Quality of anti-malarial drugs provided by public and private healthcare providers in south-east Nigeria

BACKGROUND: There is little existing knowledge about actual quality of drugs provided by different providers in Nigeria and in many sub-Saharan African countries. Such information is important for improving malaria treatment that will help in the development and implementation of actions designed to improve the quality of treatment. The objective of the study was to determine the quality of drugs used for the treatment of malaria in a broad spectrum of public and private healthcare providers. METHODS: The study was undertaken in six towns (three urban and three rural) in Anambra state, south-east Nigeria. Anti-malarials (225 samples), which included artesunate, dihydroartemisinin, sulphadoxine-pyrimethamine (SP), quinine, and chloroquine, were either purchased or collected from randomly selected providers. The quality of these drugs was assessed by laboratory analysis of the dissolution profile using published pharmacopoeial monograms and measuring the amount of active ingredient using high performance liquid chromatography (HPLC). FINDINGS: It was found that 60 (37%) of the anti-malarials tested did not meet the United States Pharmacopoeia (USP) specifications for the amount of active ingredients, with the suspect drugs either lacking the active ingredients or containing suboptimal quantities of the active ingredients. Quinine (46%) and SP formulations (39%) were among drugs that did not satisfy the tolerance limits published in USP monograms. A total of 78% of the suspect drugs were from private facilities, mostly low-level providers, such as patent medicine dealers (vendors). CONCLUSION: This study found that there was a high prevalence of poor quality drugs. The findings provide areas for public intervention to improve the quality of malaria treatment services. There should be enforced checks and regulation of drug supply management as well as stiffer penalties for people stocking substandard and counterfeit drugs

Positional cloning of rp2 QTL associates the P450 genes CYP6Z1, CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vector Anopheles funestus

Pyrethroid resistance in Anopheles funestus is threatening malaria control in Africa. Elucidation of underlying resistance mechanisms is crucial to improve the success of future control programs. A positional cloning approach was used to identify genes conferring resistance in the uncharacterised rp2 quantitative trait locus (QTL) previously detected in this vector using F6 advanced intercross lines (AIL). A 113 kb BAC clone spanning rp2 was identified and sequenced revealing a cluster of 15 P450 genes and one salivary protein gene (SG7-2). Contrary to A. gambiae, AfCYP6M1 is triplicated in A. funestus, while AgCYP6Z2 orthologue is absent. Five hundred and sixty-five new single nucleotide polymorphisms (SNPs)were identified for genetic mapping from rp2 P450s and other genes revealing high genetic polymorphisms with one SNP every 36 bp. A significant genotype/phenotype association was detected for rp2 P450s but not for a cluster of cuticular protein genes previously associated with resistance in A. gambiae. QTL mapping using F6 AIL confirms the rp2 QTL with an increase logarithm of odds score of 5. Multiplex gene expression profiling of 15 P450s and other genes around rp2 followed by individual validation using qRT–PCR indicated a significant overexpression in the resistant FUMOZ-R strain of the P450s AfCYP6Z1, AfCYP6Z3, AfCYP6M7 and the glutathione-s-transferase GSTe2 with respective fold change of 11.2,6.3, 5.5 and 2.8. Polymorphisms analysis of AfCYP6Z1 and AfCYP6Z3 identified amino acid changes potentially associated with resistance further indicating that these genes are controlling the pyrethroid resistance explained by the rp2 QTL. The characterisation of this rp2 QTL significantly improves our understanding of resistance mechanisms in A. funestus

Transduction of SIV-Specific TCR Genes into Rhesus Macaque CD8+ T Cells Conveys the Ability to Suppress SIV Replication

The SIV/rhesus macaque model for HIV/AIDS is a powerful system for examining the contribution of T cells in the control of AIDS viruses. To better our understanding of CD8(+) T-cell control of SIV replication in CD4(+) T cells, we asked whether TCRs isolated from rhesus macaque CD8(+) T-cell clones that exhibited varying abilities to suppress SIV replication could convey their suppressive properties to CD8(+) T cells obtained from an uninfected/unvaccinated animal.We transferred SIV-specific TCR genes isolated from rhesus macaque CD8(+) T-cell clones with varying abilities to suppress SIV replication in vitro into CD8(+) T cells obtained from an uninfected animal by retroviral transduction. After sorting and expansion, transduced CD8(+) T-cell lines were obtained that specifically bound their cognate SIV tetramer. These cell lines displayed appropriate effector function and specificity, expressing intracellular IFNγ upon peptide stimulation. Importantly, the SIV suppression properties of the transduced cell lines mirrored those of the original TCR donor clones: cell lines expressing TCRs transferred from highly suppressive clones effectively reduced wild-type SIV replication, while expression of a non-suppressing TCR failed to reduce the spread of virus. However, all TCRs were able to suppress the replication of an SIV mutant that did not downregulate MHC-I, recapitulating the properties of their donor clones.Our results show that antigen-specific SIV suppression can be transferred between allogenic T cells simply by TCR gene transfer. This advance provides a platform for examining the contributions of TCRs versus the intrinsic effector characteristics of T-cell clones in virus suppression. Additionally, this approach can be applied to develop non-human primate models to evaluate adoptive T-cell transfer therapy for AIDS and other diseases

Essential Domains of Anaplasma phagocytophilum Invasins Utilized to Infect Mammalian Host Cells

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLex-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLex sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLex but express the structurally similar glycan, 6-sulfo-sLex, required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLex antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLex antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilumbinding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection

Regulatory T cell-derived extracellular vesicles modify dendritic cell function

Regulatory T cells (Treg) are a subpopulation of T cells that maintain tolerance to self and limit other immune responses. They achieve this through different mechanisms including the release of extracellular vesicles (EVs) such as exosomes as shown by us, and others. One of the ways that Treg derived EVs inhibit target cells such as effector T cells is via the transfer of miRNA. Another key target for the immunoregulatory function of Tregs is the dendritic cells (DCs). In this study we demonstrate directly, and for the first time, that miRNAs are transferred from Tregs to DCs via Treg derived EVs. In particular two miRNAs, namely miR-150-5p and miR-142-3p, were increased in DCs following their interaction with Tregs and Treg derived exosomes. One of the consequences for DCs following the acquisition of miRNAs contained in Treg derived EVs was the induction of a tolerogenic phenotype in these cells, with increased IL-10 and decreased IL-6 production being observed following LPS stimulation. Altogether our findings provide data to support the idea that intercellular transfer of miRNAs via EVs may be a novel mechanism by which Tregs regulate DC function and could represent a mechanism to inhibit immune reactions in tissues

IFNγ and IL-12 restrict Th2 responses during Helminth/Plasmodium co-infection and promote IFNγ from Th2 cells

Parasitic helminths establish chronic infections in mammalian hosts. Helminth/Plasmodium co-infections occur frequently in endemic areas. However, it is unclear whether Plasmodium infections compromise anti-helminth immunity, contributing to the chronicity of infection. Immunity to Plasmodium or helminths requires divergent CD4+ T cell-driven responses, dominated by IFNγ or IL-4, respectively. Recent literature has indicated that Th cells, including Th2 cells, have phenotypic plasticity with the ability to produce non-lineage associated cytokines. Whether such plasticity occurs during co-infection is unclear. In this study, we observed reduced anti-helminth Th2 cell responses and compromised anti-helminth immunity during Heligmosomoides polygyrus and Plasmodium chabaudi co-infection. Using newly established triple cytokine reporter mice (Il4gfpIfngyfpIl17aFP635), we demonstrated that Il4gfp+ Th2 cells purified from in vitro cultures or isolated ex vivo from helminth-infected mice up-regulated IFNγ following adoptive transfer into Rag1-/- mice infected with P. chabaudi. Functionally, Th2 cells that up-regulated IFNγ were transcriptionally re-wired and protected recipient mice from high parasitemia. Mechanistically, TCR stimulation and responsiveness to IL-12 and IFNγ, but not type I IFN, was required for optimal IFNγ production by Th2 cells. Finally, blockade of IL-12 and IFNγ during co-infection partially preserved anti-helminth Th2 responses. In summary, this study demonstrates that Th2 cells retain substantial plasticity with the ability to produce IFNγ during Plasmodium infection. Consequently, co-infection with Plasmodium spp. may contribute to the chronicity of helminth infection by reducing anti-helminth Th2 cells and converting them into IFNγ-secreting cells