RESUMO
BACKGROUND: The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa. METHODOLOGY/PRINCIPAL FINDINGS: We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities. CONCLUSIONS/SIGNIFICANCE: This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer.
Assuntos
Amoeba/microbiologia , Úlcera de Buruli/epidemiologia , Úlcera de Buruli/microbiologia , Reservatórios de Doenças , Mycobacterium/isolamento & purificação , Úlcera de Buruli/transmissão , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Microbiologia Ambiental , Gana , Humanos , Viabilidade Microbiana , Dados de Sequência Molecular , Fagocitose , Análise de Sequência de DNARESUMO
BACKGROUND: Buruli ulcer (BU) is an emerging infectious disease caused by Mycobacterium ulcerans that can result in extensive necrotizing cutaneous lesions due to the cytotoxic exotoxin mycolactone. There is no specific vaccine against BU but reports show some degree of cross-reactive protection conferred by M. bovis BCG immunization. Alternatively, an M. ulcerans-specific immunization could be a better preventive strategy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used the mouse model to characterize the histological and cytokine profiles triggered by vaccination with either BCG or mycolactone-negative M. ulcerans, followed by footpad infection with virulent M. ulcerans. We observed that BCG vaccination significantly delayed the onset of M. ulcerans growth and footpad swelling through the induction of an earlier and sustained IFN-γ T cell response in the draining lymph node (DLN). BCG vaccination also resulted in cell-mediated immunity (CMI) in M. ulcerans-infected footpads, given the predominance of a chronic mononuclear infiltrate positive for iNOS, as well as increased and sustained levels of IFN-γ and TNF. No significant IL-4, IL-17 or IL-10 responses were detected in the footpad or the DLN, in either infected or vaccinated mice. Despite this protective Th1 response, BCG vaccination did not avoid the later progression of M. ulcerans infection, regardless of challenge dose. Immunization with mycolactone-deficient M. ulcerans also significantly delayed the progression of footpad infection, swelling and ulceration, but ultimately M. ulcerans pathogenic mechanisms prevailed. CONCLUSIONS/SIGNIFICANCE: The delay in the emergence of pathology observed in vaccinated mice emphasizes the relevance of protective Th1 recall responses against M. ulcerans. In future studies it will be important to determine how the transient CMI induced by vaccination is compromised.
Assuntos
Vacina BCG/administração & dosagem , Úlcera de Buruli/imunologia , Úlcera de Buruli/prevenção & controle , Imunidade Celular , Mycobacterium ulcerans/imunologia , Animais , Úlcera de Buruli/patologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Feminino , Macrolídeos , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/metabolismo , Células Th1/imunologiaRESUMO
Macrophages and neutrophils possess overlapping and complementary features associated to their common origin and subsequent specialization during myelopoiesis. That specialization results in macrophage lineage being limited in antimicrobial capacity and cytotoxicity comparatively with the neutrophil lineage. These and other features of mature macrophages and neutrophils, like different lifespan and tissue localization, promote their particular lifestyles and prompt a functional partnership for cooperation in the protective antimicrobial host defense. This partnership includes reciprocal recruitment to infected sites, cooperative effector antimicrobial activities, and pro-resolving anti-inflammatory effects. One modality of the cooperative effector antimicrobial activities involves the phagocytosis by the macrophage of apoptosing neutrophils and of nonapoptosing neutrophils expressing "eat-me" signals. This cooperative interaction results in the enhancement of the comparatively limited macrophage antimicrobial capacity by the acquisition and use of potent neutrophil microbicidal molecules. Here, data are reviewed that suggest that this is a process actively engaging the two professional phagocytes. Phagocytosis of neutrophils by macrophages at inflammatory/infectious foci accumulates two effects beneficial to the protective host immune response: help in the control of the infection and prevention of neutrophil autolysis, effects that converge to accelerate the resolution of the infection-associated inflammation.
Assuntos
Infecções/imunologia , Inflamação/prevenção & controle , Macrófagos/imunologia , Neutrófilos/imunologia , Fagocitose , Animais , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Macrófagos/microbiologia , Neutrófilos/microbiologia , Transdução de SinaisRESUMO
Emerging data suggest new facets of the concerted participation of neutrophils and macrophages in antimicrobial immunity. The classical view is that DCs and macrophages are the inducers of adaptive antimicrobial immunity, but there is evidence for neutrophil participation in this task as cytokine and chemokine producers and APCs. On the other hand, the concept that the T(H)1 response is only associated with control of infections by intracellular pathogens through activation of macrophages by IFN-gamma, and the T(H)17/IL-17 axis is only involved in protection against extracellular pathogens through mobilization and activation of neutrophils is simplistic: There is evidence suggesting that T(H)1 and T(H)17 responses, separately or in parallel, may use macrophages and neutrophils against infections by extracellular and intracellular microbial pathogens. Opsonization by pathogen-specific Igs enhances the antimicrobial capabilities of neutrophils and macrophages in infections by extracellular and intracellular microbes. The functional partnership between macrophages and neutrophils as inducers and effectors of adaptive antimicrobial immunity conforms to their affiliation with the myeloid phagocyte system and reveals a strategy based on the concurrent use of the two professional phagocytes in the adaptive defense mechanisms. Starting from a common myeloid precursor in the bone marrow, macrophages and neutrophils split during differentiation but come together at the infectious foci for a cooperative strategy that uses modulator and effector activities to attack invading microbial pathogens.
Assuntos
Imunidade Adaptativa/imunologia , Infecções/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Animais , Diferenciação Celular/imunologia , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismoRESUMO
The antimicrobial effector activity of phagocytes is crucial in the host innate defense against infection, and the classic view is that the phagocytes operating against intracellular and extracellular microbial pathogens are,respectively, macrophages and neutrophils. As a result of the common origin of the two phagocytes, they share several functionalities, including avid phagocytosis,similar kinetic behavior under inflammatory/infectious conditions, and antimicrobial and immunomodulatory activities. However, consequent to specialization during their differentiation, macrophages and neutrophils acquire distinctive, complementary features that originate different levels of antimicrobial capacities and cytotoxicity and different tissue localization and lifespan.This review highlights data suggesting the perspective that the combination of overlapping and complementary characteristics of the two professional phagocytes promotes their cooperative participation as effectors and modulators in innate immunity against infection and as orchestrators of adaptive immunity. In the concerted activities operating in antimicrobial innate immunity, macrophages and neutrophils are not able to replace each other. The common and complementary developmental,kinetic, and functional properties of neutrophils and macrophages make them the effector arms of a myeloid phagocyte system that groups neutrophils with members of the old mononuclear phagocyte system. The use by mammals of a system with two dedicated phagocytic cells working cooperatively represents an advantageous innate immune attack strategy that allows the efficient and safe use of powerful but dangerous microbicidal molecules.This crucial strategy is a target of key virulence mechanisms of successful pathogens.
Assuntos
Imunidade Inata/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Fagocitose , Animais , Infecções Bacterianas/imunologia , Comunicação Celular , Linhagem da Célula , Movimento Celular , Humanos , Inflamação , Macrófagos/parasitologia , Mamíferos/imunologia , Células Mieloides/citologia , Neutrófilos/microbiologia , Doenças Parasitárias/imunologiaRESUMO
Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative pathogen agent of an important fish septicemia. The key virulence factor of Phdp is the plasmid-encoded exotoxin AIP56, which is secreted by exponentially growing pathogenic strains. AIP56 has 520 amino acids including an N-terminal cleavable signal peptide of 23 amino acid residues, two cysteine residues and a zinc-binding region signature HEXXH that is typical of most zinc metallopeptidases. AIP56 induces in vitro and in vivo selective apoptosis of fish macrophages and neutrophils through a caspase-3 dependent mechanism that also involves caspase-8 and -9. In vivo, the AIP56-induced phagocyte apoptosis progresses to secondary necrosis with release of cytotoxic phagocyte molecules including neutrophil elastase. Fish injected with recombinant AIP56 die with a pathology similar to that seen in the natural infection.
Assuntos
Apoptose/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Photobacterium/patogenicidade , Animais , Humanos , Macrófagos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Fatores de Virulência/toxicidadeRESUMO
BACKGROUND: Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin. METHODOLOGY/PRINCIPAL FINDINGS: One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5' end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3' end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone. CONCLUSION: Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.
Assuntos
Microbiologia Ambiental , Mycobacterium ulcerans/fisiologia , Animais , Toxinas Bacterianas/metabolismo , Células Cultivadas , Feminino , Pé/microbiologia , Genótipo , Hemípteros/microbiologia , Macrolídeos , Macrófagos/microbiologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium ulcerans/classificação , Mycobacterium ulcerans/genética , Mycobacterium ulcerans/isolamento & purificação , Mycobacterium ulcerans/metabolismo , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genéticaRESUMO
In the search for pro-inflammatory genes in sea bass a TNF-alpha gene was cloned and sequenced. The sea bass TNF-alpha (sbTNF-alpha) putative protein conserves the TNF-alpha family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-alpha Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-alpha exists as two forms: a approximately 28 kDa membrane-bound form and a approximately 18.4 kDa soluble protein. The single copy sbTNF-alpha gene contains a four exon-three intron structure similar to other known TNF-alpha genes. Homology modeling of sbTNF-alpha is compatible with the trimeric quaternary architecture of its mammalian counterparts. SbTNF-alpha is constitutively expressed in several unstimulated tissues, and was not up-regulated in the spleen and head-kidney, in response to UV-killed Photobacterium damselae subsp. piscicida. However, an increase of sbTNF-alpha expression was detected in the head-kidney during an experimental infection using the same pathogen.
Assuntos
Bass/genética , Bass/metabolismo , Regulação da Expressão Gênica , Fator de Necrose Tumoral alfa/genética , Animais , Bass/microbiologia , Clonagem Molecular , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/veterinária , Interleucina-1beta/genética , Modelos Moleculares , Dados de Sequência Molecular , Photobacterium/fisiologia , Filogenia , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Fator de Necrose Tumoral alfa/químicaRESUMO
Bacterial modulation of phagocyte cell death is an emerging theme in pathogenesis. Here we describe the systemic destruction of macrophages and neutrophils by the Gram-negative Photobacterium damselae ssp. piscicida (Phdp) in fish pasteurellosis, a deadly systemic infection. Following experimental inoculation, Phdp spreads by bacteraemia and colonizes the organs, producing a septicaemic infection, and secretes the apoptogenic exotoxin AIP56 which is systemically disseminated. In experimental and natural pasteurellosis, destruction of macrophages and neutrophils by secondary necrosis following caspase-3-associated apoptosis was seen predominantly in the spleen, head kidney and gut lamina propria. Identical phagocyte destruction occurred after injection of rAIP56, but not of heat-inactivated rAIP56, or AIP56-negative Phdp strains, indicating that AIP56 is responsible for phagocyte destruction occurring in pasteurellosis. Active caspase-3 and active neutrophil elastase are present in the blood in advanced infection, indicating that phagocyte lysis by secondary necrosis is accompanied by release of tissue-damaging molecules. The AIP56-induced lysis of phagocytes represents a very efficient, self-amplifying etiopathogenic mechanism, because it results in two effects that operate in concert against the host, namely, evasion of the pathogen from a crucial defence mechanism through the destruction of both professional phagocytes, and release of tissue-damaging molecules. The induction by a bacterial exotoxin of in vivo systemic lysis of both professional phagocytes by secondary necrosis, now described for the first time, may represent an overlooked etiopathogenic mechanism operating in other infections of vertebrates.
Assuntos
Exotoxinas/fisiologia , Doenças dos Peixes/patologia , Infecções por Bactérias Gram-Negativas/patologia , Macrófagos/patologia , Neutrófilos/patologia , Animais , Apoptose , Western Blotting , Caspase 3/sangue , Caspase 3/metabolismo , Eletroforese em Gel de Poliacrilamida , Exotoxinas/genética , Exotoxinas/metabolismo , Doenças dos Peixes/sangue , Doenças dos Peixes/microbiologia , Peixes , Infecções por Bactérias Gram-Negativas/microbiologia , Imuno-Histoquímica , Rim/enzimologia , Rim/microbiologia , Rim/patologia , Elastase de Leucócito/sangue , Elastase de Leucócito/metabolismo , Macrófagos/microbiologia , Necrose , Neutrófilos/microbiologia , Photobacterium/genética , Photobacterium/patogenicidade , Baço/enzimologia , Baço/microbiologia , Baço/patologia , Virulência/genéticaRESUMO
Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging tropical skin disease. Virulent M. ulcerans secretes mycolactone, a cytotoxic exotoxin with a key pathogenic role. M. ulcerans in biopsy specimens has been described as an extracellular bacillus. In vitro assays have suggested a mycolactone-induced inhibition of M. ulcerans uptake by macrophages in which its proliferation has not been demonstrated. Therefore, and uniquely for a mycobacterium, M. ulcerans has been classified as an extracellular pathogen. In specimens from patients and in mouse footpad lesions, extracellular bacilli were concentrated in central necrotic acellular areas; however, we found bacilli within macrophages in surrounding inflammatory infiltrates. We demonstrated that mycolactone-producing M. ulcerans isolates are efficiently phagocytosed by murine macrophages, indicating that the extracellular location of M. ulcerans is not a result of inhibition of phagocytosis. Additionally, we found that M. ulcerans multiplies inside cultured mouse macrophages when low multiplicities of infection are used to prevent early mycolactone-associated cytotoxicity. Following the proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, becoming extracellular. Our data show that M. ulcerans, like M. tuberculosis, is an intracellular parasite with phases of intramacrophage and extracellular multiplication. The occurrence of an intramacrophage phase is in accordance with the development of cell-mediated and delayed-type hypersensitivity responses in BU patients.
Assuntos
Macrófagos/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium ulcerans/crescimento & desenvolvimento , Dermatopatias Bacterianas/microbiologia , Úlcera Cutânea/microbiologia , Animais , Toxinas Bacterianas/biossíntese , Células Cultivadas , Modelos Animais de Doenças , Feminino , Pé/microbiologia , Pé/patologia , Histocitoquímica , Humanos , Macrolídeos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Infecções por Mycobacterium não Tuberculosas/patologia , Fagocitose , Dermatopatias Bacterianas/patologia , Úlcera Cutânea/patologiaRESUMO
A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.
Assuntos
Apoptose , Macrófagos/fisiologia , Neutrófilos/fisiologia , Photobacterium/patogenicidade , Plasmídeos/genética , Fatores de Virulência/fisiologia , Animais , Toxinas Bacterianas/genética , Sequência de Bases , Bass , Linhagem Celular , Citrobacter rodentium/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli O157/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Imunização Passiva , Dados de Sequência Molecular , Photobacterium/genética , Proteínas Recombinantes , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
Buruli ulcer (BU) is a devastating, necrotizing, tropical skin disease caused by infections with Mycobacterium ulcerans. In contrast to other mycobacterioses, BU has been associated with minimal or absent inflammation. However, here we show that in the mouse M. ulcerans induces persistent inflammatory responses with virulence-dependent patterns. Mycolactone-positive, cytotoxic strains are virulent for mice and multiply progressively, inducing both early and persistent acute inflammatory responses. The cytotoxicity of these strains leads to progressive destruction of the inflammatory infiltrates by postapoptotic secondary necrosis, generating necrotic acellular areas with extracellular bacilli released by the lysis of infected phagocytes. The necrotic areas, always surrounded by acute inflammatory infiltrates, expand through the progressive invasion of healthy tissues around the initial necrotic lesions by bacteria and by newly recruited acute inflammatory cells. Our observations show that the lack of inflammatory infiltrates in the extensive areas of necrosis seen in advanced infections results from the destruction of continuously produced inflammatory infiltrates and not from M. ulcerans-induced local or systemic immunosuppression. Whether this is the mechanism behind the predominance of minimal or absent inflammatory responses in BU biopsies remains to be elucidated.
Assuntos
Dermatite/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium ulcerans/patogenicidade , Peritonite/microbiologia , Animais , Apoptose , Células Cultivadas , Dermatite/patologia , Feminino , Macrófagos/microbiologia , Camundongos , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium ulcerans/isolamento & purificação , Necrose/microbiologia , Necrose/patologia , Peritonite/patologia , VirulênciaRESUMO
Two isolates, belonging to a new species of a novel genus of the Phylum "Deinococcus/Thermus ", were recovered from hot spring runoffs on the Island of São Miguel in the Azores. Strains RQ-24(T) and TU-8 are the first cultured representatives of a distinct phylogenetic lineage within this phylum. These strains form orange/red colonies, spherical-shaped cells, have an optimum growth temperature of about 50 degrees C, an optimum pH for growth between about 7.5 and 9.5, and do not grow at pH below 6.5 or above pH 11.2. These organisms grow in complex media without added NaCl, but have a maximum growth rate in media with 1.0% NaCl and grow in media containing up to 6.0% NaCl. The organisms are extremely ionizing radiation resistant; 60% of the cells survive 5.0 kGy. These strains are chemoorganotrophic and aerobic; do not grow in Thermus medium under anaerobic conditions with or without nitrate as electron acceptor and glucose as a source of carbon and energy, but ferment glucose to D-lactate without formation of gas. The organisms assimilate a large variety of sugars, organic acids and amino acids. Fatty acids are predominantly iso- and anteiso-branched; long chain 1,2 diols were also found in low relative proportions; menaquinone 8 (MK-8) is the primary respiratory quinone. Peptidoglycan was not detected. Based on 16S rRNA gene sequence analysis, physiological, biochemical and chemical analysis we describe a new species of one novel genus represented by strain RQ-24(T) (CIP 108686(T)=LMG 22925(T)=DSM 17093(T)) for which we propose the name Truepera radiovictrix. We also propose the family Trueperaceae fam. nov. to accommodate this new genus.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Fontes Termais/microbiologia , Aminoácidos/metabolismo , Bactérias/citologia , Bactérias/efeitos da radiação , Fenômenos Fisiológicos Bacterianos , Sequência de Bases , Ácidos Carboxílicos/metabolismo , DNA Bacteriano/química , DNA Ribossômico/química , Deinococcus/classificação , Ácidos Graxos/análise , Ácidos Graxos/isolamento & purificação , Fermentação , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peptidoglicano/análise , Filogenia , Pigmentos Biológicos/análise , Portugal , RNA Ribossômico 16S/genética , Radiação Ionizante , Cloreto de Sódio/metabolismo , Temperatura , Thermus/classificação , Vitamina K 2/análogos & derivados , Vitamina K 2/isolamento & purificação , Microbiologia da ÁguaRESUMO
In order to identify the phagocytic cells of sea bass, the peritoneal leucocyte population of fish injected intraperitoneally with Photobacterium damselae subspecies piscicida was studied by light microscopy using cytocentrifuge preparations stained by the Antonow technique for peroxidase detection. Among the leucocytes present in the peritoneal exudate of the infected fish (macrophages, neutrophils, eosinophilic granular cells, lymphocytes and thrombocytes), macrophages and neutrophils were the only phagocytic cells. Neutrophils were easily distinguished from macrophages in Antonow stained preparations by the pattern of peroxidase positivity. Using ultrastructural cytochemistry, neutrophils were found to have abundant cytoplasmic granules positive for peroxidase and arylsulphatase and were negative for alpha-naphthyl butyrate (ANB) esterase. In contrast, ANB esterase activity was detected in macrophages. These leucocytes were typically negative for peroxidase, but ocasionally, some macrophages with peroxidase or arylsulphatase-positive vacuoles were observed. Both phagocytes had cytoplasmic granules positive for acid phosphatase. Glycogen particles were found in the cytoplasm of the two phagocytic cells, but they were much more abundant in neutrophils. Macrophages were much more abundant than neutrophils in the peritoneal cavity of non-injected sea bass but early after the intraperitoneal injection of bacteria, the number of neutrophils increased quickly and extensively. Higher numbers of intraperitoneally injected bacteria were found inside macrophages as compared to neutrophils because macrophages strongly predominated in the peritoneal population at the time of injection. However, when the bacteria were injected into peritoneal cavities with high numbers of neutrophils (attracted by a previous injection of 12% casein), the percentage of neutrophils with phagocytosed bacteria increased, approaching that of infected macrophages. Taken together, these results show that in sea bass, as in many other organisms, in addition to macrophages, neutrophils are important phagocytic cells, the relative participation of each of the two phagocytes in defense mechanisms against infection depending on the opportunity to encounter the invading infectious agents.
Assuntos
Bass , Doenças dos Peixes/patologia , Macrófagos/patologia , Neutrófilos/patologia , Cavidade Peritoneal/patologia , Peritonite/veterinária , Animais , Hidrolases de Éster Carboxílico/química , Doenças dos Peixes/imunologia , Histocitoquímica/veterinária , Injeções Intraperitoneais/veterinária , Macrófagos/imunologia , Macrófagos/ultraestrutura , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Cavidade Peritoneal/citologia , Cavidade Peritoneal/microbiologia , Peritonite/imunologia , Peritonite/patologia , Peroxidase/química , Fagócitos/imunologia , Fagócitos/patologia , Fagócitos/ultraestrutura , Fagocitose , Photobacterium/imunologia , Coloração e Rotulagem/veterináriaRESUMO
Iron accumulation in the liver in hereditary hemochromatosis (HH) has been shown to be highly variable. Some studies point to the importance of major histocompatibility complex (MHC) class I (MHC-I) and CD8(+) cells as modifiers of iron overload. In this report, using mice knockout for H2K(b-/-) and H2D(b-/-) genes, it is demonstrated that lack of classical MHC-I molecules results in a spontaneous increase of nonheme iron content in the liver (mainly located in the hepatocytes) when compared to wild-type mice. In CD8(-/-) and Rag2(-/-) mice, no spontaneous hepatic iron accumulation was observed. These results demonstrate for the first time that classical MHC-I molecules could be involved in the regulation of iron metabolism and contribute to the established genotype/phenotype discrepancies seen in HH.