inflammatory bowel disease (IBD), which primarily comprises Crohn's disease (CD) and ulcerative colitis (UC). Efficacious interventions to regulate proinflammatory signals, which play critical roles in IBD, require further scientific investigation. We recently demonstrated that rebalancing intestinal immunity via the surface layer protein A (SlpA) from Lactobacillus acidophilus NCFM potentially represents a feasible therapeutic approach to restore intestinal homeostasis. To expand on these findings, we established a new method of purifying bacterial SlpA, a new SlpA-specific monoclonal antibody, and found no SlpA-associated toxicity in mice. Thus, these data may assist in our efforts to determine the immune regulatory efficacy of SlpA in humans.
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Proteínas de Bactérias/uso terapêutico , Terapia Biológica , Enteropatias/terapia , Lactobacillus acidophilus , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/toxicidade , Microbioma Gastrointestinal , Homeostase , Intestinos/microbiologia , Lactobacillus acidophilus/química , Lactobacillus acidophilus/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência MolecularRESUMO
Ingestion of Bacillus anthracis results in rapid gastrointestinal (GI) infection, known as GI anthrax. We previously showed that during GI anthrax, there is swift deterioration of intestinal barrier function leading to translocation of gut-associated bacteria into systemic circulation. Additionally, we described dysfunction in colonic B cells. In concordance with our previous studies, here, we report early migration of the Sterne strain of B. anthracis along with other gut-resident bacteria into the infected murine liver. Additionally, despite a global decrease in the B cell population, we observed an increase in both B-1a and marginal zone (MZ)-like B cells. Both of these cell types are capable of producing immunoglobulins against common pathogens and commensals, which act as a general antibody barrier before an antigen-specific antibody response. Accumulation of these cells in the liver was associated with an increase in chemokine expression. These data suggest that the presence of Sterne and other commensals in the liver trigger migration of MZ-like B cells from the spleen to the liver to neutralize systemic spread. Further research is required to evaluate the possible cause of their failure to clear the infection within the liver, including the potential role of dysfunctional mitogen-activated protein kinase (MAPK) signaling.
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Antraz/microbiologia , Linfócitos B/microbiologia , Bacillus anthracis/metabolismo , Gastroenteropatias/microbiologia , Trato Gastrointestinal/microbiologia , Animais , Antraz/patologia , Linfócitos B/metabolismo , Colo/microbiologia , Modelos Animais de Doenças , Gastroenteropatias/patologia , Trato Gastrointestinal/patologia , Fígado/microbiologia , Camundongos , Baço/microbiologia , Esporos Bacterianos/metabolismoRESUMO
Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3(-/-) mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.
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Antígenos CD/imunologia , Proteínas de Bactérias/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Lactobacillus acidophilus/imunologia , Lectinas Tipo C/imunologia , Animais , Antígenos CD/genética , Proteínas de Bactérias/genética , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Lactobacillus acidophilus/genética , Lectinas Tipo C/genética , Lipopolissacarídeos/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Ligação Proteica/genética , Ligação Proteica/imunologia , Ácidos Teicoicos/genética , Ácidos Teicoicos/imunologiaRESUMO
We have identified a spliced mRNA transcript of CD20 (named D393-CD20) which was associated with resistance to RTX in primary B cell from patients with lymphoma and leukaemia. In the present work, we wished to investigate whether D393-CD20 variant was expressed by B cells from patients with pemphigus. Ten patients with bullous pemphigoid and twenty-five patients with pemphigus were included. All patients were responder to conventional immunosuppressive agents or rituximab (n = 11). Efficacy of B-cell activation by pokeweed mitogen was assessed by CD86 expression using a FACS Canto II flow cytometer. mRNA CD20 expression study was then performed using RT-PCR assay allowing first to discriminate wild-type (wt)-CD20 and D393-CD20 transcript. Although wt-CD20 expression was always detected, we were unable to detect D393-CD20, even after B-cell activation or RTX treatment. Our results suggest that D393-CD20 transcript may be a molecular marker of B-cell malignancies rather than autoimmune disease like pemphigus. Further study of RTX non-responders or non-escaping PV patients is thus still required to appreciate whether D393-CD20 expression may be detected under the pressure of RTX therapy.
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Antígenos CD20/genética , Linfócitos B/imunologia , Pênfigo/genética , Pênfigo/imunologia , Idoso , Processamento Alternativo , Anticorpos Monoclonais Murinos/uso terapêutico , Feminino , Marcadores Genéticos , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Pênfigo/terapia , RNA Mensageiro/genética , RituximabRESUMO
Pemphigus is a severe blistering condition of the skin and mucosa caused by autoantibodies directed against desmogleins, which are a type of keratinocyte adhesion protein. B cell depletion by rituximab has short-term efficacy against pemphigus. We aimed to assess the long-term course of pemphigus patients after B cell depletion and to understand the immunological mechanisms that mediate long-lasting remissions. We evaluated the clinical course of 22 pemphigus patients treated with rituximab after a 79-month median follow-up and compared the anti-desmoglein B cell response and B and T lymphocyte subpopulations and repertoire between patients who achieved complete remission (CR) and those who had incomplete remission (IR). Thirteen patients (59%) experienced CR during the study, including 10 patients off treatment and 3 patients with prednisone doses <10 mg/day; 9 patients had IR. A marked increase was observed in the ratio of CD19(+)CD27(-) naïve B cells to CD19(+)CD27(+) memory B cells. Indeed, patients in CR had a fourfold higher number of transitional B cells and interleukin-10-secreting regulatory B cells than those in IR. Furthermore, CR was associated with modification of the initial B cell repertoire and the disappearance of desmoglein-specific circulating immunoglobulin G-positive (IgG(+)) B lymphocytes, whereas a skewed B cell repertoire was observed in patients in IR. Thus, a blockage of B cell maturation, a prolonged repopulation with naïve B cells, and a delayed reappearance of memory B cells, which resulted in the disappearance of circulating desmoglein-specific IgG(+) B lymphocytes, contribute to the long-lasting effectiveness of rituximab for treating pemphigus.
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Anticorpos Monoclonais Murinos/uso terapêutico , Linfócitos B/imunologia , Desmogleínas/imunologia , Pênfigo/tratamento farmacológico , Humanos , Imunofenotipagem , Pênfigo/imunologia , Pênfigo/fisiopatologia , RituximabRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathogenic autoantibodies directed against nuclear Ags and immune complex deposits in damaged organs. Environmental factors have been thought to play a role in the onset of the disease. The recognition of these factors is mediated by TLRs, in particular TLR2 and TLR4 which bind pathogen-associated molecular patterns of Gram(+) and Gram(-) bacteria, respectively. We attempted to determine the role of these TLRs in SLE by creating TLR2- or TLR4-deficient C57BL/6(lpr/lpr) mice. These mice developed a less severe disease and fewer immunological alterations. Indeed, in C57BL/6(lpr/lpr)-TLR2 or -TLR4-deficient mice, glomerular IgG deposits and mesangial cell proliferation were dramatically decreased and antinuclear, anti-dsDNA, and anti-cardiolipin autoantibody titers were significantly reduced. However, the response against nucleosome remained unaffected, indicating a role of TLR2 and TLR4 in the production of Abs directed against only certain categories of SLE-related autoantigens. Analysis of B cell phenotype showed a significant reduction of marginal zone B cells, particularly in C57BL/6(lpr/lpr)-TLR4-deficient mice, suggesting an important role of TLR4 in the sustained activation of these cells likely involved in autoantibody production. Interestingly, the lack of TLR4 also affected the production of cytokines involved in the development of lupus disease.