The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Molecular markers in acute and chronic phases of human toxoplasmosis: determination of immunoglobulin G avidity by Western blotting.

We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases of Toxoplasma gondii infection.

Quantification of crotamine, a small basic myotoxin, in South American rattlesnake (Crotalus durissus terrificus) venom by enzyme-linked immunosorbent assay with parallel-lines analysis.

Intraspecific variation in Crotalus durissus terrificus venom composition was studied in relation to crotamine activity. Crotamine induces paralysis in extension of hind legs of mice and myonecrosis in skeletal muscle cells. To determine whether the venom of crotamine-negative rattlesnake contains a quantity of myotoxin incapable of inducing paralysis, we have developed a very sensitivity immunological assay method, an enzyme-linked immunoabsorbent assay (ELISA), capable of detecting 0.6 ng of purified crotamine. The parallel-lines analysis of ELISA data showed to be useful because it shows the reliability of the experimental conditions. A variation in the amount of myotoxin in the crotamine-positive venom was observed, but not less than 0.1 mg of crotamine per mg of venom. It was not possible to detect it in crotamine-negative venom even at high venom concentrations.

Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus).

A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by Laure (1975), and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon. Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.

Serodiagnosis of hepatitis C virus. Effect of new evaluation of cutoff values for enzyme-linked immunosorbent assay in Brazilian patients.

With the goal of reducing false-positive results in enzyme-linked immunosorbent assay (ELISA) serodiagnosis of hepatitis C virus in clinical practice, a study was undertaken to establish better cutoff values. We examined 277 serum samples from patients with hepatitis (non-A, non-B; B; autoimmune); subjects with antinuclear antibodies or rheumatoid factor, anticytomegalovirus or Epstein-Barr virus IgG or IgM antibodies, or parasitic disease (Chagas disease, leishmaniasis); and healthy volunteers. Concordant positive results in 2 different immunoblot assays in 250 samples were taken as indicative of true-positive, and when negative, of absence of infection. Reactivity in 3 ELISA tests were evaluated for the manufacturer recommended cutoff (CO) and for 2CO, 3CO, and 4CO; and corresponding sensitivity and specificity were calculated for single or combined pairs of ELISA tests. Although CO is adequate for blood bank screening, because it provides maximal sensitivity, the frequently observed false-positive results could be significantly reduced by increasing the cutoff value to 2CO, with no significant loss in sensitivity either in relation to pairs of immunoenzymatic tests or to a single ELISA.

Evaluation of recombinant antigens for serodiagnosis of Chagas' disease in South and Central America.

The commercially available diagnostic tests for Chagas' disease employ whole extracts or semipurified fractions of Trypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas' disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzi recombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas' disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of 125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas' disease.

Renal and hepatic interactions of acetaminophen and amikacin in the infant rat.

Results suggest that amikacin at the highest dose induces more changes on the renal function while the effect of APAP is more marked on the hepatic function. It might suggest that simultaneous administration of amikacin-APAP produces less changes that when the drugs are administered alone.

Baccilary angiomatosis: negative results using normal Balb/c and Balb/c nude mice

Baccilary angiomatosis has recently been described as a disease that can spread systematically and that is potentially fatal. It is caused by Bartonella henselae and B. quintana, and presents as especially pronounced signs and symptoms in patients suffering from acquired immunodeficiency syndrome (AIDS). To clarify the pathogenesis of the disease and to try to define the relationships among baccilary angiomatosis, cat scratch disease and Carrión's bartonellosis, the authors of this study have attempted to develop an experimental model using mice that were immunocompetent as well as those that had their cellular immunity genetically compromised. A know concentration of B. henselae was inoculated intradermally in Balb/c an isogenic mice or an athymic group of the same lineage. Blood samples were taken on days-0, 3, 7, 10, 14, 28, and 60 after inoculation for indirect immunofluorescence antibody testing. On the 21st and 60th day, one animal from each group was sacrificed and a post mortem carried out including histological evaluation of the liver, spleen, lymph nodes, skin and other organs. Hemocultures of the sacrificed animals were collected. All results of serologic response, cultures and histologic examination were negative. The authors discuss the methodology, especially the use of isogenic animals of the same lineage in B. henselae infection, with and without immunodeficiency, and the resources for the negative results of histopathology, serology and cultures.

Cloning and characterization of a gene encoding a novel immunodominant antigen of Trypanosoma cruzi.

A Trypanosoma cruzi genomic expression library was screened with a pool of sera obtained from chronic chagasic patients. The recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas' disease. The full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. The Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously described T. cruzi sequences. In contrast with other T. cruzi antigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are tightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain.

Dot-ELISA for the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluid using a new solid phase (resin-treated polyester fabric) and Cysticercus longicollis antigens.

A dot-ELISA was developed for the detection of antibodies in CSF in the immunologic diagnosis of human neurocysticercosis, using antigen extracts of the membrane and scolex of Cysticercus cellulosae (M+S-Cc) and, alternately, membrane (M) and vesicular fluid (VF) of Cysticercus longicollis (Cl) covalently bound to a new solid phase consisting of polyester fabric treated with N-methylol-acrylamide resin (dot-RT). The test was performed at room temperature, with reduced incubation times and with no need for special care in the manipulation of the support. The sensitivity rates obtained were 95.1% for antigen Cc and 97.6% for antigen Cl. Specificity was 90.6% when Cc was used, and 96.9% and 100% when M-Cl and VF-Cl were used, respectively. No significant differences in titer were observed between tests carried out with homologous and heterologous antigens. The low cost and easy execution of the dot-RT test using antigen extracts of Cysticercus longicollis indicate the test for use in the immunodiagnosis of human neurocysticercosis.

Immunoblot assay using excreted-secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute, and chronic Chagas' disease.

Immunoblotting with trypomastigote excreted-secreted antigens (TESA blot) of Trypanosoma cruzi was evaluated as a method for diagnosis of chronic and acute phases as well as congenital (in newborn children) Chagas' disease. Serum samples from acute-phase and congenital infections were considered to be positive when they reacted with ladder-like bands of 130- to 200-kDa antigens, recognized by immunoglobulin M (IgM) and IgG antibodies, while IgG from chronic-phase sera recognized a broad band antigen of 150 to 160 kDa. Nonchagasic sera were not reactive to these antigens. The study was carried out on 512 patients, 111 of whom were nonchagasic but included cases of leishmaniasis or other pathologies, and 401 chagasic patients. The latter group comprised 361 chronic cases, 36 acute cases, and 4 congenital cases in newborn children. Among the chronic cases, 256 were from areas in which T. cruzi is endemic but which differed widely in the pathogenic expression of T. cruzi infection and in parasitemia levels. These patients at the same time showed a broad range of low, medium, and high reactivity to conventional enzyme-linked immunosorbent assays and indirect immunofluorescence serotests for Chagas' disease. For these reasons they may better represent the universe of chagasic patients than would a sample of highly reactive sera obtained from chagasic patients in a single area endemic for T. cruzi. All acute and congenital cases showed positivity in the IgM and IgG TESA blots, while chronic cases were 100% positive for IgG antibodies. In nonchagasic sera, including 30 cases of visceral and muco-cutaneous leishmaniasis, the specificity index was 1.000, and no cross-reactions were observed. The TESA blot thus seems to be useful as a sensitive and specific diagnostic assay in cases of suspected acute or congenital T. cruzi infection and as a general confirmatory test for conventional Chagas' disease serology.

Organization and expression of the gene encoding an immunodominant repetitive antigen associated to the cytoskeleton of Trypanosoma cruzi.

We have studied the genomic organization and expression of the gene encoding a high molecular mass (300 kDa) repetitive antigen associated with the cytoskeleton of Trypanosoma cruzi. Protease digestion of the native protein, restriction analysis of genomic DNA and sequencing of genomic and cDNA clones indicated that most of the protein is built up by tandemly arranged, nearly identical repeats of 68 amino acids. The gene size was estimated to be approx. 9.4 kb based on the sizes of the transcript and the native protein. The nucleotide sequence conservation among the repeats indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino-acid sequence conservation. Two duplicated allelic forms of this gene were mapped in fragments of about 20 kb. In some strains an additional allele was located in a fragment of 9.4 kb. Our results suggest that this repetitive antigen is a structural protein which could be involved in the attachment of the flagellum to the cell body.

Schistosoma mansoni circulating polysaccharide and protein antigens recognized by sheep antisera in patients with different clinical forms of schistosomiasis before and after treatment.

Two sheep antisera, one of which raised against polysaccharide (Po) and other against protein (Pt) components of Schistosoma mansoni adult worms, were assessed by ELISA for their ability to detect circulating parasite antigens in patients with different clinical forms of chronic schistosomiasis mansoni. The former antiserum detected parasite antigens in liver granulomata and the latter in renal glomeruli from schistosomiasis patients and mice experimentally infected with S. mansoni. In general, the levels and/or positivity rate of circulating antigens and specific IgG antibodies were significantly higher in patients with hepatointestinal (HI) and hepatosplenic (HS) forms than in mild intestinal (I) forms. An association between Po antigens and clinical features of the disease was observed, as the level of these antigens was low (137 ng/ml) as well as the positivity rate (7.9%) in patients with I forms; values that were intermediate (593 ng/ml and 33.3%) in those with HI forms, and high (1.563 ng/ml and 50.0%) in more severe HS forms. The Pt antigens were detected in the studied clinical forms not differing statistically but, the positivity rate was significantly higher in HS forms comparatively to I forms. The antisera studied revealed distinct circulating antigen profiles, and the prognostic value of Po and Pt antigens was suggested.

Detection of antibodies in sera from Chagas' disease patients using a Trypanosoma cruzi immunodominant recombinant antigen.

A Trypanosoma cruzi DNA fragment encoding an immunodominant repetitive antigen (H49) was subcloned into a protein purification and expressed system. Purified H49 peptide reacted specifically in an enzyme-linked immunosorbent assay (ELISA) with sera from T. cruzi-infected patients, but not with sera from patients with other parasitic diseases such as leishmaniasis and T. rangeli-infection. The H49 recombinant ELISA was able to detect specific antibodies in 84% of chronic chagasic serum samples tested. One of the major advantage of the recombinant ELISA for serodiagnosis of chronic Chagas' disease resides in its high specificity (100%). Our data suggest that recombinant peptides could provide a practical basis for specific diagnosis tests for Chagas' disease.

Experimental toxoplasmosis in turkeys.

Fourteen 2-3-wk-old turkeys were inoculated orally with 10(5) or 10(4) infective oocysts of the ME 49 strain of Toxoplasma gondii. Of the 8 turkeys given 10(5) oocysts in experiment 1, 3 died or were killed 12 or 14 days after inoculation (DAI) because of respiratory distress associated with a concomitant Aspergillus-like fungus infection. The remaining 5 turkeys remained normal and were killed 62 DAI. Toxoplasma gondii was isolated in mice from the heart of all 5, from the breast muscles of 2, leg muscles of 3, and from the brains and livers of none of the turkeys. All 6 turkeys fed 10(4) oocysts in experiment 2 remained clinically normal until necropsy on 41 DAI; T. gondii was isolated from pooled tissues from each turkey. All 14 turkeys developed high antibody titers to T. gondii in the modified agglutination test (MAT) using formalinized tachyzoites. The enzyme-linked immunosorbent assay (ELISA) was as sensitive as MAT for detecting T. gondii antibodies in turkey sera. The latex agglutination and indirect hemagglutination tests were less sensitive than the MAT and ELISA. No dye-test-measurable antibodies were found in sera of any turkey.

Serologic and parasitologic responses of domestic chickens after oral inoculation with Toxoplasma gondii oocysts.

Four-week-old chickens were inoculated orally with 1,000 or 100,000 oocysts of the ME-49 or GT-1 strain of Toxoplasma gondii, and their antibody responses were measured, using the direct modified agglutination test, latex agglutination test, indirect hemagglutination test, ELISA, and the Sabin-Feldman dye test. Antibodies against T gondii were detected by use of the modified agglutination test and ELISA within 2 weeks of oocyst inoculation, and antibodies persisted until termination of the study by postinoculation day 68. The latex agglutination test was insensitive in detecting T gondii antibodies, and antibodies were not detected by use of the dye and indirect hemagglutination tests. Of tissues bioassayed in mice for tissue cysts by pepsin digestion of individual organs of chickens on postinoculation day 68, tissue cysts were found in the brain of all 5, heart of 3, and leg muscles of 2, but not in the liver and breast muscles. None of the birds developed clinical toxoplasmosis.

An unusually high prevalence of ocular toxoplasmosis in southern Brazil.

Because of the frequency of ocular toxoplasmosis and its occurrence in multiple siblings in southern Brazil, a population-based household survey was performed to better understand the epidemiologic characteristics of the disease in this region. Of 1,042 individuals examined, 184 (17.7%) were deemed to have ocular toxoplasmosis on the basis of conservative assessment of ophthalmic findings. Of those with ocular toxoplasmosis, 183 (99.5%) had specific IgG antibodies, compared with only 140 of 181 age-matched control subjects (77.4%; P less than .001). The prevalence of ocular toxoplasmosis was 0.9% in 1- to 8-year-olds, 4.3% in 9- to 12-year-olds, 14.3% in 13- to 16-year-olds, and 21.3% (95% confidence interval, 18.6% to 24.2%) in all individuals 13 years or older. The prevalence of ocular toxoplasmosis in this population was more than 30 times higher than previous estimates for the same condition elsewhere. The low prevalence in the young children we studied supplements previous data suggesting that, in this population, ocular toxoplasmosis is a sequela of postnatal rather than congenital infection.