RESUMO
DC, through the uptake, processing, and presentation of antigen, are responsible for activation of T cell responses to defend the host against infection, yet it is not known if they can directly kill invading bacteria. Here, we studied in human leprosy, how Langerhans cells (LC), specialized DC, contribute to host defense against bacterial infection. IFN-γ treatment of LC isolated from human epidermis and infected with Mycobacterium leprae (M. leprae) activated an antimicrobial activity, which was dependent on the upregulation of the antimicrobial peptide cathelicidin and induction of autophagy. IFN-γ induction of autophagy promoted fusion of phagosomes containing M. leprae with lysosomes and the delivery of cathelicidin to the intracellular compartment containing the pathogen. Autophagy enhanced the ability of M. leprae-infected LC to present antigen to CD1a-restricted T cells. The frequency of IFN-γ labeling and LC containing both cathelicidin and autophagic vesicles was greater in the self-healing lesions vs. progressive lesions, thus correlating with the effectiveness of host defense against the pathogen. These data indicate that autophagy links the ability of DC to kill and degrade an invading pathogen, ensuring cell survival from the infection while facilitating presentation of microbial antigens to resident T cells.
Assuntos
Apresentação de Antígeno , Autofagia , Células de Langerhans/imunologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Antígenos de Bactérias/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Autofagossomos/imunologia , Autofagossomos/metabolismo , Autofagossomos/microbiologia , Biópsia , Células Cultivadas , Epiderme/imunologia , Epiderme/microbiologia , Epiderme/patologia , Humanos , Interferon gama/imunologia , Células de Langerhans/microbiologia , Células de Langerhans/ultraestrutura , Hanseníase/microbiologia , Hanseníase/patologia , Lisossomos/imunologia , Lisossomos/metabolismo , Lisossomos/microbiologia , Microscopia Eletrônica de Transmissão , Mycobacterium leprae/isolamento & purificação , Cultura Primária de Células , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Regulação para Cima/imunologia , CatelicidinasRESUMO
In Arthropoda, the ovary is classified into Chelicerata-type and Mandibulata-type, based on the oocyte-growth position within the ovary. By contrast, oocytes of Diplopoda and Chilopoda grow within the hemocoelic space. However, as the position of oocyte-growth in Symphyla and Pauropoda has not been confirmed, whether the hemocoelic nature of oocyte-growth is common among myriapods remains ambiguous. This study described the ovarian structure of Hanseniella caldaria to reveal the oocyte-growth position in Symphyla. The oocyte is surrounded by the follicle epithelium, and the inner surface of the follicle epithelium, i.e., the space between follicle cells and oocytes, is lined with a basement membrane. The follicle epithelial layer continues to the ovarian epithelium via the follicle extension with a continuous layer of basement membrane. Data on the architecture of the follicle suggest that the follicle pouch opens to the hemocoel. Hence, the oocyte of H. caldaria grows within the hemocoelic space. Based on our findings in H. caldaria and previous studies in a millipede and in centipedes, the hemocoelic nature of oocyte-growth is considered as a common feature among myriapods and a synapomorphy of the Myriapoda for which morphological synapomorphies have been ambiguous.
Assuntos
Artrópodes/crescimento & desenvolvimento , Oócitos/crescimento & desenvolvimento , Animais , Artrópodes/citologia , Artrópodes/ultraestrutura , Feminino , Microscopia , Microscopia Eletrônica de Transmissão , Oócitos/citologia , Oócitos/ultraestrutura , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/ultraestrutura , FilogeniaRESUMO
Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages.
Assuntos
Autofagia/fisiologia , Hanseníase/imunologia , Pele/microbiologia , Adulto , Idoso , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Interferon gama/imunologia , Hanseníase/patologia , Macrófagos/imunologia , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mycobacterium leprae/imunologia , Reação em Cadeia da Polimerase , Pele/imunologia , Pele/patologia , Transcriptoma , Adulto JovemRESUMO
Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.
Assuntos
Recombinação Genética , Fagos de Streptococcus/classificação , Fagos de Streptococcus/genética , Streptococcus thermophilus/virologia , Fagos Bacilares , Queijo/microbiologia , Queijo/virologia , Produtos Fermentados do Leite/microbiologia , Produtos Fermentados do Leite/virologia , Empacotamento do DNA , DNA Viral , Fermentação , Microbiologia de Alimentos , Genoma Viral , Lactococcus lactis/virologia , Microscopia Eletrônica de Transmissão , Filogenia , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Fagos de Streptococcus/isolamento & purificação , Fagos de Streptococcus/ultraestrutura , Proteínas Estruturais Virais/isolamento & purificação , Iogurte/microbiologia , Iogurte/virologiaRESUMO
Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Adulto Jovem , Pele/imunologia , Pele/microbiologia , Pele/patologia , Autofagia/fisiologia , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Western Blotting , Reação em Cadeia da Polimerase , Imunofluorescência , Interferon gama/imunologia , Microscopia Eletrônica de Transmissão , Transcriptoma , Hanseníase/imunologia , Hanseníase/patologia , Macrófagos/imunologia , Mycobacterium leprae/imunologiaRESUMO
The aim of the present work was to develop and optimize surface-functionalized solid lipid nanoparticles (SLNs) for improvement of the therapeutic index of dapsone (DAP), with the application of a design of experiments. The formulation was designed to target intestinal microfold (M-cells) as a strategy to increase internalization of the drug by the infected macrophages. DAP-loaded SLNs and mannosylated SLNs (M-SLNs) were successfully developed by hot ultrasonication method employing a three-level, three-factor Box-Behnken design, after the preformulation study was carried out with different lipids. All the formulations were systematically characterized regarding their diameter, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, and loading capacity. They were also subjected to morphological studies using transmission electron microscopy, in vitro release study, infrared analysis (Fourier transform infrared spectroscopy), calorimetry studies (differential scanning calorimetry), and stability studies. The diameter of SLNs, SLN-DAP, M-SLNs, and M-SLN-DAP was approximately 300 nm and the obtained PDI was <0.2, confirming uniform populations. Entrapment efficiency and loading capacity were approximately 50% and 12%, respectively. Transmission electron microscopy showed spherical shape and nonaggregated nanoparticles. Fourier transform infrared spectroscopy was used to confirm the success of mannose coating process though Schiff's base formation. The variation of the ZP between uncoated (approximately -30 mV) and mannosylated formulations (approximately +60 mV) also confirmed the successful coating process. A decrease in the enthalpy and broadening of the lipid melting peaks of the differential scanning calorimetry thermograms are consistent with the nanostructure of the SLNs. Moreover, the drug release was pH-sensitive, with a faster drug release at acidic pH than at neutral pH. Storage stability for the formulations for at least 8 weeks is expected, since they maintain the original characteristics of diameter, PDI, and ZP. These results pose a strong argument that the developed formulations can be explored as a promising carrier for treating leprosy with an innovative approach to target DAP directly to M-cells.
Assuntos
Dapsona/administração & dosagem , Manose/química , Nanopartículas/administração & dosagem , Nanopartículas/química , Varredura Diferencial de Calorimetria , Química Farmacêutica/métodos , Dapsona/química , Dapsona/farmacocinética , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Hansenostáticos/administração & dosagem , Hansenostáticos/química , Hansenostáticos/farmacocinética , Lipídeos/química , Microscopia Eletrônica de Transmissão , Modelos Estatísticos , Nanoestruturas/química , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
PURPOSE: This study describes how protein release from polymer matrices correlate with simple measurements on the intrinsic viscosity of the polymer solutions used for casting the matrices and calculations of the solubility parameters of polymers and solvents used. METHOD: Matrices of poly(dl-lactide-co-glycolide) (PLGA) were cast with bovine serum albumin (BSA) as a model drug using different solvents (acetone, dichloromethane, ethanol and water). The amount of released protein from the different matrices was correlated with the Hildebrand and Hansen solubility parameters of the solvents, and the intrinsic viscosity of the polymer solutions. Matrix microstructure was investigated by transmission and scanning electron microscopy (TEM and SEM). Polycaprolactone (PCL) matrices were used in a similar way to support the results for PLGA matrices. RESULTS: The maximum amount of BSA released and the release profile from PLGA matrices varied depending on the solvent used for casting. The maximum amount of released BSA decreased with higher intrinsic viscosity, and increased with solubility parameter difference between the solvent and polymer used. The solvent used also had an effect on the matrix microstructure as determined by TEM and SEM. Similar results were obtained for the PCL polymer systems. CONCLUSIONS: The smaller the difference in the solubility parameter between the polymer and the solvent used for casting a polymer matrix, the lower will be the maximum protein release. This is because of the presence of smaller pore sizes in the cast matrix if a solvent with a solubility parameter close to the one of the polymer is used. Likewise, the intrinsic viscosity of the polymer solution increases as solubility parameter differences decrease, thus, simple measurements of intrinsic viscosity and solubility parameter difference, allow the prediction of protein release profiles.
Assuntos
Portadores de Fármacos/química , Ácido Láctico/química , Ácido Poliglicólico/química , Soroalbumina Bovina/administração & dosagem , Solventes/química , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Poliésteres/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Soroalbumina Bovina/química , Solubilidade , ViscosidadeRESUMO
In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus.
Assuntos
Culicidae/virologia , Vírus de Insetos/isolamento & purificação , Vírus de RNA/isolamento & purificação , Vírus não Classificados/isolamento & purificação , Sequência de Aminoácidos , Animais , Culex/virologia , Genoma Viral , Vírus de Insetos/classificação , Vírus de Insetos/genética , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Fases de Leitura Aberta , Filipinas , Filogenia , Vírus de Plantas/genética , Vírus de RNA/classificação , Vírus de RNA/genética , RNA Polimerase Dependente de RNA/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Vírion/ultraestrutura , Vírus não Classificados/classificação , Vírus não Classificados/genéticaRESUMO
A systematic ultrastructure of peripheral nerves across the spectrum of leprosy was studied with an aim to better understanding the pathogenesis of nerve involvement in leprosy using light and electron microscope. The pathogenesis of nerve destruction varies in leprosy considerably along the spectrum. The study has begun to shed new light on some aspects of the infection of Mycobacterium leprae (M. lepare) and phenomenon has opened new avenue of research and possible mechanism of pathogenesis in TT/BT/BL/LL leprosy. In tuberculoid type (TT) and borderline tuberculoid (BT) leprosy, the degenerative changes of Schwann cells (SCs) and presence of perineural and perivascular cuffing by mononuclear cells. The endoneurial blood vessel (EBV) showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen and causing ischemia. However, borderline lepromatous (BL) and lepromatous leprosy (LL) foamy macrophages and vacuolated SC contain numerous small dense materials, irregular in shape and size was prominent and, considered to be degenerated and fragmented M. Leprae. The dense materials were also found in the cytoplasm of vascular EC. It was revealed that besides SC, the EC of EBV frequently harbor M. leprae in LL. The lumen of the EBV was wide open with enlarged nucleus. In the present study, the ultrastructural characteristics suggest that hypersensitivity mechanisms are possibly responsible for nerve damage in TT/BT leprosy. However, the study indicates that the mechanisms of nerve damage in BL/LL are basically different wherein hypersensitivity appears to play a very limited role.
Assuntos
Células Endoteliais/ultraestrutura , Hanseníase/patologia , Nervos Periféricos/ultraestrutura , Células de Schwann/ultraestrutura , Células Endoteliais/microbiologia , Humanos , Microscopia Eletrônica de Transmissão , Nervos Periféricos/microbiologia , Células de Schwann/microbiologiaRESUMO
This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Viabilidade Microbiana , Mycobacterium leprae/patogenicidade , Adesinas Bacterianas/análise , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Hanseníase/microbiologia , Hanseníase/patologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Fagocitose , Proteoma/análise , Alvéolos Pulmonares/microbiologia , Alvéolos Pulmonares/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.
Assuntos
Vetores Aracnídeos/virologia , Citrus/virologia , Ácaros/virologia , Doenças das Plantas/virologia , Vírus de RNA/isolamento & purificação , Sequência de Aminoácidos , Animais , Citrus/ultraestrutura , Colômbia , Frutas , Biblioteca Gênica , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , Folhas de Planta/virologia , Vírus de RNA/classificação , Vírus de RNA/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Plântula/ultraestrutura , Plântula/virologia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
A Citrus volkameriana tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawaii. Citrus leprosis virus C (CiLV-C)-specific detection assays, however, were negative for all tissues tested. Short, bacilliform virus-like particles were observed by transmission electron microscopy in the cytoplasm of symptomatic leaves but not in healthy controls. Double-stranded (ds) RNAs ≈8 and 3 kbp in size were present in symptomatic leaf tissue but not in healthy controls. Excluding poly(A) tails, the largest molecule, RNA1, was 8,354 bp in length. The ≈3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3,169 and 3,113 bp, respectively. Phylogenetic analyses indicated that the RNA-dependent RNA polymerase (RdRp) domain located in RNA1 was most closely related to the RdRp domain of CiLV-C. A reverse-transcription polymerase chain reaction assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus plants with symptoms of hibiscus green spot, a disease associated with infection by Hibiscus green spot virus (HGSV). All nearby citrus trees tested negative with the assay; however, symptomatic H. arnottianus plants were positive. All three RNAs were present in symptomatic H. arnottianus and were >98% identical to the RNAs isolated from C. volkameriana. We contend that the virus described in this study is HGSV, and propose that it be the type member of a new virus genus, Higrevirus.
Assuntos
Citrus/virologia , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Vírus de RNA/isolamento & purificação , RNA Polimerase Dependente de RNA/genética , Sequência de Aminoácidos , Sequência de Bases , Citrus/ultraestrutura , DNA Complementar/química , DNA Complementar/genética , Genoma Viral/genética , Havaí , Hibiscus/virologia , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , Casca de Planta/virologia , Folhas de Planta/virologia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Vírus de Plantas/ultraestrutura , Estrutura Terciária de Proteína/genética , Vírus de RNA/classificação , Vírus de RNA/genética , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Vírion/ultraestruturaRESUMO
This study describes the normal morphology and morphometry of the dorsal cutaneous branch of the ulnar nerve (DCBU) in humans. Fourteen nerves of eight donors were prepared by conventional techniques for paraffin and epoxy resin embedding. Semiautomatic morphometric analysis was performed by means of specific computer software. Histograms of the myelinated and unmyelinated fiber population and the G-ratio distribution of fibers were plotted. Myelinated fiber density per nerve varied from 5,910 to 10,166 fibers/mm(2) , with an average of 8,170 ± 393 fibers/mm(2) . The distribution was bimodal with peaks at 4.0 and 9.5 µm. Unmyelinated fiber density per nerve varied from 50,985 to 127,108, with an average of 78,474 ± 6,610 fibers/mm(2) , with a unimodal distribution displaying a peak at 0.8 µm. This study thus adds information about the fascicles and myelinated and unmyelinated fibers of DCBU nerves in normal people, which may be useful in further studies concerning ulnar nerve neuropathies, mainly leprosy neuropathy.
Assuntos
Neurônios/citologia , Nervo Ulnar/anatomia & histologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pele/inervação , Adulto JovemRESUMO
AIMS: To study Microfasciculation, a perineurial response found in neuropathies, emphasizing its frequency, detailed morphological characteristics and biological significance in pure neural leprosy (PNL), post-treatment leprosy neuropathy (PTLN) and non-leprosy neuropathies (NLN). METHODS AND RESULTS: Morphological characteristics of microfascicles were examined via histological staining methods, immunohistochemical expression of neural markers and transmission electronmicroscopy. The detection of microfasciculation in 18 nerve biopsy specimens [12 PNL, six PTLN but not in the NLN group, was associated strongly with perineurial damage and the presence of a multibacillary inflammatory process in the nerves, particularly in the perineurium. Immunoreactivity to anti-S100 protein, anti-neurofilament, anti-nerve growth receptor and anti-myelin basic protein immunoreactivity was found within microfascicles. Ultrastructural examination of three biopsies showed that fibroblast-perineurial cells were devoid of basement membrane despite perineurial-like NGFr immunoreactivity. Morphological evidence demonstrated that multipotent pericytes from inflammation-activated microvessels could be the origin of fibroblast-perineurial cells. CONCLUSIONS: A microfasciculation pattern was found in 10% of leprosy-affected nerves. The microfascicles were composed predominantly of unmyelinated fibres and denervated Schwann cells (SCs) surrounded by fibroblast-perineurial cells. This pattern was found more frequently in leprosy nerves with acid-fast bacilli (AFB) and perineurial damage while undergoing an inflammatory process. Further experimental studies are necessary to elucidate microfascicle formation.
Assuntos
Hanseníase Tuberculoide/patologia , Fibras Nervosas/ultraestrutura , Nervos Periféricos/ultraestrutura , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Células de Schwann/ultraestruturaRESUMO
The leprosis disease shows a viral etiology and the citrus leprosis virus is considered its etiologic agent. The disease may show two types of cytopatologic symptom caused by two virus: nuclear (CiLV-N) and cytoplasmic (CiLV-C) types. The aim of this study was to compare the morpho-anatomical differences in the lesions caused by leprosis virus-cytoplasmic and nuclear types in Citrus sinensis (L.) Osbeck 'Pêra'. Leaf and fruit lesions were collected in Piracicaba/São Paulo (cytoplasmic type) and Monte Alegre do Sul/São Paulo and Amparo/São Paulo (nuclear type). The lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hydroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 microm thick), stained and mounted in synthetic resin. The digital images were acquired in a microscope with digital video camera. Leaf and fruit lesions caused by the two viruses were morphologically distinct. Only the lesion caused by CiLV-N virus presented three well-defined regions. In both lesions there was the accumulation of lipidic substances in necrotic areas that were surrounded by cells with amorphous or droplets protein. Only leaf and fruit lesions caused by CiLV-N virus exhibited traumatic gum ducts in the vascular bundles.
Assuntos
Citrus sinensis/virologia , Doenças das Plantas/virologia , Vírus de Plantas/classificação , Citrus sinensis/ultraestrutura , Microscopia Eletrônica de TransmissãoRESUMO
The leprosis disease shows a viral etiology and the citrus leprosis virus is considered its etiologic agent. The disease may show two types of cytopatologic symptom caused by two virus: nuclear (CiLV-N) and cytoplasmic (CiLV-C) types. The aim of this study was to compare the morpho-anatomical differences in the lesions caused by leprosis virus-cytoplasmic and nuclear types in Citrus sinensis (L.) Osbeck 'Pêra'. Leaf and fruit lesions were collected in Piracicaba/São Paulo (cytoplasmic type) and Monte Alegre do Sul/São Paulo and Amparo/São Paulo (nuclear type). The lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hydroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 μm thick), stained and mounted in synthetic resin. The digital images were acquired in a microscope with digital video camera. Leaf and fruit lesions caused by the two viruses were morphologically distinct. Only the lesion caused by CiLV-N virus presented three well-defined regions. In both lesions there was the accumulation of lipidic substances in necrotic areas that were surrounded by cells with amorphous or droplets protein. Only leaf and fruit lesions caused by CiLV-N virus exhibited traumatic gum ducts in the vascular bundles.
A doença leprose dos citros tem etiologia viral sendo o citrus leprosis virus seu agente etiológico. Demonstrou-se que há dois vírus distintos que causam sintomas de leprose em ci-tros: citoplasmático (CiLV-C) e o nuclear (CiLV-N). O objetivo desse estudo foi comparar as diferenças morfo-anatômicas nas lesões causadas por CiLV-C e por CiLV-N em laranjeira doce (Citrus sinensis (L.) Osbeck) 'Pêra'. As lesões foliares e dos frutos foram coletadas em Piracicaba/SP (tipo citoplas-mático) e em Monte Alegre do Sul/SP e Amparo/SP (tipo nuclear). As lesões foram fotografadas e em seguida fixadas em solução Karnovsky, desidratadas em série etílica, incluídas em historesina e secionadas em micrótomo rotativo. As lâminas foram coradas, analisadas e fotografadas. As lesões foliares e do fruto causadas pelos dois vírus eram morfologicamente distintas. Somente a lesão causada por CiLV-N apresentou três regiões bem definidas. Em ambas as lesões ocorreu acúmulo de substâncias lipídicas nas áreas necrosadas que se achavam envoltas por células com conteúdo protéico amorfo ou em gotas. Somente as lesões da folha e do fruto causadas pelo CiLV-N exibiram ductos traumáticos gomosos nos feixes vasculares.
Assuntos
Citrus sinensis/virologia , Doenças das Plantas/virologia , Vírus de Plantas/classificação , Citrus sinensis/ultraestrutura , Microscopia Eletrônica de TransmissãoRESUMO
Peripheral nerve biopsies from 10 Lepromatous leprosy (LL) patients who were on multidrug treatment (MDT) were investigated by light and electron microscopy. Clofazimine (CLF) has been included as an essential component of MDT, which is the standard WHO regimen for treatment of leprosy. The patients receiving continuous MDT for a long period had viable bacilli in Schwann cells (SCs) of peripheral nerves whereas they had disappeared from the skin. Our ultrastructural observations clearly indicated the presence of CLF crystals in SCs. The crystals were in the form of osmiophilic rods of various shapes and sizes. On the other hand, the blood nerve barrier was clearly noticed in endoneurial blood vessels (EBV), and the barrier seems to play an important role for penetration of antileprosy drugs especially CLF.
Assuntos
Antituberculosos/farmacocinética , Clofazimina/farmacocinética , Células Endoteliais/microbiologia , Mycobacterium leprae/efeitos dos fármacos , Nervos Periféricos/microbiologia , Células de Schwann/microbiologia , Adulto , Antituberculosos/uso terapêutico , Clofazimina/uso terapêutico , Quimioterapia Combinada , Células Endoteliais/citologia , Células Endoteliais/ultraestrutura , Humanos , Hanseníase Virchowiana/tratamento farmacológico , Microscopia , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mycobacterium leprae/ultraestrutura , Células de Schwann/citologia , Células de Schwann/ultraestruturaRESUMO
M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.
Assuntos
Citosol/fisiologia , Lisossomos/fisiologia , Mycobacterium/fisiologia , Células Mieloides/microbiologia , Fagossomos/fisiologia , Apresentação de Antígeno/fisiologia , Biomarcadores/metabolismo , Compartimento Celular/fisiologia , Morte Celular/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Citosol/ultraestrutura , Regulação Bacteriana da Expressão Gênica/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Humanos , Imuno-Histoquímica , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Lisossomos/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Mycobacterium/genética , Mycobacterium/ultraestrutura , Mycobacterium leprae/genética , Mycobacterium leprae/fisiologia , Mycobacterium leprae/ultraestrutura , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Mycobacterium tuberculosis/ultraestrutura , Células Mieloides/fisiologia , Células Mieloides/ultraestrutura , Fagossomos/ultraestruturaRESUMO
Crystal-storing histiocytosis is a rare diagnosis that to date has only been associated with 2 conditions: intracytoplasmic accumulation of crystallized immunoglobulins in patients with lymphoproliferative disorders or plasma cell dyscrasias, and histiocytic accumulations of phagocytosed clofazimine, a drug used to treat lepromatous leprosy. We describe a 78-year-old woman with a past medical history of dermatologic mastocytosis and peripheral eosinophilia who presented with diarrhea and weight loss, and was found at colonoscopy to have polyposis limited to the right and transverse colon. She eventually underwent subtotal colectomy to remove the segment of polyposis. At gross examination, the colonic mucosa contained numerous polyps ranging from 1 to 7 mm which on histologic evaluation proved to represent mucosal and submucosal collections of histiocytes whose cytoplasm was distended by numerous brightly eosinophilic crystals. An intense eosinophilic infiltrate surrounded the histiocyte collections and also mildly involved the intervening colonic mucosa and superficial submucosa. Electron microscopy confirmed the presence of intracytoplasmic material identical to Charcot-Leyden crystals within histiocytes, representing the breakdown products of degranulated eosinophils. This is the first reported case of crystal-storing histiocytosis produced by massive accumulation of Charcot-Leyden crystals in eosinophilic colitis.
Assuntos
Colite/patologia , Colo/patologia , Eosinofilia/patologia , Histiocitose/patologia , Corpos de Inclusão/patologia , Polipose Intestinal/patologia , Idoso , Degranulação Celular , Colite/complicações , Colonoscopia , Cristalização , Eosinofilia/complicações , Eosinófilos/ultraestrutura , Feminino , Histiocitose/complicações , Humanos , Corpos de Inclusão/ultraestrutura , Polipose Intestinal/complicações , Macrófagos/patologia , Microscopia Eletrônica de TransmissãoRESUMO
We show the distribution of the neural and non-neural elements in the early development of the optic nerve in the freshwater turtle, Mauremys leprosa, using light and electron microscopy. The first optic axons invaded the ventral periphery of the optic stalk in close relationship to the radial neuroepithelial processes. Growth cones were thus exclusively located in the ventral margin. As development progressed, growth cones were present in ventral and dorsal regions, including the dorsal periphery, where they intermingled with mature axons. However, growth cones predominated in the ventral part and axonal profiles dorsally, reflecting a dorsal to ventral gradient of maturation. The size and morphology of growth cones depended on the developmental stage and the region of the optic nerve. At early stages, most growth cones were of irregular shape, showing abundant lamellipodia. At the following stages, they tended to be larger and more complex in the ventral third than in intermediate and dorsal portions, suggesting a differential behavior of the growth cones along the ventro-dorsal axis. The arrival of optic axons at the optic stalk involved the progressive transformation of neuroepithelial cells into glial cells. Simultaneously with the fiber invasion, an important number of cells died by apoptosis in the dorsal wall of the optic nerve. These findings are discussed in relation to the results described in the developing optic nerve of other vertebrates.