Archaeosomes made of Halorubrum tebenquichense total polar lipids: A new source of adjuvancy

Background: Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined. Results: DNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response. Conclusion: We herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles

Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy

<p>Abstract</p> <p>Background</p> <p>Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new <it>Halorubrum tebenquichense </it>strains isolated from grey crystals (<it>GC</it>) and black mood (<it>BM</it>) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form <it>BM </it>(ARC-BM) and from <it>GC </it>(ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined.</p> <p>Results</p> <p>DNA G+C content (59.5 and 61.7% mol <it>BM </it>and <it>GC</it>, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that <it>BM </it>and <it>GC </it>isolates were two non-previously described strains of <it>H. tebenquichense</it>. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response.</p> <p>Conclusion</p> <p>We herein report the finding of new <it>H. tebenquichense </it>non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.</p

Benznidazole Therapy Modulates Interferon-γ and M2 Muscarinic Receptor Autoantibody Responses in Trypanosoma cruzi-Infected Children

OBJECTIVE: The presence of autoantibodies with adrenergic and cholinergic activity, capable of triggering neurotransmitter receptor-mediated effects, has been associated with pathogenesis in T. cruzi-infected hosts. The goal of this study was to investigate the production of anti-M2 muscarinic receptor autoantibodies (Anti-M2R AAbs) as well as the IFN-γ profile in children at the early stage of Chagas disease, and to examine whether trypanocidal chemotherapy with benzonidazole (BZ) could modify both response patterns. METHODS: This study comprised 30 T. cruzi-infected children (mean age: 13.8 years) and 19 uninfected controls (mean age: 12.7 years). Infected patients were treated with BZ and followed-up. Blood samples collected at diagnosis-T0, end of treatment-T1, and six months later-T2 were analysed by ELISA for detection of Anti-M2R AAbs and circulating levels of IFN-γ. RESULTS: At T0, anti-M2R AAbs were demonstrated in 56.7% of T. cruzi-infected patients, whereas uninfected controls were 100% negative. The average age of Anti-M2R AAbs(+) patients was higher than that from negative population. Infected children also displayed significantly stronger serum IFN-γ responses than controls. Upon BZ treatment, a significant linear decreasing trend in Anti-M2R AAb reactivity was recorded throughout the follow-up, with 29.7-88.1% decrease at T2. IFN-γ circulating levels also declined by T2. CONCLUSION: Anti-M2R AAbs and IFN-γ raise early during chagasic infection in children and are downmodulated by BZ therapy. These findings reinforce the usefulness of early BZ treatment not only to eliminate the parasite but also to reduce potentially pathogenic immune responses

Archaeosomes made of Halorubrum tebenquichense total polar lipids: A new source of adjuvancy

Background: Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined. Results: DNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response. Conclusion: We herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.Facultad de Ciencias Exacta

Elevated serum levels of macrophage migration inhibitory factor are associated with progressive chronic cardiomyopathy in patients with chagas disease

Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression.Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients

Efficacy of voriconazole in a murine model of acute Trypanosoma cruzi infection

OBJECTIVES: Antifungal triazole derivatives have been studied as possible alternatives for the treatment of Chagas' disease. Voriconazole has demonstrated in vitro activity against Trypanosoma cruzi, but its efficacy in vivo has not yet been tested. We aimed to determine the effect of voriconazole in a murine model of acute T. cruzi infection. METHODS: Treatment efficacy was evaluated by comparing parasitaemia, mortality and organ involvement (by histological examination) of infected mice. RESULTS: Treatment with voriconazole significantly lowered parasitaemia and mortality compared with controls, reduced the percentage of mice with amastigote nests in heart and skeletal muscle and moderately decreased myocardial inflammation. CONCLUSIONS: Our findings support the potential of voriconazole for the treatment of acute Chagas' disease and motivate future animal studies using varying doses and treatment schemes. Further evaluation of voriconazole for clinical use in human Chagas' patients is warranted.Fil: Gulin, Julián Ernesto Nicolás. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Eagleson, M. A.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Harvard University; Estados UnidosFil: Postan, Miriam. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cutrullis, Romina Andrea. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Freilij, Hector León. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: García Bournissen, Facundo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Petray, Patricia Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Altcheh, Jaime Marcelo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Benznidazole therapy modulates Interferon-γ and M2 Muscarinic receptor autoantibody responses in Trypanosoma cruzi-Infected children

Fil: Cutrullis, Romina A. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Moscatelli, Guillermo F. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Moroni, Samanta. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Volta, Bibiana J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Cardoni, Rita L. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Altcheh, Jaime. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Corral, Ricardo S. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Freilij, Héctor. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Petray, Patricia B. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Objective The presence of autoantibodies with adrenergic and cholinergic activity, capable of triggering neurotransmitter receptor-mediated effects, has been associated with pathogenesis in T. cruzi-infected hosts. The goal of this study was to investigate the production of anti-M2 muscarinic receptor autoantibodies (Anti-M2R AAbs) as well as the IFN-γ profile in children at the early stage of Chagas disease, and to examine whether trypanocidal chemotherapy with benznidazole (BZ) could modify both response patterns. Methods This study comprised 30 T. cruzi-infected children (mean age: 13.8 years) and 19 uninfected controls (mean age: 12.7 years). Infected patients were treated with BZ and followed-up. Blood samples collected at diagnosis-T0, end of treatment-T1, and six months later-T2 were analysed by ELISA for detection of Anti-M2R AAbs and circulating levels of IFN-γ. Results At T0, anti-M2R AAbs were demonstrated in 56.7% of T. cruzi-infected patients, whereas uninfected controls were 100% negative. The average age of Anti-M2R AAbs+ patients was higher than that from negative population. Infected children also displayed significantly stronger serum IFN-γ responses than controls. Upon BZ treatment, a significant linear decreasing trend in Anti-M2R AAb reactivity was recorded throughout the follow-up, with 29.7–88.1% decrease at T2. IFN-γ circulating levels also declined by T2. Conclusion Anti-M2R AAbs and IFN-γ raise early during chagasic infection in children and are downmodulated by BZ therapy. These findings reinforce the usefulness of early BZ treatment not only to eliminate the parasite but also to reduce potentially pathogenic immune responses

Benznidazole therapy modulates Interferon-γ and M2 Muscarinic receptor autoantibody responses in Trypanosoma cruzi-Infected children

Fil: Cutrullis, Romina A. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Moscatelli, Guillermo F. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Moroni, Samanta. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Volta, Bibiana J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Cardoni, Rita L. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Altcheh, Jaime. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Corral, Ricardo S. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Freilij, Héctor. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Petray, Patricia B. Hospital de Niños Ricardo Gutiérrez. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Objective The presence of autoantibodies with adrenergic and cholinergic activity, capable of triggering neurotransmitter receptor-mediated effects, has been associated with pathogenesis in T. cruzi-infected hosts. The goal of this study was to investigate the production of anti-M2 muscarinic receptor autoantibodies (Anti-M2R AAbs) as well as the IFN-γ profile in children at the early stage of Chagas disease, and to examine whether trypanocidal chemotherapy with benznidazole (BZ) could modify both response patterns. Methods This study comprised 30 T. cruzi-infected children (mean age: 13.8 years) and 19 uninfected controls (mean age: 12.7 years). Infected patients were treated with BZ and followed-up. Blood samples collected at diagnosis-T0, end of treatment-T1, and six months later-T2 were analysed by ELISA for detection of Anti-M2R AAbs and circulating levels of IFN-γ. Results At T0, anti-M2R AAbs were demonstrated in 56.7% of T. cruzi-infected patients, whereas uninfected controls were 100% negative. The average age of Anti-M2R AAbs+ patients was higher than that from negative population. Infected children also displayed significantly stronger serum IFN-γ responses than controls. Upon BZ treatment, a significant linear decreasing trend in Anti-M2R AAb reactivity was recorded throughout the follow-up, with 29.7–88.1% decrease at T2. IFN-γ circulating levels also declined by T2. Conclusion Anti-M2R AAbs and IFN-γ raise early during chagasic infection in children and are downmodulated by BZ therapy. These findings reinforce the usefulness of early BZ treatment not only to eliminate the parasite but also to reduce potentially pathogenic immune responses

MIF is overexpressed in the heart of chronic chagasic mice and potentiates production of TNF-α and ROS by <i>Trypanosoma cruzi</i>-infected macrophages.

<p>C3H/He mice (<i>n</i> = 5) were infected intraperitoneally with 10⁶ blood trypomastigotes of the Sylvio X10/4 clone of <i>T. cruzi</i> (upper panel) or with 50 blood trypomastigotes of the Tulahuén strain of the parasite (central panel). Hearts from infected animals and uninfected controls (lower panel) were removed at 120 days p.i. (A) Immunohistochemical analysis was performed on cardiac muscle sections using murine MIF polyclonal antibodies. Microphotographs of myocardial tissues show a representative experiment of three performed. Bar = 50 µm. (B) MIF overexpression in the heart was closely accompanied by intense inflammatory cell infiltration. The leukocyte population invading the myocardium was isolated from 20 <i>T. cruzi</i>-infected mice at 120 days p.i. and digested with collagenase and hyaluronidase. The mononuclear cell fraction was incubated with anti-mouse CD11b/MAC-1- PerCP-Cy 5.5, CD3-FITC, CD4-PE and CD8-Alexa Fluor 647 antibodies. The labeled cells (at least 5×10⁴) were fixed with 1% <i>p</i>-formaldehyde and analyzed by flow cytometry. (C), (D) Effect of exogenous MIF on <i>T. cruzi</i>-induced release of TNF-α and ROS in murine J774 macrophages. Adherent cells were infected for 24 h with culture trypomastigotes of <i>T. cruzi</i> (Tulahuén strain) at a 10∶1 parasite/cell ratio, in the presence (+ rMIF) or in the absence (− rMIF) of recombinant mouse MIF (1 µg/ml). TNF-α production was quantified in uninfected (Mock) and parasite-infected cell supernatants using a sandwich ELISA (C). For ROS measurement. J774 macrophages (10⁶) were incubated with 10 µM DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) for 30 min and then infected for 24 h with <i>T. cruzi</i> trypomastigotes with or without addition of rMIF, as described above. Uninfected (Mock) and infected cells were then fixed and analyzed by flow cytometry (D). Data are the means ± S E M of three independent experiments, each performed in triplicate. ^*<i>P</i><0.05 and ^**<i>P</i><0.01 versus cells not pre-treated with MIF; #<i>P</i><0.05 between infected and uninfected MIF-stimulated cells.</p

Correlation between serum MIF level and parameters of cardiac dysfunction in chagasic patients.

<p>(A) LV end-diastolic diameter (LVEDD), (B) LV end-systolic diameter (LVESD), (C) left atrial diameter (LAD), (D) aortic diameter (AD), (E) LV per cent fractional shortening (LVFS), and (F) serum concentration of MIF and high sensitivity C-reactive protein (HS-CRP) were analyzed in chagasic (both chronic indeterminate and cardiomyopathic) patients (<i>n</i> = 26). MIF concentration was measured by ELISA; LVEDD, LVESD, LAD, AD and LVFS were determined by echodopplercardiography; HS-CRP level was quantified by immunonephelometry. Individual results are shown for each patient; the line represents the linear regression for each comparison. Alpha level was adjusted to 0.01. The correlation coefficient (r) and <i>P</i> values for each association are indicated.</p