KAP1/TRIM28: an inhibitor of IRF5 function in inflammatory macrophages.

IRF5 plays a key role in the induction of pro-inflammatory cytokines, contributing to the plasticity and polarisation of macrophages to an M1 phenotype and initiation of a potent T(H)1-T(H)17 response. To better understand the means of IRF5 transcriptional action, we conducted a screen for IRF5-interacting partners by affinity purification coupled to mass spectrometry and identified KAP1/TRIM28 as a novel protein-protein interaction partner of IRF5. KAP1 acts as a transcriptional co-repressor, chiefly via recruitment of complexes involved in chromatin silencing, such as histone deacetylases and methyltransferases. We mapped the N-terminus of IRF5, encompassing its DNA-binding domain together with a highly intrinsically disordered region, as crucial for the IRF5-KAP1 interaction interface, and demonstrated that IRF5 can also form complexes with the methyltransferase SETDB1. Knockdown of KAP1 (TRIM28) gene expression in human M1 macrophages potentiated IRF5-mediated expression of TNF and other M1 macrophage markers. This effect may be linked to methyltransferase activity of SETDB1, such as trimethylation of lysine 9 of histone 3 (H3K9me3), deposition of which was decreased at the human TNF locus upon KAP1 knockdown. Our study furthers an understanding of the complex molecular interactions between the TRIM and IRF protein families, and highlights a role of the inhibitory properties of KAP1 in association with IRF5-mediated gene expression.

An Anopheles gambiae salivary gland promoter analysis in Drosophila melanogaster and Anopheles stephensi.

Regulatory regions driving gene expression in specific target organs of the African malaria vector Anopheles gambiae are of critical relevance for studies on Plasmodium-Anopheles interactions as well as to devise strategies for blocking malaria parasite development in the mosquito. In order to identify an appropriate salivary gland promoter we analysed the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4. An 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic Anopheles stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-rather than in the distal-lateral lobes of female glands. Surprisingly, a promoter fragment from the D7r4 gene conferred strong tissue-specific expression in Drosophila melanogaster but only low transcription levels in transgenic An. stephensi. These results imply a certain conservation of gland-specific control elements between the fruit fly and the mosquito suggesting that an increased degree of complexity, probably connected to the evolution of haematophagy, underlies the regulation of tissue-specific expression in mosquito female salivary glands.

A cluster of four D7-related genes is expressed in the salivary glands of the African malaria vector Anopheles gambiae.

Four genes expressed in the Anopheles gambiae adult female salivary glands and similar in sequence to the Aedes aegypti D7 gene were identified. The genes, called D7-related (D7r), are included in a single cluster encompassing approximately six kilobases on chromosome arm 3R. The deduced proteins contain secretory signals and they are probably injected by the mosquito into the host with the saliva during blood feeding. The region of similarity to D7 encompasses the carboxy-terminal part of the Ae. aegypti protein and the different An. gambiae D7r show a degree of similarity to each other, varying from 53% to 73%. The weak but significant similarity to members of a wide family of insect proteins, including odourant- and pheromone-binding proteins, raises the possibility that the D7r-encoded proteins may bind and/or carry small hydrophobic ligands.

The lower susceptibility to Plasmodium falciparum malaria of Fulani of Burkina Faso (west Africa) is associated with low frequencies of classic malaria-resistance genes.

The gene frequencies in 1993-94 for haemoglobin S, haemoglobin C, alpha-3.7 deletional thalassaemia, G6PDA-, HLAB*5301 were estimated in Fulani, Mossi and Rimaibé ethnic groups of Burkina Faso, West Africa. The aim of the study was to verify whether the previously reported Fulani lower susceptibility to Plasmodium falciparum malaria was associated with any of these malaria-resistance genes. Similar frequencies for haemoglobin S were recorded in the 3 ethnic groups (0.024 +/- 0.008, 0.030 +/- 0.011, 0.022 +/- 0.013; in Mossi, Rimaibé and Fulani, respectively). The Mossi and Rimaibé showed higher frequencies when compared to Fulani for haemoglobin C (0.117 +/- 0.018, 0.127 +/- 0.020, 0.059 +/- 0.020), alpha-3.7 deletional thalassaemia (0.227 +/- 0.040, 0.134 +/- 0.032, 0.103 +/- 0.028), G6PDA- (0.196 +/- 0.025, 0.187 +/- 0.044, 0.069 +/- 0.025) and HLA B*5301 (0.189 +/- 0.038, 0.202 +/- 0.041, 0.061 +/- 0.024). Among Fulani the proportion of individuals not having any of these protective alleles was more than 3-fold greater than in the Mossi-Rimaibé group (56.8% vs 16.7%; P < 0.001). These findings exclude the involvement of these genetic factors of resistance to P. falciparum in the lower susceptibility to malaria of Fulani. This evidence, in association with the previously reported higher immune reactivity to malaria of Fulani, further supports the existence in this ethnic group of unknown genetic factor(s) of resistance to malaria probably involved in the regulation of humoral immune responses.

Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s.

The sequence of a 2.3 kb long DNA segment derived from the 5'-most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako.

Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation.

Three chromosomal forms of Anopheles gambiae s.s., designated as Bamako, Mopti and Savanna, were studied for diagnostic PCR assays based on the analysis of the X-linked ribosomal DNA (rDNA). The study was performed on a 1.3 kb fragment containing part of the 28S coding region and part of the intergenic spacer region. The amplified material was cut with fourteen restriction enzymes to detect Restriction Fragment Length Polymorphisms (RFLPs). The enzymes Tru9I and HhaI produced patterns of DNA bands which differentiated Mopti from Savanna and Bamako; moreover, a distinct 'hybrid' pattern was recognized in the F1 female progeny from the cross of Mopti with either one of the other two chromosomal forms. The diagnostic significance of the PCR-RFLP assay was verified on 203 karyotyped females from field samples collected in two villages in Mali and one village in Burkina Faso. Agreement was observed between the chromosomal and the molecular identifications. No 'hybrid' molecular patterns were detected even among carriers of rare heterokaryotypes hypothetically produced by crosses between Mopti and Savanna. The results confirm previous observations indicating barriers to gene flow within An. gambiae s.s. and supporting the specific status of the taxonomic units proposed on cytogenetic ground.

Advances in the identification of Dirofilaria repens and Dirofilaria immitis by a PCR-based approach.

The differential epidemiology of D. repens and D. immitis is still poorly understood due to the lack of a diagnostic method which would make possible the routine identification of these parasites as developing larvae either in the vector or in unsuitable hosts, including man. The PCR-based method here described allows: i) the unambiguous identification of mature and immature adult worms in bioptic material, of microfilariae in blood samples and of developing larvae in mosquito vectors; ii) the analysis of samples stored either dry or in various preservation media, with the exception of formalin. The high specificity and sensitivity of the diagnosis improve the perspectives for comparative epidemiological investigations on D. repens and D. immitis in areas where the two nematodes are sympatric.

Polymerase chain reaction-identification of Dirofilaria repens and Dirofilaria immitis.

On the basis of known DNA sequences of Dirofilaria repens and D. immitis we designed specific primers for the amplification by Polymerase Chain Reaction (PCR) of the DNA from the two species. The PCR-based identification was found to be unambiguous and allowed specific diagnosis of microfilariae in blood samples, of developing larvae in the mosquito vector and of immature adults in bioptic material, overcoming the serious constraints of the morphological separation of these filarial parasites at the pre-adult stages. The technique was found to be very sensitive and applicable to samples stored either dry or in various preservation media, with the exception of formalin. The reliable identification of D. repens and D. immitis from bioptic material is expected to greatly enhance the chances of detecting human infections and to further clarify the role of the two parasites as pathogens of man. The possibility of routine identification of developing larvae in the vector will substantially improve the perspectives for epidemiological investigations, particularly in Southern European regions, such as Italy, where the two nematode species are largely sympatric.