Is the thymus a target organ in infectious diseases?

The thymus is a central lymphoid organ, in wich T cell precursors differentiale and generate most of the so-called T cell reprtoire. Along with a variety of acute infectious diseases, we and others determined important changes in both microenvironmental and lymphoid compartments of the organ. For example, one major and common feature observed in acute viral, bacterial and parasitic diseases, is a depletion of cortical thymocytes, mostly those bearing the CD4-CD8 double positive phenotype. This occurs simmultaneously to the relative enrichment in medullary CD4 or CD8 single positive cells, expressing high densities of the CD3 complex. Additionally we noticed a variety of changes in the thymic microenvironment (and particularly is epithelial component), comprising abnormal location of thymic epithelial cell subsets as well has a denser Ia-bearing cellular network. Moreover, the extracellular matrix network was altered with an intralobular increase of basement membrane proteins that positively correlated with the degree of thymocyte death. Lastly, anti-thymic cell antibodies were detected in both human and animal models of infectious diseases, and in some of them a phenomenon of molecular mimicry could be evidenced. Taken together, the data receiwed herein clearly show that the thymus should be regarded as a target in infectious diseases.

Visceral leishmaniasis in Teresina, state of Piauí, Brazil: preliminary observations on the detection and transmissibility of canine and sandfly infections

A Leishmania donovani-complex specific DNA probe was usedto confirm the widespread dissemination of amastigotes in apparently normal skinof dogs with canine visceral leishmaniasis. When Lutzomyia longipalpis were fed on abnormal skin of five naturally infected dogs 57 of 163 (35 per cent) fliesbecame infected: four of 65 flies (6 per cent) became infected when fed on apparently normal skin. The bite of a single sandfly that had fed seven days previouslyon a naturally infected dog transmitted the infection to a young dog from a non-endemic area. Within 22 days a lesion had developed at the site of the infectivebite (inner ear): 98 days after infection organisms had not disseminated throughout the skin, bone marrow, spleen or liver and the animal was still serologically negative by indirect immunofluorescence and dot-enzyme-linked immunosorbent assay. When fed Lu. longipalpis were captured from a kennel with a sick dog known to be infected, 33 out of 49 (67 per cent) of flies contained promastigotes. In contrast only two infections were detected among more than 200 sandflies captured in houses. These observations confirm the ease of transmissibility of L.chagasi from dog to sandfly to dog in Teresina. It is likely that canine VL is the major source of human VL by the transmission route dog-sandfly-human. the Lmet2 DNA probe was a useful epidemiological tool for detecting L. chagasi in sandflies.

Pulmonary biology of anti-interleukin 5 antibodies

Interleukin 5 (IL-5) is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulate in the lungs during asthma. We have studied anti IL-5 antibodies on allergic responses in mice, guinea pigs and monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single dose of 0.3 mg/kg, i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, a humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 did not cause immunosuppression in guinea pigs. Studies with this antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

Identification of snails within the Bulinus africanus group from East Africa by multiplex SNaPshotTManalysis of single nucleotide polymorphisms within the cytochrome oxidase subunit I

Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI) as detected by a novel multiplexed SNaPshotTM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshotTM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host 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.

Influence of the blood meal source on the biology of Meccus picturatus Usinger 1939 (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Aspects related to hatching, time-lapse between presenting the blood meal and beginning of feeding, feeding time, postfeed defecation delay,life time, mortality and fecundity for each stage of Meccus picturatus, life-cycle were evaluated and compared in two cohorts of M. picturatus fed on hens or rabbits. The hatching rate observed for each of the two studied groups of eggs was 78.1% (n = 2298) on the group fed on hens and 82.1% (n = 2704) on that fed on rabbits, and the average time of hatching was 20 days. Mean time-lapse for beginning feeding was under 3 min in nymphal stages and postfeed defecation delay was under 10 min in all stages, in both cohorts. Mean feeding time was significantly (P < 0.05) shorter in triatomines fed on hens than on rabbits. A similar number of nymphs of each cohort, 69 fed on hens (34.5%) and 68 fed on rabbits (34%), completed the cycle. No significantly (P > 0.05) differences were recorded among the average times from NI to adult in the cohort fed on hens (196.8 ± 15.8 days) and the average time in the cohort fed on rabbits (189.5 ± 22.9). The average span in days for each stage fed on hens was not significantly different to the average span for each stage fed on rabbits. The number of blood meals at each nymphal stage varied from 1 to 6 in both cohorts. The mortality rates were higher on fifth nymphal stage, in both cohorts. No significant (P > 0.05) differences were recorded on mortality rates on most nymphal stages of both cohorts. The average number of eggs laid per female from the cohort fed on hens in a 9-month period was 791.1, whereas the average number of eggs in the cohort fed on rabbits was 928.3.

Dried blood spots as a practical and inexpensive source for human immunodeficiency virus and hepatitis C virus surveillance

Passive surveillance of infectious diseases with a high percentage of asymptomatic cases or long incubation periods, such as acquired immunodeficiency syndrome (AIDS), does not reflect the current transmission dynamics. Thus, a multi-strategic surveillance, such as the human immunodeficiency virus (HIV) sentinel surveillance proposed by the World Health Organization (WHO), is necessary. The Brazilian HIV sentinel surveillance was started in May 1992 with this purpose. The objectives of this study were to evaluate the feasibility and costs of HIV and hepatitis C virus (HCV) surveillance using dried blood spots (DBS) collected for neonatal screening of metabolic diseases in the state of Minas Gerais, Brazil. This was accomplished through the comparison of HIV and HCV seroprevalence with previous Brazilian studies. From December 2001 to June 2002, 24,905 newborns were tested for HIV and 4211 for HCV. HIV seroprevalence was 0.25% and the 95% confidence interval (CI) was 0.18, 0.31%; and HCV seroprevalence was 0.71% and the 95% CI was 0.46, 0.97%. These numbers are similar to previous Brazilian studies. Cost in this study was approximately US$ 3.10 per sample, which was roughly one third of the cost of the same exam at the Brazilian HIV sentinel surveillance. We conclude that it is possible and more cost-effective to use DBS for infectious diseases surveillance, albeit it is still necessary to compare these results with the usual sentinel methodology in a concomitant trial.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

Single and concomitant experimental infectionsby Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the Neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

Lutzomyia longipalpis in Brazil: a complex or a single species? A mini-review

Lutzomyia longipalpis is the main vector of Leishmania infantum chagasi, the causative agent of American visceral leishmaniasis (AVL). Although there is strong evidence that Lu. longipalpis is a species complex, not all data concerning populations from Brazil support this hypothesis. The issue is still somewhat controversial for this large part of Lu. longipalpis distribution range even though that it is the Latin American region contributing to most of the cases of AVL. In this mini-review we consider in detail the current data for the Brazilian populations and conclude that Lu. longipalpis is a complex of incipient vector species with a complexity similar to Anopheles gambiae s.s. in Africa.

Brazilian flora extracts as source of novel antileishmanial and antifungal compounds

Natural products have long been providing important drug leads for infectious diseases. Leishmaniasis is a protozoan parasitic disease found mainly in developing countries, and it has toxic therapies with few alternatives. Fungal infections have been the main cause of death in immunocompromised patients and new drugs are urgently needed. In this work, a total of 16 plant species belonging to 11 families, selected on an ethnopharmacological basis, were analyzed in vitro against Leishmania (L.) chagasi, Leishmania (L.) amazonensis, Candida krusei, and C. parapsilosis. Of these plant species, seven showed antifungal activity against C. krusei, five showed antileishmanial activity against L. chagasi and four against L. amazonensis, among them species of genus Plectranthus. Our findings confirm the traditional therapeutic use of these plants in the treatment of infectious and inflammatory disorders and also offer insights into the isolation of active and novel drug prototypes, especially those used against neglected diseases as Leishmaniasis.

Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria

Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3) isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.