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Ethnopharmacological relevance Respiratory diseases and asthma, in particular, are nowadays a global health problem. In Rwanda, some traditional healers claim to treat asthma with plant-based recipes, though there is no scientific proof so far. Aim of the study Our study aimed at evaluating the toxicity and the anti-inflammatory effect of plant recipes used in Rwanda against asthma in order to select potential candidates for further characterization of the active compounds. Materials and methods Water (aqueous) and methanol-dichloromethane (organic) extracts from selected folkloric recipes were submitted for toxicity test on THP-1 derived macrophages using CellTiter-Glo Luminescent Cell Viability Assay. The evaluation of the anti-inflammatory effect of the plant extracts was carried out using the Caspase-Glo 1 Inflammasome assay on THP-1 -derived macrophages. Results Most of both organic and aqueous extract showed more than 95% of cell viability up to 200 µg/ml, except for R03Cn organic extract that inhibited 25% of the cell viability. Plant extracts inhibited caspase-1 activation in THP-1 derived macrophages in a dose-dependent manner. Four extracts (R03Cn and R07Kn aqueous extracts, R10MK and R19Sz organic extracts) strongly downregulated the activation of caspase-1 (more than 70% at 50 µg/ml). In general, organic extracts exhibited better caspase-1 inhibitory effects than their aqueous counterparts. Conclusions The inhibition of inflammasome/caspase-1 is one of key mechanisms of action in asthma. Some traditional recipes are active on this mechanism and are thus strong candidates for the treatment of asthma and other inflammasome-mediated diseases. Further investigations are needed to characterize active molecules.

Onchocerca volvulus is the nematode pathogen responsible for human onchocerciasis also known as “River blindness”, a neglected tropical disease that affects up to 18 million people worldwide. Helminths Excretory Secretory Products (ESPs) constitute a rich repertoire of molecules that can be exploited for host-parasite relationship, diagnosis and vaccine studies. Here, we report, using a range of molecular techniques including PCR, western blot, recombinant DNA technology, ELISA, high performance thin-layer chromatography and mass spectrometry that the 28 KDa cysteine-rich protein (Ov28CRP) is a reliable component of the O. volvulus ESPs to address the biology of this parasite. We showed that (1) Ov28CRP is a putative ganglioside GM2 Activator Protein (GM2AP) conserved in nematode; (2) OvGM2AP gene is transcriptionally activated in all investigated stages of the parasitic life cycle, including larval and adult stages; (3) The full-length OvGM2AP was detected in in-vitro O. volvulus ESPs of adult and larval stages; (4) the mass expressed and purified recombinant OvGM2AP purified from insect cell culture medium was found to be glycosylated at asparagine 173 and lacked N-terminal signal peptide sequence; (5) the recombinant OvGM2AP discriminated serum samples of infected and uninfected individuals; (6) OvGM2AP competitively inhibits MUG degradation by recombinant β-hexosaminidase A but not MUGS, and could not hydrolyze the GM2 to GM3; (7) humoral immune responses to the recombinant OvGM2AP revealed a negative correlation with ivermectin treatment. Altogether, our findings suggest for the first time that OvGM2AP is an antigenic molecule whose biochemical and immunological features are important to gain more insight into our understanding of host-parasite relationship, as well as its function in parasite development at large., SCOPUS: ar.j, info:eu-repo/semantics/published

Onchocerciasis is a severely debilitating yet neglected tropical disease (NTD) that creates social stigma, generates and perpetuates poverty, and leads ultimately in some cases to irreversible unilateral or bilateral blindness if untreated. Consequently, the disease is a major impediment to socioeconomic development. Many control programs have been launched for the disease with moderate successes achieved. This mitigated hit is partially due to the lingering need for reliable, non-invasive and easily applicable tools for mapping endemic regions and post-elimination surveillance. In this work, bioinformatics analyses combined with immunological assays were applied in a bid to develop potential tools for diagnosis and assessing the success of drug treatment programs. We report that (i) the O. volvulus antigen, Ov58GPCR is a G-protein coupled receptor (GPCR) conserved in related nematodes, (ii) synthetic peptides predicted to be in the extracellular domain (ECD) of Ov58GPCR are indeed immunogenic epitopes in actively-infected individuals, (iii) synthetic peptide cocktails discriminate between actively-infected individuals, treated individuals and healthy African controls, (iv) polyclonal antibodies against one of the peptides or against the bacterially-expressed ECD reacted specifically with the native antigen of O. volvulus total and surface extracts, (v) Ov58GPCR is transcribed in both larvae and adult parasite stages, (vi) IgG and IgE responses to the recombinant ECD decline with ivermectin treatment. All these findings suggest that the extracellular domain and synthetic peptides of Ov58GPCR, as well as the specific immune response generated could be harnessed in the context of disease diagnosis and surveillance., SCOPUS: ar.j, info:eu-repo/semantics/published

Contains fulltext : 96063.pdf (Publisher’s version ) (Open Access) BACKGROUND: Nanobodies are single-domain antigen binding fragments derived from functional heavy-chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti-EGFR (99m)Tc-7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of (99m)Tc-7C12. METHODS: First we compared the renal uptake of (99m)Tc-7C12 in megalin-deficient and megalin-wild-type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of (99m)Tc-7C12 was analyzed in CD-1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload + gelofusine coadministration (LysPreload + GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad + GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts. RESULTS: Renal uptake of (99m)Tc-7C12 was 44.22 +/- 3.46% lower in megalin-deficient compared with megalin-wild-type mice. In CD-1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 +/- 2.99 and 36.22 +/- 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 +/- 2.09%. Gelofusine and lysine coadministration improved tumor uptake. CONCLUSION: Megalin contributes to the renal accumulation of (99m)Tc-7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy.

Potential confounding factors in bioluminescence imaging (BLI)-quantification need to be identified to improve its accuracy and repeatability.The aim of this study was to study the degree to which plasma protein binding of BLI substrates influences signal intensity and quantification, both in vitro and in vivo.Protein binding was assessed using ultrafiltration and spectrophotometry. Effects of increasing serum protein concentrations were studied in vitro, in intact cells expressing Firefly or Renilla luciferase, and in vivo, in a doxorubicin-induced hypoalbuminemia mouse model.Increasing concentrations of serum proteins showed an important negative effect on BLI signal intensity, both for D-luciferin and coelenterazine-dependent reactions. This was due to a decrease in the free fraction of the substrate in the presence of serum proteins of up to 88%. In vivo, hypoalbuminemia was associated with higher BLI signal intensity, although this was only significant in animals with a major decrease in albumin (2 g/dL). Important kinetic differences, including earlier peak luminescence and a more rapid decline in luminescence, were observed for coelenterazine-dependent BLI under hypoalbuminemic conditions. Changes in protein concentrations can significantly influence BLI quantification and its presence decreases sensitivity in vitro. In vivo, effects on signal intensity and kinetics are only noted at severe hypoalbuminemia and can be neglected in most experimental designs. These findings again emphasize the need for careful interpretation of BLI quantification data.

Introduction: The development of specific radiolabeled probes towards molecular markers in vivo has gained interest as targeted imaging allows for a more accurate detection of diseases. We investigate the feasibility of targeted imaging of cancer antigens using the variable domain of single chain camelid antibodies (Nanobodies®) labeled with 99mTechnetium. Nanobodies against carcinoembryonic antigen (CEA) were used as a model. Methods: His6-CEA1 Nanobodies were generated and labeled with 99mTc at their His-tag using Tc(I)-tricarbonyl (Isolink, Mallinckrodt, B.V., Petten, The Netherlands). The normal biodistribution was assessed in healthy athymic mice by ex vivo analysis at 1 and 3 h. In vivo targeting was evaluated in the same mouse model bearing the CEA-positive LS174T tumour or a CEA-negative A431 (human skin carcinoma) control tumour. Pinhole SPECT imaging was performed at 3 hours after intravenous injection of 90 MBq 99mTc-His6-CEA1 using a dual-headed gamma camera equipped with pinhole collimators. Results: Radiolabeling efficiency was > 95%. General biodistribution showed intense renal uptake and marked liver accumulation. Using pinhole-SPECT, the average uptake of 99mTc- His6-CEA1 in LS174T (CEA positive) was significantly higher compared to the A431 (CEA negative) control tumour: respectively 3.2 ± 0.6 %IA/cm3 and 1.1 ± 0.2 %IA/cm3 (p < 0.05). Conclusion: This study presents effective labeling of Nanobodies with 99mTc using Tc(I)-carbonyl chemistry and shows their potential as a new type of specific probes for imaging antigen expression.

Nanobodies represent an interesting class of probes for the generic development of molecular imaging agents. We studied the relationship between tumor uptake of the epidermal growth factor receptor (EGFR)-specific nanobody (99m)Tc-7C12 and tumor burden and evaluated the possibility of using this probe to monitor tumor response to erlotinib.The specificity and affinity of (99m)Tc-7C12 was determined on A431 cells. Cells expressing firefly luciferase were used to evaluate tumor burden using bioluminescence imaging. We evaluated the effect of erlotinib on tumor burden and (99m)Tc-7C12 uptake in vitro as well as in vivo. In vivo bioluminescence imaging was performed followed by pinhole single-photon emission computed tomography/micro-computed tomography.(99m)Tc-7C12 binds specifically to the receptor with high affinity (3.67 ± 0.59 nM). Erlotinib reduced tumor uptake and cell viability in a concentration-dependent manner. Tumor uptake of (99m)Tc-7C12 showed good correlation with tumor burden. Erlotinib treatment resulted in a progressive reduction of tumor burden and tumor uptake of (99m)Tc-7C12.(99m)Tc-7C12 binds to EGFR with high affinity and specificity. Tumor uptake is correlated with tumor burden. Quantification of (99m)Tc-7C12 uptake is promising for monitoring therapy response of EGFR-expressing tumors.

Purpose : Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnositcs and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice. Methods : Fluorescence-activated cell sorting (FAC) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The nanobody was then labeled with 99mTc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of 99mTc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU 145) overexpression of EGFR. Results : FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of 99mTc-8B6 Nanobody is relatively fast (half-life, 1.5h) and mainly via the kidneys. At 3h postinjection, total kidney accumulation is high(46.6+0.9%IA) compared to total liver uptake (18.9+0.6IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2+0.5%IA/cm3) of 99mTc-8B6 Nanobody compared to DU145 xenografts (1.8+0.3%IA/cm3, pConclusion : The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with 99mTc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti-epidermal growth factor receptor (anti-EGFR) Nanobodies, (99m)Tc-7C12 and (99m)Tc-7D12.Nanobodies were labeled via their hexahistidine tail with (99m)Tc-tricarbonyl ((99m)Tc(CO)(3)) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with (99m)Tc-7C12 and (99m)Tc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six (57)Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm(3)) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either (99m)Tc-7C12 or (99m)Tc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g).Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both (99m)Tc-7C12 and (99m)Tc-7D12 (4.55+/-0.24 %IA/cm(3) and 4.62+/-0.36 %IA/cm(3), respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16+/-0.14 for (99m)Tc-7C12 and 1.49+/-0.60 for (99m)Tc-7D12). (99m)Tc-7C12 showed significantly higher kidney uptake (63.48+/-2.36 vs. 56.25+/-2.46 %IA/cm(3)) and lower liver uptake (2.55+/-0.26 vs. 4.88+/-0.86 %IA/cm(3)) than did (99m)Tc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, (99m)Tc-7C12 showed higher tumor uptake (9.11+/-1.12 %IA/g) than did (99m)Tc-7D12 (6.09+/-0.77 %IA/g). (99m)Tc-7D12 demonstrated significantly higher blood activity than did (99m)Tc-7C12, but both showed short plasma half-lives (10 min).The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, (99m)Tc-7C12 was selected for further studies.

INTRODUCTION: In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. D: -luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of D: -luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo. MATERIALS AND METHODS: Fluc-positive R1M cells were subcutaneously inoculated in nu/nu mice. Dynamic BLI was performed after IV or IP administration of D: -luciferin. Maximal photon emission (PE(max)) was calculated. For repeatability assessment, every acquisition was repeated after 4 h and analysed using Bland-Altman method. A second group of animals was serially imaged, alternating IV and IP administration up to 21 days. When mice were killed, PE(max) after IV administration was correlated with histological cell number. RESULTS: The coefficients of repeatability were 80.2% (IV) versus 95.0% (IP). Time-to-peak is shorter, and its variance lower for IV (p < 0.0001). PE(max) was 5.6 times higher for IV. A trend was observed towards lower photon emission per cell in larger tumours. CONCLUSION: IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden.