Molecular detection of Treponema pallidum sp. pallidum in blood samples of VDRL-seroreactive women with lethal pregnancy outcomes: a retrospective observational study in northern Brazil

INTRODUCTION: Although control measures of maternal and congenital syphilis are available in Brazil, difficulties exist within the healthcare network in providing a laboratory diagnosis of the infection during the prenatal period. The objective of this study was to confirm the presence of Treponema pallidum by PCR in women with positive VDRL serology and lethal pregnancy outcomes, i.e., abortion, stillbirth and neonatal death. METHODS: A retrospective study was conducted on VDRLseroreactive women with lethal pregnancy outcomes admitted to the Fundação Santa Casa de Misericórdia do Pará (FSCM-PA) between January and July 2004. Serum samples and DNA from whole blood were obtained at the time of screening by the VDRL test. These samples were analyzed by IgG ELISA, IgM FTA-Abs and simple PCR (polA). RESULTS: During the study period, 0.7% (36/4,912) of women with lethal pregnancy outcomes presented a positive VDRL test. The polAgene was amplified in 72.7% (24/33) of these women, with 55.6% (20/36) and 94.4% (34/36) presenting IgM and IgG antibodies against T. pallidum, respectively. Comparison of these results showed a significant difference, with agreement between the PCR and IgM FTA-Abs results, suggesting that maternal syphilis was an active infection. No basic cause of death of the conceptus was reported in 97.2% (35/36) of cases. Among women who were submitted to the VDRL test during the prenatal period, only four of the nine seroreactive patients underwent treatment. CONCLUSIONS: The high frequency of syphilis in the group studied indicates the fragility of the service of infection diagnosis, treatment and monitoring, compromising epidemiological control.

Thiamin, selenium, and copper levels in patients with idiopathic dilated cardiomyopathy taking diuretics

OBJECTIVE: To analyze the association of thiamin, selenium, and copper serum levels with cardiac function in patients with idiopathic dilated cardiomyopathy using diuretics, and also to compare them with levels in control patients with no evidence of disease. METHODS: The study comprised 30 patients with heart disease and 30 healthy control individuals. Thiamin was analyzed by measuring the activity of erythrocytic transketolase and the effect of thiamin pyrophosphate. Selenium and copper serum levels were measured by hydride generation and flame atomic absorption spectrophotometry, respectively. RESULTS: Thiamin deficiency was observed in 10% of the control individuals and in 33% of the patients with heart disease (p=0.02). The mean selenium and copper serum levels in control individuals and patients with heart disease were, respectively, 73.2±9.9 µg/L (56.5 to 94.5 µg/L) and 72.3±14.3 µg/L (35.5 to 94 µg/L) (p=0.77); 1.1±0.4mg/L (0.6 to 1.8mg/L) and 1.2± 0.4mg/L (0.6 to 2.2mg/L) (p=0.27). No association between the levels of these nutrients and cardiac function was observed. CONCLUSION: Thiamin deficiency was significantly more frequent in patients with heart disease. No significant difference was observed between the mean selenium and copper serum levels in control individuals and in patients with heart disease. The results suggest possible benefits with thiamin replacement in patients taking diuretics.

Estudos experimentais sôbre a origem do milho

1) It may seem rather strange that, in spite of the efforts of a considerable number of scientists, the problem of the origin of indian corn or maize still has remained an open question. There are no fossil remains or archaeological relics except those which are quite identical with types still existing. (Fig. 1). The main difficulty in finding the wild ancestor- which may still exist - results from the fact that it has been somewhat difficult to decide what it should be like and also where to look for it. 2) There is no need to discuss the literature since an excellent review has recently been published by MANGELSDORF and REEVES (1939). It may be sufficient to state that there are basically two hypotheses, that of ST. HILAIRE (1829) who considered Brazilian pod corn as the nearest relative of wild corn still existing, and that of ASCHERSON (1875) who considered Euchlaena from Central America as the wild ancestor of corn. Later hypotheses represent or variants of these two hypotheses or of other concepts, howewer generally with neither disproving their predecessors nor showing why the new hypotheses were better than the older ones. Since nearly all possible combinations of ideas have thus been put forward, it har- dly seems possible to find something theoretically new, while it is essential first to produce new facts. 3) The studies about the origin of maize received a new impulse from MANGELSDORF and REEVES'S experimental work on both Zea-Tripsacum and Zea-Euchlaena hybrids. Independently I started experiments in 1937 with the hope that new results might be obtained when using South American material. Having lost priority in some respects I decided to withold publication untill now, when I can put forward more concise ideas about the origin of maize, based on a new experimental reconstruction of the "wild type". 4) The two main aspects of MANGELSDORF and REEVES hypothesis are discussed. We agree with the authors that ST. HILAIRE's theory is probably correct in so far as the tunicata gene is a wild type relic gene, but cannot accept the reconstruction of wild corn as a homozygous pod corn with a hermaphroditic tassel. As shown experimentally (Fig. 2-3) these tassels have their central spike transformed into a terminal, many rowed ear with a flexible rachis, while possessing at the same time the lateral ear. Thus no explanation is given of the origin of the corn ear, which is the main feature of cultivated corn (BRIEGER, 1943). The second part of the hypothesis referring to the origin of Euchlaena from corn, inverting thus ASCHERSON's theory, cannot be accepted for several reasons, stated in some detail. The data at hand justify only the conclusion that both genera, Euchlaena and Zea, are related, and there is as little proof for considering the former as ancestor of the latter as there is for the new inverse theory. 5) The analysis of indigenous corn, which will be published in detail by BRIEGER and CUTLER, showed several very primitive characters, but no type was found which was in all characters sufficiently primitive. A genetical analysis of Paulista Pod Corn showed that it contains the same gene as other tunicates, in the IV chromosome, the segregation being complicated by a new gametophyte factor Ga3. The full results of this analysis shall be published elsewhere. (BRIEGER). Selection experiments with Paulista Pod Corn showed that no approximation to a wild ancestor may be obtained when limiting the studies to pure corn. Thus it seemed necessary to substitute "domesticated" by "wild type" modifiers, and the only means for achieving this substitution are hybridizations with Euchlaena. These hybrids have now been analysed init fourth generation, including backcrosses, and, again, the full data will be published elsewhere, by BRIEGER and ADDISON. In one present publication three forms obtained will be described only, which represent an approximation to wild type corn. 6) Before entering howewer into detail, some arguments against ST. HILAIRE's theory must be mentioned. The premendelian argument, referring to the instability of this character, is explained by the fact that all fertile pod corn plants are heterozygous for the dominant Tu factor. But the sterility of the homozygous TuTu, which phenotypically cannot be identified, is still unexplained. The most important argument against the acceptance of the Tunicata faetor as wild type relic gene was removed recently by CUTLER (not yet published) who showed that this type has been preserved for centuries by the Bolivian indians as a mystical "medicine". 7) The main botanical requirements for transforming the corn ear into a wild type structure are stated, and alternative solutions given. One series of these characters are found in Tripsacum and Euchlaena : 2 rows on opposite sides of the rachis, protection of the grains by scales, fragility of the rachis. There remains the other alternative : 4 rows, possibly forming double rows of female and male spikelets, protection of kernels by their glumes, separation of grains at their base from the cob which is thin and flexible. 8) Three successive stages in the reconstruction of wild corn, obtained experimentally, are discussed and illustrated, all characterized by the presence of the Tu gene. a) The structure of the Fl hybrids has already been described in 1943. The main features of the Tunicata hybrids (Fig. -8), when compared with non-tunicate hybrids (Fig. 5-6), consist in the absence of scaly protections, the fragility of the rachis and finally the differentiation of the double rows into one male and one female spikelet. As has been pointed out, these characters represent new phenotypic effects of the tunicate factor which do not appear in the presence of pure maize modifiers. b) The next step was observed among the first backcross to teosinte (Fig. 9). As shown in the photography, Fig. 9D, the features are essencially those of the Fl plants, except that the rachis is more teosinte like, with longer internodes, irregular four-row-arrangement and a complete fragility on the nodes. c) In the next generation a completely new type appeared (Fig. 10) which resembles neither corn nor teosinte, mainly in consequence of one character: the rachis is thin and flexible and not fragile, while the grains have an abscission layer at the base, The medium sized, pointed, brownish and hard granis are protected by their well developed corneous glumes. This last form may not yet be the nearest approach to a wild grass, and I shall try in further experiments to introduce other changes such as an increase of fertile flowers per spikelet, the reduction of difference between terminal and lateral inflorescences, etc.. But the nature of the atavistic reversion is alveadwy such that it alters considerably our expectation when looking for a still existing wild ancestor of corn. 9) The next step in our deductions must now consist in an reversion of our question. We must now explain how we may obtain domesticated corn, starting from a hypothetical wild plant, similar to type c. Of the several changes which must have been necessary to attract the attention of the Indians, the following two seem to me the most important: the disappearance of all abscission layers and the reduction of the glumes. This may have been brought about by an accumulation of mutations. But it seems much more probable to assume that some crossing with a tripsacoid grass or even with Tripsacum australe may have been responsible. In such a cross, the two types of abscission layer would be counterbalanced as shown by the Flhybrids of corn, Tripsacum and Euchlaena. Furthermore in later generations a.tu-allele of Tripsacum may become homozygous and substitute the wild tunicate factor of corn. The hypothesis of a hybrid origin of cultivated corn is not completely new, but has been discussed already by HARSHBERGER and COLLINS. Our hypothesis differs from that of MANGELSDORF and REEVES who assume that crosses with Tripsacum are responsible only for some features of Central and North American corn. 10) The following arguments give indirects evidence in support of our hypothesis: a) Several characters have been observed in indigenous corn from the central region of South America, which may be interpreted as "tripsacoid". b) Equally "zeoid" characters seem to be present in Tripsacum australe of central South-America. c) A system of unbalanced factors, combined by the in-tergeneric cross, may be responsible for the sterility of the wild type tunicata factor when homozygous, a result of the action of modifiers, brought in from Tripsacum together with the tuallele. d) The hybrid theory may explain satisfactorily the presence of so many lethals and semilethals, responsible for the phenomenon of inbreeding in cultivated corn. It must be emphasized that corn does not possess any efficient mechanism to prevent crossing and which could explain the accumulation of these mutants during the evolutionary process. Teosinte which'has about the same mechanism of sexual reproduction has not accumulated such genes, nor self-sterile plants in spite of their pronounced preference for crossing. 11) The second most important step in domestication must have consisted in transforming a four rowed ear into an ear with many rows. The fusion theory, recently revived byLANGHAM is rejected. What happened evidently, just as in succulent pXants (Cactus) or in cones os Gymnosperms, is that there has been a change in phyllotaxy and a symmetry of longitudinal rows superimposed on the original spiral arrangement. 12) The geographical distribution of indigenous corn in South America has been discussed. So far, we may distinguish three zones. The most primitive corn appears in the central lowlands of what I call the Central Triangle of South America: east of the Andies, south of the Amazone-Basin, Northwest of a line formed by the rivers São Prancisco-Paraná and including the Paraguay-Basin. The uniformity of the types found in this extremely large zone is astonishing (BRIEGER and CUTLER). To the west, there is the well known Andian region, characterized by a large number of extremely diverse types from small pop corn to large Cuszco, from soft starch to modified sweet corn, from large cylindrical ears to small round ears, etc.. The third region extends along the atlantic coast in the east, from the Caribean Sea to the Argentine, and is characterized by Cateto, an orange hard flint corn. The Andean types must have been obtained very early, and undoubtedly are the result of the intense Inca agriculture. The Cateto type may be obtained easily by crosses, for instance, of "São Paulo Pointed Pop" to some orange soft corn of the central region. The relation of these three South American zones to Central and North America are not discussed, and it seems essential first to study the intermediate region of Ecuador, Colombia and Venezuela. The geograprical distribution of chromosome knobs is rapidly discussed; but it seems that no conclusions can be drawn before a large number of Tripsacum species has been analysed.

Sobre os efeitos vasomotôres exercidos pela agua distilada e pelas soluções anisotonicas de clorêto de sodio

The writer reports experiments done with distilled water and hypotonic and hypertonic salt solutions of definite osmotic concentrations. The experiments were performed according to the Laewen-Trendelenburg technic using the vascular system of the frog's hind legs, and according to the Pissemski-Krawkow method using the capillaries of the rabbit's ear. Both preparations react to distilled water by marked vaso-constriction, the same phenomenon taking place in the case of the hypotonic salt solutions. The lower the concentration pf the hypotonic salt solution the stronger the vaso-constriction obtained. With hypertonic salt solutions was observed a strong but rather transient vaso-dilatation followed by secondary vaso-constriction. The later results were found only in the experiments with the frog's hind legs.

T cell responses to repeat and non-repeat regions of the circumsporozoite protein detected in volunteers immunized with Plasmodium falciparum sporozoites

The design of malarial vaccine based on the circumsporozoite (CS) protein, a majuor surface antigen of the sporozoite stage of the malaria parasite, requires the identification of T and B cell epitopes for inclusion in recombinant or synthetic vaccine candidates. We have investigated the specificity and function of a series of T cell clones, derived from volunteers immunized with Plasmodium falciparum sporozoites in an effort to identify relevant epitopes in the immune response to the pre-erythrocytic stages of the parasite. CD4+ T cell clones were obtained wich specifically recognized a repetitive epitope located in the 5'repeat region of the CS protein. This epitope, when conjugated to the 3'repeat region in a synthetic MAPs construct, induced high titers of antisporozoite antibodies in C57B1 mice. A second T cell epitope, which mapped to aa 326-345 of the carboxy terminal, was recognized by lytic, as well as non-lytic, CD4+ T cells derived from the sporozoite-immunized volunteers. The demonstration of CD4+ CTL in the volunteers, and the recent studies inthe rodent model (Renia et al., 1991; Tsuji et al., 1990), suggested that CS-specific CD4+ T cells, in addition to their indirect role as helper cells in the induction of antibody and CD8 + effector cells, may also play a direct role in protection against sporozoite challenge by targeting EEF within the liver.

Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase

Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

Immune mechanisms underlying the premunition against Plasmodium falciparum malaria

The most unique characteristic of a parasite when it is in its normal host is the ability to make itself tolerated, which clearly indicates that it has sophisticated means to ensure the neutrality of its host. This is true also in the case of Plasmodium falciparum, since after numerous malaria attacks an equilibrium is reached with a chronic stage of infection, characterized by a relatively low parasitemia, and low or no disease (Sergent & Parrot 1935). We shall briefly review the main characteristics of this state of "premunition", and present data suggesting that the underlying mechanisms of defense rely on the cooperation between cell and antibodies, leading to an antibody dependent cellular inhibition of the intra-erythrocytic growth of the parasite.

Increase of a Calcium Independent Transglutaminase Activity in the Erythrocyte during the Infection with Plasmodium falciparum

We have studied the activity of a calcium dependent transglutaminase (EC 2.3.2.13) during the growth of the parasite Plasmodium falciparum inside the infected human erythrocyte. There is only one detectable transglutaminase in the two-cell-system, and its origin is erythrocytic. No activity was detected in preparations of the parasite devoid of erythrocyte cytoplasm. The Michaelis Menten constants (Km) of the enzyme for the substrates N'N'dimethylcaseine and putrescine were undistinguishable whether the cell extracts used in their determination were obtained from normal or from infected red cells. The total activity of transglutaminase in stringently synchronized cultures, measured at 0.5mM Ca2+, decreased with the maturation of the parasite. However, a fraction which became irreversibly activated and independent of calcium concentration was detected. The proportion of this fraction grew with maturation; it represented only 20% of the activity in 20 hr-old-trophozoites while in 48-hr-schizonts it was more than 85% of the total activity. The activation of this fraction of transglutaminase did not depend on an increase in the erythrocyte cytoplasmic calcium, since most of the calcium was shown to be located in the parasite.

Plasmodium yoelii: identification of a gene encoding a putative ADP-ribosylation factor-1 GTPase-activating Protein, PyAG1

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.

Comparative developmental and susceptibility to insecticide of Bolivian and Brazilian populations of Triatoma infestans

The triatomine bug Triatoma infestans probably originated in Bolivia and dispersed passively over wide areas of South America, where it is the principal vector of Trypanosoma cruzi. In the region of its probable origin this species shows colonization in two different ecotopes, so that it may be encountered in sylvatic as well as in artificial habitats. The sylvatic colonization pattern is not observed in the rest of its range, where T. infestans is exclusive to man-made habitats. The objective of this study was to compare several aspects of two T. infestans populations, one from Minas Gerais (Brazil) and the other from the Cochabamba Valley (Bolivia), with a view to elucidate the factors associated with the different colonization patterns observed for this species. The differences between the developmental cycle, weight, capacity to ingest blood and mortality rate of first instar nymphs should indicate more fragility of Brazilian population that may be related to its elimination possibility.

Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone

Chloroquine has been the mainstay of malaria chemotherapy for the past five decades, but resistance is now widespread. Pyrimethamine or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance. Both pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors, the proguanil acting primarily through its major metabolite cycloguanil. Resistance to these drugs arises due to specific point mutations in the dhfr gene. Cross resistance between cycloguanil and pyrimethamine is not absolute. It is, therefore, important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that DHFR inhibitor with less mutation rate is favored in drug combinations. Hence, we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil. Using erythrocytic stages of P. falciparum cultures, progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence. Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil. It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs.

Haematozoan parasites of the lizard Ameiva ameiva (Teiidae) from Amazonian Brazil: a preliminary note

Three different haematozoan parasites are described in the blood of the teiid lizard Ameiva ameiva Linn. from North Brazil: one in the monocytes and the other two in erythrocytes. The leucocytic parasite is probably a species of Lainsonia Landau, 1973 (Lankesterellidae) as suggested by the presence of sporogonic stages in the internal organs, morphology of the blood forms (sporozoites), and their survival and accumulation in macrophages of the liver. One of the erythrocytic parasites produces encapsulated, stain-resistant forms in the peripheral blood, very similar to gametocytes of Hemolivia Petit et al., 1990. The other is morphologically very different and characteristically adheres to the host-cell nucleus. None of the parasites underwent development in the mosquitoes Culex quinquefasciatus and Aedes aegypti and their behaviour in other haematophagous hosts is under investigation. Mixed infections of the parasites commonly occur and this often creates difficulties in relating the tissue stages in the internal organs to the forms seen in the blood. Concomitant infections with a Plasmodium tropiduri-like malaria parasite were seen and were sometimes extremely heavy.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Erythrovirus B19 infection in acquired immunodeficiency syndrome: screening by histopathology, immunohistochemistry, and in situ hybridization

Erythrovirus B19 infects erythrocytic progenitors, transiently interrupting erythropoiesis. In AIDS patients it causes chronic anemia amenable to treatment. We looked for evidences of B19 infection in stored bone marrow material from patients with acquired immunodeficiency syndrome. Histological sections were made from stored paraffin blocks from 33 autopsies (39 blocks) and 35 biopsies (45 blocks, 30 patients) performed from 1988 to 2002. They were examined after hematoxylin-eosin (HE) staining, immunohistochemical (IHC), and in situ hybridization. HE revealed intra-nuclear inclusion bodies ("lantern cells") suggesting B19 infection in 19 sections corresponding to 19 of 63 patients examined with this test. Seven of 78 sections subjected to immunohistochemistry were positive, corresponding to 7 of 58 patients examined with this test. Fourteen sections corresponding to 13 of the 20 HE and/or IHC positive patients were subjected to in situ hybridization, with six positives results. Among the 13 patients subjected to the three techniques, only one gave unequivocal positive results in all and was considered a true positive. The frequency of B19 infection (1/63 patients) in the material examined can be deemed low.

Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.