Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference.
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Actins↗
Biomedical subjects
C A Schmidt
Publications and source records attributed to C A Schmidt.
Incorporation of bromophenol blue enhances visibility of polyacrylamide gels.
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Bromphenol Blue↗
A prospective study of human herpesvirus type 6 detected by polymerase chain reaction after liver transplantation.
Human herpesvirus type 6 (HHV-6) causes roseola infantum (exanthema subitum) upon primary infection in young children. Thereafter it persists lifelong in the organism. Like other herpesviruses, HHV-6 can be reactivated in periods of immunosuppression - e.g., after organ transplantation. In order to study the incidence and the time to reactivation after orthotopic liver transplantation (OLT) we tested buffy coat lysates before and up to 10 weeks after transplantation for the presence of HHV-6 DNA by polymerase chain reaction (PCR). Forty-six patients (male n=27, female n=19) with a median age of 48 years (range 20-66) were studied. Altogether, 30 of 287 (10.5%) buffy coat samples were PCR-positive. Before OLT 2 of 21 (9.5%) patients were positive. This ratio is not different from healthy blood donor controls. After OLT 13 of 46 (23.8%) patients were positive on one or more occasions. However, there was no statistically significant difference before and after OLT. Ten patients were analyzed for HHV-6 variants by restriction enzyme digestion of PCR products. One patient carried variant A and 9 variant B. In conclusion, HHV-6 can be detected in buffy coat cells after OLT. Our observations do not argue in favor of a reactivation.
Adult↗
Acute myeloid and T-cell acute lymphoblastic leukaemia with aberrant antigen expression exhibit similar TCRdelta gene rearrangements.
TCR delta gene recombination patterns were analysed by Southern blot, polymerase chain reaction and nucleotide sequencing in acute myeloid leukaemias, with coexpression of lymphoid antigens (Ly+ AML, n=10) as well as in early T-cell acute lymphoblastic leukaemias with (My+ T-ALL, n=10) and without coexpression of myeloid antigens (My(-) T-ALL, n=9). These 29 acute leukaemias exhibiting TCR delta gene rearrangements were selected from 66 Ly+ AML, 14 My+ T-ALL and 12 My(-) T-ALL cases. Similar recombination patterns, namely D delta 2J delta1 and V delta 1J delta1 gene rearrangements, were observed in Ly+ AML and My+T-ALL. In contrast to V delta2 D delta3 rearrangements in B-cell precursor ALL, these rearrangements require activation of a T-cell-specific TCR delta enhancer. Comparison of My+ T-ALL and Ly+ AML with My(-) T-ALL exhibited a higher incidence of incomplete D delta 2J delta1 rearrangements in My+ T-ALL and Ly+ AML. Since a D delta 2J delta1 rearrangement is an early event in TCR delta recombination, these leukaemias seem to be arrested at an earlier stage of differentiation. Similar patterns of TCR delta rearrangements in My+ T-ALL and Ly+ AML suggest existence of a common myeloid/T-lymphoid progenitor cell. Although weak or missing expression of terminal deoxynucleotidyl transferase (T delta T) was found in 7/10 Ly+ AML cases, no difference was observed in numbers of N-nucleotides inserted in junctional regions when comparing with 3/10 cases exhibiting TdT expression. Since TdT activity is necessary for N-nucleotide addition, this finding suggests down-regulation of T delta T expression after rearrangement took place in these Ly+ AML cases.
Acute Disease↗
Comparison of polymerase chain reaction from plasma and buffy coat with antigen detection and occurrence of immunoglobulin M for the demonstration of cytomegalovirus infection after liver transplantation.
We compared the value of PCR on plasma with PCR on buffy coat leukocytes, Ag assay, and the determination of IgM antibodies by ELISA for the diagnosis and follow-up of cytomegalovirus infection. Thirty patients were followed after liver transplantation (LTX). We compared the tests to assess their clinical usefulness. Fourteen of 30 (46%) patients were both positive in plasma and buffy coat PCR and Ag test. Sixteen patients were negative in both procedures. There was a 97.2% concordance between PCRs done from plasma or buffy coat. The concordance of results of PCR and Ag test in single samples was 94.3%. Discordant results were found in 5.6% of samples. Discordance was observed in the early and the late phase of CMV infection and was due to positive PCRs preceding positive Ag tests for 1-3 weeks in one-half of the patients. IgM antibodies were first observed after a median period of 8 weeks (range, 6-11 weeks) after LTX. Positive PCRs and Ag tests preceded clinical manifestation of CMV disease by a 1 week median (range, 0-3 weeks), whereas positive IgM ELISAs occurred after a median period of 2.5 weeks (range, 0-4 weeks) after the onset of CMV disease. The sensitivity and specificity of both PCR and Ag test were identical, 100% and 76%, respectively. However, for the IgM ELISA, the sensitivity was only 66%, and the specificity was 84%. In conclusion, plasma or buffy coat PCR and Ag test are equally reliable procedures for early detection and monitoring of CMV infection. PCR can become positive earlier than the Ag test, but it is technically more demanding to perform. The demonstration of IgM antibodies is of little practical help because an antibody response occurs too late in relation to infection.
Adult↗
Human herpesvirus type 7 in blood donors: detection by the polymerase chain reaction.
In order to evaluate the prevalence of human herpesvirus type 7 (HHV-7) in adult blood donors oral lavage fluid, buffy coat, and urine samples from 112 persons were examined by the polymerase chain reaction (PCR) at one time point. In addition, 11 donors were studied longitudinally over 11 weeks. When the results of the initial and the longitudinal study were combined HHV-7 DNA was found in samples from 109 of 112 (97.3%) adult blood donors. On the basis of different sensitivity levels of the first and the nested PCR differences were detected in the viral DNA load in the samples. It was found that lavage fluid regularly carried significantly higher DNA concentrations than buffy coat. Out of 112 donors, 102 (91.1%) and 8 (7.1%) were positive in the first, less sensitive PCR in lavage fluid and buffy coat, respectively (P < .0001). After nested PCR, 107 (95.5%) and 74 (66.1%) were positive in lavage fluid and buffy coat, respectively (P < .0001). Urine samples were found positive only sporadically. The longitudinal study showed that the oral lavage fluid of most of the donors consistently carried HHV-7 over up to 53 weeks, whereas buffy coat samples were positive less often. In conclusion, HHV-7 is found frequently in adult blood donors in the oral lavage fluid and buffy coat, which are, therefore, potential sources of HHV-7 transmission.
Adult↗
TCR delta gene rearrangements in acute myeloid leukemia with T-lymphoid antigen expression.
In this review we present our data concerning T-cell receptor (TCR) delta gene rearrangements in acute myeloid leukemia with coexpression of T-lymphoid features (CD2/CD4/CD7; Ly+ AML). We found a correlation between TCR delta gene rearrangements and coexpression of these T-lymphoid features. Ten of 66 Ly+ AML and only one of 44 AML cases without this coexpression exhibited TCR delta gene rearrangements (p = .028). In contrast, no correlation was observed between terminal deoxynucleotidyl transferase (TdT) expression and the occurrence of TCR delta gene rearrangements in AML. Rearrangements were found in two of 25 AML with and seven of 71 AML cases without TdT expression. Interestingly, nucleotide sequencing of junctional sites revealed up to 36 N-nucleotides in cases without or with only weak TdT expression indicating downregulation of TdT expression after the TCR rearrangement took place. Complete V delta 1J delta 1 and incomplete D delta 2J delta 1 gene rearrangements were observed most frequently in Ly+ AML. These recombination patterns were similar to patterns observed in acute T-lymphoblastic leukemia with coexpression of myeloid features (My+ T-ALL) suggesting transformation of a common myeloid/T-lymphoid progenitor cell in these cases.
Acute Disease↗
PCR-SSCP: nonradioisotopic detection with biotinylated primers and streptavidin-alkaline phosphatase conjugate.
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Alkaline Phosphatase↗
Subcellular distribution of the alpha and beta topoisomerase II-DNA complexes stabilized by VM-26.
Studies were done to determine (a) the subcellular distribution of the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II, and (b) the extent to which each isozyme forms complexes with DNA in tumor cells incubated with and without VM-26. Western blotting revealed that topoisomerase II beta was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5-100 microM VM-26 for 30 min protected the beta isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase II beta detected in nuclei of CEM cells incubated for 30 min with 25 microM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase II beta in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase II beta from degradation during cell fractionation. The stabilization of topoisomerase II beta by VM-26 allowed subsequent studies of the subcellular distribution of the topoisomerase II isozymes. Both isozymes were detected in the nonmatrix (high salt-soluble) fraction of nuclei from CEM cells, but only topoisomerase II alpha was present in the nuclear matrix. VM-26 stabilized binding of the alpha and beta topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase II alpha to matrix DNA. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function, and that both the alpha and beta isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26 is a useful way to stabilize topoisomerase II beta during cell fractionation.
Amsacrine↗
Long-term follow-up of hepatitis B virus-infected recipients after orthotopic liver transplantation.
The outcome after OLT was studied in 53 patients with chronic hepatitis B virus (HBV)* infection, 15 of whom had, in addition, evidence of hepatitis delta virus (HDV) superinfection. Nine of 53 patients received short-term immunoprophylaxis with anti-hepatitis B surface (HBs) hyperimmunoglobulin up to 1 week after OLT and 44 of 53 patients received long-term unlimited immunoprophylaxis. Eight of 9 (89%) patients with short-term immunoprophylaxis showed reactivation of replication with HBV DNA in serum > 10 pg/ml independently of the preoperative HBV DNA level and HBsAg reappeared in all cases. Four (44%) patients in this group lost their graft because of fulminant hepatitis or cirrhosis and required retransplantation, and 2 patients (22%) died after reinfection in the second graft. Nineteen of 44 (43%) patients with long-term immunoprophylaxis developed HBV values > 10 pg/ml after transplant and 12 of 44 (27%) became HBsAg+ again. Most of them had quantifiable HBV DNA levels before OLT. Retransplantation was required in 5 of 44 (11%) patients and 4 of them died after HBV recurrence. The frequency of HBV reactivation and the development of viral hepatitis after OLT were associated with the preoperative presence of HBV, as determined by the molecular hybridization assay. With nested polymerase chain reaction, all 53 patients were HBV-DNA+ in the serum before and after OLT. with just one exception, none of the patients with HDV superinfection died, in spite of increased HDV replication after OLT. The data indicate that long-term immunoprophylaxis with anti-HBs hyperimmunoglobulin after OLT improves the prognosis in HBV-infected patients. The preoperative detection of HBV DNA in serum by molecular hybridization assay is correlated with graft infection and represents a prognostic parameter. The presence of HDV may have a protective effect after OLT.
Adult↗
Expression of axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis.
We previously reported the cloning, and characterization of a receptor tyrosine kinase, axl, from two patients with chronic myelogenous leukemia. Herein, we describe the expression pattern of axl in normal and malignant hematopoietic tissue axl message is detected in normal human bone marrow but not significantly in normal blood leukocytes. Cell separation experiments showed that axl is expressed in hematopoietic CD34+ progenitor and marrow stromal cells, at low levels in peripheral monocytes, but not in lymphocytes or granulocytes. Consistent with the normal pattern of axl expression, axl RNA was found predominantly in diseases of the myeloid lineage: 39 of 66 (59%) patients with myeloproliferative disorders (acute myeloid leukemia, chronic myeloid leukemia (CML) in chronic phase, CML in myeloid blast crisis, and myelodysplasia) showed significant axl transcription, as compared with 1 of 45 (2%) lymphoid leukemias (chronic lymphocytic leukemia, acute lymphocytic leukemia, and CML in lymphoid blast crisis). Treatment of K562 cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), administration of interferon alpha (IFN alpha) to normal monocytes, and treatment of U937 cells with TPA and IFN tau significantly induced axl expression, supporting a role for this kinase in the intracellular signaling of myeloid cells through a variety of biochemical pathways. These results suggest that the axl kinase may be operative in normal and malignant myeloid biology.
Base Sequence↗
Herpesvirus type 6 in patients undergoing bone marrow transplantation: serologic features and detection by polymerase chain reaction.
To evaluate the potential role of human herpesvirus type 6 (HHV-6) infection in patients after bone marrow transplantation (BMT) we sequentially analyzed buffy coat leukocytes, oral lavage fluid, and urine from 57 patients for the presence of HHV-6 DNA by polymerase chain reaction (PCR) before and after 60 BMTs. Twenty-four patients undergoing autologous BMT and 36 with allogeneic BMT were studied. Thirty-six patients (60%) were PCR positive in one or more tests. The majority of PCR-positive patients had positive results only sporadically, in 1 (n = 23) or 2 weeks (n = 5). Six patients were positive in 3 to 5 weeks. In 2 patients, we found a high frequency of positive tests, in 7 of 7 and 10 of 10 weeks analyzed. Twenty-four patients (40%) remained PCR negative throughout the post-BMT period. There was a significant correlation between the results of HHV-6 PCR and the occurrence of acute graft-versus-host disease (aGVHD). In grade II-IV, 6 of 8 (75%) patients had 2 or more positive PCR tests, compared with 5 of 25 (20%) patients without or with grade I aGVHD (P = .01). There was no difference in the outcome of PCR tests with respect to the type of BMT or pre-BMT HHV-6 enzyme-linked immunosorbent assay titers. Restriction enzyme analysis of PCR amplificates from 18 patients showed HHV-6 variant B in 16 (88.9%) and variant A in 2 cases (11.1%). We conclude that HHV-6 DNA can be detected in 60% of the patients after BMT. HHV-6 DNA can be detected more frequently in patients with moderate and severe aGVHD than in patients without aGVHD or with mild aGVHD.
Adolescent↗
Detection of allelic loss within the beta 1-interferon gene in childhood acute lymphoblastic leukemia using differential PCR.
Deletion of the short arm of chromosome 9p involving the beta 1-interferon (IFN) gene has been implicated in the process of malignant transformation in lymphomas and acute lymphoblastic leukemias. Since cytogenetic analysis is frequently unsuccessful in clinical samples, we used a recently described differential PCR technique to detect losses within the beta 1-IFN gene in 86 acute leukemias. Using differential PCR, no beta 1-IFN deletion was detected in 44 acute myeloid leukemia (AML) and eight control samples. However, five of 42 acute lymphoblastic leukemia (ALL) probes (12%) exhibited loss of the beta 1-IFN gene (three common ALL, two T-ALL). Cytogenetic analysis was performed independently in three of these five cases and revealed abnormalities of chromosome 9p in two samples. Two of five T-ALL cases exhibited a loss within the beta 1-IFN gene, compared with 3/29 c-ALLs, suggesting a predominance of IFN gene loss in T-ALLs. These data indicate that PCR can be used for rapid detection of gene dosage phenomena in clinical leukemia samples.
Acute Disease↗
Overexpression of the Raf-1 proto-oncogene in human myeloid leukemia.
The Raf-1 protein, a cytoplasmic serine/threonine kinase, plays an important role in signal transduction pathways. In order to examine the role of Raf-1 in human myeloid leukemia, we determined raf-1 mRNA expression by Northern blot analysis in blast cell samples from 27 acute myeloid leukemia (AML) cases and peripheral blood mononuclear cells from six healthy donors. A normal raf-1 transcript size was detected in all cases investigated. However, overexpression of raf-1 mRNA was found in 2 of 27 AML cases, both of which were erythroleukemias (AML, FAB M6).
Acute Disease↗
A potential role for human herpesvirus type 6 in nervous system disease.
Human herpesvirus type 6 (HHV-6) is a new representative of the herpesvirus family which was associated with a spectrum of diseases, including myalgic encephalitis, meningitis and the chronic fatigue syndrome. We set out to study the potential role of HHV-6 in multiple sclerosis (MS) (n = 21), facial palsy (FP) (n = 19) and Guillain-Barré-syndrome (GBS) (n = 7). Results were compared with a control group (CG) (n = 16). We analyzed paired samples of serum and cerebrospinal fluid (CSF) with the polymerase chain reaction (PCR) for the presence of HHV-6 DNA. The studies were complemented by ELISA determination of serum antibodies against HHV-6. In the MS group we detected HHV-6 DNA in the CSF from three of 21 (14.3%) patients but not in the corresponding serum samples. In FP, GBS and controls CSF and serum PCRs were negative in all cases. HHV-6 serum antibody titers were significantly higher in MS compared with FP, GBS and controls. These findings suggest that HHV-6 may play a role in MS.
Adolescent↗
Molecular characterization of illegitimate TCR delta gene rearrangements in acute myeloid leukaemia.
Recently, we and others have shown the occurrence of TCR delta gene rearrangements in acute myeloid leukaemia (AML). In this study we describe the molecular characteristics of these rearrangements by the polymerase chain reaction (PCR) and the direct sequencing of PCR products. 11 rearrangements were characterized in blast cell samples from six patients. We found a heterogenous pattern of TCR delta gene rearrangements with involvement of V delta 1-5 regions. These findings differ from observations in T-ALL and B-cell precursor ALL, where predominantly usage of V delta 1 and V delta 2 regions has been described. Furthermore, extensive diversity of junctional sites was observed, including addition of up to 37 N nucleotides, nucleotide deletions at junction sites of V delta and J delta segments and usage of up to three D delta segments. The D delta 3 fragment was the most frequently used diversity element and was found in 10 rearrangements. Nine of the 11 rearrangements were non-functional, either incomplete or out of the reading frame. Therefore a functional TCR delta cannot be expressed in these myeloid blast cells.
Adult↗
Detection of herpesvirus type 6 by polymerase chain reaction in blood donors: random tests and prospective longitudinal studies.
In order to evaluate the prevalence of HHV-6 in blood donors, we examined 112 persons by polymerase chain reaction (PCR) and ELISA. HHV-6 antibodies could be detected in 107/111 (96.4%) of the donors. The median ELISA antibody level was 0.451 (range 0.056-0.914). 14 individuals (12.5%) were PCR positive in either oral lavage fluid, urine or buffy coat. Six persons (5.4%) were PCR positive in buffy coat samples. The prospective longitudinal analysis of 11 donors for periods between 7 and 13 weeks revealed that 4/6 persons who were initially PCR negative had positive tests in 9/63 weeks studied. Two persons were consistently PCR positive over the whole observation period of 12 and 13 weeks. HHV-6 variants could be determined in 14 persons as variant A in nine and variant B in five cases. These observations emphasize the high prevalence of HHV-6 and suggest that some blood donors carry detectable concentrations of the virus and therefore may be a source for transmission of HHV-6. The finding of positive PCR in antibody negative individuals suggests that antibody determination may not be sufficient to identify potentially infectious persons.
Antibodies, Viral↗
Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens.
DNA fragments from eight different reading frames of human cytomegalovirus (HCMV) were generated by PCR and subsequently cloned and expressed in Escherichia coli in fusion with glutathione S-transferase. The recombinant viral antigens were evaluated in immunoblot analyses. The most reactive antigens were purified and further evaluated in ELISAs. For this, sera from healthy blood donors and immunocompetent individuals with acute HCMV infection, and follow-up sera from transplant recipients with acute primary HCMV infection were used. The results of our experiments indicate that only three particular recombinant polypeptides from two viral proteins are necessary for serodiagnosis. While a fragment covering amino acids (aa) 495 to 691 of pp150 (150/1) was the most suitable antigen for the identification of infected individuals in general, immunoglobulin M antibodies against the C-terminal parts of pp150 (aa 862 to 1048; 150/7) and p52 (aa 297 to 433; 52/3) proved to be excellent serological markers to monitor acute HCMV infection. The selected recombinant antigens enable the improvement of serodiagnosis of HCMV-related diseases, especially during the early stages of infection.
Antibodies, Viral↗