RESUMEN
BACKGROUND: Morphea (localized scleroderma) and eosinophilic fasciitis (EF) are rare fibrosing disorders which may present a diagnostic challenge. While histopathologic features are often distinct, in some cases there may be overlap. T-cells contribute to etiopathogenesis of both autoimmune conditions. We sought to determine whether T-cell immune polarization enables histopathologic distinction. MATERIALS & METHODS: We retrospectively examined clinicopathologically confirmed cases of morphea (n = 12) and EF (n = 8) using immunohistochemistry for CD3, CD8, and dual staining for CD4 with T-bet, GATA-3, STAT-3 or BNC-2 (transcription factors reported to be specific and mutually exclusive for Th1, Th2, Th17 and Th22 cells, respectively) to characterize the T-cell infiltrate. RESULTS: No significant difference in CD3+ cells was identified (P = .195), however, the CD4/CD8+ T-cell ratio was significantly greater in morphea compared to EF (1.2 and 0.6, respectively; P = .034). Th1/Th2 was significantly lower in morphea compared to EF (1.7 and 2.7, respectively; P = .027). The percent of Th17+ cells was significantly higher in EF (P = 0.041). No significant difference in percent of Th22+ cells was identified. CONCLUSION: Morphea and EF may be histopathologically distinguished based on helper T-cell subtype polarization. These findings offer novel insight into our understanding of disease pathogenesis and support a role for Th1/Th2 immune regulation and Th17 inhibition in anti-fibrotic therapeutic strategy.
Asunto(s)
Antígenos de Diferenciación/inmunología , Eosinofilia , Fascitis , Esclerodermia Localizada , Células TH1 , Células Th17 , Adulto , Anciano , Anciano de 80 o más Años , Eosinofilia/inmunología , Eosinofilia/patología , Fascitis/inmunología , Fascitis/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Esclerodermia Localizada/inmunología , Esclerodermia Localizada/patología , Células TH1/inmunología , Células TH1/patología , Células Th17/inmunología , Células Th17/patologíaRESUMEN
In this study we investigated why bloodstream forms of Trypanosoma brucei gambiense cross human brain microvascular endothelial cells (BMECs), a human blood-brain barrier (BBB) model system, at much greater efficiency than do T. b. brucei. After noting that T. b. gambiense displayed higher levels of cathepsin L-like cysteine proteases, we investigated whether these enzymes contribute to parasite crossing. First, we found that T. b. gambiense crossing of human BMECs was abrogated by N-methylpiperazine-urea-Phe-homopheylalanine-vinylsulfone-benzene (K11777), an irreversible inhibitor of cathepsin L-like cysteine proteases. Affinity labeling and immunochemical studies characterized brucipain as the K11777-sensitive cysteine protease expressed at higher levels by T. b. gambiense. K11777-treated T. b. gambiense failed to elicit calcium fluxes in BMECs, suggesting that generation of activation signals for the BBB is critically dependant on brucipain activity. Strikingly, crossing of T. b. brucei across the BBB was enhanced upon incubation with brucipain-rich supernatants derived from T. b. gambiense. The effects of the conditioned medium, which correlated with ability to evoke calcium fluxes, were canceled by K11777, but not by the cathepsin B inhibitor CA074. Collectively, these in vitro studies implicate brucipain as a critical driver of T. b. gambiense transendothelial migration of the human BBB.
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Señalización del Calcio/fisiología , Movimiento Celular/fisiología , Cisteína Endopeptidasas/metabolismo , Trypanosoma/enzimología , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/parasitología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Endoteliales/parasitología , Estrenos/farmacocinética , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Naftalenos/farmacología , Fenilalanina/análogos & derivados , Piperazinas , Proteínas Protozoarias/metabolismo , Pirrolidinonas/farmacocinética , Compuestos de Tosilo , Trypanosoma/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei gambiense/enzimología , Trypanosoma brucei gambiense/metabolismo , Trypanosoma brucei rhodesiense/enzimología , Trypanosoma brucei rhodesiense/metabolismo , Compuestos de Vinilo/farmacologíaRESUMEN
OBJECTIVE: Where human African trypanosomiasis (HAT) patients are seen, failure to microscopically diagnose infections by Trypanosoma brucei gambiense in blood smears and/or cerebrospinal fluid (CSF) in the critical early stages of the disease is the single most important factor in treatment failure, a result of delayed treatment onset or its absence. We hypothesized that the enhanced sensitivity of detergent-enhanced loop-mediated isothermal amplification (LAMP) will allow for point of care (POC) detection of African trypanosomes in the CSF of HAT patients where the probability for detecting a single parasite or parasite DNA molecule in 1 µL of CSF sample is negligible by current methods. METHODOLOGY: We used LAMP targeting the multicopy pan-T. brucei repetitive insertion mobile element (RIME LAMP) and the Trypanosoma brucei gambiense 5.8S rRNA-internal transcribed spacer 2 gene (TBG1 LAMP). We tested 1 µL out of 20 µL sham or Triton X-100 treated CSFs from 73 stage-1 and 77 stage-2 HAT patients from the Central African Republic and 100 CSF negative controls. RESULTS: Under sham conditions, parasite DNA was detected by RIME and TBG1 LAMP in 1.4% of the stage-1 and stage-2 gambiense HAT CSF samples tested. After sample incubation with detergent, the number of LAMP parasite positive stage-2 CSF's increased to 26%, a value which included the 2 of the 4 CSF samples where trypanosomes were identified microscopically. Unexpected was the 41% increase in parasite positive stage-1 CSF's detected by LAMP. Cohen's kappa coefficients for RIME versus TBG1 LAMP of 0.92 (95%CI: 0.82-1.00) for stage-1 and 0.90 (95%CI: 0.80-1.00) for stage-2 reflected a high level of agreement between the data sets indicating that the results were not due to amplicon contamination, data confirmed in χ2 tests (p<0.001) and Fisher's exact probability test (p = 4.7e-13). CONCLUSION: This study detected genomic trypanosome DNA in the CSF independent of the HAT stage and may be consistent with early CNS entry and other scenarios that identify critical knowledge gaps for future studies. Detergent-enhanced LAMP could be applicable for non-invasive African trypanosome detection in human skin and saliva or as an epidemiologic tool for the determination of human (or animal) African trypanosome prevalence in areas where chronically low parasitemias are present.
Asunto(s)
Líquido Cefalorraquídeo/parasitología , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Índice de Severidad de la Enfermedad , Trypanosoma/aislamiento & purificación , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , República Centroafricana , Niño , Preescolar , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/genética , Detergentes/metabolismo , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , ARN Ribosómico 5.8S/genética , Sensibilidad y Especificidad , Trypanosoma/genética , Adulto JovenRESUMEN
The loop-mediated isothermal amplification (LAMP) assay with its advantages of cost effectiveness, rapidity, and simplicity, has evolved as a sensitive and specific method for the detection of African trypanosomes. Highly sensitive LAMP reactions specific for Trypanosoma brucei rhodesiense or that recognize but do not discriminate between Trypanosoma brucei brucei, T. b. rhodesiense, Trypanosoma brucei gambiense, and Trypanosoma evansi have been developed. A sensitive LAMP assay targeting the T. b. gambiense 5.8S ribosomal RNA internal transcribed spacer 2 (5.8S-ITS2) gene is also available but this assay does not target binding sites that span the CCCA (C3A) (557-560 bps) insertion site that further differentiates T. b. gambiense from T. b. brucei Here we describe 5.8S-ITS2-targeted LAMP assay that fit these criteria. The LAMP primer sets containing the T. b. gambiense-specific C3A tetranucleotide at the start of the outer forward primer sequences showed high specificity and sensitivity down to at least 0.1 fg T. b. gambiense genomic DNA.
Asunto(s)
Genes Protozoarios/genética , ARN Ribosómico 5.8S/genética , Trypanosoma brucei gambiense/genética , Tripanosomiasis Africana/diagnóstico , Diagnóstico Precoz , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Tripanosomiasis Africana/parasitologíaRESUMEN
Using an in vitro model of the human blood-brain barrier consisting of human brain microvascular endothelial cells we recently demonstrated that Trypanosoma brucei gambiense bloodstream-forms efficiently cross these cells via a paracellular route while Trypanosoma brucei brucei crosses these cells poorly. Using a combination of techniques that include fluorescence activated cell sorting, confocal and electron microscopy, we now show that some T.b. gambiense blood stream form parasites have the capacity to enter human brain microvascular endothelial cells. The intracellular location of the trypanosomes was demonstrated in relation to the endothelial cell plasma membrane and to the actin cytoskeleton. These parasites may be a terminal stage within a lysosomal compartment or they may be viable trypanosomes that will be able to exit the brain microvascular endothelial cells. This process may provide an additional transcellular route by which the parasites cross the blood-brain barrier.
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Barrera Hematoencefálica/parasitología , Endotelio Vascular/parasitología , Trypanosoma brucei gambiense/fisiología , Tripanosomiasis Africana/parasitología , Animales , Barrera Hematoencefálica/ultraestructura , Encéfalo/irrigación sanguínea , Células Cultivadas , Infecciones Protozoarias del Sistema Nervioso Central/parasitología , Infecciones Protozoarias del Sistema Nervioso Central/patología , Células Endoteliales/parasitología , Células Endoteliales/ultraestructura , Endotelio Vascular/ultraestructura , Interacciones Huésped-Parásitos , Humanos , Microcirculación/parasitología , Microscopía Confocal , Trypanosoma brucei gambiense/aislamiento & purificación , Tripanosomiasis Africana/patologíaRESUMEN
Targeted therapies for pulmonary adenocarcinoma (ACA) necessitate specific subtyping and molecular testing of non-small cell lung carcinomas (NSCLC). However, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has decreased the tissue available for these assessments. While EBUS-TBNA specimens have previously been reported to successfully subtype NSCLC, allow immunohistochemistry (IHC), and support molecular diagnostics, no studies have documented the extent to which all objectives are possible in a single sample. Of 107 consecutive EBUS-TBNA specimens that were eligible for molecular testing, 98.8% had enough tissue for IHC, 80.2% received a definitive subtype, and 71.0% had both sufficient tissue to attempt molecular testing and technical success on multigene next-generation sequencing and ALK fluorescence in situ hybridization assays. Both subtyping and molecular diagnostics were possible in 57.9% of patients. The mean number of immunostains performed did not differ between patients with or without successful molecular testing (4.4 versus 4.6, P = .88). Only 40% of patients with insufficient tissue underwent repeat sampling. These findings indicate that a majority of EBUS-TBNA specimens provide sufficient tissue for subtyping pulmonary NSCLC, performing IHC, and completing multiple gene analyses. Although priorities must be assessed for each case individually, performance of IHC does not detract from completion of molecular diagnostics in general. Because most patients never undergo repeat sampling, the tissue yield of EBUS-TBNA should be improved to maximize evaluation for targeted therapies.
Asunto(s)
Biopsia con Aguja/métodos , Broncoscopía/métodos , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Ultrasonografía Intervencional/métodos , Adulto , Anciano , Anciano de 80 o más Años , Biopsia con Aguja Fina , Carcinoma de Pulmón de Células no Pequeñas/genética , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/genética , Masculino , Microdisección , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a "LAMPole") that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Inmunoglobulina G/sangre , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Humanos , RatonesRESUMEN
PURPOSE: To report the clinical and immunopathologic features and the response to therapy in a series of six patients with cicatrizing conjunctivitis due to lichen planus. DESIGN: Retrospective case series. METHODS: All six patients were seen in an ocular pemphigoid clinic. Clinical, immunopathologic, and serologic features were evaluated and therapeutic response in each patient was monitored. RESULTS: All six patients had evidence of conjunctival scarring. Five patients had lichen planus of the oral mucosa and gingiva; one patient had involvement of the skin. Histologic findings consisted of thickened epithelium and an interface lymphocytic infiltrate along the lamina propria. In three patients, electron microscopy of the conjunctiva revealed thickening, fragmentation, and duplication of the basement membrane zone. Direct immunofluorescence examination of the conjunctiva and oral mucosa demonstrated linear and shaggy fibrinogen deposition along the basement membrane zone, confirming the diagnosis of lichen planus. All six patients were placed on immunosuppressive therapy with control of the disease. However, only one patient was able to discontinue the anti-inflammatory medication and have the lichen planus remain in remission. CONCLUSIONS: Lichen planus should be included in the differential diagnosis of cicatrizing conjunctivitis. Performing appropriate investigations to distinguish conjunctival lichen planus from other autoimmune diseases such as mucous membrane pemphigoid is critical to managing the patient with cicatrizing conjunctivitis appropriately. Oral cyclosporine effectively controlled the conjunctival lichen planus in four of the six cases.
Asunto(s)
Cicatriz/etiología , Conjuntivitis/etiología , Liquen Plano/complicaciones , Administración Oral , Anciano , Anciano de 80 o más Años , Membrana Basal/metabolismo , Cicatriz/diagnóstico , Cicatriz/tratamiento farmacológico , Conjuntivitis/diagnóstico , Conjuntivitis/tratamiento farmacológico , Ciclosporina/uso terapéutico , Femenino , Fibrinógeno/metabolismo , Técnica del Anticuerpo Fluorescente Directa , Humanos , Inmunosupresores/uso terapéutico , Liquen Plano/diagnóstico , Liquen Plano/tratamiento farmacológico , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
BACKGROUND: Anti-desmoplakin (DP) antibodies are present in paraneoplastic pemphigus (PNP) as a component of a complex humoral autoimmune reaction characterized by antibodies against proteins of the plakin family, desmogleins, and an unidentified 170 kd protein. Anti-DP antibodies have also been rarely identified in other blistering diseases. The significance of anti-DP antibodies in the pathogenesis of bullous diseases is unclear. OBSERVATION: We studied 3 patients with severe and chronic mucosal dominant pemphigus vulgaris (PV). In addition to anti-desmoglein 3 antibodies, these patients had anti-DP autoantibodies, demonstrable by immunofluorescence (IF), immunoprecipitation (IP), and indirect immunoelectromicroscopy (IIEM). This finding suggested these patients may have had PNP and not PV. However, antibodies against periplakin, envoplakin, bullous pemphigoid antigen 1 (BPAG 1), plectin, and 170 kd PNP antigen could not be detected using IP and immunoblotting. Extensive and repeated investigations for an underlying neoplasm throughout the follow-up period were consistently negative for all patients. CONCLUSION: This study demonstrates that anti-DP antibodies without the presence of any other anti-plakin antibodies are not specific for PNP, and are present in some cases of PV. Cellular disadhesion induced by anti-desmoglein antibodies can trigger an epitope-spreading phenomenon with a secondary formation of autoantibodies against desmoplakins, intracellular desmosomal antigens. The role of anti-DP antibodies in the pathogenesis of these PV patients is still unclear. The presence of anti-DP antibodies will produce a false positive serologic interpretation for the diagnosis of PNP especially if one uses only indirect IF on murine bladder, the most commonly employed screening test to identify PNP. More specific immunologic tests are required in this subset of patients with PV.
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Autoanticuerpos/análisis , Enfermedades Autoinmunes/inmunología , Proteínas del Citoesqueleto/inmunología , Pénfigo/inmunología , Autoantígenos/análisis , Cadherinas/inmunología , Niño , Desmogleína 3 , Desmogleínas , Desmoplaquinas , Desmosomas , Femenino , Humanos , Pruebas Inmunológicas , Masculino , Persona de Mediana Edad , Membrana MucosaRESUMEN
The trypanocidal effects of aqueous gold(III) and palladium(II) and their metalloporphyrin derivatives on Trypanosoma brucei brucei growth in culture have been studied using an Alamar Blue indicator assay. All the experiments were conducted in the dark. As previously described for mercury(II), cadmium(II) and lead(II) porphyrins [Chem.-Biol. Interact. 139 (2002) 177], the toxicity of the metalloporphyrin complex of palladium(II) to T. b. brucei parasites was much higher compared to the aqueous free palladium(II) and free base porphyrin. Palladium(II) porphyrin, free palladium(II), and the free base porphyrin were trypanocidal to T. b. brucei at concentrations >1.5 x 10(-6), >6.1 x 10(-6) and >1.9 x 10(-5) M, respectively. While gold(III) porphyrin was effective against the parasites at concentrations >4.8 x 10(-6) M, its aqueous gold(III) was toxic at concentrations as low as 2.0 x 10(-7) M due to the generation of free radicals in the presence of this metal ion which enhanced its toxicity to the T. b. brucei parasites. Although some cell division was observed in some of the cells treated with palladium(II) porphyrin, some dividing cells had no nucleus due to unequal division and delivery of the nuclei into the daughter cells. As a result, the rate of cell division decreased with time and cell death occurred within 24 h. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. Of all the porphyrins and free metal ions tested, only mercury(II) porphyrin and aqueous gold(III) ion were toxic to the trypanosomes in the 10(-7) M range. The chemotherapeutic potential of these observations is discussed.
Asunto(s)
Antiprotozoarios/toxicidad , Compuestos de Oro/toxicidad , Metaloporfirinas/toxicidad , Paladio/toxicidad , Trypanosoma brucei brucei/efectos de los fármacos , Animales , Antiprotozoarios/química , División Celular/efectos de los fármacos , Compuestos de Oro/química , Técnicas In Vitro , Concentración 50 Inhibidora , Longevidad/efectos de los fármacos , Metaloporfirinas/química , Paladio/química , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
BACKGROUND: Discoid lupus erythematosus (DLE) demonstrates both cutaneous and mucosal manifestations. Mucosal involvement is typically limited to the oral and anogenital mucosa. Conjunctival involvement in DLE is rare, especially in the absence of accompanying cutaneous disease. OBJECTIVE: We describe the clinical and immunopathologic features of two cases of cicatrizing conjunctivitis due to DLE. METHODS: In each patient, the clinical, immunopathologic, and serologic features were reviewed and the results recorded. RESULTS: Both patients presented with cicatrizing conjunctivitis suggestive of mucous membrane pemphigoid. Patient 1 had no history of typical DLE skin lesions. Patient 2 had a history of cutaneous and oral DLE prior to presentation. Histologic, electron microscopic, and direct immunofluorescence examination confirmed the diagnosis DLE in both patients. CONCLUSION: In patients presenting with cicatrizing conjunctivitis, DLE should be considered in the differential diagnosis. Performing appropriate investigations to distinguish conjunctival DLE from other autoimmune diseases with conjunctival involvement such as mucous membrane pemphigoid is critical in selecting an appropriate treatment regimen, in offering an accurate prognosis, and in monitoring for signs and symptoms of disease progression.
Asunto(s)
Cicatriz/etiología , Conjuntivitis/etiología , Lupus Eritematoso Discoide/complicaciones , Adulto , Anciano , Conjuntivitis/complicaciones , Diagnóstico Diferencial , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Lupus Eritematoso Discoide/diagnóstico , Lupus Eritematoso Discoide/metabolismo , Lupus Eritematoso Discoide/patología , Microscopía Electrónica , Pruebas SerológicasRESUMEN
The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.
Asunto(s)
Barrera Hematoencefálica/parasitología , Células Endoteliales/parasitología , Trypanosoma brucei brucei/fisiología , Trypanosoma brucei gambiense/fisiología , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/ultraestructura , Calcio/metabolismo , Línea Celular , Impedancia Eléctrica , Células Endoteliales/ultraestructura , Humanos , Técnicas In Vitro , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , TransfecciónRESUMEN
Malaria (Plasmodium spp.) and human African trypanosomiasis (Trypanosoma brucei spp.) are vector borne, deadly parasitic diseases. While chemotherapeutic agents for both diseases are available, difficulty in disease eradication and development of drug resistance require that new therapies targeting unexplored pathways or exploiting novel modes of action be developed. Intracellular Plasmodium and extracellular Trypanosoma brucei may have unique and essential requirements for divalent metal ions, beyond that deemed physiological for the host. Membrane Active Chelators (MACs), biologically active only in a hydrophobic lipid environment, are able to bind metal ions at elevated non-physiological concentrations in the vicinity of cell membranes. A dose-response relationship study using validated viability assays revealed that two MAC drugs, DP-b99 and DP-460, were cytotoxic for these parasites in vitro. The 50% effective concentration (EC50) values for DP-b99 and DP-460 were 87 µM and 39 µM for Trypanosoma brucei brucei and 21 µM and 28 µM for erythrocytic Plasmodium falciparum, respectively. Furthermore, drug potency was maintained for at least 24h in serum containing medium at 37°C. While the exact mechanism of action of MACs against intracellular malaria and extracellular African trypanosome parasites has yet to be determined, their potential as antiparasitic agents warrants further investigation.
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Antimaláricos/farmacología , Ácido Egtácico/análogos & derivados , Plasmodium/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma/efectos de los fármacos , Animales , Ácido Egtácico/química , Ácido Egtácico/farmacología , Estructura MolecularRESUMEN
Human African trypanosomiasis or sleeping sickness is a vector-borne parasitic disease that has a major impact on human health and welfare in sub-Saharan countries. Based mostly on data from animal models, it is currently thought that trypanosome entry into the brain occurs by initial infection of the choroid plexus and the circumventricular organs followed days to weeks later by entry into the brain parenchyma. However, Trypanosoma brucei bloodstream forms rapidly cross human brain microvascular endothelial cells in vitro and appear to be able to enter the murine brain without inflicting cerebral injury. Using a murine model and intravital brain imaging, we show that bloodstream forms of T. b. brucei and T. b. rhodesiense enter the brain parenchyma within hours, before a significant level of microvascular inflammation is detectable. Extravascular bloodstream forms were viable as indicated by motility and cell division, and remained detectable for at least 3 days post infection suggesting the potential for parasite survival in the brain parenchyma. Vascular inflammation, as reflected by leukocyte recruitment and emigration from cortical microvessels, became apparent only with increasing parasitemia at later stages of the infection, but was not associated with neurological signs. Extravascular trypanosomes were predominantly associated with postcapillary venules suggesting that early brain infection occurs by parasite passage across the neuroimmunological blood brain barrier. Thus, trypanosomes can invade the murine brain parenchyma during the early stages of the disease before meningoencephalitis is fully established. Whether individual trypanosomes can act alone or require the interaction from a quorum of parasites remains to be shown. The significance of these findings for disease development is now testable.
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Encéfalo/parasitología , Trypanosoma brucei brucei/fisiología , Animales , Sangre/parasitología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/parasitología , Encéfalo/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Especificidad de la Especie , Factores de Tiempo , Trypanosoma brucei brucei/metabolismoRESUMEN
BACKGROUND: The loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 10(3) per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay. METHODOLOGY/PRINCIPAL FINDINGS: For proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 10(3) parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards. CONCLUSIONS/SIGNIFICANCE: This simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.
Asunto(s)
ADN Protozoario/sangre , ADN Protozoario/líquido cefalorraquídeo , Detergentes/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Trypanosoma brucei rhodesiense/aislamiento & purificación , Tripanosomiasis Africana/parasitología , Animales , Cartilla de ADN , Electroforesis en Gel de Agar , Humanos , Sensibilidad y Especificidad , Trypanosoma brucei rhodesiense/genética , Tripanosomiasis Africana/sangre , Tripanosomiasis Africana/líquido cefalorraquídeoRESUMEN
BACKGROUND: Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Galpha(q) with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. CONCLUSIONS/SIGNIFICANCE: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q)-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.
Asunto(s)
Células Endoteliales/parasitología , Receptor PAR-2/fisiología , Trypanosoma brucei rhodesiense/patogenicidad , Animales , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Línea Celular , Silenciador del Gen , Humanos , ARN Interferente Pequeño/genética , Receptor PAR-2/antagonistas & inhibidores , Receptor PAR-2/genética , Transducción de SeñalRESUMEN
Borrelia burgdorferi and Anaplasma phagocytophilum coinfect and are transmitted by Ixodes species ticks. Clinical indicators suggest that A. phagocytophilum coinfection contributes to the severity, dissemination, and, possibly, sequelae of Lyme disease. Previous in vitro studies showed that spirochete penetration through human brain microvascular endothelial cells of the blood-brain barrier is facilitated by endothelial cell-derived matrix metalloproteases (MMPs). A. phagocytophilum-infected neutrophils continuously release MMPs and other vasoactive biomediators. We examined B. burgdorferi infection of brain microvascular barriers during A. phagocytophilum coinfection and showed that coinfection enhanced reductions in transendothelial electrical resistance and enhanced or synergistically increased production of MMPs (MMP-1, -3, -7, -8, and -9), cytokines (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), and chemokines (IL-8 and macrophage inflammatory protein 1alpha) known to affect vascular permeability and inflammatory responses.
Asunto(s)
Anaplasma phagocytophilum/fisiología , Borrelia burgdorferi/fisiología , Citocinas/biosíntesis , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Endotelio Vascular/citología , Metaloproteinasas de la Matriz/biosíntesis , Barrera Hematoencefálica , Encéfalo , Línea Celular , Células Endoteliales/inmunología , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiología , Humanos , Enfermedad de Lyme/metabolismo , Enfermedad de Lyme/microbiología , Neutrófilos/microbiologíaRESUMEN
Neurological manifestations of Lyme disease in humans are attributed in part to penetration of the blood-brain barrier (BBB) and invasion of the central nervous system (CNS) by Borrelia burgdorferi. However, how the spirochetes cross the BBB remains an unresolved issue. We examined the traversal of B. burgdorferi across the human BBB and systemic endothelial cell barriers using in vitro model systems constructed of human brain microvascular endothelial cells (BMEC) and EA.hy 926, a human umbilical vein endothelial cell (HUVEC) line grown on Costar Transwell inserts. These studies showed that B. burgdorferi differentially crosses human BMEC and HUVEC and that the human BMEC form a barrier to traversal. During the transmigration by the spirochetes, it was found that the integrity of the endothelial cell monolayers was maintained, as assessed by transendothelial electrical resistance measurements at the end of the experimental period, and that B. burgdorferi appeared to bind human BMEC by their tips near or at cell borders, suggesting a paracellular route of transmigration. Importantly, traversal of B. burgdorferi across human BMEC induces the expression of plasminogen activators, plasminogen activator receptors, and matrix metalloproteinases. Thus, the fibrinolytic system linked by an activation cascade may lead to focal and transient degradation of tight junction proteins that allows B. burgdorferi to invade the CNS.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Borrelia burgdorferi/metabolismo , Enfermedad de Lyme/metabolismo , Péptido Hidrolasas/metabolismo , Barrera Hematoencefálica/microbiología , Células Endoteliales/metabolismo , Humanos , Enfermedad de Lyme/microbiologíaRESUMEN
BACKGROUND: Sarcoidosis is a chronic granulomatous disease of unknown etiology. Cutaneous disease is common and includes two clinicopathologic categories: granulomatous infiltration or a reactive phenomenon. In the granulomatous infiltrative group, clinical manifestations can be variable. Ulcers in sarcoidosis are uncommonly recognized and have been categorized previously under the rubric of atrophic, necrobiosis-like, or ulcerative sarcoidosis. PATIENTS AND METHODS: We evaluated retrospectively sarcoidosis patients presenting to the Johns Hopkins Department of Dermatology between June 1989 and May 2002. Multiple skin biopsies were performed for histopathologic evaluation. Investigation for extracutaneous manifestations, including routine serologic assays, chest radiography, pulmonary function tests, electrocardiogram, and angiotensin-converting enzyme level, and referral for ophthalmologic examination were performed in all patients. RESULTS: Of 147 consecutive patients presenting with cutaneous sarcoidosis, seven demonstrated ulcerative-atrophic sarcoidosis lesions. All patients were African-American (five females and two males). All patients had ulcers surrounded by atrophic necrobiosis lipoidica-like plaques on the pretibial areas. All patients had other mucocutaneous manifestations of sarcoidosis, with the majority having evidence of internal disease. Combined immunosuppressive and immunomodulatory therapy was effective in controlling the cutaneous manifestations of all patients with ulcerative sarcoidosis. CONCLUSIONS: The ulcerative variant is a poorly defined subset of cutaneous sarcoidosis. Trauma, superimposed on atrophic plaques, appears to be the principal mechanism of this rare variant of cutaneous sarcoidosis.