Impact of repeated NeemAzal®-treated blood meals on the fitness of Anopheles stephensi mosquitoes

Background: Herbal remedies are widely used in many malaria endemic countries to treat patients, in particular in the absence of anti-malarial drugs and in some settings to prevent the disease. Herbal medicines may be specifically designed for prophylaxis and/or for blocking malaria transmission to benefit both, the individual consumer and the community at large. Neem represents a good candidate for this purpose due to its inhibitory effects on the parasite stages that cause the clinical manifestations of malaria and on those responsible for infection in the vector. Furthermore, neem secondary metabolites have been shown to interfere with various physiological processes in insect vectors. This study was undertaken to assess the impact of the standardised neem extract NeemAzal® on the fitness of the malaria vector Anopheles stephensi following repeated exposure to the product through consecutive blood meals on treated mice. Methods: Batches of An. stephensi mosquitoes were offered 5 consecutive blood meals on female BALB/c mice treated with NeemAzal® at an azadirachtin A concentration of 60, 105 or 150 mg/kg. The blood feeding capacity was estimated by measuring the haematin content of the rectal fluid excreted by the mosquitoes during feeding. The number of eggs laid was estimated by image analysis and their hatchability assessed by direct observations. Results: A dose and frequency dependent impact of NeemAzal® treatment on the mosquito feeding capacity, oviposition and egg hatchability was demonstrated. In the 150 mg/kg treatment group, the mosquito feeding capacity was reduced by 50% already at the second blood meal and by 50 to 80% in all treatment groups at the fifth blood meal. Consequently, a 50 – 65% reduction in the number of eggs laid per female mosquito was observed after the fifth blood meal in all treatment groups. Similarly, after the fifth treated blood meal exposure, hatchability was found to be reduced by 62% and 70% in the 105 and 150 mg/kg group respectively. Conclusions: The findings of this study, taken together with the accumulated knowledge on neem open the challenging prospects of designing neem-based formulations as multi-target phytomedicines exhibiting preventive, parasite transmission-blocking as well as anti-vectorial properties. Keywords: Malaria, Vectors, Neem, Azadirachtin, Transmission-blocking, Anti-vectoria

Salinomycin and Other Ionophores as a New Class of Antimalarial Drugs with Transmission-Blocking Activity

The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads

Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds

BACKGROUND: Medicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernonia amygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodium berghei and to validate the findings on field isolates of Plasmodium falciparum. METHODS: Aqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V. amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P. falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926. RESULTS: Ver-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An. stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 μg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines. CONCLUSIONS: Vernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal

The Sin3A/MAD1 Complex, through Its PAH2 Domain, Acts as a Second Repressor of Retinoic Acid Receptor Beta Expression in Breast Cancer Cells

Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARβ is a tumor-suppressor gene silenced by epigenetic mechanisms such as DNA methylation in breast, cervical and non-small cell lung cancers. An increased expression of RARβ has been associated with improved breast cancer-specific survival. The PAH2 domain of the scaffold protein SIN3A interacts with the specific Sin3 Interaction Domain (SID) of several transcription factors, such as MAD1, bringing chromatin-modifying proteins such as histone deacetylases, and it targets chromatin for specific modifications. Previously, we have established that blocking the PAH2-mediated Sin3A interaction with SID-containing proteins using SID peptides or small molecule inhibitors (SMI) increased RARβ expression and induced retinoic acid metabolism in breast cancer cells, both in in vitro and in vivo models. Here, we report studies designed to understand the mechanistic basis of RARβ induction and function. Using human breast cancer cells transfected with MAD1 SID or treated with the MAD SID peptide, we observed a dissociation of MAD1, RARα and RARβ from Sin3A in a coimmunoprecipitation assay. This was associated with increased RARα and RARβ expression and function by a luciferase assay, which was enhanced by the addition of AM580, a specific RARα agonist; EMSA showed that MAD1 binds to E-Box, similar to MYC, on the RARβ promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RARβ activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RARβ expression. These data suggest that SIN3A/MAD1 acts as a second RARβ repressor and may be involved in fine-tuning retinoid sensitivity

The Sin3A/MAD1 Complex, through Its PAH2 Domain, Acts as a Second Repressor of Retinoic Acid Receptor Beta Expression in Breast Cancer Cells

Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARβ is a tumor-suppressor gene silenced by epigenetic mechanisms such as DNA methylation in breast, cervical and non-small cell lung cancers. An increased expression of RARβ has been associated with improved breast cancer-specific survival. The PAH2 domain of the scaffold protein SIN3A interacts with the specific Sin3 Interaction Domain (SID) of several transcription factors, such as MAD1, bringing chromatin-modifying proteins such as histone deacetylases, and it targets chromatin for specific modifications. Previously, we have established that blocking the PAH2-mediated Sin3A interaction with SID-containing proteins using SID peptides or small molecule inhibitors (SMI) increased RARβ expression and induced retinoic acid metabolism in breast cancer cells, both in in vitro and in vivo models. Here, we report studies designed to understand the mechanistic basis of RARβ induction and function. Using human breast cancer cells transfected with MAD1 SID or treated with the MAD SID peptide, we observed a dissociation of MAD1, RARα and RARβ from Sin3A in a coimmunoprecipitation assay. This was associated with increased RARα and RARβ expression and function by a luciferase assay, which was enhanced by the addition of AM580, a specific RARα agonist; EMSA showed that MAD1 binds to E-Box, similar to MYC, on the RARβ promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RARβ activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RARβ expression. These data suggest that SIN3A/MAD1 acts as a second RARβ repressor and may be involved in fine-tuning retinoid sensitivity

A user friendly method to assess Anopheles stephensi (Diptera: Culicidae) vector fitness: fecundity

Fecundity, bloodmeal size, and survival are among the most important parameters in the overall fitness of mosquitoes. Impact of an intervention that affects fecundity can be assessed by directly counting the eggs laid by exposed mosquitoes, which is usually done manually. We have developed a macroinstruction, which can be used to count thousands of Anopheles stephensi Liston eggs in a few minutes, to provide an alternative and adaptable method to egg counting as a measure of fecundity. The macro was developed using a scanner and a computer running AxioVision Rel. 4.8 software, a freely accessible software compatible with Windows XP/7/Vista. Using this semiautomated method, it is possible to reduce time, avoid human error and bias, and obtain improved consistency in studies measuring mosquito fecundity

Transmission blocking effects of Azadirachta indica limonoids on early sporogonic development of Plasmodium: activity and bioavailability of seed fractions and isolated compounds

Azadirachta indica (Meliaceae) possesses a wide spectrum of biological properties, conferred to the plant by secondary metabolites. A. indica seeds contain abundantly limonoid molecules such as azadirone, nimbin, salanin and azadirachtins (A to L), azadirachtin A (AzaA) being one of the most bio-active molecules. AzaA has been shown to inhibit Plasmodium berghei microgamete formation and an AzaA rich commercial kernel extract (NeemAzal®) was found to completely block the transmission of P. berghei to Anopheles stephensi females when administered to gametocytemic mice at an AzaA dose of 50 mg/kg before exposure to mosquitoes. The present study was aimed at i) elucidating early sporogonic stage specific effects of A. indica seed fractions and their main constituents; ii) assessing the bioavailability of a fraction rich in AzA and the isolated AzaA molecule through a biological response-based assay. Ex vivo and in vitro assays were performed with the murine malaria parasites P. berghei ANKA strain and P. berghei CTRPp.GFP. Fractions were obtained from A. indica seeds collected in Burkina Faso and from NeemAzal® (NA, provided by Trifolio-M GmbH, Lahnau, Germany) by column chromatography. Constituents were identified by NMR spectroscopy. NA, AzaA, nimbin and salannin rich fractions from unripe seeds tested at 50 µg/ml, revealed inhibitory activity on early sporogonic stages in vitro. Nimbin and salannin were found to interfere with ookinete maturation while NA and AzaA showed multiple effects on early sporogonic development. The IC50 value determined for NA was 6.8 µg/ml (CI95: 5.95- 7.86), about half of that of AzaA IC50 (12.4 µg/ml; CI95: 11.0- 14.04). The stronger activity of NA, when compared to AzaA, appeared not to be due to an additive or synergistic effect of other azadirachtins (B, D and I) present in NA, since the addition of these compounds at 50 µM to AzaA did not evidence any decrease of the IC50. Also, bioavailability of AzaA, administered as constituent of NA, compared to pure AzaA appeared to be increased. Ex vivo exflagellation tests using blood sampled from mice treated with NA at an AzaA dosage of 150 mg/kg, revealed a half life of NA anti-plasmodial compounds of up to 7 hours. Accumulated evidence on bioavailability and anti-plasmodial activity of limonoids against Plasmodium stages developing in the human and mosquito host, suggests A. indica as a valid resource for the design of limonoid dosed, transmission blocking phytomedicines

Estimation of tuberculosis incidence at subnational level using three methods to monitor progress towards ending TB in India, 2015–2020

Objectives We verified subnational (state/union territory (UT)/district) claims of achievements in reducing tuberculosis (TB) incidence in 2020 compared with 2015, in India.Design A community-based survey, analysis of programme data and anti-TB drug sales and utilisation data.Setting National TB Elimination Program and private TB treatment settings in 73 districts that had filed a claim to the Central TB Division of India for progress towards TB-free status.Participants Each district was divided into survey units (SU) and one village/ward was randomly selected from each SU. All household members in the selected village were interviewed. Sputum from participants with a history of anti-TB therapy (ATT), those currently experiencing chest symptoms or on ATT were tested using Xpert/Rif/TrueNat. The survey continued until 30 Mycobacterium tuberculosis cases were identified in a district.Outcome measures We calculated a direct estimate of TB incidence based on incident cases identified in the survey. We calculated an under-reporting factor by matching these cases within the TB notification system. The TB notification adjusted for this factor was the estimate by the indirect method. We also calculated TB incidence from drug sale data in the private sector and drug utilisation data in the public sector. We compared the three estimates of TB incidence in 2020 with TB incidence in 2015.Results The estimated direct incidence ranged from 19 (Purba Medinipur, West Bengal) to 1457 (Jaintia Hills, Meghalaya) per 100 000 population. Indirect estimates of incidence ranged between 19 (Diu, Dadra and Nagar Haveli) and 788 (Dumka, Jharkhand) per 100 000 population. The incidence using drug sale data ranged from 19 per 100 000 population in Diu, Dadra and Nagar Haveli to 651 per 100 000 population in Centenary, Maharashtra.Conclusion TB incidence in 1 state, 2 UTs and 35 districts had declined by at least 20% since 2015. Two districts in India were declared TB free in 2020