Leishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties. In this review, we focus on 3 main aspects of sand fly salivary molecules: (1) structure and composition of salivary glands, including the properties of salivary molecules related to hemostasis and blood feeding, (2) immunomodulatory properties of salivary molecules and the diverse impacts of these molecules on leishmaniasis, ranging from disease exacerbation to vaccine development, and (3) use of salivary molecules for field applications, including monitoring host exposure to sand flies and the risk of Leishmania transmission. Studies showed interesting differences between salivary proteins of Phlebotomus and Lutzomyia species, however, no data were ever published on salivary proteins of Sergentomyia species. In the last 15 years, numerous studies have characterized sand fly salivary proteins and, in parallel, have addressed the impact of such molecules on the biology of the host–sand fly–parasite interaction. The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas.

In East Africa, Phlebotomus orientalis serves as the main vector of Leishmania donovani, the causative agent of visceral leishmaniasis (VL). Phlebotomus orientalis is present at two distant localities in Ethiopia; Addis Zemen where VL is endemic and Melka Werer where transmission of VL does not occur. To find out whether the difference in epidemiology of VL is due to distant compositions of P. orientalis saliva we established colonies from Addis Zemen and Melka Werer, analyzed and compared the transcriptomes, proteomes and enzymatic activity of the salivary glands. Two cDNA libraries were constructed from the female salivary glands of P. orientalis from Addis Zemen and Melka Werer. Clones of each P. orientalis library were randomly selected, sequenced and analyzed. In P. orientalis transcriptomes, we identified members of 13 main protein families. Phylogenetic analysis and multiple sequence alignments were performed to evaluate differences between the P. orientalis colonies and to show the relationship with other sand fly species from the subgenus Larroussius. To further compare both colonies, we investigated the humoral antigenicity and cross-reactivity of the salivary proteins and the activity of salivary apyrase and hyaluronidase. This is the first report of the salivary components of P. orientalis, an important vector sand fly. Our study expanded the knowledge of salivary gland compounds of sand fly species in the subgenus Larroussius. Based on the phylogenetic analysis, we showed that P. orientalis is closely related to Phlebotomus tobbi and Phlebotomus perniciosus, whereas Phlebotomus ariasi is evolutionarily more distinct species. We also demonstrated that there is no significant difference between the transcriptomes, proteomes or enzymatic properties of the salivary components of Addis Zemen (endemic area) and Melka Werer (non-endemic area) P. orientalis colonies. Thus, the different epidemiology of VL in these Ethiopian foci cannot be attributed to the salivary gland composition. Phlebotomus orientalis is the vector of visceral leishmaniasis (VL) caused by Leishmania donovani in Northeast Africa. Immunization with sand fly saliva or with individual salivary proteins has been shown to protect against leishmaniasis in different hosts, warranting the intensive study of salivary proteins of sand fly vectors. In our study, we characterize the salivary compounds of P. orientalis, thereby broadening the repertoire of salivary proteins of sand fly species belonging to the subgenus Larroussius. In order to find out whether there is any connection between the composition of P. orientalis saliva and the epidemiology of VL in two distinct Ethiopian foci, Addis Zemen and Melka Werer, we studied the transcriptomes, proteomes, enzymatic activities, and the main salivary antigens in two P. orientalis colonies originating from these areas. We did not detect any significant difference between the saliva of female sand flies originating in Addis Zemen (endemic area) and Melka Werer (non-endemic area). Therefore, the different epidemiology of VL in these Ethiopian foci cannot be related to the distant salivary gland protein composition. Identifying the sand fly salivary gland compounds will be useful for future research focused on characterizing suitable salivary proteins as potential anti-Leishmania vaccine candidates.

Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species. The sand fly Phlebotomus orientalis is the main vector of Leishmania donovani, the causative agent of visceral leishmaniasis in East Africa. During bloodfeeding, sand flies inject saliva into the host skin and repeated bites result in a specific antibody response in the bitten hosts. Antibody responses are directed against sand fly salivary proteins and the levels of these antibodies reflect the intensity of exposure to biting sand flies. The antibody reactions can be measured using salivary gland homogenates (SGHs), but for large-scale testing its use is impractical because of the amount of work required to obtain sufficient quantities of SGH. Recombinant proteins prepared based on the antigens in the sand fly saliva can substitute whole SGH in large-scale studies. We tested five recombinant proteins from Ph. orientalis saliva expressed in Escherichia coli and demonstrated that the yellow-related protein rPorSP24 can replace the SGH in estimating exposure to sand flies of dogs, goats, and sheep in Ethiopia. Immune reactions to vector saliva in endemic areas, provides useful information on levels of exposure and, thereby, on the effectiveness of vector control programs.

Small non-coding RNA molecules (miRNAs) play an important role in the epigenetic regulation of gene expression. As these molecules have been repeatedly implicated in human cancers, they have been suggested as biomarkers of the disease. Additionally, miRNA levels have been shown to be affected by environmental pollutants, including airborne contaminants. In this review, we searched the current literature for miRNAs involved in lung cancer, as well as miRNAs deregulated as a result of exposure to air pollutants. We then performed a synthesis of the data and identified those molecules commonly deregulated under both conditions. We detected a total of 25 miRNAs meeting the criteria, among them, miR-222, miR-21, miR-126-3p, miR-155 and miR-425 being the most prominent. We propose these miRNAs as biomarkers of choice for the identification of human populations exposed to air pollution with a significant risk of developing lung cancer.

This study presents a toxicological evaluation of two types of carbon dots (CD), similar in size (<10 nm) but differing in surface charge. Whole-genome mRNA and miRNA expression (RNAseq), as well as gene-specific DNA methylation changes, were analyzed in human embryonic lung fibroblasts (HEL 12469) after 4 h and 24 h exposure to concentrations of 10 and 50 µg/mL (for positive charged CD; pCD) or 10 and 100 µg/mL (for negative charged CD, nCD). The results showed a distinct response for the tested nanomaterials (NMs). The exposure to pCD induced the expression of a substantially lower number of mRNAs than those to nCD, with few commonly differentially expressed genes between the two CDs. For both CDs, the number of deregulated mRNAs increased with the dose and exposure time. The pathway analysis revealed a deregulation of processes associated with immune response, tumorigenesis and cell cycle regulation, after exposure to pCD. For nCD treatment, pathways relating to cell proliferation, apoptosis, oxidative stress, gene expression, and cycle regulation were detected. The expression of miRNAs followed a similar pattern: more pronounced changes after nCD exposure and few commonly differentially expressed miRNAs between the two CDs. For both CDs the pathway analysis based on miRNA-mRNA interactions, showed a deregulation of cancer-related pathways, immune processes and processes involved in extracellular matrix interactions. DNA methylation was not affected by exposure to any of the two CDs. In summary, although the tested CDs induced distinct responses on the level of mRNA and miRNA expression, pathway analyses revealed a potential common biological impact of both NMs independent of their surface charge.

Road traffic emissions consist of gaseous components, particles of various sizes, and chemical compounds that are bound to them. Exposure to vehicle emissions is implicated in the etiology of inflammatory respiratory disorders. We investigated the inflammation-related markers in human bronchial epithelial cells (BEAS-2B) and a 3D model of the human airways (MucilAir™), after exposure to complete emissions and extractable organic matter (EOM) from particles generated by ordinary gasoline (E5), and a gasoline-ethanol blend (E20; ethanol content 20% v/v). The production of 22 lipid oxidation products (derivatives of linoleic and arachidonic acid, AA) and 45 inflammatory molecules (cytokines, chemokines, growth factors) was assessed after days 1 and 5 of exposure, using LC-MS/MS and a multiplex immunoassay, respectively. The response observed in MucilAir™ exposed to E5 gasoline emissions, characterized by elevated levels of pro-inflammatory AA metabolites (prostaglandins) and inflammatory markers, was the most pronounced. E20 EOM exposure was associated with increased levels of AA metabolites with anti-inflammatory effects in this cell model. The exposure of BEAS-2B cells to complete emissions reduced lipid oxidation, while E20 EOM tended to increase concentrations of AA metabolite and chemokine production; the impacts on other inflammatory markers were limited. In summary, complete E5 emission exposure of MucilAir™ induces the processes associated with the pro-inflammatory response. This observation highlights the potential negative health impacts of ordinary gasoline, while the effects of alternative fuel are relatively weak.