High frequency of trypanosomatids in gallery forest bats of a Neotropical savanna.

Bats are well-known hosts of trypanosomatids, though information about their role as reservoirs of these protozoans in the Brazilian savanna is poorly known. We aimed to analyze the occurrence of trypanosomatid species in bats occurring in remnants of gallery forests of Brasília, Federal District of Brazil. We sampled bats using mist nets in six sites, and we collected blood, wing fragments and oral swab samples from all captured individuals. Trypanosomatids were identified in the captured bats through sequencing of the SSUrRNA region and kDNA qPCR. We found no parasite in blood smears of 146 individuals of 14 species captured, but blood cultures were positive for nine bats. We detected trypanosomatids molecularly in 111 (76%) specimens of all bat species in the studied areas. Most of the infected bats had Leishmania-like DNA detected in blood and swab samples of the oral mucosa. We distinguished three species of Trypanosoma (Trypanosoma dionisii, T. rangeli and T. cruzi) in Carollia perspicillata. SSUrRNA PCR of oral samples is a non-invasive and practical method for identification of trypanosomatid species in bats. Our results support our belief that bats could be potential reservoirs for Trypanosoma and Leishmania-like species in the enzootic cycle of these parasites in gallery forests of the Brazilian Cerrado biome.

Combined phylogenetic and morphometric information to delimit and unify the Triatoma brasiliensis species complex and the Brasiliensis subcomplex.

"Triatoma brasiliensis species complex" was defined as a monophyletic group of the species: T. brasiliensis, T. juazeirensis, T. melanica, and T. sherlocki. An alternative grouping scheme proposed the concept of "Brasiliensis subcomplex" which included the former species together with T. melanocephala, T. petrocchiae, T. lenti, T. tibiamaculata, and T. vitticeps. To evaluate the relationship among these taxa we combined the results obtained with four mitochondrial genes (12S, 16S, COI and Cytb, adding to 1811bp) and geometric morphometric analysis of wings and heads. Panstrongylus megistus was included in the analysis as it was previously found related to T. tibiamaculata, T. melanocephala and T. vitticeps. The results of both molecular and morphometric approaches clearly grouped the species analyzed into two monophyletic units, supported by both genetic and wing variability. The first one (G1) comprises the four species originally included in the T. brasiliensis species complex plus T. lenti and T. petrocchiae. The second group (G2) was composed by T. melanocephala, T. tibiamaculata and T. vitticeps, and remarkably, P. megistus if considering wing variability and phylogenetic results. Nevertheless, geometric morphometrics of heads provided a quantitative measurement that discriminates Panstrongylus from the Triatoma species based on the position of the antennal insertion relative to eyes, as it is used as the generic distinctive character. The discrepancy among approaches questions the validity of this character to define Panstrongylus genus. Independently of the chosen group definition -"T. brasiliensis species complex" or "Brasiliensis subcomplex"-we propose to delimit it to species of G1 that are all associated with the Caatinga biome in the Brazilian Northeast. G2 are the ones associated with the Atlantic Forest biome.

Biological, morphological and morphometric studies of Triatoma melanocephala Neiva & Pinto, 1923 (Hemiptera: Reduviidae: Triatominae).

Triatoma melanocephala Neiva & Pinto is found in the Brazilian states of Bahia, Paraíba, Pernambuco, Rio Grande do Norte, and Sergipe. In addition to the species' specific description, eight other articles on this insect were found in the literature. In this study, data was obtained on the morphology, morphometry, and life cycle of T. melanocephala, since this vector is of epidemiological and taxonomic importance. The specimens studied were obtained from a colony that has been kept at the Triatomine Insectarium of the College of Pharmaceutical Sciences of São Paulo State University's in Brazil. The morphological studies were performed using scanning electron microscopy. These studies characterized the eggs, the external adult female genitalia, and the ninth ventral abdominal segments of male and female nymphs. The morphometric studies characterized the five nymphal instars and the adult stage by measuring the head, thorax, abdomen, antennae, and mouthparts parameters. The life cycle of T. melanocephala was developed starting by 15 couples in the fifth instar. They were fed on Swiss mice every two weeks and observed daily. During daily observation, minimum temperature, maximum temperature, and relative humidity of the laboratory were measured. The results of the biological, morphometric, and morphological studies have increased the knowledge available on T. melanocephala.

A novel association between Rhodnius neglectus and the Livistona australis palm tree in an urban center foreshadowing the risk of Chagas disease transmission by vectorial invasions in Monte Alto City, São Paulo, Brazil.

After several public notifications of domiciliary invasions, palm trees were investigated in downtown Monte Alto City, São Paulo State, Brazil, in proximity to the city hall building, the main church, condominiums and marketing establishments. One hundred seventy four palm trees of 10 species were investigated, in which 72 specimens of Rhodnius neglectus, a potential Chagas disease vector, were captured via manual methods. All insects were collected from dead leaves, organic debris and bird nests in the only three Livistona australis palm trees in the central park square. This was the first record of R. neglectus colonizing this palm species. Although no Trypanosoma cruzi was found by abdominal compression followed by light microscopy, the poor nutritional status of the bugs hampered the examination of gut contents for parasite detection. Furthermore, the central crowns of the trees, which shelter bats (Chiroptera: Mammalia), could not be carefully searched for insects due to difficult access. This new finding highlights the sudden alteration in insect behavior, probably as a result of man's interference. This report aims to warn those involved in the health system about this new threat, justifying detailed research of the area to evaluate the magnitude of this emerging public health issue.

Cross-mating experiments detect reproductive compatibility between Triatoma sherlocki and other members of the Triatoma brasiliensis species complex.

Phylogenetic approaches based on mitochondrial DNA variation (fragments of Cyt B and 16S ribosomal RNA) have revealed Triatoma sherlocki as the most recent species addition to the Triatoma brasiliensis species complex; a monophyletic group which includes T. brasiliensis, Triatoma melanica, and Triatoma juazeirensis. T. sherlocki is the most differentiated among all species of this complex: it is unable to fly, possesses longer legs than the other members, and has reddish tonality in some parts of its exochorion. We question whether these species are reproductively compatible because of this pronounced morphological differentiation, and therefore, we present a series of cross breeding experiments that test compatibility between T. sherlocki and other members of the T. brasiliensis complex. We extended our analyses to include crosses between T. sherlocki and Triatoma lenti, because the latter has been suggested as a possible member of this complex. T. sherlocki male×T. lenti female pairs failed to produce hybrids. All other crosses of T. sherlocki and members of T. brasiliensis species complex, as well as backcrosses, produced viable offspring through the third generation. This study stresses the importance of searching for the features that may isolate members of the T. brasiliensis species complex.

Dispersion capacity of Triatoma sherlocki, Triatoma juazeirensis and laboratory-bred hybrids.

Flight dispersion is recognized as one of the most important mechanisms for triatomine house infestation. Triatoma sherlocki and T. juazeirensis are closely related species that occur within the same ecotope and their possible reproductive boundaries are unknown. T. sherlocki has shorter wings than T. juazeirensis; a characteristic that possibly implies in reduced flight dispersion, however, this species has been found to invade and colonize homes in Bahia, Brazil. Here, we tested the flight potential of T. sherlocki, compared to that of T. juazeirensis and laboratory-bred hybrids. Insects were kept in an apparatus designed to distinguish flyers from nonflyers. Fifty-one and 53% of T. juazeirensis and hybrids were flyers respectively, whereas no T. sherlocki were recorded to fly. Morphometric analysis of the main structures associated with the locomotor abilities showed that hybrids exhibited intermediate size for most of characters. The width of pronotum of both hybrids and T. juazeirensis was significantly larger than T. sherlocki. We suggested that lack of flight ability of T. sherlocki is possibly a result of reduced wing size and distinct shape, combined with undeveloped flight muscles in a shorter thoracic box. The mobility of T. sherlocki might be compensated by its significantly longer legs, and may possibly increase its ability to invade human dwellings by active dispersion. What is more, this study showed that hybrids between T. sherloki and T. juazeirensis have intermediate morphological characters that may give them higher fitness than their parents, and thus may advance the process of house infestation by either fight or walking in case of an eventual natural hybridization.

Phylogeny of Triatoma sherlocki (Hemiptera: Reduviidae: Triatominae) inferred from two mitochondrial genes suggests its location within the Triatoma brasiliensis complex.

The phylogenetic position of Triatoma sherlocki within triatomines group was inferred by analyzing mtDNA fragments of Cyt B and 16S ribosomal RNA by using maximum parsimony and Bayesian analysis. Despite being differentiated from members of the T. brasiliensis complex on morphologic grounds, molecular phylogenetic analysis suggests T. sherlocki is a member of this complex; moreover, it was placed as a sister species of T. melanica. These suggestions were supported by robust credibility rates. Hence, we show evidence for the paraphyletic group of the "Triatoma brasiliensis complex," which should be composed of T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, T. juazeirensis, T. melanica, and T. sherlocki.

Convenient and Efficient Synthesis of a Lanthanide-Coordinated, Diethylene Triamine Pentaacetic Acid Labeled Biopolymer as an Assay for the Cholecystokinin B Receptor.

To develop an assay for the cholecystokinin B receptor with an Eu(3+)-labeled cholecystokinin peptide via a diethylene triamine pentaacetic acid chelating linker, a commercial dianhydride diethylene triamine pentaacetic acid precursor was directly attached to the N-terminus of cholecystokinin peptides by a solid-phase synthesis method with a satisfactory yield and purity after reverse-phase high-performance liquid chromatography separation. Lanthanide was then coordinated to the peptide via a diethylene triamine pentaacetic acid bifunctional agent. This method is a useful approach to the large-scale synthesis of lanthanide(3+)-coordinated, diethylene triamine pentaacetic acid labeled biopolymers. This research provides not only a simple and convenient method for the preparation of lanthanide-based peptide ligand libraries but also possible lanthanide-based high-throughput screening of peptide receptors with a timeresolved fluorescence assay system. Five biopolymers were synthesized and characterized with high-resolution electrospray ionization in this study.

ROBITOM-robot for biopsy and therapy of the mamma.

MR-Mammography reaches a high sensitivity in detecting breast carcinomas of 3 mm in size at least. In cooperation with the Institute of Diagnostic and Interventional Radiology of the Friedrich-Schiller-University of Jena, a manipulator has been developed by the IMB, which combines the advantages of MRM imaging with a minimal invasive biopsy and a possible subsequent therapy. Referring to this ROBITOM I was introduced in November 1999 as worldwide first, precise operating manipulator system in the ISO center of a closed MR, at RSNA in Chicago. Clinical trials started at 22. November 2000. The experiences and results of these tests were brought into the following prototype ROBITOM II, that is currently developed at the IMB. The completion of this Prototype is planned at the end of 2002.

Robotic system for biopsy and therapy of breast lesions in a high-field whole-body magnetic resonance tomography unit.

RATIONALE AND OBJECTIVES: With magnetic resonance mammography, significant progress has been achieved in the diagnosis of small breast cancers. However, biopsy and therapy of suspicious lesions must take place at a later time. Diagnosis and simultaneous biopsy and therapy in one single examination would considerably reduce costs, strain on the patient, and side effects. METHODS: ROBITOM (Robotic system for biopsy and interventional therapy of mammary lesions) is used to approach a lesion found in the breast in an image-controlled manner under a high magnetic field (eg, 1.5 T). The robotic system works in the direct vicinity of the isocenter of a magnet and consists of a trocar, coaxial sleeve, biopsy needle, laser applicator, and a control and driving unit. It contains a rack, a driving unit along the three coaxes of space, and a gripping unit for instruments or biopsy sample removal. The system has six degrees of freedom. RESULTS: In vitro experiments in pig liver including eight targets (vitamin E capsules, 4 mm in diameter) were performed. All eight capsules were hit precisely by the robotic biopsy system. The procedure was performed directly in the isocenter of a 1.5-T whole-body scanner. CONCLUSIONS: The system allows the coordinates of a lesion in the breast to be approached in a high magnetic field without shifting the patient. A combination of imaging with biopsy and subsequent therapy (eg, laser therapy or cryotherapy) seems to be feasible in the future.

The medical engineering program of Forschungszentrum Karlsruhe.

The research activities of the Forschungszentrum Karlsruhe on minimally-invasive surgery (MIS) have for several years improved techniques and instrumentation for different types of MIS. Many types of instruments and robotic devices have been developed and new techniques implemented. In this paper we present the most recent results from our different projects, such as endoscopic heart surgery, tracking systems, a camera guidance device, telemanipulator systems, minimally-invasive breast biopsy in closed-bore MRI, endoscopic training simulators and developments using smart materials (e.g. Nitinol).

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Aggregation of resin-bound peptides during solid-phase peptide synthesis. Prediction of difficult sequences.

Nonrandom incomplete aminoacylation of a pendent peptide chain on an insoluble polymeric support during solid-phase peptide synthesis is sequence-dependent and is caused by aggregation of peptide chains, manifested by a decreased swelling capacity. The volume of the swollen peptidyl-resin after each coupling during the syntheses of 87 sequence unrelated peptides was measured, and for each amino acid an aggregation parameter, , was derived that reflects the propensity of the swollen volume of peptidyl-resin to decrease during peptide synthesis. These aggregation parameters were used to predict potentially difficult sequences.

Identification of seroreactive epitopes of human papillomavirus type 18 E7 protein by synthetic peptides.

Nine everlapping peptides covering the entire sequence of early protein E7 of human papillomavirus type 18 (HPV-18) were synthesized and tested as antigens with pools of selected human sera in ELISA. Peptides denoted 18/E7-2, 18/E7-3, and 18/E7-5 (amino acid positions 11-33, 21-40, and 41-60, respectively) were reactive with pooled sera originating from HPV-18 DNA-positive cervical cancer patients but not with sera from HPV-16 DNA-positive cervical cancer patients or from condyloma acuminata patients. This suggested that the epitopes contained in these peptides were HPV-18 type-specific, relative to HPV types 16, 11, and 6. On the other hand, 18/E7-1 (aa 1-23) and 18/E7-6 (aa 51-70) peptides were cross-reactive. The prevalence of antibodies reactive with 18/E7-2, 18/E7-3, and 18/E7-5 peptides in cervical carcinoma patients was very low. Thus, the utilization of these peptides for monitoring HPV-18 infection seems to be rather limited.

Human breast milk contains procathepsin D--detection by specific antibodies.

The presence of the zymogen of cathepsin D in human milk was detected using antibodies specific for the proenzyme and by the proteolytic activity at low pH. The antibodies were raised against a synthetic propeptide of human cathepsin D and were tested using immunoprecipitations and western blots of samples from different breast cancer cell lines as well as cytosol fractions of human breast cancer tissues. In all experiments these antibodies recognized specifically procathepsin D. Procathepsin D from human milk was partially activated at low pH. The activity was monitored using hemoglobin 14C proteolytic assay, and it was abolished by pepstatin A--a specific inhibitor of aspartic proteinases. Western blots did not reveal presence of cathepsin B or cathepsin H. These data indicate specific secretion of cathepsin D in human breast milk.

Epitope mapping of the human papillomavirus type 16 E4 protein by means of synthetic peptides.

Eight overlapping icosapeptides covering the entire sequence of the E4 protein of human papillomavirus type 16 (HPV-16), were prepared and tested for their reactivity with human sera in IgG-specific ELISA. The strongest reactivity of sera from HPV-16 DNA-positive invasive cervical carcinoma (INCA) patients was detected with the peptide denoted 16/E4-6, covering amino acids 51 to 70. Subsequently nearly 200 sera were tested for the presence of the 16/E4-6-specific antibody. Reactivity was more frequent in cervical intraepithelial neoplasia patients and INCA patients than in matched control subjects. Sera from INCA patients were also tested for antibody reactive with peptide 16/E7-2 covering the major type-specific reactive region of the HPV-16 E7 protein. Only four of 13 sera possessing the 16/E4-6-specific antibody were reactive with the 16/E7-2 peptide.

[Penile block in adults]. / Bloc pénien chez l'adulte.

A block of the penile nerves provides a sensory blockade of the penis. In adults, surgery can thus be carried out on the foreskin, glans, corpus cavernosum, corpus spongiosum or penile urethra. The two dorsal nerves of the penis can be blocked by two different routes. In the median technique, only one injection is performed in the subpubic space, near the posterior inferior aspect of the symphysis. In the bilateral technique, each penile nerve is blocked separately at the level of the penile root. Whichever technique is used, additional subcutaneous infiltration of the penile root improves the quality of analgesia. Bupivacaine without adrenaline is used at a concentration of 0.25% or 0.5%. In the median technique, bilateral diffusion of the anaesthetic solution has been demonstrated in ten patients by adding contrast medium to the anaesthetic solution. On the other hand, contralateral diffusion was only found in six of ten patients after an unilateral injection. These results substantiate the value of the bilateral technique in the adult. Both techniques were used in a group of 80 patients, aged 17 to 87 years. In 47 patients no other agent was administered, while the remaining 33 had either additional sedation or a general anaesthetic. Among the latter, three had a partial failure of the block. Postoperative analgesia, which was of excellent quality, covered an average of 10 hours. Neither local nor general incident occurred. Penile block is a reliable technique for regional anaesthesia. Because it is easy to carry out, and comfortable for the patient, this technique may be suggested to adults requiring penile surgery.