A quick, simple method for purifying <i>Leishmania mexicana</i> amastigotes in large numbers

Hart, David T.; Vickerman, K.; Coombs, Graham H.

doi:10.1017/s0031182000066889

Cited by 150 publications

(72 citation statements)

References 13 publications

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“…C57BL/6, TLR2 −/− , and C3He/J (TLR4 −/− ) mice were obtained from the Indian Institute of Science, and IL-10 −/− mice were a kind gift from Gobardhan Das (International Center for Genetic Engineering and Biotechnology, New Delhi, India (42), were used for this study. Amastigotes were obtained from the spleen of infected hamsters (43), and they were subsequently transformed into promastigotes and maintained (44).…”

Section: Methodsmentioning

confidence: 99%

Antimony-resistant but not antimony-sensitiveLeishmania donovaniup-regulates host IL-10 to overexpress multidrug-resistant protein 1

Mukherjee

Mukhopadhyay

Bannerjee

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The molecular mechanism of antimony-resistant Leishmania donovani (Sb R LD)-driven up-regulation of IL-10 and multidrug-resistant protein 1 (MDR1) in infected macrophages (Mϕs) has been investigated. This study showed that both promastigote and amastigote forms of Sb R LD, but not the antimony-sensitive form of LD, express a unique glycan with N-acetylgalactosamine as a terminal sugar. Removal of it either by enzyme treatment or by knocking down the relevant enzyme, galactosyltransferase in Sb R LD (KD Sb R LD), compromises the ability to induce the above effects. Infection of Mϕs with KD Sb R LD enhanced the sensitivity toward antimonials compared with infection with Sb R LD, and infection of BALB/c mice with KD Sb R LD caused significantly less organ parasite burden compared with infection induced by Sb R LD. The innate immune receptor, Toll-like receptor 2/6 heterodimer, is exploited by Sb R LD to activate ERK and nuclear translocation of NF-κB involving p50/c-Rel leading to IL-10 induction, whereas MDR1 up-regulation is mediated by PI3K/Akt and the JNK pathway. Interestingly both recombinant IL-10 and Sb R LD up-regulate MDR1 in Mϕ with different time kinetics, where phosphorylation of PI3K was noted at 12 h and 48 h, respectively, but Mϕs derived from IL-10 −/− mice are unable to show MDR1 up-regulation on infection with Sb R LD. Thus, it is very likely that an IL-10 surge is a prerequisite for MDR1 up-regulation. The transcription factor important for IL-10-driven MDR1 up-regulation is c-Fos/c-Jun and not NF-κB, as evident from studies with pharmacological inhibitors and promoter mapping with deletion constructs.visceral leishmaniasis | antimony resistance | glycoconjugate | antimony efflux K ala-azar or visceral leishmaniasis, which is caused by the protozoan parasite Leishmania donovani (LD), is (re)emerging and spreading worldwide, essentially because of humanmade and environmental changes, immunosuppression, and drug resistance (1, 2). To combat the disease, organic pentavalent antimonials were introduced in the Indian subcontinent almost 9 decades ago (3) with dramatic clinical success. However, the toxicity, together with the high treatment failure rate (up to 65%) and the emergence of resistance in Bihar State, India, made the drug obsolete in the Indian subcontinent (4, 5). Nevertheless, it is still used as a first-line treatment in Africa and Latin America (6). Interestingly, 78% of the recent clinical isolates from the hyperendemic zone of Bihar State still showed in vitro resistance to antimonials (7), which might be explained by an increased fitness of the corresponding parasites (8).Recently, we have demonstrated three unique characteristics of antimony-resistant LD (Sb R LD). First, it shows a higher concentration of terminal glycoconjugates (N-acetylgalactosamine residues) on its surface than antimony-sensitive LD (Sb S LD) (7).Second, on infection of macrophages (Mϕs), Sb R LD induces a surge of IL-10 (7, 9). Third, we also observed that Sb R LD modulates host cells to overexpress the ATP-b...

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Section: Methodsmentioning

confidence: 99%

Antimony-resistant but not antimony-sensitiveLeishmania donovaniup-regulates host IL-10 to overexpress multidrug-resistant protein 1

Mukherjee

Mukhopadhyay

Bannerjee

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Mice were injected i.v. via the tail vein with100 l of PBS/mouse with 1 ϫ 10 8 promastigotes suspended in PBS using a 22-gauge needle as reported elsewhere (43).…”

Section: Animalsmentioning

confidence: 99%

Ubiquitin Conjugation of Open Reading Frame F DNA Vaccine Leads to Enhanced Cell-Mediated Immune Response and Induces Protection against Both Antimony-Susceptible and -Resistant Strains ofLeishmania donovani

Sharma

Madhubala

2009

The Journal of Immunology

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Resistance of Leishmania donovani to sodium antimony gluconate has become a critical issue in the current, prolonged epidemic in India. Hence, there is an urgent need for a vaccine that is protective against both antimony-susceptible and -resistant strains of L. donovani. The multigene LD1 locus located on chromosome 35 of Leishmania is amplified in ∼15% of the isolates examined. The open reading frame F (ORFF), a potential vaccine candidate against visceral leishmaniasis, is part of the multigene LD1 locus. ORFF was expressed as a chimeric conjugate of ubiquitin to elicit an Ag-specific cell-mediated immune response. Analysis of the cellular immune responses of ubiquitin-conjugated ORFF (UBQ-ORFF) DNA-immunized, uninfected BALB/c mice demonstrated that the vaccine induced enhanced IFN-γ-producing CD4+ and CD8+ T cells compared with nonubiquitinated ORFF DNA vaccine. Higher levels of IL-12 and IFN-γ and the low levels of IL-4 and IL-10 further indicated that the immune responses with UBQ-ORFF were mediated toward the Th1 rather than Th2 type. Infection of immunized mice with either the antimony-susceptible (AG83) or -resistant (GE1F8R) L. donovani strain showed that UBQ-ORFF DNA vaccine induced higher protection when compared with ORFF DNA. UBQ-ORFF DNA-immunized and -infected mice showed a significant increase in IL-12 and IFN-γ and significant down-regulation of IL-10. High levels of production of nitrite and superoxide, two macrophage-derived oxidants that are critical in controlling Leishmania infection, were observed in protected mice. The feasibility of using ubiquitinated-conjugated ORFF DNA vaccine as a promising immune enhancer for vaccination against both antimony-susceptible and -resistant strains of L. donovani is reported.

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“…Los parásitos intracelulares producidos de esta manera presentan características antigénicas, morfológicas y ultraestruturales similares a las formas intracelulares que se producen en infecciones M1 vivo (20,31). En contraste, Lamy informa la transformación de Leishmania a temperaturas bajas, mediante la infección a macrófagos, indicando que la temperatura no es un factor crítico en el proceso de transformación de promastigote a amastigote (3)(4)(5)(6).…”

Section: Discussionunclassified

Efecto de la temperatura en la transformación de promastigotes en amastigotes de Leishmania amazonensis in vitra

Echeverry

Winograd

1997

biomedica

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ResumenAunque el efecto de la temperatura en la transformación axénica de Leishmania ha sido ampliamente estudiado, el papel que esta variable juega en la transformación del parásito intracelular se desconoce. El objetivo del presente trabajo fue evaluar si la transformación intracelular de promastigotes en amastigotes ocurre a bajas temperaturas. Cuando se infectaron macrófagos murinos de la línea celular J-774 a dos temperaturas diferentes (24 y 33"C), se observaron formas intracelulares de Leishmania amazonensis a las dos temperaturas. Los parásitos intracelulares producidos se compararon mediante criterios morfológicos, inmunológicos y ultraestructurales. Los resultados indican que los parásitos obtenidos en infecciones a 33% son similares morfológica, inmunológica y ultraestructuralmente con las formas intracelulares obtenidas in vivo conocidas como amastigotes; sin embargo, los parásitos producidos a temperaturas más bajas (24OC) no tienen similitud antigénica ni ultraestructural con los anteriores. Se concluye que la temperatura cumple un papel relevante en la transformación de la forma extracelular de Leishmania amazonensis hacia la forma intracelular durante su invasión a macrófagos murinos. SummaryAlthough it is well known that the effect of temperature is important in the transformation of Leishmania promastigotes into amastigotes under axenic conditions, the contribution of this variable in the transformation of intracellular parasites is presently unknown. When J-774 murine cell line macrophages were infected with Leishrnania amazonensisat two different temperatures (24 and 33OC), intracellular parasites were observed at both temperatures. The resulting intracellular parasites were compared by morphological, immunological and structural criteria. The results indicate that while parasites produced at 33°C have characteristics similar to those previoulsy reported in the literature, infection of macrophages at lower temperatures resulted in intracellular parasites with no evidence of transformation into amastigotes. These results indicate that temperature is essential for the intracellular transformation of promastigotes into amastigotes.Los rnicroorganismos agrupados en el género extracelular (promastigote) que habita el intestino Leishmania requieren un hospedero invertebrado del insecto y mediante su picadura a un vertebrado -flebotomíneos del género Lutzomia-y uno penetra en su torrente sanguíneo, teniendo como vertebrado para completar su ciclo de vida. blanco las célulasfagocíticas del tipo macrófago. Leishmaniasepresentacornounacélulaflagelada Al interactuar con estas células, se hace intra-

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A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers

Cited by 150 publications

References 13 publications

Antimony-resistant but not antimony-sensitiveLeishmania donovaniup-regulates host IL-10 to overexpress multidrug-resistant protein 1

Antimony-resistant but not antimony-sensitiveLeishmania donovaniup-regulates host IL-10 to overexpress multidrug-resistant protein 1

Ubiquitin Conjugation of Open Reading Frame F DNA Vaccine Leads to Enhanced Cell-Mediated Immune Response and Induces Protection against Both Antimony-Susceptible and -Resistant Strains ofLeishmania donovani

Efecto de la temperatura en la transformación de promastigotes en amastigotes de Leishmania amazonensis in vitra

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