The design and development of a study protocol to investigate Onchocerca volvulus, Loa loa and Mansonella perstans-mediated modulation of the metabolic and immunological profile in lean and obese individuals in Cameroon.

BACKGROUND: Life-style metabolic diseases are steadily rising, not only in developed countries, but also in low- and middle-income countries, presenting a global health problem. Metabolic disorders like type 2 diabetes and cardiovascular diseases are among the ten leading causes of death defined by the WHO in 2019. Results from animal and observational human studies suggest a connection between the decline in human helminth infections and rise of life-style-associated metabolic diseases in developing regions. This trial was designed to investigate filarial infections and their impact on metabolic diseases in Cameroon. We hypothesize that the induction of regulatory immune responses during filarial infection reduces obesity-induced low-grade inflammatory immune responses and thereby improves metabolic parameters, whereas anthelmintic treatment abolishes this protective effect. METHODS/DESIGN: Participants infected with Mansonella perstans, Onchocerca volvulus and/or Loa loa being lean (BMI <25), overweight (BMI >25 and <30) or clinically obese (BMI ≥30) from Littoral regions of Cameroon will be evaluated for their parasitological, immunological, metabolic and biochemical profile before and after treatment of their parasitic infections. Anthropomorphic measurements and a detailed questionnaire will complement our analysis. The investigation will assess blood immune cell populations, serum adipokines and cytokines that could be influenced by the parasite infection and/or metabolic diseases. Further, parameters like blood glucose, homeostatic model assessment of insulin resistance (HOMA-IR), circulating lipids and circulating makers of liver function will be monitored. Parameters will be assessed before treatment, 12 and 18 months after treatment. CONCLUSION: The focus of this study is to obtain a comprehensive metabolic profile of the participants in rural areas of Cameroon and to investigate the relationship between filarial immunomodulation and metabolic diseases. This study will elucidate the effect of anti-filarial treatment on the metabolic and immunological parameters that partake in the development of insulin resistance, narrowing in on a potential protective effect of filarial infections on metabolic diseases. TRIAL REGISTRATION: doi.org/10.1186/ISRCTN43845142, ISRCTN43845142 February 2020 Trial title Effects of filarial parasite infection on type 2 diabetes Issue date: 27.10.22, V.1.

Histopathological evaluation of Onchocerca volvulus nodules by microscopy and by digital image analysis for the study of macrofilaricidal drug efficacy.

Background: Novel drugs or drug combinations that kill or permanently sterilize adult Onchocerca volvulus worms would be very helpful for treatment and elimination of onchocerciasis. In absence of a reliable biomarker for viable adult worms, histopathological assessment of worms within onchocercal nodules is a standard method to determine macrofilaricidal activity. The goal of the present study was to determine the agreement between two independent experts in the analysis of nodule sections and to assess the value of digital imaging as a means of standardizing the analysis. Material and methods: Two expert microscopists independently assessed 605 nodules by direct microscopy. At least two sections with two different stains hematoxylin & eosin (H&E, APR immunostain) of paraffin-embedded, ethanol-fixed whole-nodule cross-sections were analyzed. After variables were identified prone to observer discrepancies, we performed a second study to compare consolidated results for 100 nodules obtained by the two readers by microscopy and by analysis of scanned, high resolution digital images (20x magnification). The last data set analyzed was a quality panel of 100 nodules that has been previously examined by microscopy, and included additional immunostains for Wolbachia endobacteria. These slides were digitalized, read by the two assessors and results were compared with original microscopy results. Results: The degree of agreement between assessors varied for different parameters. Agreement for female worm counts in nodules was approximately 80%, while agreement regarding female worm viability was 98%. There were no major differences observed between results obtained by microscopy or digital images. Good agreement for important parameters was also observed for the nodules of the quality panel. Conclusion: Nodule analysis by experienced microscopists was reproducible with regard to important parameters such as identification of living female worms or detection of normal embryogenesis. Assessments varied more for other parameters, and we recommend continued use of two independent readers for detailed analyzes. Analysis of scanned images provided similar results to direct microscopy. This facilitates training and comparison of nodule findings by readers in different locations. Analysis of high quality digital images that can be viewed remotely should improve the quality and availability of nodule assessments that are primary endpoints for onchocerciasis clinical trials.

Eosinophils Suppress the Migration of T Cells Into the Brain of Plasmodium berghei-Infected Ifnar1-/- Mice and Protect Them From Experimental Cerebral Malaria.

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA (PbAAma1OVA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that Ifnar1-/- mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+ T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected Ifnar1-/- mice and wild type mice suffering from ECM. Importantly, CD8+ T cells were increased in the spleens of ECM-protected Ifnar1-/- mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+ T cell infiltration into the brain and increased ECM induction in PbAAma1OVA-infected Ifnar1-/- mice. However, eosinophil-depletion did not reduce the CD8+ T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+ T cell migration and proliferation during PbAAma1OVA-infection in Ifnar1-/- mice and thereby are contributing to the protection from ECM.

Protection of Batf3-deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T-cell responses and immune regulation.

Excessive inflammatory immune responses during infections with Plasmodium parasites are responsible for severe complications such as cerebral malaria (CM) that can be studied experimentally in mice. Dendritic cells (DCs) activate cytotoxic CD8+ T-cells and initiate immune responses against the parasites. Batf3-/- mice lack a DC subset, which efficiently induces strong CD8 T-cell responses by cross-presentation of exogenous antigens. Here we show that Batf3-/- mice infected with Plasmodium berghei ANKA (PbA) were protected from experimental CM (ECM), characterized by a stable blood-brain barrier (BBB) and significantly less infiltrated peripheral immune cells in the brain. Importantly, the absence of ECM in Batf3-/- mice correlated with attenuated responses of cytotoxic T-cells, as their parasite-specific lytic activity as well as the production of interferon gamma and granzyme B were significantly decreased. Remarkably, spleens of ECM-protected Batf3-/- mice had elevated levels of regulatory immune cells and interleukin 10. Thus, protection from ECM in PbA-infected Batf3-/- mice was associated with the absence of strong CD8+ T-cell activity and induction of immunoregulatory mediators and cells.