New concepts in systemic autoimmunity testing.

Diagnosis of systemic autoimmune diseases is highly complex, and it is becoming increasingly difficult to make assumptions about the functional roles and diagnostic significance of autoantibodies. The latter is mainly due to the fact that results from different assay systems are not interchangeable. A laboratory "gold standard" which helps the clinician to differentiate irrelevant autoimmune phenomena from significant autoimmune diseases at an early stage, is clearly missed. To meet this challenge, a rheuma entrance screening (RES) assay toolbox is proposed based on fully-automated enzyme immunoassay (EIA) technology on one system for the clinical and routine laboratory. The RES concept is intended to cover the most important syndromes of systemic rheumatic diseases, i.e. collagenosis, early rheumatoid arthritis, early osteoarthritis, anti-phospholipid syndrome and inflammation. The serological part of diagnosis of these diseases comprises testing for anti-nuclear antibodies (ANA), rheumatoid factor (RF), low levels of C-reactive protein (CRP), and disease-specific anti-phospholipid antibodies, e.g. anti-beta-2 glycoprotein I (anti-beta2 GPI). To eliminate the known problems of varying assay systems in this field, a novel, objective, rapid and reproducible approach to screen for such analytes in patient serum or plasma more efficiently is the application of EIAs on the fully-automated immunoassay analyser COBAS CORE (Roche Diagnostics GmbH, Mannheim, Germany). The combined use of the RES (COBAS CORE HEp2 ANA EIA, COBAS CORE RF EIA Quant, COBAS CORE CRP EIA Quant and COBAS CORE Anti-beta2 GPI EIA) is intended for patients sent to the laboratory with the primary suspicion of harbouring a systemic rheumatic disease.

The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR.

The superoxide dismutase gene has been identified as a target in screening for the presence of mycobacteria on the genus level and differentiating relevant mycobacterial species from one another by PCR. Consensus primers deduced from known superoxide dismutase gene sequences allowed the amplification of DNAs from a variety of bacteria, fungi, and protozoa. Selected amplicons from Actinomyces viscosus, Corynebacterium diphtheriae, Corynebacterium pseudodiphtheriticum, Mycobacterium avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. scrofulaceum, M. simiae, M. tuberculosis, M. xenopi, and Nocardia asteroides were subsequently cloned and sequenced. The alignment of those sequences facilitated the selection of primers targeting conserved regions present in myobacterial species but absent in nonmycobacterial species and thus allowed the genus-specific amplification of all 28 different mycobacterial species tested. A pool of genus-specific allowed the genus-specific amplification of all 28 different mycobacterial species tested. A pool of genus-specific probes recognized 23 of the 28 mycobacterial species and did not cross-react with any of the 96 nonmycobacterial species tested. In addition, probes recognizing species-specific variable regions within the superoxide dismutase genes of M. avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. scrofulaceum, M. simiae, the M. tuberculosis complex, and M. xenopi were identified. All probes recognized only the species from which they were derived and did not cross-react with any other mycobacterial species or with any of the nonmycobacterial species tested. We conclude that the superoxide dismutase gene is a suitable target for amplifying mycobacteria by PCR on the genus level, confirming correct amplification by genus-specific probes, and differentiating relevant species from one another by a set of species-specific probes.

A Plasmodium falciparum-specific reverse target capture assay.

Plasmodium falciparum DNA is detected with an assay modeled according to the reverse target capture assay described by Morrissey et al. [19] for the detection of Listeria cells. A poly(A)-tailed oligonucleotide (pWZ34), derived from the partial sequence of a 4-kb repetitive unit of P. falciparum, functions as a capture probe and the labelled 21-bp repetitive units specific for P. falciparum serve as a reporter probe. Both probes are complementary to non-overlapping regions of the target DNA and in the presence of high concentration of chaotropic salts, hybridization efficiently takes place at relatively low temperatures (15 min. 37 degrees C). The addition of poly(dT)-derivatized ferromagnetic beads allows the formation of A:T base pairing between the tailed beads and the tailed capture probe. Upon applying magnetic force, the target-capture-reporter-probe complex attached to the beads is removed from the reaction mixture, leaving the bulk of unreacted reporter molecules behind. Subsequent washings of the immobilized complex reduces the amount of non-specifically bound reporter probe. After elution of the complex from the beads a new cycle of capture, washing and release of the target-capture-reporter-probe complex is initiated by the additions of unused (dT)-tailed beads. After 3 cycles, the signal-to-noise ratio with 0.1 pg of P. falciparum DNA as a target was as high as 21-27, with a background of 8-10 cpm. The assay is unique in its speed, well suited for large sample numbers, and allows the manipulation of the background at will by simply increasing the number of capture rounds.

Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates.

Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.

Detection of pyrimethamine resistance in Plasmodium falciparum by mutation-specific polymerase chain reaction.

The point mutation at nucleotide 323 within the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum, which distinguishes pyrimethamine-sensitive from drug-resistant isolates, can be discriminated by the polymerase chain reaction using mutation-specific primers. The technique makes use of the principle that short oligonucleotides with a perfect match at their 3' ends, complementary to the mutation to be detected, will initiate the polymerization by Taq polymerase far more efficiently than primers with a single mismatch in this position. The Asn-108 codon was detected using a primer of 17 nucleotides with an adenosine at its 3' end, the Thr-108 codon with a 14-mer primer ending with a cytosine and the Ser-108 codon with a 16-mer containing guanidine at the critical 3' end. By selecting appropriate counterprimers, the size of the amplification products is either indicative of pyrimethamine-resistant parasites of the 7G8 type, or of pyrimethamine-sensitive parasites of the FCR-3 type or 3D7 type. The fragments obtained can be easily separated in a single lane after agarose gel electrophoresis. Coded P. falciparum DNA samples were typed unambiguously using these primers as were reconstituted parasitized blood samples stored as high salt lysates. Sensitivity, speed and specificity make this assay a realistic alternative to in vitro drug testing to monitor the resistance of P. falciparum to inhibitors of the dihydrofolate reductase.

Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum.

The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogenous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogenous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.

Comparison of thick films, in vitro culture and DNA hybridization probes for detecting Plasmodium falciparum malaria.

Using blood from volunteers with sporozoite induced malaria, a comparison was made of the sensitivity and specificity of Giemsa stained thick film examination, in vitro culture, and 4 different DNA probes for detecting parasitemia. Between 9 and 13 days after sporozoite inoculation, patent parasitemia (4-550 parasites/microliters) was detected by thick film examination of 0.5 microliters blood in 7 volunteers. Cultures of 1 ml blood obtained 7 days after sporozoite inoculation were positive in all volunteers who eventually developed patent parasitemia. The DNA hybridization probes detected parasites in only 5-28% of smear- or culture-positive samples.

High salt lysates: a simple method to store blood samples without refrigeration for subsequent use with DNA probes.

Blood specimens to be tested for the presence of Plasmodium falciparum using specific DNA probes can be stored as high salt lysates (HSL) without refrigeration. The lysates are prepared from 100 microliter blood samples by a simple 3-step procedure using 2 volumes of H2O to lyse the erythrocytes (step I), 1 volume of a detergent/EDTA mix to lyse the parasites (step II), followed by the addition of 1 volume cesium trifluoroacetate (CsTFA) (step III). The parasite DNA was found to be undegraded, as shown by the unaltered pattern of repetitive sequences obtained after storage of up to 1 month at 37 degrees C, due to the inhibition of DNA degrading enzymes by the cesium salt. The bulk of protein can be removed from the samples by a 1-step precipitation. The addition of 0.3 volumes of a mixture of ethanol: chloroform: isoamyl alcohol (2.5:1:0.04 v/v) precipitates greater than 90% of the proteins from the lysates, leaving greater than 86% of the parasite DNA in the supernatant. The reduced protein content of the samples, when applied to solid supports, results in an increased signal: background ratio on autoradiograms.

Purification of the bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum.

The bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum has been purified to homogeneity using a sequence of separation steps including phenyl-Superose, gel filtration, dye affinity matrix, hydroxyapatite, and anion exchange chromatography. The specific activity of dihydrofolate reductase increased approximately 24,000-fold from 3.3 units mg-1 protein to 79,000 units mg-1 protein after five successive chromatographic steps with a yield of 31%. Both enzyme activities coeluted as a symmetric peak in highly purified preparations, indicating the existence of a bifunctional enzyme complex in P. falciparum. The apparent molecular weight of the native complex was approximately 120,000 as determined by gel filtration. When individual fractions of the anion exchange column were subject to polyacrylamide electrophoresis under denaturing conditions, the increase in intensity of a single band correlated with the amount of both the thymidylate synthase and dihydrofolate reductase activity. Further purification led to an electrophoretically pure protein (yield 2.6%) with an apparent molecular weight of 67,000, suggesting that the bifunctional enzyme complex from P. falciparum is composed of two subunits of identical size and charge.

Kinetic and molecular properties of the dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant clones of the human malaria parasite Plasmodium falciparum.

Dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADPH+-oxidoreductase; EC 1.5.1.3) was partially purified by affinity chromatography from three clones of the human malaria parasite Plasmodium falciparum. The three clones were representative of pyrimethamine-sensitive (clone 3D7) and pyrimethamine-resistant (clone HB3 and clone 7G8) parasites with ID50 values of 0.53 nM (3D7), 210 nM (HB3), and 540 nM (7G8), when tested in vitro against the drug. The specific activities of the partially purified DHFR differed by less than a factor of 2 between the sensitive clone 3D7 (442 +/- 39 nmol min-1 mg-1 protein) and the resistant clones HB3 (634 +/- 25 nmol min-1 mg-1 protein) and 7G8 (565 +/- 85 nmol min-1 mg-1 protein). The number of catalytic sites in partially purified DHFR from the three clones was similar and ranged from 151 to 194 pmol mg-1 protein. The Km value for NADPH was similar in all three clones (4.5-11.6 microM). The Km value for dihydrofolate was altered 13-fold comparing the sensitive clone 3D7 (3.2 +/- 0.6 microM) with the resistant clone HB3 (42.6 +/- 1.6 microM), with the Km for the resistant clone 7G8 falling in between (11.9 +/- 1.2 microM). The inhibition constants for pyrimethamine increased from 0.19 +/- 0.08 nM (3D7) to 2.0 +/- 0.3 nM (HB3) to 8.9 +/- 0.8 nM (7G8). The inhibition by pyrimethamine of the sensitive clone 3D7 was noncompetitive and competitive for the two other clones. The titration of partially purified DHFR with pyrimethamine revealed a 500-fold increase in the concentration of the drug needed to inhibit the DHFR activity by 50%, when the sensitive clone 3D7 (0.18 +/- 0.02 nM) was compared to the resistant clone 7G8 (95 +/- 16 nM). From the comparison of the specific activities and the catalytic center activities with the Km values for the substrate and the inhibition constants for pyrimethamine, both of which are altered in the resistant clones, we conclude that the molecular mechanism for pyrimethamine resistance in the three clones studied is not based on an overproduction of the DHFR but is due to a decreased affinity to antifolates by a structurally altered enzyme.

Detection of Plasmodium falciparum DNA using repetitive DNA clones as species specific probes.

Repetitive sequences were identified in genomic libraries of Plasmodium falciparum and analyzed for their potential use as specific DNA probes. Nucleotide sequencing revealed inserts composed of 21 base pair tandem repeats. Clone 26 containing an insert of 147 base pairs in M13mp18 was used in three different approaches as a probe to detect P. falciparum DNA: the replicative form of clone 26 was labeled by nick translation; the single strand DNA of clone 26 was labeled by primer extension and a two step sandwich assay was employed hybridizing single strand unlabeled clone 26 DNA to the target DNA (first step) and using nick translated M13 DNA in a second step to detect the vector part of clone 26. The most sensitive probes detected 25 pg of P. falciparum DNA after 2 h of film exposure, 3 pg after 14 h and 0.78 pg after 40 h. Hybridization to genomic blots of Plasmodium vivax and human DNA using clone 26 as a probe revealed that the 21 base pair repeats specifically hybridized with P. falciparum DNA while failing to react with either human or P. vivax DNA.

Characterisation and translation studies of messenger RNA from the human malaria parasite Plasmodium falciparum and construction of a cDNA library.

RNA was isolated from trophozoites, schizonts and mixed populations of Plasmodium falciparum. 5% of the total was poly(A+) message, of average length 1.2 kb (10-12 kb maximum) and a poly(A) content of 10%. The mRNA fractions could be translated in vitro by reticulocyte lysates supplemented either with exogenous or P. falciparum tRNA. The patterns from two independent isolates, one cloned (T9-94) and one uncloned (K1) were virtually identical. Major translation products from 16-230 kDa have been measured. The most abundant is lactate dehydrogenase (34.8 kDa). Trophozoite mRNA codes principally for proteins of less than or equal to 93 kDa, while schizont mRNA codes for additional proteins of higher molecular mass. There are marked similarities between the in vitro translation products and proteins synthesised in vivo in synchronous cultures. A number of schizont mRNA translation products (principally those of 230, 203, 185, 170, 115, 101 and 71 kDa) are specifically precipitated without post-translational modification by sera from humans exposed to malaria. A cDNA library has been constructed in phage lambda from total poly(A+) RNA and partially characterised. About 10% of the clones derive from abundant mRNA sequences. Putative actin clones have been isolated from this library and the parasite actin mRNA sized at approx. 2.8 kb.

The accumulation of lactic acid and its influence on the growth of Plasmodium falciparum in synchronized cultures.

Synchronization of Plasmodium falciparum cultured in vitro results in a one-step growth pattern that allows the study of stage-specific metabolic activities of the parasites. Lactic acid (LA) was selected as a metabolic marker, and the concentration of this end product found in spent media was correlated with the different erythrocytic stages of the parasites. When the medium was changed at 12 h intervals, cultures containing predominantly trophozoites produced 3.66 +/- 0.55 mumol LA per 12 h per 10(7) parasitized cells (n = 26), an amount of LA that is about 8 to 20 times higher than that found in corresponding cultures containing predominantly ring forms. Depending on the stage of development, parasitized red blood cells produced between 5 and 100 times more LA than uninfected erythrocytes (3.72 +/- 0.62 mumol LA per 12 hours per 10(9) red blood cells) (n = 41) when cultured under identical conditions. The intraerythrocytic development of the parasites was not impaired by exposure to extracellular concentrations of LA up to 12 mM over a 12 h period. The growth resulting in such cultures was described as uninhibited and was characterized by a multiplication index of 10 or higher. Above the threshold of 12 mM of LA, progressive inhibition of parasite development occurred. The stage-specific LA production reported can be used to predict the amount of LA that will have accumulated at the end of a subsequent 12 h incubation period during synchronized in vitro growth of Plasmodium falciparum. Using these values, it is possible to establish an optimal medium exchange schedule, thereby assuring uninhibited growth and a correspondingly high parasite yield.

Antigens of the erythrocytes stages of the human malaria parasite Plasmodium falciparum detected by monoclonal antibodies.

A range of 22 mouse anti-P. falciparum monoclonal antibodies have been characterized by indirect immunofluorescence and immunoprecipitation. On the basis of these studies, 5 groups of antibodies and 6 classes of antigen were defined. Group I antibodies give, bright, uniform, generalised staining of all blood stages including gametocytes. Three of these antibodies precipitate a metabolically labelled molecule(s) of 35 kDa. One precipitates a 50 kDa antigen. Group II antibodies, which give strong localised immunofluorescence in merozoites, and a weak diffuse pattern in earlier stages, precipitate biosynthetically labelled molecules of 160 kDa. Group III antibodies react with all asexual stages. With merozoites they produce intense staining around the perimeter, both in fixed and unfixed preparations. They precipitate biosynthetic molecules of 190 kDa. Group IV antibodies are identical to Group III except they are stage restricted to schizonts and merozoites. They also precipitate 190 kDa antigens. These, however, in contrast to group III, are readily accessible to 125I-lactoperoxidase labelling. One antibody also precipitates a set of smaller peptides. Finally, Group V antibodies produce very bright ill-defined staining of pigment-containing parasites, as well as of inclusions in the red cell. They precipitate a series of molecules of 160, 60 and 35 kDa which are readily accessible to 125I. The 160 kDa molecule is also labelled by [35S]methionine. These results are discussed in the context of the development of a malaria vaccine and immunodiagnostic tests.

Plasmodium falciparum: modifications of the in vitro culture conditions improving parasitic yields.

We attempted to optimize some of the variables involved in the in vitro culturing of Plasmodium falciparum. Irrespective of the isolates used, suspension cultures in glucose-enriched RPMI-1640 medium buffered with TES yielded about twice the amount of parasites than could be obtained from static, thin-layer cultures with HEPES-buffered RPMI-1640 without additional glucose. In suspension cultures, methylcellulose (1 mg/ml) was added to protect the erythrocytes. In addition the erythrocytes were found to be more suitable for culturing P. falciparum when stored as a concentrate in saline-adenine-glucose than as whole blood in citrate-phosphate-dextrose. With a cloned isolate of P. falciparum (Tak9/clone 96) a further stimulation of the final parasitemia could be achieved by supplementing the medium with hypoxanthine (50 micrograms/ml) and reduced glutathione (600 micrograms/ml). Moreover, we identified hypoxanthine and glutathione as two of the factors critical for the ability of human serum to support the growth of the parasites. These modifications give a two- to four-fold increase in the final parasitemia over the original Trager-Jensen culture method, thus allowing more parasites to be isolated for biochemical studies.

The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation.

The DNA of Plasmodium falciparum has been purified and fragmented with the restriction endonucleases EcoRI and HindIII. The fragments have been incorporated in vitro into derivatives of bacteriophage lambda to make libraries in which most of the parasite DNA is represented. By Southern hybridisation we have been able to recover from these libraries specific clones containing (a) repetitive DNA sequences, (b) rRNA gene(s) and (c) sequences homologous to an actin gene probe. Parasite DNA from two independent sources differs markedly in the pattern of its repetitive DNA visualised by hybridisation to our repetitive clone. By contrast, the rRNA genes of the two isolates prove to be carried on identically sized fragments.

Isolation and characterisation of ribosomal RNA from the human malaria parasite Plasmodium falciparum.

Ribosomal RNA isolated from the malaria parasite Plasmodium falciparum consists of two species with molecular weight of 1.49 +/- 0.09 X 10(6) and 0.78 +/- 0.02 X 10(6) present in equimolar quantities. Their molecular weights are comparable with those of other protozoa but quite distinct from those of the human host. The overall base composition of the rRNA (40% G+C) is close to that of the rodent parasite Plasmodium berghei, unlike the latter, however, P. falciparum has no nick in the RNA from its large ribosomal subunit.

Characterization of a R plasmid-associated, trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene.

The trimethoprim-resistant dihydrofolate reductase associated with the R plasmid R388 was isolated from strains that over-produce the enzyme. It was purified to apparent homogeneity by affinity chromatography and two consecutive gel filtration steps under native and denaturing conditions. The purified enzyme is composed of four identical subunits with molecular weights of 8300. A 1100 bp long DNA segment which confers resistance to trimethoprim was sequenced. The structural gene was identified on the plasmid DNA by comparing the amino acid composition of the deduced proteins with that of the purified enzyme. The gene is 234 bp long and codes for 78 amino acids. No homology can be found between the deduced amino acid sequence of the R388 dihydrofolate reductase and those of other prokaryotic or eukaryotic dihydrofolate reductases. However, it differs in only 17 positions from the enzyme associated with the trimethoprim-resistance plasmid R67.

Isolation of a small DNA fragment carrying the gene for a dihydrofolate reductase from a trimethoprim resistance factor.

DNA fragments of the R factor R388 which renders E. coli resistant to trimethoprim by inducing a trimethoprim resistant dihydrofolate reductase (Amyes and Smith, 1974) were inserted into plasmids and screened for the expression of the trimethoprim resistance gene. By means of a two step deletion procedure a 1770 bp EcoRI/BamH1 fragment was isolated which conferred drug resistance and which was found to induce the synthesis of the same dihydrofolate reductase as the parental R factor. Gene dosage experiments indicated that the induction was due to the presence of a dihydrofolate reductase structural gene on the 1770 bp fragment. The gene could be assigned to a segment which was less than 1200 bp long. The 1770 bp fragment and a recombinant plasmid consisting of pSF2124 and part of R388 were mapped with several restriction nucleases. The R factor induced enzyme was partially purified from a strain carrying a multicopy recombinant plasmid into which the 1770 bp fragment was inserted and which induced high levels of dihydrofolate reductase. The enzyme was found to be stable at 100 degrees. Some aspects of the synthesis of dihydrofolate reductase are discussed.