Anna Rosa Sannella

Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNγ, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFβ, TGFβRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFβ and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

New uses for old drugs. Auranofin, a clinically established antiarthritic metallodrug, exhibits potent antimalarial effects in vitro: Mechanistic and pharmacological implications

The clinically established gold‐based antiarthritic drug auranofin (AF) manifests a pronounced reactivity toward thiol and selenol groups of proteins. In particular, AF behaves as a potent inhibitor of mammalian thioredoxin reductases causing severe intracellular oxidative stress. Given the high sensitivity of Plasmodium falciparum to oxidative stress, we thought that auranofin might act as an effective antimalarial agent. Thus, we report here new experimental results showing that auranofin and a few related gold complexes strongly inhibit P. falciparum growth in vitro. The observed antiplasmodial effects probably arise from direct inhibition of P. falciparum thioredoxin reductase. The above findings and the safe toxicity profile of auranofin warrant rapid evaluation of AF for malaria treatment in animal models.

Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction

A molecular assay has been developed for the specific detection and genetic characterisation of Plasmodium falciparum gametocytes in the blood of malaria infected individuals. The assay is based on the reverse transcription and polymerase chain reaction (RT-PCR) amplification of the messenger RNA of gene pfg377, a sexual-stage specific transcript abundantly produced in maturing gametocytes. The gene contains four regions of repetitive sequences, of which region 3 was shown to be the most polymorphic in laboratory clones and field isolates of the parasite. Analysis of samples of malaria infected blood by RT-PCR specific for region 3 has enabled identification of multiple gametocyte-producing clones within single infections. The assay is able to detect gametocytes below the threshold of microscopic detection, and is highly specific for its gametocyte targets also in the presence of a vast excess of asexual forms.

Outstanding plasmodicidal properties within a small panel of metallic compounds: hints for the development of new metal-based antimalarials

A variegate group of metallodrugs was evaluated in vitro for antimalarial activity through the pLDH test. The panel comprised one mononuclear gold(III) complex, (Aubipy), three dinuclear gold(III) compounds (Auoxo4, Auoxo5 and Auoxo6), three ruthenium(III) complexes (NAMI A, PMRU20, PMRU27), one ruthenium(II) complex (PMRU52), one bismuth(III) compound (Bismuth citrate), antimony trichloride (SbCl(3)) and arsenic trioxide (As(2)O(3)). This panel, although relatively small, was built up in such a way to include a variety of metal centers, structural motifs and metal coordination environments. In general, the tested compounds turned out to contrast effectively Plasmodium falciparum growth in vitro. In two cases, i.e. NAMI A and antimony trichloride, IC(50) values in the high nanomolar range were measured. Notably, the antiplasmodial effects appear not to be correlated to in vitro anticancer properties. The mechanistic and pharmacological implications of the obtained results are discussed.

A rare Cryptosporidium parvum genotype associated with infection of lambs and zoonotic transmission in Italy

Abstract An outbreak of cryptosporidiosis occurred in a mixed sheep/cattle farm of Central Italy in October 2011. A total of 450 ovines (250 sheep and 200 lambs) and 140 bovines (130 cows and 10 calves) were housed in two separated units, at the time of the outbreak. About half of the lambs had diarrhea due to Cryptosporidium sp. with a mortality rate of 80%; calves were not infected. Genomic DNA was extracted from an archived slide and from fecal specimens, and the parasite was identified as Cryptosporidium parvum by PCR and sequence analysis at the CpA135 gene. Genotyping at the GP60 gene showed the presence of a very rare genotype, IIaA20G2R1. Shortly after the outbreak was identified, the son of the farms owner, aged 18 months, experienced an acute gastroenteritis and was hospitalized due to recurrent episodes of diarrhea, fever, vomiting and lack of appetite. The feces tested negative for bacteria and viruses, whereas cryptosporidiosis was diagnosed by microscopy and an immunochromatographic test. Molecular typing identified the C. parvum genotype IIaA20G2R1 in the feces of the child. This is the first case of transmission of cryptosporidiosis in Italy involving lambs as source of oocysts infectious to humans.

Pigs as Natural Hosts of Dientamoeba fragilis Genotypes Found in Humans

The world is home to more than 1 billion pigs, which produce large quantities of feces. We know that some organisms in pig feces can cause human disease, and now we might have another to add to the list. Little is known about where the common intestinal parasite Dientamoeba fragilis comes from and how it is spread. However, recent molecular analysis confirmed that the organism found in pigs is indeed the same as the one found in humans. Therefore, pigs (or their feces) might be a source of this parasitic infection in humans.

Selected gold compounds cause pronounced inhibition of Falcipain 2 and effectively block P. falciparum growth in vitro

A number of structurally diverse gold compounds were evaluated as possible inhibitors of Falcipain 2 (Fp2), a cysteine protease from P. falciparum that is a validated target for the development of novel antimalarial drugs. Remarkably, most tested compounds caused pronounced but reversible inhibition of Fp2 with K(i) values falling in the micromolar range. Enzyme inhibition is basically ascribed to gold binding to catalytic active site cysteine. The same gold compounds were then tested for their ability to inhibit P. falciparum growth in vitro; important parasite growth inhibition was indeed observed. However, careful analysis of the two sets of data failed to establish any direct correlation between enzyme inhibition and reduction of P. falciparum growth suggesting that Fp2 inhibition represents just one of the various mechanisms through which gold compounds effectively antagonize P. falciparum replication.

Genetic Confirmation of Quinine-Resistant Plasmodium falciparum Malaria Followed by Postmalaria Neurological Syndrome in a Traveler from Mozambique

ABSTRACT A case of quinine-resistant Plasmodium falciparum malaria, followed by a postmalaria neurological syndrome and a recurrence episode, is described. Genetic characterization of the P. falciparum isolate obtained by analysis of msp1 and msp2 amplicons revealed the coexistence of two genotypes causing the first malaria episode and the presence of a unique isolate responsible for the recurrence.

Plasmodium falciparum soluble extracts potentiate the suppressive function of polyclonal T regulatory cells through activation of TGFβ‐mediated signals

Increased numbers of T regulatory cells (Tregs), key mediators of immune homeostasis, were reported in human and murine malaria and it is current opinion that these cells play a role in balancing protective immunity and pathogenesis during infection. However, the mechanisms governing their expansion during malaria infection are not completely defined. In this article we show that soluble extracts of Plasmodium falciparum (PfSEs), but not equivalent preparation of uninfected erythrocytes, induce the differentiation of polyclonally activated CD4+ cells in Tregs endowed with strong suppressive activity. PfSEs activate latent TGFβ bound on the membrane of Treg cells, thus allowing the cytokine interaction with TGFβ receptor, and inducing Foxp3 gene expression and TGFβ production. The activation of membrane‐bound latent TGFβ by PfSEs is significantly reduced by a broad‐spectrum metalloproteinases inhibitor with Zn++‐chelating activity, and completely inhibited by the combined action of such inhibitor and antibodies to a P. falciparum thrombospondin‐related adhesive protein (PfTRAP). We conclude that Pf‐Zn++‐dependent proteinases and, to a lesser extent, PfTRAP molecules are involved in the activation of latent TGFβ bound on the membrane of activated Treg cells and suggest that, in malaria infection, this mechanism could contribute to the expansion of Tregs with different antigen specificity.

Comparative Proteomics and Functional Analysis Reveal a Role of Plasmodium falciparum Osmiophilic Bodies in Malaria Parasite Transmission

An essential step in the transmission of the malaria parasite to the Anopheles vector is the transformation of the mature gametocytes into gametes in the mosquito gut, where they egress from the erythrocytes and mate to produce a zygote, which matures into a motile ookinete. Osmiophilic bodies are electron dense secretory organelles of the female gametocytes which discharge their contents during gamete formation, suggestive of a role in gamete egress. Only one protein with no functional annotation, Pfg377, is described to specifically reside in osmiophilic bodies in Plasmodium falciparum. Importantly, Pfg377 defective gametocytes lack osmiophilic bodies and fail to infect mosquitoes, as confirmed here with newly produced pfg377 disrupted parasites. The unique feature of Pfg377 defective gametocytes of lacking osmiophilic bodies was here exploited to perform comparative, label free, global and affinity proteomics analyses of mutant and wild type gametocytes to identify components of these organelles. Subcellular localization studies with fluorescent reporter gene fusions and specific antibodies revealed an osmiophilic body localization for four out of five candidate gene products analyzed: the proteases PfSUB2 (subtilisin 2) and PfDPAP2 (Dipeptidyl aminopeptidase 2), the ortholog of the osmiophilic body component of the rodent malaria gametocytes PbGEST and a previously nonannotated 13 kDa protein. These results establish that osmiophilic bodies and their components are dispensable or marginally contribute (PfDPAP2) to gamete egress. Instead, this work reveals a previously unsuspected role of these organelles in P. falciparum development in the mosquito vector.