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BACKGROUND: Many insect-borne pathogens appear to manipulate the odors of their hosts in ways that influence vector behaviors. In our prior work, we identified characteristic changes in volatile emissions of cultured Plasmodium falciparum parasites in vitro and during natural human falciparum malaria. In the current study, we prospectively evaluate the reproducibility of these findings in an independent cohort of children in Blantyre, Malawi. METHODS: We enrolled febrile children under evaluation for malaria and collected breath from children with and without malaria, as well as healthy controls. Using gas-chromatography/mass spectrometry, we characterized breath volatiles associated with malaria. By repeated sampling of children with malaria before and after antimalarial use, we determined how breath profiles respond to treatment. In addition, we investigated the stage-specificity of biomarkers through correlation with asexual and sexual stage parasitemia. RESULTS: Our data provide robust evidence that P. falciparum infection leads to specific, reproducible changes in breath compounds. While no individual compound served as adequate classifier in isolation, selected volatiles together yielded high sensitivity for diagnosis of malaria. Overall, the results of our predictive models suggest the presence of volatile signatures that reproducibly predict malaria infection status and determine response to therapy, even in cases of low parasitemia.
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Low-field, portable MR imaging may expedite patient management in the setting of critical illness. We successfully implemented low-field MR imaging at the Queen Elizabeth Central Hospital in Malawi; a low-resource setting. We present our experience of low-field, portable MR imaging start-up and use in Malawi; the first of its kind in Sub-Saharan Africa, together with complementary troubleshooting mechanisms that may be used especially in similar resource-constrained contexts.
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Enfermedad Crítica , Hospitales , Humanos , Imagen por Resonancia Magnética , MalauiRESUMEN
The emergence of multidrug-resistant organisms and the failure to eradicate infection by a number of important pathogens has led to increased efforts to develop vaccines to prevent infectious diseases. However, the nature of the immune response to vaccination with a given antigen can be complex and unpredictable. An example is the galactose- and N-acetylgalactosamine-inhibitable lectin, a surface antigen of Entamoeba histolytica that has been identified as a major candidate in a vaccine to prevent amebiasis. Vaccination with the lectin can induce protective immunity to amebic liver abscess in some animals, but others of the same species exhibit exacerbations of disease after vaccination. To better understand this phenomenon, we used recombinant proteins corresponding to four distinct domains of the molecule, and synthetic peptides to localize both protective and exacerbative epitopes of the heavy chain subunit of the lectin. We show that protective immunity after vaccination can be correlated with the development of an antibody response to a region of 25 amino acid residues of the lectin, and have confirmed the importance of the antibody response to this region by passive immunization studies. In addition, we show that exacerbation of disease can be linked to the development of antibodies that bind to an NH2-terminal domain of the lectin. These findings are clinically relevant, as individuals who are colonized with E. histolytica but are resistant to invasive disease have a high prevalence of antibodies to the protective epitope(s), compared to individuals with a history of invasive amebiasis. These studies should enable us to develop an improved vaccine for amebiasis, and provide a model for the identification of protective and exacerbative epitopes of complex antigens.
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Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/inmunología , Entamoeba histolytica/inmunología , Entamebiasis/inmunología , Epítopos/inmunología , Lectinas/inmunología , Absceso Hepático Amebiano/inmunología , Vacunas Antiprotozoos , Vacunas Sintéticas , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/biosíntesis , Entamebiasis/prevención & control , Femenino , Gerbillinae , Humanos , Lectinas/biosíntesis , Lectinas/química , Absceso Hepático Amebiano/prevención & control , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/química , Ratones , Ratones SCID , Datos de Secuencia Molecular , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunologíaRESUMEN
Depletion of CD4+ cells using anti-CD4 monoclonal antibodies leads to allograft tolerance. Here we show that anti-CD4-mediated tolerance to pancreatic islets of Langerhans transplanted from an A/J (IEk) donor to a diabetic C57B1/6 (B6) (IE-) recipient occurs in the absence of clonal deletion of the potentially IE-reactive V beta 11+ T cells. Instead, a state of clonal anergy is induced in both the CD4+V beta 11+ and CD8+V beta 11+ T cell subsets. This clonal anergy can be partially overcome in vitro by the addition of recombinant interleukin 2.
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Antígenos CD4/inmunología , Tolerancia Inmunológica/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Animales , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos CD8 , Supervivencia de Injerto/inmunología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
BACKGROUND AND PURPOSE: Validation of diffusion-weighted images obtained on 0.35T MR imaging in Malawi has facilitated meaningful review of previously unreported findings in cerebral malaria. Malawian children with acute cerebral malaria demonstrated restricted diffusion on brain MR imaging, including an unusual pattern of restriction isolated to the subcortical white matter. We describe the patterns of diffusion restriction in cerebral malaria and further evaluate risk factors for and outcomes associated with an isolated subcortical white matter diffusion restriction. MATERIALS AND METHODS: Between 2009 and 2014, comatose Malawian children admitted to the hospital with cerebral malaria underwent admission brain MR imaging. Imaging data were compiled via NeuroInterp, a RedCap data base. Clinical information obtained included coma score, serum studies, and coma duration. Electroencephalograms were obtained between 2009 and 2011. Outcomes captured included death, neurologic sequelae, or full recovery. RESULTS: One hundred ninety-four/269 (72.1%) children with cerebral malaria demonstrated at least 1 area of diffusion restriction. The most common pattern was bilateral subcortical white matter involvement (41.6%), followed by corpus callosum (37.5%), deep gray matter (36.8%), cortical gray matter (17.8%), and posterior fossa (8.9%) involvement. Sixty-one (22.7%) demonstrated isolated subcortical white matter diffusion restriction. These children had lower whole-blood lactate levels (OR, 0.9; 95% CI, 0.85-0.98), were less likely to require anticonvulsants (OR, 0.6; 95% CI, 0.30-0.98), had higher average electroencephalogram voltage (OR, 1.01; 95% CI, 1.00-1.02), were less likely to die (OR, 0.09; 95% CI, 0.01-0.67), and were more likely to recover without neurologic sequelae (OR, 3.7; 95% CI, 1.5-9.1). CONCLUSIONS: Restricted diffusion is common in pediatric cerebral malaria. Isolated subcortical white matter diffusion restriction is a unique imaging pattern associated with less severe disease and a good prognosis for full recovery. The underlying pathophysiology may be related to selective white matter vulnerability.
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Imagen de Difusión por Resonancia Magnética/métodos , Malaria Cerebral/fisiopatología , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Encéfalo/fisiopatología , Preescolar , Femenino , Humanos , Malaria Cerebral/diagnóstico por imagen , Malaria Cerebral/patología , Malaui , Masculino , Pronóstico , Estudios ProspectivosRESUMEN
BACKGROUND: Plasmodium falciparum malaria infects 300-500 million people every year, causing 1-2 million deaths annually. Evidence of a coagulation disorder, activation of endothelial cells (EC) and increase in inflammatory cytokines are often present in malaria. OBJECTIVES: We have asked whether interaction of parasitized red blood cells (pRBC) with EC induces tissue factor (TF) expression in vitro and in vivo. The role of phosphatidylserine-containing pRBC to support the assembly of blood coagulation complexes was also investigated. RESULTS: We demonstrate that mature forms of pRBC induce functional expression of TF by EC in vitro with productive assembly of the extrinsic Xnase complex and initiation of the coagulation cascade. Late-stage pRBC also support the prothrombinase and intrinsic Xnase complex formation in vitro, and may function as activated platelets in the amplification phase of the blood coagulation. Notably, post-mortem brain sections obtained from P. falciparum-infected children who died from cerebral malaria and other causes display a consistent staining for TF in the EC. CONCLUSIONS: These findings place TF expression by endothelium and the amplification of the coagulation cascade by pRBC and/or activated platelets as potentially critical steps in the pathogenesis of malaria. Furthermore, it may allow investigators to test other therapeutic alternatives targeting TF or modulators of EC function in the treatment of malaria and/or its complications.
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Coagulación Sanguínea , Células Endoteliales/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Malaria Cerebral/sangre , Plasmodium falciparum/aislamiento & purificación , Tromboplastina/metabolismo , Adolescente , Animales , Encéfalo/irrigación sanguínea , Encéfalo/parasitología , Encéfalo/patología , Química Encefálica , Células Cultivadas , Niño , Preescolar , Células Endoteliales/química , Células Endoteliales/parasitología , Células Endoteliales/patología , Factor V/metabolismo , Factor Xa/metabolismo , Femenino , Humanos , Inmunohistoquímica , Lactante , Malaria Cerebral/metabolismo , Malaria Cerebral/parasitología , Malaria Cerebral/patología , Masculino , Microcirculación/citología , Microcirculación/metabolismo , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Tromboplastina/análisis , Factores de TiempoRESUMEN
We studied the effects of anti-CD4 treatment of diabetic ACI rats on the induction of tolerance to allogeneic (Lewis) islet allografts. When given as a 4-day treatment regimen, OX38, a mouse anti-rat CD4 antibody, caused depletion of greater than 80% of CD4+ cells from the peripheral blood of treated rats. After induction of diabetes (a single high-dose bolus of streptozocin) and 3 days after the initiation of anti-CD4 immunotherapy, recipient ACI rats were transplanted with fully allogeneic (Lewis) islets of Langerhans via the portal circulation. These transplanted islets were capable of returning the anti-CD4-treated ACI recipients to normoglycemia, which was maintained indefinitely in the absence of further immunosuppression. In contrast, treatment of recipient rats with OX8, an anti-CD8 monoclonal antibody (MoAb), induced only a slight prolongation of graft survival (less than or equal to 30 days). Further characterization of the cellular requirements for the induction of long-term transplantation survival revealed that successful pretransplantation anti-CD4 therapy could be ablated by the coincident treatment of recipient rats with depleting levels of anti-CD8 MoAb. These data point to the necessity of a regulator CD8+ cell in the induction of anti-CD4-mediated transplantation survival in this rat model of islet transplantation.
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Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/cirugía , Diabetes Mellitus Experimental/terapia , Modelos Animales de Enfermedad , Antígenos de Histocompatibilidad Clase I/inmunología , Terapia de Inmunosupresión , Inmunoterapia , Masculino , Ratas , Ratas Endogámicas ACI , Ratas Endogámicas Lew , Estreptozocina , Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
BACKGROUND: A procoagulant state is implicated in cerebral malaria (CM) pathogenesis, but whether disseminated intravascular coagulation (DIC) is present or associated with a fatal outcome is unclear. OBJECTIVES: To determine the frequency of overt DIC, according to ISTH criteria, in children with fatal and non-fatal CM. METHODS/PATIENTS: Malawian children were recruited into a prospective cohort study in the following diagnostic groups: retinopathy-positive CM (n = 140), retinopathy-negative CM (n = 36), non-malarial coma (n = 14), uncomplicated malaria (UM), (n = 91), mild non-malarial febrile illness (n = 85), and healthy controls (n = 36). Assays in the ISTH DIC criteria were performed, and three fibrin-related markers, i.e. protein C, antithrombin, and soluble thrombomodulin, were measured. RESULTS AND CONCLUSIONS: Data enabling assignment of the presence or absence of 'overt DIC' were available for 98 of 140 children with retinopathy-positive CM. Overt DIC was present in 19 (19%), and was associated with a fatal outcome (odds ratio [OR] 3.068; 95% confidence interval [CI] 1.085-8.609; P = 0.035]. The levels of the three fibrin-related markers and soluble thrombomodulin were higher in CM patients than in UM patients (all P < 0.001). The mean fibrin degradation product level was higher in fatal CM patients (71.3 µg mL(-1) [95% CI 49.0-93.6]) than in non-fatal CM patients (48.0 µg mL(-1) [95% CI 37.7-58.2]; P = 0.032), but, in multivariate logistic regression, thrombomodulin was the only coagulation-related marker that was independently associated with a fatal outcome (OR 1.084 for each ng mL(-1) increase [95% CI 1.017-1.156]; P = 0.014). Despite these laboratory derangements, no child in the study had clinically evident bleeding or thrombosis. An overt DIC score and high thrombomodulin levels are associated with a fatal outcome in CM, but infrequently indicate a consumptive coagulopathy.
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Coagulación Intravascular Diseminada/etiología , Malaria Cerebral/sangre , Malaria Falciparum/sangre , Biomarcadores/análisis , Glucemia/análisis , Niño , Preescolar , Coma/sangre , Coma/etiología , Femenino , Fiebre/sangre , Fibrina/biosíntesis , Pruebas Hematológicas , Humanos , Lactante , Lactatos/sangre , Malaria Cerebral/mortalidad , Malaria Falciparum/mortalidad , Malaui , Masculino , Parasitemia/sangre , Parasitemia/mortalidad , Estudios Prospectivos , Hemorragia Retiniana/sangre , Hemorragia Retiniana/parasitología , Factores de Riesgo , Trombomodulina/análisisRESUMEN
In the present report a monoclonal antibody designated OX-38 directed against the rat CD4 molecule was tested for its ability to prolong the survival of heterotopic vascularized rat heart allografts transplanted across major histocompatibility barriers. Fluorescence-activated cell-sorter analysis showed that administration of OX-38 selectively depleted 80-95% of CD4+ cells from peripheral blood of treated rats. The immunosuppressive effects of OX-38 in vivo were verified by suppression of an antibody response against OX-38 itself as a heterologous protein immunogen. Recipient rats received OX-38 antibody as a single agent given in pretransplant regimens. Nine of 12 treated rats have maintained heterotopic abdominal heart allografts for greater than 175 days. Control rats that did not receive antibody therapy rejected their grafts within 14 days. Rats that maintained heart allografts for greater than 100 days accepted second donor strain hearts but rejected third-party heart grafts transplanted into the femoral space. Anti-CD4-induced allograft unresponsiveness persisted for at least 90 days following surgical removal of donor tissue and retransplantation of a second donor-matched heart. These results indicated that transient, pretransplant therapy with monoclonal antibodies directed against the CD4+ lymphocyte induced specific, long-lasting unresponsiveness to fully MHC-mismatched cardiac allografts in rats without additional immunosuppression.
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Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Corazón/inmunología , Animales , Formación de Anticuerpos/inmunología , Antígenos de Diferenciación de Linfocitos T/fisiología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8 , Antígenos de Histocompatibilidad/inmunología , Terapia de Inmunosupresión , Depleción Linfocítica , Masculino , Ratas , Ratas Endogámicas ACI , Ratas Endogámicas BN , Ratas Endogámicas Lew , Trasplante HomólogoRESUMEN
We have used serum from patients with amebic liver abscess to investigate the role of antibody in the prevention of invasive amebiasis using the severe combined immunodeficient (SCID) mouse model of Entamoeba histolytica infection. The SCID mice were passively immunized with serum or purified antibody from patients with amebic liver abscess 24 hr prior to the direct intrahepatic challenge with 10(6) virulent E. histolytica trophozoites. This treatment reduced the mean abscess size in these animals from 24.5% to 3.5% (P < 0.0001). These results demonstrate that human anti-amebic antibodies are capable of exerting a protective effect in an animal model of amebic liver abscess formation.
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Anticuerpos Antiprotozoarios/inmunología , Entamoeba histolytica/inmunología , Absceso Hepático Amebiano/prevención & control , Animales , Humanos , Inmunización Pasiva , Ratones , Ratones SCIDRESUMEN
INTRODUCTION: Mondana Clinic is a small rural clinic located in the Napo river region of the Amazon basin in Ecuador. Since its opening in 1997 the clinic has grown to be the primary health care facility for approximately 3000 individuals. METHODS: A retrospective study was performed tabulating the ambulatory diagnosis, age, sex, and domicile of patients over a 9 month period in 1999. RESULTS: During the study period there were 765 patient visits that resulted in at least one diagnosis. Of the patient visits, 175 (22.8%) resulted in multiple diagnoses. Women accounted for 58% of the patient visits, which is similar to the 60% of ambulatory patient visits made in the USA by women. The age distribution showed 66% of patients were under 25 years of age. When comparing diagnoses of males with females, several differences were noted. As expected, urinary tract infections were approximately four-fold more common in females than in males. Gastritis and headaches were also more common reasons for patient visits in the female population than in the male. Conversely, lacerations, abrasions, and contusions ranked higher in the male than in the female population for patient visits. CONCLUSION: This study is the first to provide public health information for this region that will prove useful to the health professionals and funding agencies working in the region. Furthermore, it provides a baseline for comparison with other regions in Ecuador and South America in general, as well as comparisons with data-rich countries such as the USA.
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BACKGROUND AND PURPOSE: There have been few neuroimaging studies of pediatric CM, a common often fatal tropical condition. We undertook a prospective study of pediatric CM to better characterize the MRI features of this syndrome, comparing findings in children meeting a stringent definition of CM with those in a control group who were infected with malaria but who were likely to have a nonmalarial cause of coma. MATERIALS AND METHODS: Consecutive children admitted with traditionally defined CM (parasitemia, coma, and no other coma etiology evident) were eligible for this study. The presence or absence of malaria retinopathy was determined. MRI findings in children with ret+ CM (patients) were compared with those with ret- CM (controls). Two radiologists blinded to retinopathy status jointly developed a scoring procedure for image interpretation and provided independent reviews. MRI findings were compared between patients with and without retinopathy, to assess the specificity of changes for patients with very strictly defined CM. RESULTS: Of 152 children with clinically defined CM, 120 were ret+, and 32 were ret-. Abnormalities much more common in the patients with ret+ CM were markedly increased brain volume; abnormal T2 signal intensity; and DWI abnormalities in the cortical, deep gray, and white matter structures. Focal abnormalities rarely respected arterial vascular distributions. Most of the findings in the more clinically heterogeneous ret- group were normal, and none of the abnormalities noted were more prevalent in controls. CONCLUSIONS: Distinctive MRI findings present in patients meeting a stringent definition of CM may offer insights into disease pathogenesis and treatment.
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Encéfalo/patología , Imagen por Resonancia Magnética/estadística & datos numéricos , Malaria Cerebral/epidemiología , Malaria Cerebral/patología , Enfermedad Aguda , Preescolar , Femenino , Humanos , Malaui/epidemiología , Masculino , Prevalencia , Reproducibilidad de los Resultados , Medición de Riesgo , Sensibilidad y EspecificidadRESUMEN
Advanced medical imaging technologies are generally unavailable in low income, tropical settings despite the reality that neurologic disorders are disproportionately common in such environments. Through a series of donations as well as extramural research funding support, an MRI facility opened in Blantyre, Malawi in July 2008. Resulting opportunities for studying common tropical disorders, such as malaria and schistosomiasis, in vivo are promising. The subsequent improvements in local patient care were expected and exceptional and include major revisions in basic care protocols that may eventually impact care protocols at facilities in the region that do not have recourse to MRI. In addition, advanced neuroimaging technology has energized the medical education system, possibly slowing the brain drain. Advanced technologies, though potentially associated with significant fiscal opportunity costs, may bring unexpected and extensive benefits to the healthcare and medical education systems involved.
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Investigación Biomédica , Educación Médica , Imagen por Resonancia Magnética , Calidad de la Atención de Salud , Atención a la Salud , Humanos , Interpretación de Imagen Asistida por Computador , Malaui , NeuroimagenRESUMEN
Amebic liver abscess is characterized by extensive areas of dead hepatocytes that form cavities surrounded by a thin rim of inflammatory cells and few Entamoeba histolytica trophozoites. E. histolytica produces pore-forming proteins and proteinases, but how trophozoites actually kill host cells has been unclear. Here, we report that E. histolytica induces apoptosis in both inflammatory cells and hepatocytes in a severe combined immunodeficient (SCID) mouse model of amebic liver abscess. By studying infection in C57/BL6.lpr and C57/BL6.gld mice, we found that E. histolytica-induced apoptosis does not require the Fas/Fas ligand pathway of apoptosis, and by using mice with a targeted deletion of the tumor necrosis factor receptor I gene, we have shown that E. histolytica-induced apoptosis is not mediated by tumor necrosis factor alpha. Our data indicate that apoptosis plays a prominent role in the host cell death seen in amebic liver abscess in a mouse model of disease and suggest that E. histolytica induces cell death without using two common pathways for apoptosis.
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Apoptosis , Entamoeba histolytica/patogenicidad , Absceso Hepático Amebiano/patología , Animales , Antígenos CD/genética , Absceso Hepático Amebiano/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones SCID , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/metabolismoRESUMEN
Evidence from in vitro studies suggests that gamma interferon (IFN-gamma) and nitric oxide (NO) are important in host defense against the protozoan parasite Entamoeba histolytica. We used SCID mice with targeted disruption of the IFN-gamma receptor gene and mice with targeted disruption of the gene encoding inducible NO synthase to show that IFN-gamma plays a role in the innate immunity to amebic liver abscess seen in SCID mice while NO is required for control of amebic liver abscess in immunocompetent mice.
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Interferón gamma/inmunología , Absceso Hepático Amebiano/inmunología , Óxido Nítrico/inmunología , Animales , Secuencia de Bases , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Entamoeba histolytica/inmunología , Entamoeba histolytica/patogenicidad , Interferón gamma/deficiencia , Interferón gamma/genética , Absceso Hepático Amebiano/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo IIRESUMEN
Animal models of liver abscess formation with Entamoeba histolytica suggest that the neutrophil is the first cell of the host immune system to interact with the invading ameba. In vitro studies have suggested that lysis of neutrophils by virulent amebae may exacerbate the damage seen in amebic liver abscesses. To investigate the role of neutrophils in vivo, we used the severe combined immunodeficient (SCID) mouse model of amebic liver abscess formation and compared liver damage in neutrophil-depleted and control mice. We found that neutrophil-depleted animals have significantly larger amebic liver abscesses at early stages of infection and that abscesses in neutrophil-depleted SCID mice lack the prominent inflammatory cell ring seen in amebic liver abscesses in control SCID mice. These data suggest that neutrophils play a protective role in the early host response to amebic infection of the liver.
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Entamoeba histolytica/inmunología , Entamebiasis/inmunología , Absceso Hepático Amebiano/inmunología , Ratones SCID/inmunología , Neutrófilos/inmunología , Animales , Inmunidad Celular , RatonesRESUMEN
The protozoan parasite Entamoeba histolytica causes amebic dysentery and amebic liver abscess, diseases associated with significant morbidity and mortality worldwide. E. histolytica infection appears to involve the initial attachment of amebic trophozoites to intestinal epithelial cells, followed by lysis of these cells and subsequent invasion into the submucosa. A recent in vitro study (L. Eckmann, S. L. Reed, J. R. Smith, and M. F. Kagnoff, J. Clin. Invest. 96:1269-1279, 1995) demonstrated that incubation of E. histolytica trophozoites with epithelial cell lines results in epithelial cell production of inflammatory cytokines, including interleukin-1 (IL-1) and IL-8, suggesting that intestinal epithelial cell production of cytokines might play a role in the inflammatory response and tissue damage seen in intestinal amebiasis. To determine whether intestinal epithelial cell production of IL-1 and IL-8 occurs in response to E. histolytica infection in vivo and as an approach to studying the specific interactions between amebic trophozoites and human intestine, we used a SCID mouse-human intestinal xenograft (SCID-HU-INT) model of disease, where human intestinal xenografts were infected with virulent E. histolytica trophozoites. Infection of xenografts with E. histolytica trophozoites resulted in extensive tissue damage, which was associated with the development of an early inflammatory response composed primarily of neutrophils. Using oligonucleotide primers that specifically amplify human IL-1beta and IL-8, we could demonstrate by reverse transcription PCR that mRNA for both IL-1beta and IL-8 is produced by human intestinal xenografts in response to amebic infection. The increase in human intestinal IL-1beta and IL-8 in response to invasive amebiasis was confirmed by enzyme-linked immunosorbent assays specific for human IL-1beta and IL-8. Using immunohistochemistry, we confirmed that human intestinal epithelial cells were the source of IL-8 in infected xenografts and established that IL-8 production can occur at sites distal to areas of intestinal mucosal damage. These results demonstrate that human intestinal epithelial cells can produce inflammatory cytokines in response to infection in vivo and establish the SCID-HU-INT model as a system for studying the interactions between E. histolytica and human intestine.
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Disentería Amebiana/inmunología , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Intestinos/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Epitelio/inmunología , Epitelio/metabolismo , Trasplante de Tejido Fetal , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunohistoquímica , Inflamación , Interleucina-1/genética , Interleucina-1/inmunología , Interleucina-8/genética , Interleucina-8/inmunología , Intestinos/patología , Intestinos/trasplante , Ratones , Ratones SCID , Neutrófilos/inmunología , Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Transcripción Genética , Trasplante HeterólogoRESUMEN
Shigellae infect human intestine and cause intense inflammation and destruction of colonic and rectal mucosa. To model the interactions of shigella with human intestine in vivo, we have studied shigella infection in human intestinal xenografts in severe combined immunodeficient mice (SCID-HU-INT mice). Inoculation of shigella into human intestinal xenografts caused severe inflammation and mucosal damage, which was apparent as soon as 4 h following infection. Shigella infection was associated with human intestinal production of interleukin-1B (IL-1B) and IL-8 and a marked neutrophil influx into the graft. Depletion of neutrophils from SCID-HU-INT mice reduced inflammation in the human intestinal xenograft in response to shigella infection but failed to significantly alter tissue damage. However, the number of intracellular bacteria was more than 20-fold higher in the human intestinal xenografts from neutrophil-depleted SCID-HU-INT mice. Infection of human intestinal xenografts with an attenuated vaccine strain of shigella (CVD1203) induced lower levels of IL-1B and IL-8 than wild-type shigella and caused only moderate damage to the intestinal permeability barrier. Our studies establish the SCID-HU-INT mouse as a viable model for studying the interactions between shigella and human intestine and indicate that neutrophils are important for controlling the invasion of human intestine by shigella.
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Disentería Bacilar/inmunología , Intestinos/microbiología , Neutrófilos/fisiología , Animales , Movimiento Celular , Disentería Bacilar/microbiología , Humanos , Interleucina-1/biosíntesis , Interleucina-8/biosíntesis , Intestinos/inmunología , Intestinos/trasplante , Ratones , Ratones SCID , Modelos Animales , Permeabilidad , Trasplante HeterólogoRESUMEN
BACKGROUND & AIMS: Entamoeba histolytica infection of the intestine can induce severe gut inflammation. The aims of this study were to assess the role of the host inflammatory response in the tissue damage observed with amebiasis and the role of the intestinal epithelial cell in initiating that response. METHODS: E. histolytica infection was established in human intestinal xenografts in severe combined immunodeficient (SCID-HU-INT) mice. Human intestinal epithelial cell inflammatory responses to amebic infection were inhibited by the intraluminal administration of an antisense oligonucleotide to the human p65 subunit of nuclear factor kappaB, and the role of neutrophils in tissue damage observed with amebiasis was studied by depleting neutrophils from SCID-HU-INT mice. RESULTS: Administration of the antisense oligonucleotide blocked the production of human interleukin 1beta and interleukin 8 by intestinal epithelial cells and inhibited neutrophil influx into the E. histolytica-infected intestinal xenografts. Inhibition of the gut inflammatory response by the antisense oligonucleotide or the depletion of neutrophils from SCID-HU-INT mice blocked the increase in intestinal permeability observed with amebic infection. CONCLUSIONS: Intestinal epithelial cells initiate an inflammatory response with resulting neutrophil-mediated tissue damage in response to E. histolytica infection; this inflammatory cascade can be blocked by inhibiting the transcription of genes regulated by nuclear factor kappaB.
Asunto(s)
Amebiasis/inmunología , Animales , Humanos , Mucosa Intestinal/inmunología , Ratones , Ratones SCID , FN-kappa B/genética , Neutrófilos/inmunología , Oligonucleótidos Antisentido , PermeabilidadRESUMEN
The protozoan parasite Entamoeba histolytica causes intestinal inflammation and ulceration. Amoebic trophozoites activate the transcription factor NF-kappa B in human intestinal epithelial cells, initiating an inflammatory response programme with resultant damage to the intestinal tissue. Amoebic cysteine proteinases have been proposed as important virulence factors for amoebiasis. To test the role of amoebic cysteine proteinases in the pathogenesis of amoebic colitis, human intestinal xenografts in SCID mice were infected with E. histolytica trophozoites expressing an antisense message to ehcp5. The cysteine proteinase-deficient amoeba failed to induce intestinal epithelial cell production of the inflammatory cytokines interleukin (IL)-1B and IL-8, and caused significantly less gut inflammation and damage to the intestinal permeability barrier. The critical role of amoebic cysteine proteinases in human gut inflammation and tissue damage may be explained by our discovery that amoebic cysteine proteinases possess IL-1B converting enzyme (ICE) activity. This ICE activity could contribute to intestinal inflammation by activating human pIL-1B released by damaged intestinal cells. These results demonstrate for the first time that amoebic cysteine proteinases are a key virulence factor in amoebic colitis, and provide a novel mechanism for their activity.