Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 197
Filtrar
Mais filtros


Intervalo de ano de publicação
1.
J Dermatol ; 46(10): 853-858, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432529

RESUMO

In patients with lepromatous leprosy, Mycobacterium leprae is often observed inside the human microvascular endothelial cells (HMVEC) surrounding Schwann cells (SC) at the site of lesions in the peripheral nerves. Based on this observation, it is considered that the nasal mucous may be the invasion pathway for M. leprae and HMVEC serve as an important reservoir for the bacteria before they invade SC. In light of previous research which revealed that Mce1A protein mediates bacterial invasion into nasal epithelial cells and HMVEC, we conducted a study to determine whether the invasion of M. leprae into HMVEC can be suppressed by blocking the Mce1A protein. In this study, we analyzed bacterial invasive activity by adding recombinant Escherichia coli, which express the active region (InvX:72 a.a.) of Mce1A protein on their external membrane, into cultured HMVEC, using the adhesin involved in the diffuse adherence mechanism. The number of bacteria that invaded into the cells was then measured by a colony counting method. The active region of Mce1A was divided into four sections, and hyperimmune antisera was prepared for each section for analyzing the inhibitory effect against invasion. The invasive activity was suppressed by antibodies against InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. This suggests that the InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. of Mce1A protein play an important role in the invasion of M. leprae into HMVEC and that it may be possible to suppress entry of M. leprae in HMVEC with antibodies against these regions.


Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Células Endoteliais/microbiologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Animais , Anticorpos Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Linhagem Celular , Contagem de Colônia Microbiana , Humanos , Soros Imunes/imunologia , Soros Imunes/isolamento & purificação , Hanseníase/microbiologia , Hanseníase/prevenção & controle , Mycobacterium leprae/patogenicidade , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
2.
PLoS Negl Trop Dis ; 12(12): e0007001, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30566440

RESUMO

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.


Assuntos
Vetores Aracnídeos/fisiologia , Ixodes/microbiologia , Ixodidae/microbiologia , Hanseníase/transmissão , Mycobacterium leprae/fisiologia , Animais , Vetores Aracnídeos/microbiologia , Linhagem Celular , Feminino , Humanos , Ixodes/fisiologia , Ixodidae/fisiologia , Hanseníase/microbiologia , Masculino , Mycobacterium leprae/genética , Coelhos
3.
PLoS Negl Trop Dis ; 12(6): e0006532, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29953440

RESUMO

Mycobacterium leprae (M. leprae) is a human pathogen and the causative agent for leprosy, a chronic disease characterized by lesions of the skin and peripheral nerve damage. Zoonotic transmission of M. leprae to humans by nine-banded armadillos (Dasypus novemcinctus) has been shown to occur in the southern United States, mainly in Texas, Louisiana, and Florida. Nine-banded armadillos are also common in South America, and residents living in some areas in Brazil hunt and kill armadillos as a dietary source of protein. This study examines the extent of M. leprae infection in wild armadillos and whether these New World mammals may be a natural reservoir for leprosy transmission in Brazil, similar to the situation in the southern states of the U.S. The presence of the M. leprae-specific repetitive sequence RLEP was detected by PCR amplification in purified DNA extracted from armadillo spleen and liver tissue samples. A positive RLEP signal was confirmed in 62% of the armadillos (10/16), indicating high rates of infection with M. leprae. Immunohistochemistry of sections of infected armadillo spleens revealed mycobacterial DNA and cell wall constituents in situ detected by SYBR Gold and auramine/rhodamine staining techniques, respectively. The M. leprae-specific antigen, phenolic glycolipid I (PGL-I) was detected in spleen sections using a rabbit polyclonal antibody specific for PGL-I. Anti-PGL-I titers were assessed by ELISA in sera from 146 inhabitants of Belterra, a hyperendemic city located in western Pará state in Brazil. A positive anti-PGL-I titer is a known biomarker for M. leprae infection in both humans and armadillos. Individuals who consumed armadillo meat most frequently (more than once per month) showed a significantly higher anti-PGL-I titer than those who did not eat or ate less frequently than once per month. Armadillos infected with M. leprae represent a potential environmental reservoir. Consequently, people who hunt, kill, or process or eat armadillo meat are at a higher risk for infection with M. leprae from these animals.


Assuntos
Antígenos de Bactérias/imunologia , Tatus/microbiologia , Reservatórios de Doenças/microbiologia , Glicolipídeos/imunologia , Hanseníase/transmissão , Carne/microbiologia , Mycobacterium leprae/isolamento & purificação , Adulto , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Brasil/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Glicolipídeos/genética , Glicolipídeos/isolamento & purificação , Humanos , Hanseníase/epidemiologia , Hanseníase/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Reação em Cadeia da Polimerase , Coelhos , Risco , Baço/microbiologia , Adulto Jovem , Zoonoses
4.
Front Immunol ; 9: 642, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29666623

RESUMO

Background: It has been shown earlier that there is a rise in the levels of autoantibodies and T cell response to cytoskeletal proteins in leprosy. Our group recently demonstrated a rise in both T and B cell responses to keratin and myelin basic protein in all types of leprosy patients and their associations in type 1 reaction (T1R) group of leprosy. Objectives: In this study, we investigated the association of levels of autoantibodies and lymphoproliferation against myosin in leprosy patients across the spectrum and tried to find out the mimicking proteins or epitopes between host protein and protein/s of Mycobacterium leprae. Methodology: One hundred and sixty-nine leprosy patients and 55 healthy controls (HC) were enrolled in the present study. Levels of anti-myosin antibodies and T-cell responses against myosin were measured by ELISA and lymphoproliferation assay, respectively. Using 2-D gel electrophoresis, western blot and MALDI-TOF/TOF antibody-reactive spots were identified. Three-dimensional structure of mimicking proteins was modeled by online server. B cell epitopes of the proteins were predicted by BCPREDS server 1.0 followed by identification of mimicking epitopes. Mice of inbred BALB/c strain were hyperimmunized with M. leprae soluble antigen (MLSA) and splenocytes and lymph node cells of these animals were adoptively transferred to naïve mice. Results: Highest level of anti-myosin antibodies was noted in sera of T1R leprosy patients. We observed significantly higher levels of lymphoproliferative response (p < 0.05) with myosin in all types of leprosy patients compared to HC. Further, hyperimmunization of inbred BALB/c strain of female mice and rabbit with MLSA revealed that both hyperimmunized rabbit and mice evoked heightened levels of antibodies against myosin and this autoimmune response could be adoptively transferred from hyperimmunized to naïve mice. Tropomyosin was found to be mimicking with ATP-dependent Clp protease ATP-binding subunit of M. leprae. We found four mimicking epitopes between these sequences. Conclusion: These data suggest that these mimicking proteins tropomyosin and ATP-dependent Clp protease ATP-binding subunit of M. leprae or more precisely mimicking epitopes (four B cell epitopes) might be responsible for extensive tissue damage during type1 reaction in leprosy.


Assuntos
Autoantígenos/imunologia , Epitopos de Linfócito B/imunologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Peptídeos/imunologia , Linfócitos T/imunologia , Tropomiosina/imunologia , Animais , Autoanticorpos/metabolismo , Autoimunidade , Reações Cruzadas , Feminino , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Mimetismo Molecular , Coelhos
5.
s.l; s.n; 2018. 25 p. ilu, tab, graf.
Não convencional em Inglês | SES-SP, HANSEN, SESSP-ILSLPROD, SES-SP, SESSP-ILSLACERVO, SES-SP | ID: biblio-1025298

RESUMO

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.


Assuntos
Humanos , Animais , Masculino , Feminino , Coelhos , Vetores Aracnídeos/fisiologia , Vetores Aracnídeos/microbiologia , Linhagem Celular , Ixodes/fisiologia , Ixodes/microbiologia , Ixodidae/fisiologia , Ixodidae/microbiologia , Hanseníase/microbiologia , Hanseníase/transmissão , Mycobacterium leprae/fisiologia , Mycobacterium leprae/genética
6.
Transbound Emerg Dis ; 64(4): 1045-1058, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26799551

RESUMO

Tuberculous mycobacterial diseases such as leprosy and tuberculosis are ancient diseases that currently continue threatening human health in some countries. Non-tuberculous mycobacterial (NTM) infections cause a series of well-defined pathological entities, as well as some opportunistic diseases that have also increased worldwide, being more common among immunocompromised patients but rising also in immunocompetent individuals. Reports on natural infections by mycobacteria in rabbits are scarce and mainly involve NTM such as Mycobacterium avium subsp. avium in pigmy rabbits in the United States and Mycobacterium avium subsp. paratuberculosis in wild rabbits in Europe. Rabbits have been used as laboratory animals through the years, both to generate immunological reagents and as infection models. Mycobacterial infection models have been developed in this animal species showing different susceptibility patterns to mycobacteria in laboratory conditions. The latent tuberculosis model and the cavitary tuberculosis model have been widely used to elucidate pathogenic mechanisms and to evaluate chemotherapy and vaccination strategies. Rabbits have also been used as bovine paratuberculosis infection models. This review aimed to gather both wildlife and experimental infection data on mycobacteriosis in rabbits to assess their role in the spread of these infections as well as their potential use in the experimental study of mycobacterial pathogenesis and treatment.


Assuntos
Animais Selvagens , Infecções por Mycobacterium/veterinária , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Coelhos/microbiologia , Animais , Ciência dos Animais de Laboratório , Infecções por Mycobacterium/microbiologia
8.
Einstein (Sao Paulo) ; 13(3): 395-403, 2015.
Artigo em Inglês, Português | MEDLINE | ID: mdl-26466063

RESUMO

OBJECTIVE: To describe and to characterize the relaxing effect of an extract of the bark of Combretum leprosum on isolated arterial rings from different animals. METHODS: Rings (3 to 4mm) from rabbit, rat, or porcine arteries rings were suspended in an organ bath (Krebs, 37°C, 95%O2/5%CO2) to record isometric contractions. After the stabilization period (2 to 3 hours) contractions were induced by the addition of phenylephrine (0.1 to 0.3µM) or U46619 (10 to 100nM), and Combretum leprosum extract was added on the plateau of the contractions. Experiments were performed to determine the potency, duration, reversibility, and to get insights on the potential mechanism involved in extract-induced relaxations. RESULTS: In all rings tested, Combretumleprosum extract (1.5µg/mL) was able to cause relaxations, which were strictly endothelium-dependent. In rabbit or rat thoracic aorta rings, the relaxations were reversed by vitamin B12a or L-NG-nitroarginine. In porcine right coronary arteries and rabbit abdominal aorta, extract caused both L-NG-nitroarginine-sensitive and L-NG-nitroarginine-resistant relaxations. In rabbit thoracic aorta, the extract was relatively potent (EC50=0.20µg/mL) and caused relaxations; intriguingly the endothelium continued to produce relaxing factors for a long period after removing the extract. The magnitude of extract-induced relaxations was significantly reduced in the absence of extracellular Ca2+; in addition, the TRPs channels blocker ruthenium red (10µM) was able to revert extract-induced relaxations. Phytochemical analyses indicated that the extract was rich in polyphenol-like reacting substances. CONCLUSIONS: Combretum leprosum extract contains bioactive compounds capable of promoting Ca2+-dependent stimulation of endothelial cells which results in a prolonged production of relaxing factors.


Assuntos
Combretum/química , Fatores Relaxantes Dependentes do Endotélio/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Óxido Nítrico/farmacologia , Acetilcolina/farmacologia , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/fisiologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Artéria Carótida Primitiva/efeitos dos fármacos , Artéria Carótida Primitiva/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Feminino , Cobaias , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/fisiologia , Camundongos , Relaxamento Muscular/fisiologia , Casca de Planta/química , Coelhos , Ratos Wistar , Suínos , Fatores de Tempo
9.
Artigo em Inglês | MEDLINE | ID: mdl-26347855

RESUMO

The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.


Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Parede Celular/química , Mycobacterium/química , Sinais Direcionadores de Proteínas , Animais , Endopeptidase K/metabolismo , Escherichia coli/química , Escherichia coli/genética , Camundongos , Microscopia Confocal , Mycobacterium/citologia , Mycobacterium/genética , Proteólise , Coelhos , Vesículas Secretórias/química
10.
Einstein (Säo Paulo) ; 13(3): 395-403, July-Sep. 2015. graf
Artigo em Inglês | LILACS | ID: lil-761966

RESUMO

Objective To describe and to characterize the relaxing effect of an extract of the bark of Combretum leprosum on isolated arterial rings from different animals.Methods Rings (3 to 4mm) from rabbit, rat, or porcine arteries rings were suspended in an organ bath (Krebs, 37°C, 95%O2/5%CO2) to record isometric contractions. After the stabilization period (2 to 3 hours) contractions were induced by the addition of phenylephrine (0.1 to 0.3µM) or U46619 (10 to 100nM), and Combretum leprosum extract was added on the plateau of the contractions. Experiments were performed to determine the potency, duration, reversibility, and to get insights on the potential mechanism involved in extract-induced relaxations.Results In all rings tested, Combretumleprosum extract (1.5μg/mL) was able to cause relaxations, which were strictly endothelium-dependent. In rabbit or rat thoracic aorta rings, the relaxations were reversed by vitamin B12a or L-NG-nitroarginine. In porcine right coronary arteries and rabbit abdominal aorta, extract caused both L-NG-nitroarginine-sensitive and L-NG-nitroarginine-resistant relaxations. In rabbit thoracic aorta, the extract was relatively potent (EC50=0.20µg/mL) and caused relaxations; intriguingly the endothelium continued to produce relaxing factors for a long period after removing the extract. The magnitude of extract-induced relaxations was significantly reduced in the absence of extracellular Ca2+; in addition, the TRPs channels blocker ruthenium red (10µM) was able to revert extract-induced relaxations. Phytochemical analyses indicated that the extract was rich in polyphenol-like reacting substances.ConclusionsCombretum leprosum extract contains bioactive compounds capable of promoting Ca2+-dependent stimulation of endothelial cells which results in a prolonged production of relaxing factors.


Objetivo Descrever e caracterizar os relaxamentos induzidos por um extrato das cascas de Combretum leprosum em anéis de artérias de diferentes espécies de animais.Métodos Anéis (3 a 4mm) de artérias de coelho, rato e porco foram montados em cubas para órgão isolado (Krebs, 37°C, 95%O2/5%CO2) para registro das contrações isométricas. Após um período de estabilização (2 a 3 horas), as contrações foram induzidas com fenilefrina (0,1 a 0,3µM) ou U46619 (10 a 100nM); no platô dessas contrações, adicionamos o extrato Combretum leprosum. Diferentes protocolos foram realizados para determinar potência, duração, reversibilidade e mecanismo dos relaxamentos induzidos pelo extrato.Resultados Em todas as preparações testadas, o extrato de Combretum leprosum (1,5µg/mL) provocou relaxamentos dependentes de endotélio. Em aorta torácica de coelho ou rato, os relaxamentos foram revertidos pela vitamina B12a ou L-NG-nitro-arginina. Em anéis de aorta abdominal de coelho e de artérias coronárias de porco, o extrato causou relaxamentos sensíveis e resistentes à L-NG-nitro-arginina. Em aorta torácica de coelho, o extrato foi relativamente muito potente (EC50=0,20μg/mL) e quando causou relaxamentos; intrigantemente o endotélio continuou a produzir fatores relaxantes por um longo período após remoção do extrato. A magnitude dos relaxamentos induzidos pelo extrato foi significativamente reduzida em ausência Ca2+ extracelular; ademais, o vermelho de rutênio (10μM), um bloqueador de canais TRPs, foi capaz de reverter os relaxamentos induzidos pelo extrato. Análises preliminares indicaram que o extrato continha compostos com reatividade química semelhante à polifenóis.Conclusão O extrato de Combretum leprosum contem compostos bioativos capazes de promover estimulação dependente de Ca2+ das células endoteliais a qual resulta numa produção prolongada de fatores relaxantes.


Assuntos
Animais , Feminino , Cobaias , Masculino , Camundongos , Coelhos , Combretum/química , Fatores Relaxantes Dependentes do Endotélio/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Óxido Nítrico/farmacologia , Acetilcolina/farmacologia , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/fisiologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Artéria Carótida Primitiva/efeitos dos fármacos , Artéria Carótida Primitiva/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/fisiologia , Relaxamento Muscular/fisiologia , Casca de Planta/química , Ratos Wistar , Suínos , Fatores de Tempo
11.
Microbes Infect ; 17(4): 247-57, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25576930

RESUMO

Autoantibodies against various components of host are known to occur in leprosy. Nerve damage is the primary cause of disability associated with leprosy. The aim of this study was to detect the level of autoantibodies and lympho-proliferative response against myelin basic protein (MBP) in leprosy patients (LPs) and their correlation with clinical phenotypes of LPs. Further, probable role of molecular mimicry in nerve damage of LPs was investigated. We observed significantly high level of anti-MBP antibodies in LPs across the spectrum and a positive significant correlation between the level of anti-MBP antibodies and the number of nerves involved in LPs. We report here that 4 B cell epitopes of myelin A1 and Mycobacterium leprae proteins, 50S ribosomal L2 and lysyl tRNA synthetase are cross-reactive. Further, M. leprae sonicated antigen hyperimmunization was responsible for induction of autoantibody response in mice which could be adoptively transferred to naive mice. For the first time our findings suggest the role of molecular mimicry in nerve damage in leprosy.


Assuntos
Doenças Desmielinizantes/microbiologia , Hanseníase/microbiologia , Lisina-tRNA Ligase/fisiologia , Mimetismo Molecular/fisiologia , Mycobacterium leprae/patogenicidade , Proteína Básica da Mielina/fisiologia , Proteínas Ribossômicas/fisiologia , Animais , Doenças Desmielinizantes/complicações , Doenças Desmielinizantes/etiologia , Humanos , Hanseníase/complicações , Hanseníase/etiologia , Camundongos , Camundongos Endogâmicos BALB C/sangue , Coelhos
12.
J Physiol Pharmacol ; 65(5): 641-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25371523

RESUMO

The objective of the current study was to check whether countercurrent transfer of dopamine occurs in the cavernous sinus of the rabbit and whether the rabbit can be used as an animal model to study cavernous sinus function. After exsanguination of the animal, oxygenated and warmed (37°C) Hanseneleit-Krebs buffer with autologous or homologous blood (in a 3:1 or 1:1 ratio) was pumped through both common carotid arteries into the head (60 ml/min; 80-100 mm Hg) and radiolabeled dopamine (3(H)-DA, 10 µCi) was infused into the cavernous sinus through the angular oculi vein. Cerebral blood from the basilar artery was collected from the cannulated vertebral artery during 3(H)-DA infusion and for 10 minutes after completion of infusion. Selected brain tissue samples were collected after completion of the head perfusion. It was demonstrated that dopamine can penetrate from the rabbit's cavernous sinus to the internal carotid artery supplying the brain. Dopamine permeation was greater when the rabbit head was perfused with buffer and blood in a 3:1 ratio than with 1:1 (P<0.01). When the head was perfused with buffer and blood in a 3:1 ratio, significant radioactivity was found in samples collected from the brain basilar artery during and after 3(H)-DA infusion (P<0.001). The radioactivity was identified as 34.13 ± 2.7% unmetabolized 3(H)-DA and 65.9 ± 2.7% its metabolites. Significant radioactivity was also found in some brain tissue samples in both groups (P<0.05). The concentration of free radiolabeled dopamine particles in the dialysate of blood plasma and plasma diluted with buffer did not differ significantly. Because the structures of the cavernous sinus and cavernous fragment of the internal carotid artery of the rabbit are similar to those in humans, it suggests that rabbits can serve as a model for experimental physiological studies of cavernous sinus function and retrograde dopamine transfer in the cavernous sinus should be considered as an important link in the genesis of Attention Deficit Hyperactivity Disorder (ADHD) and Parkinson's disease.


Assuntos
Encéfalo/metabolismo , Seio Cavernoso/metabolismo , Dopamina/farmacologia , Animais , Encéfalo/irrigação sanguínea , Artérias Carótidas/metabolismo , Artérias Cerebrais/metabolismo , Dopamina/sangue , Cabeça , Masculino , Perfusão , Coelhos
13.
J Biol Chem ; 289(49): 33850-61, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25301946

RESUMO

Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265-2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca(2+) but increased the Ca(2+)-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.


Assuntos
Cisteína/química , Proteínas Musculares/química , Músculo Esquelético/metabolismo , Ácido Oleico/química , Ácido Palmítico/química , Fenilalanina/química , Processamento de Proteína Pós-Traducional , Proteolipídeos/química , Sequência de Aminoácidos , Animais , Evolução Biológica , Cristalografia por Raios X , Cisteína/metabolismo , Expressão Gênica , Hidroxilamina/química , Cinética , Lipoilação , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/classificação , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Fenilalanina/metabolismo , Filogenia , Proteolipídeos/classificação , Proteolipídeos/genética , Proteolipídeos/metabolismo , Coelhos , Retículo Sarcoplasmático , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Suínos , Termodinâmica
14.
PLoS One ; 9(8): e106222, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25170947

RESUMO

BACKGROUND: The diagnosis of leprosy is primarily based on clinical manifestations, and there is no widely available laboratory test for the early detection of this disease, which is caused by Mycobacterium leprae. In fact, early detection and treatment are the key elements to the successful control of leprosy. METHODOLOGY/PRINCIPAL FINDINGS: Peptide ligands for antibodies from leprosy patients were selected from phage-displayed peptide libraries. Three peptide sequences expressed by reactive phage clones were chemically synthesized. Serological assays that used synthetic peptides were evaluated using serum samples from leprosy patients, household contacts (HC) of leprosy patients, tuberculosis patients and endemic controls (EC). A pool of three peptides identified 73.9% (17/23) of multibacillary (MB) leprosy patients using an enzyme-linked immunosorbent assay (ELISA). These peptides also showed some seroreactivities to the HC and EC individuals. The peptides were not reactive to rabbit polyclonal antisera against the different environmental mycobacteria. The same peptides that were conjugated to the carrier protein bovine serum albumin (BSA) induced the production of antibodies in the mice. The anti-peptide antibodies that were used in the Western blotting analysis of M. leprae crude extracts revealed a single band of approximately 30 kDa in one-dimensional electrophoresis and four 30 kDa isoforms in the two-dimensional gel. The Western blotting data indicated that the three peptides are derived from the same bacterial protein. CONCLUSIONS/SIGNIFICANCE: These new antigens may be useful in the diagnosis of MB leprosy patients. Their potentials as diagnostic reagents must be more extensively evaluated in future studies using a large panel of positive and negative sera. Furthermore, other test approaches using peptides should be assessed to increase their sensitivity and specificity in detecting leprosy patients. We have revealed evidence in support of phage-displayed peptides as promising biotechnological tools for the design of leprosy diagnostic serological assays.


Assuntos
Anticorpos Antibacterianos/sangue , Hanseníase/sangue , Hanseníase/diagnóstico , Mycobacterium leprae , Biblioteca de Peptídeos , Animais , Anticorpos Antibacterianos/química , Bovinos , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Coelhos
15.
J Virol Methods ; 193(2): 548-53, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23911294

RESUMO

Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.


Assuntos
Proteínas do Capsídeo/imunologia , Citrus/virologia , Testes Imunológicos/métodos , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Animais , Anticorpos Antivirais , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Códon/genética , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Expressão Gênica , Vírus de Plantas/imunologia , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
16.
Cell Immunol ; 278(1-2): 63-75, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23121977

RESUMO

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.


Assuntos
Proteínas de Bactérias/química , Chaperonina 60/química , Queratina-10/química , Hanseníase/imunologia , Hanseníase/prevenção & controle , Mycobacterium leprae/imunologia , Transferência Adotiva , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Proteínas de Bactérias/imunologia , Estudos de Casos e Controles , Chaperonina 60/imunologia , Reações Cruzadas , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica , Humanos , Imunização , Queratina-10/imunologia , Hanseníase/microbiologia , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Mimetismo Molecular , Coelhos , Índice de Gravidade de Doença , Pele/imunologia , Pele/microbiologia , Baço/citologia , Baço/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/microbiologia
17.
J Parasitol ; 98(5): 977-84, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22559265

RESUMO

Four specimens of wild leporids, Lepus capensis Linnaeus, 1758, in Longde County, Ningxia Hui Autonomous Region (NXHAR), and 40 domestic rabbits, Oryctolagus cuniculus (Linnaeus, 1758) (20 from Xiji County, NXHAR, and 20 from Jingning County, Gansu Province) were examined for the presence of helminth parasites. Two new cestode species of Schizorchis Hansen, 1948 (Anoplocephalidae), were found: Schizorchis sinensis sp. n. from L. capensis and Schizorchis oryctolagi sp. n. from domestic rabbits. Schizorchis sinensis sp. n. is distinguished from all the species in the genus from pikas, Ochotona spp. (Lagomorpha: Ochotonidae) by having much longer strobila, a larger cirrus sac, a greater number of segments, and more numerous testes. The morphology of S. oryctolagi n. sp. is most similar to that of S. sinensis. It is distinguished from the latter species by its relatively small strobila, larger number of segments, smaller size of eggs, and much larger cirrus sac, which is substantially extending in a medial direction. In addition, a genital papilla is present in S. sinensis , but absent in S. oryctolagi. These findings indicate that species of Schizorchis are widespread in leporid mammals, contrary to the widespread opinion that the latter genus is specific to ochotonid lagomorph mammals. They are congruent, however, with the hypothesis of a close phylogenetic relationship between species of Schizorchis and Mosgovoyia. The occurrence of Schizorchis spp. in leporids is considered relict, vestigial from wider geographical and host ranges of this parasite genus during the Pleistocene.


Assuntos
Cestoides/classificação , Infecções por Cestoides/veterinária , Lagomorpha/parasitologia , Animais , Cestoides/anatomia & histologia , Cestoides/isolamento & purificação , Infecções por Cestoides/parasitologia , China , Coelhos
19.
J Drugs Dermatol ; 10(3): 274-8, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21369644

RESUMO

Erythema nodosum leprosum (ENL) is an inflammatory reaction that may occur in multibacillary leprosy patients, and thalidomide is the treatment of choice. Its cause and the mechanism by which thalidomide suppresses ENL are not known. In the skin lesions, im- mune complexes and split products of complement are found. The activation of complement could precipitate ENL, and thalidomide could suppress the inflammation by inhibiting the activation of complement. To determine if thalidomide could suppress the activation of complement, we first incubated normal serum with thalidomide and with M. leprae or zymosan. The amount of residual functional complement was then assessed by determining the dilution of serum required to lyses sheep erythrocytes sensitized by rabbit antibodies (CH50 Assay). M. leprae and zymosan activated complement. The residual complement activity in the serum incubated with M. leprae or with zymosan was equivalent to that incubated with M. leprae or zymosan in the presence of thalidomide, hydrolyzed thalidomide and metabolites of thalidomide. Thalidomide did not inhibit the activation of complement by zymosan, a known initiator of complement activation by the alternative pathway, or by M. leprae.


Assuntos
Ativação do Complemento/efeitos dos fármacos , Eritema Nodoso/tratamento farmacológico , Hansenostáticos/farmacologia , Hanseníase Virchowiana/tratamento farmacológico , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/imunologia , Talidomida/farmacologia , Animais , Proteínas do Sistema Complemento/análise , Eritema Nodoso/imunologia , Eritema Nodoso/fisiopatologia , Humanos , Hanseníase Virchowiana/imunologia , Hanseníase Virchowiana/fisiopatologia , Fígado/enzimologia , Camundongos , Mycobacterium leprae/metabolismo , Coelhos , Ovinos
20.
Int J Dermatol ; 50(3): 255-61, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21342156

RESUMO

An animal model of granulomatous hypersensitivity has been developed, which reproduces some features of the pathologies of important chronic granulomatous disorders, including tuberculosis, tuberculoid leprosy, sarcoidosis, berylliosis, Crohn's disease, and sensitivity to zirconium. The lesions consist of focal collections of epithelioid cells surrounded by lymphocytes to form tubercles. The epithelioid cell has a secretory function and is not phagocytic. Plasmacytoid dendritic cells are precursors of epithelioid cells, which are therefore part of the innate immune system. Subplasmalemmal linear densities are also present in these cells. This autoimmune model has been induced in rabbits using a non-myelin sensory peripheral antigen to reproduce the features of tuberculoid leprosy. The antigen is probably present only in human tissue. A granuloma antigen, which is tissue specific similar to that in peripheral nerves, could be present in sarcoidosis and Crohn's disease. In multiple sclerosis, mononuclear cells in the brain parenchyma are not phagocytic and are therefore similar to epithelioid cells. The induction of tolerance leading to the development of a vaccine to prevent the lesions in multiple sclerosis, sarcoidosis, and Crohn's disease is possible after purification of the granuloma antigen.


Assuntos
Modelos Animais de Doenças , Granuloma , Hipersensibilidade , Hanseníase Tuberculoide , Coelhos , Animais , Células Epitelioides/imunologia , Células Epitelioides/patologia , Células Epitelioides/fisiologia , Granuloma/imunologia , Granuloma/patologia , Granuloma/fisiopatologia , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Hipersensibilidade/fisiopatologia , Hanseníase Tuberculoide/imunologia , Hanseníase Tuberculoide/patologia , Hanseníase Tuberculoide/fisiopatologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA