Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

Additives and Protein-DNA Combinations Modulate the Humoral Immune Response Elicited by a Hepatitis C Virus Core-encoding Plasmid in Mice

Humoral and cellular immune responses are currently induced against hepatitis C virus (HCV) core following vaccination with core-encoding plasmids. However, the anti-core antibody response is frequently weak or transient. In this paper, we evaluated the effect of different additives and DNA-protein combinations on the anti-core antibody response. BALB/c mice were intramuscularly injected with an expression plasmid (pIDKCo), encoding a C-terminal truncated variant of the HCV core protein, alone or combined with CaCl2, PEG 6000, Freund's adjuvant, sonicated calf thymus DNA and a recombinant core protein (Co.120). Mixture of pIDKCo with PEG 6000 and Freund's adjuvant accelerated the development of the anti-core Ab response. Combination with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core antibody response elicited by Co.120 alone or combined with pIDKCo was different regarding that induced by the pIDKCo alone. Our data indicate that the antibody response induced following DNA immunization can be modified by formulation strategies.

Risk Zones of Human Leishmaniases in the Western Mediterranean Basin: Correlations between Vector Sand Flies, Bioclimatology and Phytosociology

Correspondence analysis was applied to sand fly sampling in 865 stations from the Western Mediterranean basin. The position of each of 24 species was determined with respect to the bioclimatic belts. Thus, the multidimensional analyses manifest clear correlations between bioclimatic belts and their expression in the area, the phytosociological groupings, and vector species of visceral and cutaneous leishmaniases. The transfer of these data to usual maps allows to delimit the geographical distribution of these diseases in the Western Mediterranean basin and contributes to the determination, in a rational manner, of the high risk zones.

An Adenovirus Vector Containing the Suicide Gene Thymidine Kinase for a Broad Application in Cancer Gene Therapy

Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK) gene, followed by administration of the antiviral drug ganciclovir (GCV). The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK) driven by three strong promoters (P CMV IE, SV40 and EN1) and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.

Aggregation mediated by faeces and footprints in Triatoma pseudomaculata (Heteroptera: Reduviidae), a Chagas disease vector

The behavioural response of Triatoma pseudomaculata to chemical substances present in their faeces or cuticle (footprints) was analyzed. Groups of larvae were simultaneously exposed to a clean filter paper and to another paper impregnated with a chemical stimulus in a circular arena. In these choice experiments, the insects aggregated significantly around papers impregnated with dry faeces. In addition, the bugs also showed a significant aggregation response to papers impregnated with compounds derived from their cuticle that were deposited by contact on the substrate. These results indicate that chemical compounds that affect the behaviour of T. pseudomaculata are present in the faeces and in the cuticle of this species. Results are discussed in relation to chemical communication in the Triatominae, as well as to the potential use of these substances in traps or sensors for the detection of this species.

Protective immunity of single and multi-antigen DNA vaccines against schistosomiasis

We evaluated the usefulness of the combination of three plasmids encoding tegumental (pECL and pSM14) and muscular (pIRV5) antigens of the Schistosoma mansoni on improving the protective immunity over the use of a single antigen as DNA vaccines. Female BALB/c mice were inoculated twice with 25 µg DNA plasmid within two weeks interval. The challenge was performed with 80 cercarias of a regional isolate of S. mansoni (SLM) one week after the last immunization. Six weeks after challenge, all mice were perfused for worm load determination. The following groups were analyzed: saline; empty vector; monovalent formulations of pECL; pSM14 and pIRV5 and also double combinations of pECL/pIRV5 and pIRV5/pSM14 and a triple combination of pECL/pIRV5/pSM14. The protection was expressed as a percentage of worm loads in each group compared with the saline group. The results obtained were 41% (p < 0.05); 52% (p < 0.05); 51% (p < 0.05); 48% (p < 0.05); 55% (p < 0.05); 45% (p < 0.05); 65% (p < 0.05) for each group respectively.

The epidemiologic importance of Triatoma brasiliensis as a Chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999

To clarify the epidemiologic importance of Triatoma brasiliensis, the most important Chagas disease vector in the Northeastern of Brazil, capture data related to this species, its distribution, capture index, and percentages of natural infection by Trypanosoma cruzi were examined in 12 different Brazilian states. The Brazilian National Health Foundation collected these data from 1993 to 1999, a period during which a total of 1,591,280 triatomines (21 species) were captured in domiciles within the geographic range of T. brasiliensis. Of this total, 422,965 (26.6%) were T. brasiliensis, 99.8% of which were collected in six states, and 54% in only one state (Ceará). The percentage of bugs infected with T. cruzi varied significantly among states, ranging from 0% (Goiás, Maranhão, Sergipe, and Tocantins) to more than 3% (Alagoas, Minas Gerais, and Rio Grande do Norte) with an average of 1.3%. This latter value represents a dramatic reduction in the natural infection percentages since 1983 (6.7%) suggesting that, despite the impossibility of eradicating this native species, the control measures have significantly reduced the risk of transmission. However, the wide geographic distribution of T. brasiliensis, its high incidence observed in some states, and its variable percentages of natural infection by T. cruzi indicate the need for sustained entomological surveillance and continuous control measures against this vector.

Recent observations on the sand fly (Diptera: Psychodidae) fauna of the State of Rondônia, Western Amazônia, Brazil: the importance of Psychdopygus davisi as a vector of zoonotic cutaneous leishmaniasis

Sand flies were collected in the central region of the state of Rondônia (W 64º30' to 63º00' and S 10º00'to 11º00') using Shannon and CDC light traps from October 1997 to August 2000. A total of 85,850 specimens representing 78 named species were captured. Of these 14 were new records for Rondônia. The proportion of males/females was 1/1.131. Trypanosomatids, that are presently being identified, were detected in 11 species. Leishmania (Viannia) naiffi was recorded from Psychodopygus davisi and P. hirsutus. In the present study the dominant species was P. davisi (39.6%) followed by Lutzomyia whitmani (13.1%), P. carrerai (11.6%), and P. hirsutus (10.2%). The importance of P. davisi as a vector of zoonotic cutaneous leishmaniasis is discussed.

Hemilucilia segmentaria (Fabricius, 1805) (Diptera: Calliphoridae) as new biological vector of eggs of Dermatobia hominis (Linnaeus Jr., 1781) (Diptera: Oestridae) in Reserva Biológica do Tinguá, Rio de Janeiro, Brazil

The aim of this note was to record for the first time the finding of Hemilucilia segmentaria acting as biological vector of Dermatobia hominis, during a study of the diversity of Calliphoridae at Reserva Biológica do Tinguá, Rio de Janeiro, Brazil. The insects were captured using traps baited with chicken vicera, for a period of 28-30 h twice per month. In the period of one year, 1987 insects were collected, 7.5% of which belonged to the H. segmentaria; of these a female was captured in May 2001, carrying a mass of 20 eggs on the left side of its abdomen.

Molluscicidal effect of nicotinanilide and its intermediate compounds against a freshwater snail Lymnaea luteola, the vector of animal schistosomiasis

The molluscicidal effect of nicotinanilide was evaluated and compared with niclosamide (2',5-dichloro-4'-nitrosalicylanilide, ethanolamide salt) against different stages of the freshwater snail Lymnaea luteola i.e., eggs, immature, young mature, and adults. Calculated values of lethal concentrations (LC50 and LC90 ) showed that both nicotinanilide and niclosamide as toxic against eggs, immature, and adults. The young mature stage of the snails was comparatively more tolerant to both molluscicides than the other stages. The toxicity of the intermediate compounds of nicotinanilide against the young mature stage of the snails showed them as ineffective. The mortality pattern of the snails exposed to LC90 concentration of these molluscicides showed niclosamide to kill faster (within 8 to 9 h) than nicotinanilide (26 to 28 h). In view of the above studies it may be concluded that both molluscicides are toxic against all the stages of the L. luteola snails.

Evaluation of cholinesterase level in an endemic population exposed to malathion suspension formulation as a vector control measure

The manuscript describes a study on the blood cholinesterase (ChE) level in an exposed population at different interval of time after spraying with malathion suspension (SRES) use for kala-azar vector control in an endemic area of Bihar, India. The toxicity of a 5% malathion formulation in the form of a slow release emulsified suspension (SRES) was assessed by measuring serum ChE levels in spraymen and in the exposed population.The study showed a significant decrease in ChE levels in the spraymen (p < 0.01) after one week of spraying and in exposed population one week and one month after of spraying (p < 0.01), but was still within the normal range of ChE concentration, one year after spraying, the ChE concentration in the exposed population was the same as prior to spraying (p > 0.01). On no occasion was the decrease in ChE level alarming. A parallel examination of the clinical status also showed the absence of any over toxicity or any behavioural changes in the exposed population. Hence, it may be concluded that 5% malathion slow release formulation, SRES, is a safe insecticide for use as a vector control measure in endemic areas of kala-azar in Bihar, India so long as good personal protection for spraymen is provided to minimize absorption and it can substitute the presently used traditional DDT spray.

HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi

This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.

Triatoma rubrovaria (Blanchard, 1843) (Hemiptera - Reduviidae - Triatominae) IV: bionomic aspects on the vector capacity of nymphs

Triatoma rubrovaria has become the most frequently captured triatomine species since the control of T. infestans in the state of Rio Grande do Sul (RS), Brazil. The aim of this study was to evaluate aspects of the vectorial competence of T. rubrovaria using nymphs raised in laboratory under environmental conditions of temperature and humidity and fed on mice. The average developmental period of T. rubrovaria was 180.1 days. The percentage of defecation shortly after feeding was still higher than previous studies in which samples of T. rubrovaria subjected to a slight starvation period before the blood meal were used. The obtained results support former indication that T. rubrovaria presents bionomic characteristics propitious to be a good vector of Trypanosoma cruzi to man. Therefore its domiciliary invasion process must be continuously monitored.