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1.
Front Immunol ; 12: 647987, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34248935

RESUMO

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.


Assuntos
Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Chaperonina 60/administração & dosagem , Chaperonina 60/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Lactococcus lactis/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Mycobacterium leprae/enzimologia , Administração Oral , Animais , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Feminino , Inflamação/tratamento farmacológico , Inflamação/imunologia , Lactococcus lactis/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia
2.
Chem Biol Interact ; 345: 109537, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34062171

RESUMO

The Breast Cancer Resistance Protein (BCRP/ABCG2) is an ATP-binding cassette efflux transporter that is expressed in the apical membrane of cells from relevant tissues involved in drug pharmacokinetics such as liver, intestine, kidney, testis, brain and mammary gland, among others. Tolfenamic acid is an anti-inflammatory drug used as an analgesic and antipyretic in humans and animals. Recently, tolfenamic acid has been repurposed as an antitumoral drug and for use in chronic human diseases such as Alzheimer. The aim of this work was to study whether tolfenamic acid is an in vitro Abcg2 substrate, and to investigate the potential role of Abcg2 in plasma exposure, secretion into milk and tissue accumulation of this drug. Using in vitro transepithelial assays with cells transduced with Abcg2, we showed that tolfenamic acid is an in vitro substrate of Abcg2. The in vivo effect of this transporter was tested using wild-type and Abcg2-/- mice, showing that after oral and intravenous administration of tolfenamic acid, its area under the plasma concentration-time curve in Abcg2-/- mice was between 1.7 and 1.8-fold higher compared to wild-type mice. Abcg2-/- mice also showed higher liver and testis accumulation of tolfenamic acid after intravenous administration. In this study, we demonstrate that tolfenamic acid is transported in vitro by Abcg2 and that its plasma levels as well as its tissue distribution are affected by Abcg2, with potential pharmacological and toxicological consequences.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Vacinas Bacterianas/sangue , Vacinas Bacterianas/farmacocinética , ortoaminobenzoatos/sangue , ortoaminobenzoatos/farmacocinética , Animais , Vacinas Bacterianas/farmacologia , Transporte Biológico , Camundongos , Distribuição Tecidual , ortoaminobenzoatos/farmacologia
3.
mSphere ; 6(3)2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33952660

RESUMO

Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro Here, we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis, M. marinum, or M. leprae In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo, translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals.IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis.


Assuntos
Citosol/microbiologia , Mycobacterium/imunologia , Mycobacterium/patogenicidade , Fagossomos/microbiologia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Transdução de Sinais/imunologia , Animais , Tatus/microbiologia , Translocação Bacteriana , Citosol/imunologia , Feminino , Humanos , Hanseníase/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Mycobacterium/classificação , Fagossomos/imunologia , Pele/microbiologia , Pele/patologia , Células THP-1 , Peixe-Zebra
4.
Cell Host Microbe ; 29(5): 740-741, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33984275

RESUMO

In a recent Science paper, Jain et al. (2021) discover that a fungus contributes to delayed wound healing in mice and is enriched in inflamed tissue from Crohn's disease patients. This culprit is not a well-known pathogen, but cheese yeast Debaryomyces hansenii, highlighting the importance of further studying fungi-host interactions.


Assuntos
Queijo , Doença de Crohn , Debaryomyces , Animais , Fungos , Humanos , Camundongos , Cicatrização
5.
Immunity ; 54(5): 856-858, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33979584

RESUMO

Intestinal microbiome perturbation characterizes Crohn's disease (CD), though specific contributors to pathophysiology remain elusive. In a recent issue of Science, Jain et al. show that Debaryomyces hansenii impairs intestinal healing in mice via effects on type I interferon signaling and chemokine CCL5 expression in macrophages and that it is also prevalent in the inflamed mucosa of CD patients.


Assuntos
Doença de Crohn/imunologia , Doença de Crohn/microbiologia , Mucosa Intestinal/microbiologia , Cicatrização/imunologia , Animais , Quimiocina CCL5/imunologia , Microbioma Gastrointestinal/imunologia , Humanos , Interferon Tipo I/imunologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Camundongos , Micoses/imunologia , Micoses/microbiologia , Saccharomycetales/imunologia , Transdução de Sinais/imunologia
6.
Mol Med Rep ; 23(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33880583

RESUMO

Humulus japonicus (HJ) is a traditional herbal medicine that exhibits anti­inflammatory, antimicrobial and anti­tumor effects that is used for the treatment of hypertension, pulmonary disease and leprosy. Recently, it has also been reported that HJ demonstrates neuroprotective properties in animal models of neurodegenerative diseases. The current study hypothesised that the administration of HJ would exhibit therapeutic effects in autism spectrum disorder (ASD), a neurodevelopmental disorder with lifelong consequences. The BTBR T+ Itpr3tf/J mouse model of ASD was used to investigate the anti­autistic like behavioural effects of HJ. Chronic oral administration of the ethanolic extract of HJ significantly increased social interaction, attenuated repetitive grooming behaviour and improved novel­object recognition in BTBR mice. Anti­inflammatory effects of HJ in the brain were analysed using immunohistochemistry and reverse­transcription quantitative PCR analysis. Microglia activation was markedly decreased in the striatum and hippocampus, and pro­inflammatory cytokines, including C­C Motif Chemokine Ligand 2, interleukin (IL)­1ß and IL­6, were significantly reduced in the hippocampus following HJ treatment. Moreover, HJ treatment normalised the phosphorylation levels of: N­methyl­D­aspartate receptor subtype 2B and calcium/calmodulin­dependent protein kinase type II subunit α in the hippocampus of BTBR mice. The results of the present study demonstrated that the administration of HJ may have beneficial potential for ameliorating behavioural deficits and neuroinflammation in ASD.


Assuntos
Transtorno Autístico/tratamento farmacológico , Humulus/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno Autístico/genética , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Hipocampo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fosforilação/efeitos dos fármacos
8.
Immunol Rev ; 301(1): 157-174, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33660297

RESUMO

Leprosy is a chronic granulomatous infectious disease caused by the pathogen, Mycobacterium leprae, and the more recently discovered, M. lepromatosis. Described in 1873, M. leprae was among the first microorganisms to be proposed as a cause of a human infectious disease. As an obligate intracellular bacterium, it has still not thus far been reproducibly cultivated in axenic medium or cell cultures. Shepard's mouse footpad assay, therefore, was truly a breakthrough in leprosy research. The generation of immunosuppressed and genetically engineered mice, along with advances in molecular and cellular techniques, has since offered more tools for the study of the M. leprae-induced granuloma. While far from perfect, these new mouse models have provided insights into the immunoregulatory mechanisms responsible for the spectrum of this complex disease.


Assuntos
Hanseníase , Animais , Modelos Animais de Doenças , Camundongos , Mycobacterium leprae , Pele
9.
Science ; 371(6534): 1154-1159, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33707263

RESUMO

Alterations of the mycobiota composition associated with Crohn's disease (CD) are challenging to link to defining elements of pathophysiology, such as poor injury repair. Using culture-dependent and -independent methods, we discovered that Debaryomyces hansenii preferentially localized to and was abundant within incompletely healed intestinal wounds of mice and inflamed mucosal tissues of CD human subjects. D. hansenii cultures from injured mice and inflamed CD tissues impaired colonic healing when introduced into injured conventionally raised or gnotobiotic mice. We reisolated D. hansenii from injured areas of these mice, fulfilling Koch's postulates. Mechanistically, D. hansenii impaired mucosal healing through the myeloid cell-specific type 1 interferon-CCL5 axis. Taken together, we have identified a fungus that inhabits inflamed CD tissue and can lead to dysregulated mucosal healing.


Assuntos
Doença de Crohn/microbiologia , Doença de Crohn/patologia , Debaryomyces/isolamento & purificação , Debaryomyces/fisiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Anfotericina B/farmacologia , Animais , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Quimiocina CCL5/metabolismo , Colo/microbiologia , Colo/patologia , Doença de Crohn/imunologia , Debaryomyces/crescimento & desenvolvimento , Feminino , Microbioma Gastrointestinal , Vida Livre de Germes , Humanos , Íleo/microbiologia , Íleo/patologia , Inflamação , Interferon Tipo I/metabolismo , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
10.
Science ; 371(6534): 1102-1103, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33707253
11.
Nature ; 591(7850): 438-444, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33627868

RESUMO

Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and early restricted progenitors1-6. LEPR+ cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors7-12, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin13,14, distinguishes peri-arteriolar LEPR+ cells poised to undergo osteogenesis from peri-sinusoidal LEPR+ cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR+osteolectin+ cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin+ cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin+ cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin+ cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin+ cells depleted osteolectin+ cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing.


Assuntos
Arteríolas , Linfopoese , Osteogênese , Nicho de Células-Tronco , Tecido Adiposo/citologia , Envelhecimento , Animais , Células da Medula Óssea/citologia , Osso e Ossos/citologia , Feminino , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Lectinas Tipo C/metabolismo , Linfócitos/citologia , Masculino , Camundongos , Receptores para Leptina/metabolismo , Fator de Células-Tronco , Células Estromais/citologia
12.
J Leukoc Biol ; 110(4): 693-710, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33404106

RESUMO

The inflammatory and anti-inflammatory Mϕs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting Mϕ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory Mϕ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated Mϕs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human Mϕ subsets are resistant to the cytotoxic effects of SMs, we show that M1 Mϕs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 Mϕs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 Mϕs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human Mϕs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.


Assuntos
Apoptose , Inibidores de Caspase/farmacologia , Polaridade Celular , Macrófagos/citologia , Oligopeptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Janus Quinases/metabolismo , Cinética , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Necroptose/efeitos dos fármacos , Fenótipo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Bauru; s.n; 2021. 38 p. ilus, graf.
Tese em Português | SES-SP, CONASS, HANSEN, SESSP-ILSLPROD, SES-SP, SESSP-ILSLACERVO, SES-SP, SESSP-ESPECIALIZACAOSESPROD, SES-SP | ID: biblio-1179170

RESUMO

Na hanseníase, o modelo de Shepard de inoculação por Mycobacterium leprae (M. leprae) em patas de camundongos vem sendo utilizado em diversos estudos sobre a resposta imune, avaliação de novas drogas e esquemas terapêuticos, além da dinâmica da infecção precoce e crônica. Para estudar o papel do microambiente granulomatoso na hanseníase, além de modelos animais convencionais ou imunocomprometidos, o desenvolvimento de modelos murinos de granuloma não infeccioso pode adicionar parâmetros patogênicos a serem comparados no desenvolvimento da doença. Preparações de nitrocelulose estão entre as formas de desenvolvimento de granulomas não imunogênicos em experimentação animal. O presente estudo investigou a formação de granulomas não infecciosos induzidos por partículas de nitrocelulose, em comparação a lesões induzidas por M. leprae. Grupos de camundongos nude e BALB/c, foram constituídos e inoculados, conforme a técnica de Shepard, com suspensão de M. leprae (ML), suspensão de nitrocelulose (NT), associação de M. leprae com nitrocelulose (ML/NT) e veículo controle (CTRL). Após 07, 14, 21 e 28 dias, amostras foram coletadas e analisadas histopatologicamente pelas colorações, HematoxilinaEosina e Fite-Faraco. Os grupos experimentais demonstraram a formação de granulomas em ambas os fenótipos murinos. Principalmente nos grupos NT e ML/NT, as lesões foram caracterizadas por infiltrado inflamatório mononuclear, predominantemente macrofágico, com presença de células epitelioides, eventuais macrófagos vacuolizados e ausência de células gigantes multinucleadas. As lesões induzidas exclusivamente por M. leprae pareceram menos exuberantes que àquelas observadas nos demais grupos, indicando que a nitrocelulose intensificou a resposta macrofágica nos espécimes avaliados e sugerindo que esse composto pode ser utilizado não só para o desenvolvimento de granulomas não imunogênicos, mas também na exacerbação da resposta imune em granulomas induzidos por agentes infecciosos, como M. leprae.


In leprosy, the Shepard model of inoculation by Mycobacterium leprae (M. leprae) in mouse footpad has been used in several studies on the immune response, evaluation of new drugs and therapeutic schemes, in addition to the dynamics of early and chronic infection. To study the role of the granulomatous microenvironment in leprosy, in addition to conventional or immunocompromised animal models, the development of murine models of non-infectious granuloma can add pathogenic parameters to be compared in the development of the disease. Nitrocellulose preparations are among the ways of developing non-immunogenic granulomas in animal experimentation. The present study investigated the formation of non-infectious granulomas induced by nitrocellulose particles, in comparison to lesions induced by M. leprae. Groups of athymic nude and BALB/c mice were set up and inoculated, according to Shepard technique, with M. leprae suspension (ML), nitrocellulose suspension (NT), M. leprae association with nitrocellulose (ML/NT) and control vehicle (CTRL). After 07, 14, 21 and 28 days, samples were collected and histopathologically analyzed by Hematoxylin-Eosin and FiteFaraco staining. The experimental groups demonstrated the formation of granulomas in both murine strains. Mainly in the NT and ML/NT groups, the lesions were characterized by mononuclear inflammatory infiltrate, predominantly macrophagic, with the presence of epithelioid cells, eventual vacuolated macrophages and absence of multinucleated giant cells. The lesions induced exclusively by M. leprae seemed less exuberant than those observed in the other groups, indicating that nitrocellulose intensified the macrophage response in the specimens evaluated, and suggesting that this compound can be used not only in the development of non-immunogenic granulomas, but also in exacerbation of the immune response in granulomas induced by infectious agents, such as M. leprae.


Assuntos
Camundongos , Granuloma/induzido quimicamente , Hanseníase/microbiologia , Mycobacterium leprae/crescimento & desenvolvimento , Camundongos Endogâmicos BALB C
14.
Bull Exp Biol Med ; 169(6): 836-839, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33098510

RESUMO

Leprosy was modeled in an experiment on BALB/c, BALB/cNude, CBA, and C57BL/6ТNF-/- mice using three Mycobacterium leprae strains obtained from patients with a diagnosis of A30 according to ICD-10 from different regions of the Russian Federation. Proliferation of M. leprae of the used strains showed a temporal-quantitative dependence on the used mouse line. CBA and BALB/cNude mice were optimal for strain R and BALB/c and BALB/cNude lines were optimal for strain I. BALB/cNude mice infected with strain I had low lifespan. M. leprae strain M showed low proliferation activity in BALB/cNude and C57BL/6ТNF-/- mice.


Assuntos
Imunidade Adaptativa , Imunidade Inata , Hanseníase/imunologia , Longevidade/imunologia , Mycobacterium leprae/patogenicidade , Fator de Necrose Tumoral alfa/imunologia , Animais , DNA Bacteriano/genética , Modelos Animais de Doenças , Especificidade de Hospedeiro , Humanos , Hanseníase/genética , Hanseníase/microbiologia , Hanseníase/patologia , Longevidade/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Nus , Mycobacterium leprae/genética , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/imunologia , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética
15.
PLoS Negl Trop Dis ; 14(10): e0008850, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33075048

RESUMO

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae). In lepromatous leprosy (LL), skin macrophages, harboring extensive bacterial multiplication, gain a distinctive foamy appearance due to increased intracellular lipid load. To determine the mechanism by which M. leprae modifies the lipid homeostasis in host cells, an in vitro M. leprae infection system, using human macrophage precursor THP-1 cells and M. leprae prepared from the footpads of nude mice, was employed. RNA extracted from skin smear samples of patients was used to investigate host gene expressions before and after multidrug therapy (MDT). We found that a cluster of peroxisome proliferator-activated receptor (PPAR) target genes associated with adipocyte differentiation were strongly induced in M. leprae-infected THP-1 cells, with increased intracellular lipid accumulation. PPAR-δ and PPAR-γ expressions were induced by M. leprae infection in a bacterial load-dependent manner, and their proteins underwent nuclear translocalization after infection, indicating activation of PPAR signaling in host cells. Either PPAR-δ or PPAR-γ antagonist abolished the effect of M. leprae to modify host gene expressions and inhibited intracellular lipid accumulation in host cells. M. leprae-specific gene expressions were detected in the skin smear samples both before and after MDT, whereas PPAR target gene expressions were dramatically diminished after MDT. These results suggest that M. leprae infection activates host PPAR signaling to induce an array of adipocyte differentiation-associated genes, leading to accumulation of intracellular lipids to accommodate M. leprae parasitization. Certain PPAR target genes in skin lesions may serve as biomarkers for monitoring treatment efficacy.


Assuntos
Células Espumosas/microbiologia , Hanseníase/metabolismo , Macrófagos/microbiologia , Mycobacterium leprae/fisiologia , PPAR delta/metabolismo , PPAR gama/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adipócitos/microbiologia , Animais , Diferenciação Celular , Células Espumosas/metabolismo , Humanos , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Hanseníase/genética , Hanseníase/microbiologia , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Camundongos , Camundongos Nus , Mycobacterium leprae/efeitos dos fármacos , PPAR delta/genética , PPAR gama/genética , Pele/metabolismo , Pele/microbiologia
16.
Immunity ; 53(4): 878-894.e7, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33053333

RESUMO

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inflamação/genética , RNA Citoplasmático Pequeno/genética , Pele/patologia , Animais , Linhagem Celular , DNA Complementar/genética , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcrição Genética/genética , Transcriptoma/genética
17.
PLoS Negl Trop Dis ; 14(9): e0008583, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936818

RESUMO

BACKGROUND: Subclinical infection with Mycobacterium leprae is one potential source of leprosy transmission, and post-exposure prophylaxis (PEP) regimens have been proposed to control this source. Because PEP trials require considerable investment, we applied a sensitive variation of the kinetic mouse footpad (MFP) screening assay to aid in the choice of drugs and regimens for clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: Athymic nude mice were inoculated in the footpad (FP) with 6 x 103 viable M. leprae and treated by gastric gavage with a single dose of Rifampin (SDR), Rifampin + Ofloxacin + Minocycline (SD-ROM), or Rifapentine + Minocycline + Moxifloxacin (SD-PMM) or with the proposed PEP++ regimen of three once-monthly doses of Rifampin + Moxifloxacin (RM), Rifampin + Clarithromycin (RC), Rifapentine + Moxifloxacin (PM), or Rifapentine + Clarithromycin (PC). At various times post-treatment, DNA was purified from the FP, and M. leprae were enumerated by RLEP quantitative PCR. A regression analysis was calculated to determine the expected RLEP value if 99.9% of the bacilli were killed after the administration of each regimen. SDR and SD-ROM induced little growth delay in this highly susceptible murine model of subclinical infection. In contrast, SD-PMM delayed measurable M. leprae growth above the inoculum by 8 months. The four multi-dose regimens delayed bacterial growth for >9months post-treatment cessation. CONCLUSIONS/SIGNIFICANCE: The delay in discernable M. leprae growth post-treatment was an excellent indicator of drug efficacy for both early (3-4 months) and late (8-9 months) drug efficacy. Our data indicates that multi-dose PEP may be required to control infection in highly susceptible individuals with subclinical leprosy to prevent disease and decrease transmission.


Assuntos
Infecções Assintomáticas/terapia , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Mycobacterium leprae/efeitos dos fármacos , Profilaxia Pós-Exposição/métodos , Animais , Carga Bacteriana/efeitos dos fármacos , Claritromicina/uso terapêutico , Combinação de Medicamentos , Hanseníase/transmissão , Camundongos , Camundongos Nus , Minociclina/uso terapêutico , Moxifloxacina/uso terapêutico , Mycobacterium leprae/crescimento & desenvolvimento , Rifampina/análogos & derivados , Rifampina/uso terapêutico
18.
Elife ; 92020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32955439

RESUMO

Acid-base conditions modify artery tone and tissue perfusion but the involved vascular-sensing mechanisms and disease consequences remain unclear. We experimentally investigated transgenic mice and performed genetic studies in a UK-based human cohort. We show that endothelial cells express the putative HCO3--sensor receptor-type tyrosine-protein phosphatase RPTPγ, which enhances endothelial intracellular Ca2+-responses in resistance arteries and facilitates endothelium-dependent vasorelaxation only when CO2/HCO3- is present. Consistent with waning RPTPγ-dependent vasorelaxation at low [HCO3-], RPTPγ limits increases in cerebral perfusion during neuronal activity and augments decreases in cerebral perfusion during hyperventilation. RPTPγ does not influence resting blood pressure but amplifies hyperventilation-induced blood pressure elevations. Loss-of-function variants in PTPRG, encoding RPTPγ, are associated with increased risk of cerebral infarction, heart attack, and reduced cardiac ejection fraction. We conclude that PTPRG is an ischemia susceptibility locus; and RPTPγ-dependent sensing of HCO3- adjusts endothelium-mediated vasorelaxation, microvascular perfusion, and blood pressure during acid-base disturbances and altered tissue metabolism.


Assuntos
Isquemia/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Animais , Bicarbonatos/metabolismo , Bancos de Espécimes Biológicos , Células Endoteliais/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Reino Unido , Vasodilatação/genética
19.
Int J Mycobacteriol ; 9(3): 268-273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32862159

RESUMO

Background: Murine leprosy is a chronic granulomatous disease caused by Mycobacterium lepraemurium (MLM) in mice and rats. The disease evolves with the development of cellular anergy that impedes the production of interferon gamma (IFNγ), tumor necrosis factor-alpha (TNFα), and nitric oxide (NO) required to kill the microorganism. In this study we investigated whether histone deacetylase inhibitors (HDACi) (valproic acid and sodium butyrate [NaB]) and the immunomodulator transfer factor in dialyzable leukocyte extracts (DLE) can prevent anergy in murine leprosy. Methods: Five groups of six Balb/c mice were intraperitoneally inoculated with 2 × 107 MLM. Thirty-days post inoculation, treatment was started; one group received no treatment, one was treated with rifampicin-clofazimine (R-C), one with sodium valproate (VPA), one with NaB, and one with DLE. The animals were monitored for the evidence of disease for 96 days. After euthanasia, their spleens were removed and processed for histologic, bacteriologic, and cytokine studies. Results: R-C completely controlled the ongoing disease. DLE and NaB significantly reduced the development of lesions, including granuloma size and the number of bacilli; VPA was less effective. DLE, NaB, and VPA reverted the anergic condition in diverse grades and allowed the expression of IFNγ, TNFα, and inducible NO synthase, also in diverse grades. Conclusion: Anergy in leprosy and murine leprosy allows disease progression. In this study, anergy was prevented, in significant degree, by DLE (an immunomodulator) and NaB (HDACi). VPA was less effective. These results suggest potential beneficial effects of DLE and NaB in the ancillary treatment of leprosy.


Assuntos
Ácido Butírico/administração & dosagem , Extratos Celulares/farmacologia , Anergia Clonal/imunologia , Inibidores de Histona Desacetilases/administração & dosagem , Hanseníase/imunologia , Ácido Valproico/administração & dosagem , Animais , Extratos Celulares/imunologia , Diálise , Feminino , Leucócitos/química , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium lepraemurium/efeitos dos fármacos , Mycobacterium lepraemurium/imunologia
20.
PLoS Negl Trop Dis ; 14(7): e0007871, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628669

RESUMO

Leprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogen's obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.


Assuntos
Genoma Bacteriano , Hanseníase/microbiologia , Redes e Vias Metabólicas , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Animais , Humanos , Camundongos , Camundongos Nus , Mycobacterium leprae/crescimento & desenvolvimento
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