Carlo Severini

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.

Malaria in Maremma, Italy.

BACKGROUND In August, 1997, a woman with no history of travel to malarious regions developed Plasmodium vivax malaria. She lived in a rural area of Italy where indigenous Anophyles labranchiae mosquitoes were present. METHODS AND FINDINGS An environmental investigation was done within a 3 km radius of the patients house. Adult mosquitoes and larvae were collected and examined by PCR with the gene for plasmodium circumsporozoite protein as target. About 200 people living in the area were interviewed to detect possible carriers of P. vivax. FINDINGS None of the mosquitoes captured were carrying any malarial organisms. The house-to-house investigation identified a 7-year-old girl who had had a feverish illness a few days after her arrival in Italy from India, and who, 3 months later, still had P. vivax in her blood; she and her mother had antimalarial antibodies. INTERPRETATION These investigations suggest that the index case of malaria was caused by local anopheline mosquitoes infected with exogenous P. vivax.

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.

Phylogenetic relationships of seven palearctic members of the maculipennis complex inferred from ITS2 sequence analysis

The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined from seven palearctic mosquitoes species belonging to the Anopheles maculipennis species complex, namely An. atroparvus, An. labranchiae, An. maculipennis, An. messeae, An. melanoon, An. sacharovi and An. martinius. The length of the ITS2 ranged from 280 to 300 bp, with a GC content of 49.4–54.1%. With the exception of An. messeae, negligible levels of intraspecific polymorphism and no intrapopulation variation were observed. The phylogenetic relationships among the members of the maculipennis complex were inferred by maximum‐parsimony analysis of the paup program and the neighbour‐joining and maximun‐likelihood analysis of the phylip program. All of the trees obtained were almost identical in topology, although the relationships among three species, i.e. An. maculipennis, An. messeae and An. melanoon, remained unresolved. The phylogenies were in good agreement with the previous gene–enzyme and polytene chromosome banding pattern studies.

Identification of the Plasmodium vivax mdr-Like Gene (pvmdr1) and Analysis of Single-Nucleotide Polymorphisms among Isolates from Different Areas of Endemicity

Because of the lack of methods for continuous in vitro culture of Plasmodium vivax, little is known about drug-resistance mechanisms in this malaria-causing parasite. Therefore, identification of all the genes potentially involved in drug resistance and of molecular markers related to drug resistance would provide a framework for studying the incidence and spread of drug-resistant P. vivax strains. We have identified the P. vivax orthologue of the pfmdr1 gene (pvmdr1), which was shown to have a role in the drug resistance of Plasmodium falciparum. Comparison of the alignments of both nucleotide and amino acid sequences of pvmdr1 with those of other Plasmodium multidrug-resistance genes revealed an open-reading frame of 4392 base pairs encoding a deduced protein of 1464 amino acids. Nucleotide polymorphisms at 2 codons of the pvmdr1 gene--Y976F and F1076L--were found in 14 of 23 P. vivax isolates from different areas of endemicity, including Thailand, Indonesia, Turkey, Azerbaijan, and French Guyana.

The β-tubulin gene of Babesia and Theileria parasites is an informative marker for species discrimination☆

A fragment of the beta-tubulin gene was polymerase chain reaction (PCR) amplified from genomic DNAs of Babesia bovis, Babesia bigemina, Babesia divergens, Babesia major, Babesia caballi, Babesia equi, Babesia microti, Theileria annulata and Theileria sergenti. Single amplification products were obtained for each of these species, but the size of the amplicons varied from 310 to 460 bp. Sequence analysis revealed that this variation is due to the presence of a single intron, which ranged from 20 to 170 bp. The extensive genetic variability at the beta-tubulin locus has been exploited to develop two types of species identification assays. The first assay can be used on samples containing mostly parasite DNA, like those prepared from infected erythrocytes. Following PCR amplification, the species identification is obtained directly from the size of the products (for Babesia species infecting human or horse) or using a simple PCR-restriction fragment length polymorphism (RFLP) protocol (for Babesia species infecting cattle). The second assay can be used on samples prepared from whole blood, that contain both parasite and host DNAs. In this case, due to the strong conservation of the beta-tubulin gene, co-amplification of a gene fragment from the host DNA was observed. A nested PCR assay was developed for the specific amplification of parasite DNA, using a primer designed to span the exon-intron boundary. Direct identification of Babesia species infecting human and horse is again obtained after the electrophoretic separation of the amplification products, while for Babesia and Theileria species infecting cattle, differentiation is based on a nested PCR-RFLP protocol. These methods may be used for the simultaneous identification of horses and cattle carrying multiple parasites by means of a single PCR or using the PCR-RFLP protocol.

Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains

Background Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes. Methods/Principal Findings Through recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported. Conclusions/Significance The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations.

Artemisinin and artemisinin plus curcumin liposomal formulations: Enhanced antimalarial efficacy against Plasmodium berghei-infected mice

The therapeutic efficacies of novel liposomal delivery systems based on artemisinin or artemisinin-based combination therapy with curcumin have been investigated and reported in this study. The developed liposomal formulations had proper characteristics as drug carriers for parental administration in terms of particle size, polydispersity, encapsulation efficacy and ζ-potential. Their physical and chemical stabilities were also evaluated. Furthermore, the in vivo antimalarial activity of artemisinin-based liposomal formulations was tested in Plasmodium berghei NK-65 infected mice, a suitable model for studying malaria because the infection presents structural, physiological and life cycle analogies with the human disease. Artemisinin, alone or in combination with curcumin, was encapsulated in conventional and PEGylated liposomes and its in vivo performance was assessed by comparison with the free drug. Mice were treated with artemisinin at the dosage of 50 mg/kg/days alone or plus curcumin as partner drug, administered at the dosage of 100 mg/kg/days. Artemisinin alone began to decrease parasitaemia levels only 7 days after the start of the treatment and it appeared to have a fluctuant trend in blood concentration which is reflected in the antimalarial effectiveness. By contrast, treatments with artemisinin-loaded conventional liposomes (A-CL), artemisinin-curcumin-loaded conventional liposomes (AC-CL), artemisinin-loaded PEGylated liposomes (A-PL), artemisinin-curcumin-loaded PEGylated liposomes (AC-PL) appeared to have an immediate antimalarial effect. Both nanoencapsulated artemisinin and artemisinin plus curcumin formulations cured all malaria-infected mice within the same post-inoculation period of time. Additionally, all formulations showed less variability in artemisinin plasma concentrations which suggested that A-CL, AC-CL, A-PL and AC-PL give a modified release of drug(s) and, as a consequence, a constant antimalarial effect during time. In particular, A-PL seems to give the most pronounced and statistically significant therapeutic effect in this murine model of malaria. The enhanced permanency in blood of A-PL suggests the use of these nanosystems as suitable passive targeted carriers for parasitic infections; this strong effect of formulation is added up to the mechanism of action of artemisinin which acts in the erythrocyte cycle stage of human host as a blood schizonticide.

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.

Genetic diversity of Plasmodium vivax isolates from Azerbaijan

BackgroundPlasmodium vivax, although causing a less serious disease than Plasmodium falciparum, is the most widespread of the four human malarial species. Further to the recent recrudescence of P. vivax cases in the Newly Independent States (NIS) of central Asia, a survey on the genetic diversity and dissemination in Azerbaijan was undertaken. Azerbaijan is at the crossroads of Asia and, as such, could see a rise in the number of cases, although an effective malaria control programme has been established in the country.MethodsThirty-six P. vivax isolates from Central Azerbaijan were characterized by analysing the genetic polymorphism of the circumsporozoite protein (CSP) and the merozoite surface protein 1 (MSP-1) genes, using PCR amplifications and amplicons sequencing.ResultsAnalysis of CSP sequences showed that all the processed isolates belong to the VK 210 type, with variations in the alternation of alanine residue (A) or aspartic acid residue (D) in the repeat motif GDRA(A/D)GQPA along the sequence. As far as MSP-1 genotyping is concerned, it was found that the majority of isolates analysed belong to Belem and Sal I types. Five recombinant isolates were also identified. Combined analysis with the two genetic markers allowed the identification of 19 plasmodial sub-types.ConclusionThe results obtained in the present study indicate that there are several P. vivax clones circulating in Azerbaijan and, consequently, a careful malaria surveillance could be of paramount importance to identify, at early stage, the occurrence of possible P. vivax malaria outbreaks.