Serial quantitative PCR analysis of bone marrow samples from breast cancer patients to monitor systemic micrometastases.

BACKGROUND: We have previously developed a quantitative calibrated PCR assay to measure cytokeratin 19 (CK19) expression in haematopoietic tissue in order to detect systemic micrometastases. PATIENTS AND METHODS: Serial measurements of CK19 expression in bone marrow of patients with primary breast cancer were performed at operation, at 3 weeks and 6 months postoperatively. RESULTS: Reference values for CK19 expression were established by analysing bone marrow samples from 48 healthy female volunteers or patients without epithelial cancer. Samples from breast cancer patients with CK19 values above the upper reference limit were considered positive. Bone marrow samples taken at operation were positive in 29 out of 141 patients (20.6%) and remained positive in 12, turned negative in 4 and were unavailable in 13 at 6 months postoperatively. CONCLUSION: Serial measurements increase the reliability of detecting micrometastases perioperatively. Further studies are in progress to evaluate the relationship between elevated CK19 values and clinical outcome.

Fluorescence-based method for measuring and determining the mechanisms of recombination in quantitative PCR.

We quantitatively measured the amount of recombinant molecules formed during PCR when the break point cluster region (BCR) cDNA was coamplified with a homologous internal standard using Taq polymerase. The products were analysed under denaturing conditions using capillary electrophoresis followed by detection of the fluorescently labelled products and the recombinant molecules were differentiated by their size. Early termination of chain synthesis and reannealing of incomplete fragments, to each other as well as to BCR and internal standard, is one mechanism for generating recombinants during PCR since prolonging extension time reduced, but did not totally suppress recombinant molecule formation. Template switching by the extending chain is another mechanism since recombinant molecules could be detected even after only one round of primer extension. The latter mechanism is probably facilitated by increasing number of templates. Thus, the large increase of recombinant molecules formed in plateau phase is mediated by direct amplification of the recombinants and de novo synthesis by template switching. The effect of additives on recombination could be quantitatively measured and both betaine and DMSO were effective in suppressing recombination. Thus, prolonging extension time, reducing the number of amplification cycles and incorporating additives in the PCR reaction, reduced recombinant molecule formation.

Polymorphism of the virulence regulon and allelic variations of the sic gene among the emm1 isolates of group A Streptococcus from western Norway.

With the objective of finding genetic markers of invasiveness, 43 isolates of group A streptococcus, isolated in western Norway and from both severe invasive disease and superficial infections, were studied initially by restriction fragment length polymorphism of the virulence regulon (virR -RFLP). Polymorphism that seemed to be related to the severity of infection was observed within the emm1 sequence type, which included 11 invasive and seven non-invasive isolates. These emm1 isolates were further investigated by restriction mapping of the virR and sequence analysis of a polymorphic region, which revealed the presence of a hypervariable sic gene. Of the nine distinct sic alleles, seven were found in single isolates, of which only two were from patients with invasive disease. The other two alleles were shared among nine invasive and two non-invasive isolates. The presence of only two sic allotypes in nine of the 11 invasive isolates, as compared to a different allele in each of the five non-invasive, contemporary isolates supports the hypothesis that selection of the sic variants occurs at mucosal surfaces and implicates mainly two clones among the invasive emm1 isolates.

Quantitative analysis of cytokeratin 20 gene expression using RT-PCR and capillary electrophoresis with fluorescent DNA detection.

OBJECTIVE: We developed a quantitative reverse-transcription polymerase chain reaction (RT-PCR) to determine CK20 expression in colorectal tumor and hematopoietic tissue. DESIGN AND METHODS: Our method incorporates a calibrated PCR with an internal competitor and an external standard. RESULTS: The RT-PCR assay is sensitive detecting 10 target molecules of CK20 in solution with one round of 38 amplification cycles. Genomic DNA contamination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high throughput of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow from 20 healthy volunteers revealed a low level of CK20 expression in all samples. CONCLUSION: To study the clinical significance of CK20 expression as a marker of systemic metastatic disease it is essential to measure CK20 mRNA levels in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described.

Molecular characterization and allelic distribution of the phage-mediated hyaluronidase genes hylP and hylP2 among group A streptococci from western Norway.

Forty-two isolates of group A streptococcus from patients with invasive and non-invasive diseases in western Norway, belonging to the emm sequence types emml, emm3, emm6, emm22, emm28, emm75 and emm78 were screened by PCR for the phage-mediated hyaluronidase genes hylP and hylP2. The amplified genes were characterized by nucleotide sequencing and/or by PCR-RFLP, with the objective of looking for possible associations between alleles of these two genes and invasiveness. The hylP was amplified from all isolates and two main alleles were found hylP-emm3 in all emm3 isolates and hylP-emm6A in all emm6 isolates, the latter possibly generated by an intergenic recombination between hylP and hylP2. The isolates of the other sequence types had either of these two alleles, or both. Only 27 isolates gave amplicons of the appropriate size with the primers targeting hylP2. Sequencing of these amplicons showed two main types: one was similar to the published hylP2 and the other (hylP-emm6B) was probably a variant of hylP. PCR-RFLP revealed the presence of both hylP-emm6B and hylP2 in at least six of the emm6 isolates. The alleles of both hylP and hylP2 seemed to have emm sequence type preferences. No association between invasiveness and specific phage-mediated hyaluronidase genes/alleles or the production of extracellular hyaluronidase was observed.

Emm gene polymorphism among temporally clustered group A streptococcal isolates in western Norway.

Nineteen group A streptococcal isolates obtained in western Norway from patients with invasive disease during a period of high morbidity and mortality were examined for clonality and emm gene polymorphism. These isolates belonged to the prevalent serotypes during the outbreak, namely T1, T3 or T6. Restriction fragment length polymorphism and sequencing of the emm genes were used to compare these isolates with 14 isolates of the same serotype but from non-invasive infections. The restriction analysis did not identify specific invasive clones. The emm genes in three of the four T3 isolates from invasive disease had nucleotide substitutions inducing a charge difference in the N-terminal part of the M protein. The 4 T6 isolates had a longer emm amplicon when compared to 15 isolates from superficial infections and also showed nucleotide substitutions that could induce conformational changes in the hypervariable end of the M protein. Restriction analysis of the emm amplicon of the T6 isolates in order to estimate the number of A- and C-repeats is described. The emm gene sequence served as an epidemiological marker within the serotypes T3 and T6, but the significance of the emm polymorphism displayed by the isolates from invasive disease is uncertain at this stage.

Distribution and sequence variations of selected virulence genes among group A streptococcal isolates from western Norway.

In order to compare the distribution of selected virulence genes among group A streptococci recovered from invasive disease and superficial infections, 42 isolates were screened for mga, speB, speA, ssa and ska, by PCR. The isolates were predominantly of the sequence types emm1, emm3 and emm6, but also included a few of the types emm22, emm28, emm75 and emm78. The phage-mediated speA seemed to be prevalent in emm types 1 and 3, and its distribution was not related to disease severity. The other genes were present in all isolates. The mga, speB and speA were further studied by sequence analysis. Although allotypic associations with invasiveness were not found, allelic specificity to the emm sequence type was observed. In addition, the mga sequences indicated two lineages, related to opacity factor production. A possible recombination between these two main divergent mga genes was observed in isolates of the types emm22 and emm75. A logical nomenclature of the alleles of mga and speB is suggested.

Plasma total homocysteine response to oral doses of folic acid and pyridoxine hydrochloride (vitamin B6) in healthy individuals. Oral doses of vitamin B6 reduce concentrations of serum folate.

Plasma total homocysteine response was compared in four groups of healthy individuals given orally divided doses of vitamin supplementations for a duration of 5 weeks. The vitamin supplements; A, 0.3 mg folic acid; B, 120 mg vitamin B6; C, combination of 0.3 mg folic acid and 120 mg vitamin B6 or D, 0.6 mg folic acid reduced the concentrations of plasma total homocysteine 20, 17, 32 and 24%, respectively. However, the intergroup comparisons did not show a significant difference in the effects of vitamin supplements. Multivariate analysis with correction for differences in pre-supplement values indicated a significant effect of vitamin B6 supplementation on plasma total homocysteine and serum folate. Our data show that plasma total homocysteine concentrations are reduced with low to medium divided doses of folic acid alone or in combination with vitamin B6.

Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway.

Two-hundred and forty healthy sheep and 32 cases of natural scrapie in Norway were analysed for disease-linked polymorphisms in the prion protein (PrP) gene. Scrapie was strongly associated with the presence of a valine polymorphism at codon 136 (V136), as 68.8% of the cases were homozygous (VV136) and 15.6% were valine/alanine heterozygous (VA136). All cases were homozygous arginine/arginine at codon 154 (RR154), except two which were homozygous histidine/histidine (HH154). All cases except two were homozygous glutamine/glutamine at codon 171 (QQ171), the two exceptions being heterozygous glutamine/arginine (QR171). More than 80% of all scrapie cases in Norway have occurred in a Cheviot-related crossbred type of sheep called Rygja. This type of sheep, which is largely restricted to the south-western coast, carries the V136 allele at a higher frequency than do other breeds of Norwegian sheep. Polymorphisms at codons 138 and 151 are also described.

Sensitive and quantitative one-step polymerase chain reaction using capillary electrophoresis and fluorescence detection for measuring cytokeratin 19 expression.

An improved quantitative assay to measure cytokeratin 19 (CK19) expression has been developed. The assay utilizes reverse transcription and a one-step polymerase chain reaction (PCR), with capillary electrophoresis and fluorescent labelling, to separate and detect the PCR products. Calibration curves were constructed from a serial dilution of CK19 cDNA coamplified with a fixed amount of CK19 internal standard, which was found to be linear between 10 and 500 molecules. Quantitative measurement of CK19 in samples was carried out by coamplifying the cDNA with a fixed amount of internal standard. The values were calculated from the calibration curve. The integrity of RNA and cDNA synthesis was checked by quantitative measurement of the breakpoint cluster region (BCR) gene expression. The assay is sensitive, detecting < 10 CK19 transcripts, and reproducible with a coefficient of variation of approximately 10%. CK19 expression showed overlapping values when measured in samples from peripheral blood and bone marrow in operable breast cancer patients, in healthy volunteers or patients without epithelial cancer and in blood samples from patients with metastatic breast cancer. As the assay is easier to perform than traditional quantitative competitive PCR assays, it might be useful for quantitative measurement of other specific transcripts.

Crossreactions and sequence homologies between recombinant polypeptides from Leishmania aethiopica and human IgG and IgM.

Recombinant DNA fragments M (154 bp) and G (206 bp), coding for recombinant polypeptides that crossreact with human IgM and IgG, have been isolated from a genomic library of Leishmania aethiopica. Epitope scanning of the two recombinant polypeptides, using overlapping octapeptides, revealed several crossreactive epitopes present in both recombinant proteins. By comparing amino acid sequences, similar sequences in human mu and gamma immunoglobulin heavy chains were identified. One of the parasite octapeptides is identical to an octapeptide in gamma1 covering the Gm(a) allotypic marker. Expression of both the M and G fragments was detected in the parasites by RT-PCR of total mRNA, using primers specific for these fragments. Preliminary data showed that the presence of autoimmune anti-IgG antibodies was more pronounced in sera from patients with diffuse cutaneous leishmaniasis than in sera from patients with localised cutaneous leishmaniasis. We suggest that these immunoglobulin-crossreacting epitopes potentially might contribute to the induction of rheumatoid factors and be involved in the interplay between the parasite and the host immune system.

Identification and characterization of human B-cell epitopes in recombinant antigens of Leishmania aethiopica.

Recombinant DNA fragments from Leishmania aethiopica that code for epitopes which react with human antibodies have been characterized by cross-hybridization studies and DNA sequence analysis. Twenty clones could be grouped into seven different groups (I-VII), probably representing seven different L. aethiopica antigens. The DNA sequences of representative clones from the seven groups have been obtained and the amino acid sequence of the respective recombinant antigens established. The recombinant antigens have been analysed by epitope scanning with patient sera, and octapeptides that contain potential B-cell epitopes have been identified in all seven recombinant antigens. These octapeptides have further been tested with additional patient sera and control sera, and three octapeptides (HAFCHEEG, YHSSVVHD and SYAPCSLK) were found to contain major epitopes recognizing specific antibodies in nine, seven and four, respectively, of the twenty sera tested. Fifteen of the twenty sera reacted with one or more of these three octapeptides.

[Diagnosis and follow-up in chronic myeloid leukemia. Detection and quantification of specific transcripts with the help of reverse transcriptase-polymerase chain reaction]. / Diagnostikk og oppfølging ved kronisk myelogen leukemi. Påvisning og kvantitering av spesifikke transkripter ved hjelp av revers transkriptase-polymerasekjedereaksjon.

The Philadelphia chromosome in cells of patients with chronic myeloid leukemia and acute lymphoblastic leukemia can be detected by reverse transcriptase-polymerase chain reaction (RT-PCR). We have tested two new methods for this purpose. For diagnostic purposes, three different BCR-ABL translocations (b3a2, b2a2 and ela2) can be detected in a multiprimed, one step PCR reaction. By using a competitor DNA construct and a two-step, nested PCR reaction, a quantitative measure of the number of specific BCR-ABL transcripts can be estimated. We tested five patients with chronic myeloid leukemia. All of them showed positive BCR-ABL translocations in the diagnostic test. Patients with other myeloproliferative disorders, used as controls, were all negative. Quantitative measurements of specific BCR-ABL mRNA showed that as few as ten transcripts could be quantified in the assay. The analysis showed that coefficients of variation between 15% and 30% were obtained for specific transcripts per micrograms RNA, whereas specific BCR-ABL per normal ABL showed a coefficient of variation of 10%. These new methods to detect BCR-ABL translocation by RT-PCR should provide easy and sensitive diagnosis, and possibilities of monitoring residual disease or relapse.

The Mycobacterium leprae antigen 85 complex gene family: identification of the genes for the 85A, 85C, and related MPT51 proteins.

The genes for two novel members (designated 85A and 85C) of the Mycobacterium leprae antigen 85 complex family of proteins and the gene for the closely related M. leprae MPT51 protein were isolated. The complete DNA sequence of the M. leprae 85C gene and partial sequences of the 85A and MPT51 genes are presented. As in M. tuberculosis, the M. leprae 85A, 85C, and previously identified 85B component genes are not closely linked on the genome. However, the MPT51 genes of both species localize close to the respective 85A component genes. Like the 85B component, the M. leprae 85A-MPT51 and 85C antigens are recognized by T cells from healthy contacts and leprosy patients.

Rapid C-reactive protein (CRP) measurements in the diagnosis of acute appendicitis.

C-reactive protein (CRP) has been measured in plasma of patients with acute appendicitis and in controls without appendicitis to test the accuracy and diagnostic performance of a new rapid test kit for CRP (NycoCard CRP). The values obtained for CRP by the rapid test correlated well (Rs = 0.92) with the reference method for measuring CRP. The sensitivity, specificity and predictive values were calculated at different cut-off values. At values > 10 mg l-1 a sensitivity of 58% and a negative predictive value of 72% were found. Higher values of sensitivity were observed for men than for women, 69% and 44% respectively. Patients with acute appendicitis who had had symptoms for more than 24 h, had elevated CRP values (cut-off > 10 mg l-1) in more than 80% of cases. Our study shows that the rapid CRP test and the reference CRP test gave an almost identical result.

Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES.

In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T-cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae-specific GroES-derived peptide in a DR2-restricted fashion. No expression of the groEL gene from this operon was detected in E. coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.

Isolation and characterization of recombinant antigens from Leishmania aethiopica that react with human antibodies.

A genomic expression library of Leishmania aethiopica was constructed in lambda gt11 and screened with patient sera and sera from healthy people living in an area of endemicity. Forty-five recombinant clones were isolated and partly characterized. Clone-specific antibodies were prepared and used with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis to estimate the molecular masses of the parasite-derived antigens containing the reactive epitope(s). Antigens with apparent molecular masses of 90, 85, 63, 50, 41, 25 and 24 kDa as well as several antigens with lower molecular masses were detected. The clone-specific antibodies from patients with diffuse cutaneous leishmaniasis reacted with high-molecular-weight antigens (30,000 less than Mr less than 90,000), whereas antibodies from patients with localized cutaneous leishmaniasis recognized low-molecular-weight antigens (Mr less than 25,000). Nine different purified recombinant antigens were obtained from lysogens in Escherichia coli Y1089 by immunoaffinity chromatography on anti-beta-galactosidase columns and were subsequently tested with patient sera. It is suggested that some of these recombinant antigens might be used for immunodiagnostic purposes.