Pneumocystis carinii organisms obtained from rats, ferrets, and mice are antigenically different

Pneumocystis carinii infections were developed in animals immunosuppressed by dexamethasone treatment either from activation of latent infection (ferret) or by transtracheal inoculation with P. carinii-infected lung tissue from the homologous species (rat or mouse). Convalescent-phase antisera were obtained by stopping dexamethasone treatment after 2 to 4 weeks and allowing animals 5 to 8 weeks for recovery. P. carinii harvests from infected lungs were purified by differential filtration, solubilized in buffer containing urea, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol, subjected to SDS-polyacrylamide gel electrophoresis, and blotted to polyvinylidene difluoride sheets for Western immunoblot analysis. These lung preparations are hereafter referred to as P. carinii antigens. Convalescent-phase antisera from each animal species were reacted on Western blots of P. carinii antigens prepared from organisms isolated from rats, ferrets, or mice. Each combination of P. carinii antigens and antisera from the same species of animal reacted with three or more P. carinii antigen proteins. Convalescent-phase mouse antisera reacted with P. carinii antigens from mice but not rats or ferrets. Convalescent-phase rat antisera reacted with P. carinii antigens from rats and mice but not ferrets, and convalescent-phase ferret antisera showed reactions with ferret and mouse P. carinii antigens but not rat antigens. These findings indicate antigenic differences among P. carinii strains infecting these animals.     

Factors associated with successful mobilization of peripheral blood progenitor cells in 200 patients with lymphoid malignancies

To examine the repertoire of Pneumocystis carinii antigens recognized by antibody-secreting B cells from tracheobronchial lymph nodes isolated immediately following recovery from P. carinii pneumonia, monoclonal antibodies (MAbs) were produced from these cells. In contrast to previous studies of systemic immunity, P. carinii gpA was not the immunodominant antigen recognized by these B cells. Forty-nine (91%) of 54 P. carinii-specific hybridoma culture supernatants reacted with P. carinii antigens other than gpA. Many of the resulting MAbs recognized a previously uncharacterized antigen expressed on the surface of both cysts and trophozoites. Western blotting using one of the cloned MAbs revealed reactivity with a broad range of antigenic material, with the most intense reactivity in the 50- to 65-kDa region of the blot. The antigens identified by these MAbs merit further investigation regarding protective immunity to P. carinii because they were recognized by B cells in the context of recovery from P. carinii pneumonia.     

A 25-week placebo-controlled study of eptastigmine in patients with Alzheimer disease

Studies of Pneumocystis carinii have been limited by our inability to propagate it in continuous culture. In this context, studies of P. carinii antigens have provided significant insight into the biology of this organism. The mannose-rich surface major surface glycoprotein of P. carinii termed glycoprotein A (gpA) is the best studied of these P. carinii antigens. Significant genetic and immunologic diversity exists between the gpA molecules expressed by P. carinii derived from different mammalian sources. The molecular and biochemical nature of gpA and other P. carinii antigens including p55 are reviewed. In addition, available information concerning the role of P. carinii gpA and other antigens in host-organism interactions are also discussed.     

DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora

Samples of ambient air collected with three different types of spore traps in a rural location were examined for the presence of Pneumocystis carinii by screening for P. carinii-specific DNA sequences by DNA amplification. Eleven spore trap samples were analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of P. carinii f. sp. carinii and P. carinii f. sp. hominis. The samples were collected over a 3-year period during the months of May to September, with a range of sampling times from 9 to 240 h. One air sample from an animal facility housing P. carinii-infected rats was also examined. P. carinii-specific amplification products were obtained from samples from each of the spore traps. The amplification products from eight air samples were cloned and sequenced. The majority of the recombinants from each of these samples had sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis, and a number of clones had single-base differences. These data suggest that sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis can be detected in samples of air collected in a rural location and that P. carinii may be a component of the air spora of rural Oxfordshire.     

Glycoprotein A is the immunodominant antigen of Pneumocystis carinii in mice following immunization

A rapid and simple dual enzyme-linked immunosorbent assay was developed to quantitate the relative frequency of monoclonal antibodies produced against Pneumocystis carinii glycoprotein A (gpA) or other antigens of P. carinii after immunization with whole organisms. In BALB/cj mice, P. carinii gpA was clearly the immunodominant antigen, with a quarter of all hybridomas secreting antibody directed against this molecule.     

Cloning and nucleotide sequence of a specific DNA fragment from Paracoccidioides brasiliensis

We cloned and sequenced a species-specific 110-bp DNA fragment from Paracoccidioides brasiliensis. The DNA fragment was generated by PCR with primers complementary to the rat beta-actin gene under a low annealing temperature. Comparison of the nucleotide sequence, after excluding the primers, with those in the GenBank database identified approximately 60% homology with an exon of a major surface glycoprotein gene from Pneumocystis carinii and a fragment of unknown function in Saccharomyces cerevisiae chromosome VIII. By Southern hybridization analysis, the 32P-labelled fragment detected 1.0- and 1.9-kb restriction fragments within whole-cell genomic DNA of P. brasiliensis digested with HindIII and PstI, respectively, but failed to hybridize to genomic DNAs from Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Saccharomyces cerevisiae, Pneumocystis carinii, rat tissue, or humans under low-stringency hybridization conditions. Additionally, the specific DNA fragment from three different P. brasiliensis isolates (Pb18, RP18, RP17) was amplified by PCR with primers mostly complementary to nonactin sequences of the 110-bp DNA fragment. In contrast, there were no amplified products from other fungus genomic DNAs previously tested, including Histoplasma capsulatum. To date, this is the first species-specific DNA fragment cloned from P. brasiliensis which might be useful as a diagnostic marker for the identification and classification of different P. brasiliensis isolates.     

Detection of Pneumocystis carinii sequences in serum by polymerase chain reaction

The clinical significance of the detection of Pneumocystis carinii DNA was evaluated, as well as the detection of circulating P. carinii antigen from serum using previously collected samples. Fourteen serum samples from 13 patients were diagnosed positively for P. carinii DNA by polymerase chain reaction (PCR). Ten of 14 episodes (71.4%) of pulmonary complications were compatible with P. carinii pneumonia. Two patients were definitely diagnosed as having had P. carinii pneumonia at autopsy. All patients positive for circulating antigens were also positive for P. carinii DNA, suggesting that the detection of P. carinii DNA by PCR is more sensitive compared to the detection of circulating antigens by the Ouchterlony method. It is concluded that the detection of P. carinii DNA in serum by PCR provides useful information for identifying P. carinii pneumonia.     

Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.     

Spread of amikacin resistance in Acinetobacter baumannii strains isolated in Spain due to an epidemic strain

Sixteen amikacin-resistant clinical Acinetobacter baumannii isolates from nine different hospitals in Spain were investigated to determine whether the high incidence of amikacin-resistant A. baumannii was due to the dissemination of an amikacin-resistant strain or to the spread of an amikacin resistance gene. The epidemiological relationship studied by repetitive extragenic palindromic PCR and low-frequency restriction analysis of chromosomal DNA showed that the same clone was isolated in eight of nine hospitals, although other clones were also found. The strains were studied for the presence of the aph(3')-VIa and aac(6')-I genes, which encode enzymes which inactivate amikacin, by PCR. All 16 clinical isolates had positive PCRs with primers specific for the amplification of the aph(3')-VIa gene, whereas none had a positive reaction for the amplification of the aac(6')-I gene. Therefore, the high incidence of amikacin resistance among clinical A. baumannii isolates in Spain was mainly due to an epidemic strain, although the spread of the aph(3')-VI gene cannot be ruled out.     

Pneumocystis carinii host origin defines the antibody specificity and protective response induced by immunization

To determine how the known host species-specific antigenic variation of Pneumocystis carinii would affect immune recognition, mice were immunized with either mouse- or ferret-derived P. carinii, with subsequent analysis of the immune response and the ability of the mice to resist infection after immunosuppression and challenge. Immunization with mouse-derived P. carinii produced a strong immune response to mouse but not ferret P. carinii. These mice were completely protected from P. carinii pneumonia when challenged by intratracheal inoculation with mouse P. carinii. In contrast, immunization with ferret P. carinii produced a limited antibody response to mouse P. carinii and had no protective effect. These results show that P. carinii from different host species are immunologically distinct, and any possible use of immunotherapy for P. carinii pneumonia in humans must take into consideration these biologically significant antigenic differences in P. carinii of animal origin.     

Molecular analysis of bacterial isolates and total community DNA from kraft pulp mill effluent treatment systems

Chloroaliphatics are major components of bleached kraft mill effluents. Gene probes and oligonucleotide primers were developed to monitor kraft pulp mill effluent treatment systems for the presence of key genes (dehalogenases) responsible for the dehalogenation of chloroaliphatic organics. The primers were used for polymerase chain reaction (PCR) analysis of genomic DNA extracted from dehalogenating bacterial isolates and from total community DNA extracted from water and sediments of mill effluent treatment system. PCR amplification with oligonucleotide primers designed from dhlB, encoding the haloacid dehalogenase from Xanthobacter autotrophicus, revealed the presence of dehalogenase genes in both aerated lagoons and stabilization basins. Similarly, positive results were obtained with mmoX primers designed from the soluble methane monooxygenase gene of Methylococcus capsulatus Bath. The haloacetate dehalogenase encoding gene (dehH2) from Moraxella sp. was typically not detected in mill effluent treatment systems unless the biomass was selectively enriched. DNA sequence analysis of several PCR fragaments revealed significant similarity to known dehalogenase amd methane monooxygenase genes. The results indicated a broad distribution of known dehalogenation genes and bacteria with chloroorganic-degrading potential in the mill effluent treatment systems.     

Determination of copy number of rRNA genes in Pneumocystis carinii f. sp. hominis

Differential PCR was performed to determine the copy number of rRNA genes in Pneumocystis carinii f. sp. hominis. Two different reference genes, thymidylate synthase (TS) and beta-tubulin (BTU) genes, were used. Primers for the internal transcribed spacer (ITS) region of nuclear rRNA genes and either the TS or BTU gene were mixed together to perform PCR on seven different bronchoalveolar lavage specimens from patients with P. carinii pneumonia. The radioactivity derived from the incorporated radioactive nucleotides of each PCR product band was then used to calculate the copy number of the ITS relative to that of the TS or BTU gene. The copy number ratio between the ITS and the TS gene was determined to be 0.8, and that between the ITS and the BTU gene was also 0.8. These results suggest that the ITS has the same copy number as the TS or BTU gene. Since the copy number of the TS or BTU gene is presumed to be 1, the results also suggest that P. carinii f. sp. hominis has only one copy of the ITS and thus one copy of the nuclear rRNA genes. Therefore, two types of ITS sequences derived from a specimen would indicate that the patient is infected by two types of P. carinii f. sp. hominis.     

Differentiation of Chlamydia spp. by sequence determination and restriction endonuclease cleavage of RNase P RNA genes

The amplification of DNA from Chlamydia trachomatis by PCR with degenerated primers yielded a 345-bp fragment of the putative RNase P RNA gene. From the deduced DNA sequence of this gene in C. trachomatis, a modified primer pair was designed. The primer pair was subsequently used to obtain the corresponding gene products from Chlamydia pneumoniae and Chlamydia psittaci. Sequence comparisons revealed similarities of 76.6% between C. trachomatis and C. pneumoniae, 79.5% between C. trachomatis and C. psittaci, and 84.7% between C. pneumoniae and C. psittaci. Furthermore, the three species were differentiated by fragment length polymorphism analysis after restriction enzyme cleavage of the PCR products. Sequence variations among 14 serotypes of C. trachomatis were confined to one purine base substitution in the putative RNase P RNA gene of lymphogranuloma venereum strains L1 to L3. Complete sequence similarity was found for nine strains of C. pneumoniae of different geographic origins. Taken together, our results indicate a possibility of the general application of this method in clinical bacteriology. Analysis of the secondary structures of the putative RNase P RNA genes from the different Chlamydia species suggested that a novel structural element in the domain of RNase P RNA is involved in base pairing with the 3'-terminal CCA motif of a tRNA precursor. This structure has not previously been found among RNase P RNAs of members of the division Bacteria.     

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24S alpha ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24S alpha rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage 1 was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite.     

Age-dependent inaccuracy of asthma death certification in Northern England, 1991-1992

OBJECTIVE: To test the usefulness of a commercial DNA hybridization assay for the detection of high-risk (HR) human papillomavirus (HPV) types in archival cervical smears and to compare the sensitivity with that of polymerase chain reaction (PCR) using consensus primers. STUDY DESIGN: Stained material was scraped from archival slides and the pellet volume noted. DNA was extracted using silica/guanidinium isothiocyanate and the quality checked by amplification of the beta-globin gene. HR-HPV DNA was detected using a commercial hybrid capture assay (HCA) and the results compared with an in-house amplification system with consensus primers. RESULTS: Of 156 archival smears stored for 12-13 years, 20 were positive by HCA using an HR probe cocktail. Ninety-eight were also tested by PCR, and 35 were positive. The percentage of HPV-positive samples increased with the increasing size of the pellet. HR-HCA detected more positives in samples with high grade squamous intraepithelial lesion (moderate/severe dyskaryosis). CONCLUSION: Both hybridization by HCA and amplification by PCR could be used to detect genital HPV in archival smears. The general primers PCR detected more positives than HR-HCA but included HPV 6/11. While variation in sample size and prolonged storage may reduce the quality of DNA, the use of archival material for longitudinal studies of HPV presence is potentially worthwhile.     

Severe recurrent ureteral stricture: the combined use of an anterograde and retrograde approach in the prone split-leg position without X-rays

We have isolated cDNA fragments that were originated from P450 2E1 in rat brain by PCR analysis. Their size matched up to what we expected based on the reported sequence of rat liver P450 2E1 mRNA. Internal structure of the longest first-round PCR products were investigated by Southern blot analysis and "nested" PCR. Their results confirmed that PCR products actually originated from P450 2E1 mRNA in rat brain. RT-PCR was also carried out using P450 2E1 specific primers and the size of the product was exactly as we expected for P450 2E1. These experimental evidences should clarify the presence of P450 2E1 in rat brain.     

Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.     

Synthesis and biodistribution of Bowman-Birk soybean protease inhibitor conjugate with amphiphilic polyester

We describe an adaptation of competitive RT-PCR to quantitate rat IFN-gamma mRNA expression. An IFN-gamma DNA mimic that shared the same primers and had an identical sequence to the target mRNA except for deletion of 66 nucleotides was created by a simple PCR amplification from target cDNA. To reduce variations of initial RNA concentrations, beta-actin cDNAs from each target RNA sample were normalized using the densitometric data. A known amount of pretitrated DNA competitor was then used to analyze the relative levels of target cDNA in different samples by PCR co-amplification. The amplification efficiency for both target and competitor remained constant throughout the PCR reaction, and the ratio of target to competitor PCR product remained proportional to the initial ratio of target to competitor. Relative mRNA levels among samples determined by this method were comparable to levels determined by northern blot analysis. They were also comparable to levels of IFN-gamma protein estimated by ELISA. We conclude that this method can be used to estimate the relative abundance of the target mRNA. This method is adaptable to quantitation of other cytokines and is particularly valuable if there are numerous samples or if the amount of initial mRNA is limited.