Successful long-term maintenance of Mansonella perstans in an in vitro culture system

Njouendou, Abdel Jélil; Ritter, Manuel; Ndongmo, Winston Patrick Chounna; Kien, Chi Anizette; Gandjui, Narcisse Victor T.; Fombad, Fanny Fri; Tayong, Dizzle Bita; Pfarr, Kenneth; Layland, Laura E.; Hoerauf, Achim; Wanji, Samuel

doi:10.1186/s13071-017-2515-8

Cited by 26 publications

(26 citation statements)

References 31 publications

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“…Interestingly, we established an in vitro culture of viable M . perstans adult worms [ 25 ] which then were used to prepare worm antigen extract for the assessment of M . perstans -specific responses.…”

Section: Resultsmentioning

confidence: 99%

“…To determine the ability of M . perstans worm antigen extract [ 25 ] to elicit M . perstans -specific responses we stimulated freshly isolated peripheral whole blood cells from Mp MF+ individuals and compared them to responses from healthy European NEN volunteers in vitro .…”

Section: Resultsmentioning

confidence: 99%

“…To investigate the development of M . perstans life stages, we established a culture system to maintain worms in vitro as previously described [ 25 ]. In brief, M .…”

Section: Methodsmentioning

confidence: 99%

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Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses

et al. 2018

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The filarial nematode Mansonella perstans is endemic throughout Africa, northern South America and the Caribbean. Interestingly, M. perstans-infected individuals present no distinct clinical picture associated with certain pathology. Due to its relatively silent nature, research on this tropical disease has been neglected, especially M. perstans-driven immune responses. A hindrance in obtaining data on M. perstans-specific responses has been the inability to obtain adult worms since their habitats in serous cavities are difficult to access. Thus, in this study, for the first time, we used Mansonella perstans worm antigen extract as stimulant to obtain filarial-specific recall and immunoglobulin responses from M. perstans microfilaremic individuals (Mp MF+) from Cameroon. Moreover, systemic immune profiles in sera and immune cell composition in peripheral blood from Mp MF+ and amicrofilaremic individuals (Mp MF-) were obtained. Our data reveal that Mp MF+ individuals showed significantly reduced cytokine (IL-4, IL-6 and IL-12p70) and chemokine levels (IL-8 and RANTES), but significantly higher MIP-1β as well as increased M. perstans-specific IgG4 levels compared to Mp MF- individuals. In contrast, upon re-stimulation with worm antigen extract, IFN-γ, IL-13, IL-10 and IL-17A secretion was enhanced in cell cultures from Mp MF+ individuals when compared to those from cultures of healthy European individuals. Moreover, analysis of immune cell composition in peripheral blood from Mp MF+ individuals revealed increased type 2 helper T (Th2), natural killer (NK), regulatory B and T cell (Breg and Treg) subsets but decreased type 1 regulatory T (Tr1) cells. In summary, this study deciphers for the first time, M. perstans-specific immune responses using worm antigen extract and shows that patent M. perstans infections have distinct Th2, Breg and Treg subsets accompanied with reduced systemic innate and adaptive immune responses and dominant filarial-specific IgG4 levels.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses

et al. 2018

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“…The viability of the parasites was assessed daily, by visual inspection (by two individuals) under an inverted microscope until they die. Their motility was scored on a 4-point scale [ 18 , 19 ]: 0, no movement or immotile; 1, intermittent shaking of head and tail; 2, sluggish (shaking of the whole worm on a spot); 3, vigorous movement (shaking of the whole worm and migration from one spot to was considered).…”

Section: Methodsmentioning

confidence: 99%

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa

et al. 2018

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BackgroundSuitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa.MethodsIn vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco’s modified Eagle’s medium (DMEM) and Iscove’s modified Dulbecco’s medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval.ResultsOf the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4–19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility.ConclusionsThe findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2852-2) contains supplementary material, which is available to authorized users.

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“…Traditional 2-dimensional (2-D) culture systems were shown to support the early stages of development for few filarial nematodes in vitro , with some of these parasitic worms the cultures could be maintained long-term. The reported in vitro survival rates ranged from 33% after 77 days for Mansonella perstans L3 [ 17 ], 69% after 30 days for Brugia malayi L3 [ 18 ], and 60–90% for Loa loa L3 over 15 to 17.8 days in culture [ 19 , 20 ]. Recently, we described a novel 2-D in vitro culturing system based on a feeder layer of human umbilical vein endothelial cells (HUVEC) that has successfully supported the development of O .…”

Section: Introductionmentioning

confidence: 99%

Preliminary evaluations of 3-dimensional human skin models for their ability to facilitate in vitro the long-term development of the debilitating obligatory human parasite Onchocerca volvulus

et al. 2020

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Onchocerciasis also known as river blindness is a neglected tropical disease and the world's second-leading infectious cause of blindness in humans; it is caused by Onchocerca volvulus. Current treatment with ivermectin targets microfilariae and transmission and does not kill the adult parasites, which reside within subcutaneous nodules. To support the development of macrofilaricidal drugs that target the adult worm to further support the elimination of onchocerciasis, an in-depth understanding of O. volvulus biology especially the factors that support the longevity of these worms in the human host (>10 years) is required. However, research is hampered by a lack of access to adult worms. O. volvulus is an obligatory human parasite and no small animal models that can propagate this parasite were successfully developed. The current optimized 2-dimensional (2-D) in vitro culturing method starting with O. volvulus infective larvae does not yet support the development of mature adult worms. To overcome these limitations, we have developed and applied 3-dimensional (3-D) culture systems with O. volvulus larvae that simulate the human in vivo niche using in vitro engineered skin and adipose tissue. Our proof of concept studies have shown that an optimized indirect co-culture of in vitro skin tissue supported a significant increase in growth of the fourth-stage larvae to the pre-adult stage with a median length of 816–831 μm as compared to 767 μm of 2-D cultured developed larvae. Notably, when larvae were co-cultured directly with adipose tissue models, a significant improvement for larval motility and thus fitness was observed; 95% compared to 26% in the 2-D system. These promising co-culture concepts are a first step to further optimize the culturing conditions and improve the long-term development of adult worms in vitro. Ultimately, it could provide the filarial research community with a valuable source of O. volvulus worms at various developmental stages, which may accelerate innovative unsolved biomedical inquiries into the parasite’s biology.

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Successful long-term maintenance of Mansonella perstans in an in vitro culture system

Cited by 26 publications

References 31 publications

Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses

Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa

Preliminary evaluations of 3-dimensional human skin models for their ability to facilitate in vitro the long-term development of the debilitating obligatory human parasite Onchocerca volvulus

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