[Prophylaxis of infection in neutropenic patients. Guidelines of the Working Party on Infections in Hematology and Oncology].
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Antibiotic Prophylaxis↗
Biomedical subjects
C A Schmidt
Publications and source records attributed to C A Schmidt.
Enhanced p73 expression during differentiation and complex p73 isoforms in myeloid leukemia.
The p53 homologue p73 is expressed in at least six different isoforms (alpha, beta, gamma, delta, epsilon, and zeta), but unlike p53 it has rarely been found mutated in human cancers. However, altered expression of this gene has been reported in cancer cells. In order to understand if p73 is involved in normal and malignant development of myeloid cells, we investigated the expression pattern of the different p73 isoforms in progenitor and mature normal myeloid cells as well as in cells derived from acute and chronic myeloid leukemias. The results show that expression of p73 is markedly enhanced during differentiation of myeloid leukemic cells and that leukemic blasts from patients show an increased expression of the shorter p73 isoforms (gamma, delta, epsilon, zeta). In particular the epsilon isoform is only expressed in leukemic cells and completely absent in mature myeloid cells. Altogether our data suggest that p73 is involved in myeloid differentiation and its altered expression is involved in leukemic degeneration.
Acute Disease↗
Applicability of an absolute quantitative procedure to monitor intra-individual bcr/abl transcript kinetics in clinical samples from chronic myelogenous leukemia patients.
Chimeric bcr/abl fusion proteins are thought to be the molecular 'pathogen' of chronic myelogenous leukaemia (CML). Expression levels of the respective fusion RNAs reflect disease progression as well as remission upon therapeutic intervention in CML patients. However, there is no quick and reliable method that would allow the quantitative routine monitoring of bcr/abl hybrid transcripts. A fluorescent probe-based PCR assay (TaqMan) has been described to quantitfy the exact amount of target sequences. We have established TaqMan real-time RT-PCRs for M-bcr/abl (b2a2, b2a3, b3a2, b3a3) and m-bcr/abl (e1a2) fusion transcripts as well as for beta-actin. All PCRs quantified as little as 10 copies/100 ng total cDNA. In order to investigate whether this procedure is appropriate for routine diagnostic monitoring, we performed retrospective measurements on 9 documented CML disease courses. Our data show that ongoing or relapsing CML is paralleled by increasing peripheral levels of bcr/abl fusion RNAs. Furthermore, sucessful anti-leukemic treatment is reflected by decreasing absolute bcr/abl transcript numbers. In contrast with conventional bcr/abl PCR techniques we could distinguish single positive values and gradually increasing copy numbers.
Disease Progression↗
Influence of DNA polymerases on quantitative PCR results using TaqMan probe format in the LightCycler instrument.
Real-time fluorescence polymerase chain reaction (PCR) techniques are increasingly used to quantitate target sequences for diagnostic and research purposes. Currently, the so called TaqMan probe chemistry is mostly used as fluorogenic system. This probe format is strictly dependent on the 5'-exonuclease activity of DNA polymerase as fragmentation of the probe during the reaction is essential for this assay. Based on our experience that dramatic differences in quantitative PCR results may be due to different DNA polymerases we performed a detailed comparison of 15 enzymes. We found that clear differences exist between polymerases of different manufacturers. Thus, three out of seven polymerases which were declared to possess 5'-exonuclease activity appeared to be completely unsuitable for this method while the remaining had significantly different reaction efficiencies. We conclude that different DNA polymerases may determine the entire analytical performance of TaqMan assays suggesting that DNA polymerase testing is of special importance when this probe format is used.
Calibration↗
Elevated p21 mRNA level in skeletal muscle of DMD patients and mdx mice indicates either an exhausted satellite cell pool or a higher p21 expression in dystrophin-deficient cells per se.
Abnormalities in proliferation and differentiation of the dystrophin-deficient muscle are a controversial aspect of the pathogenesis of Duchenne muscular dystrophy (DMD). Analyses of molecules involved in cell cycle modulation do not exist in this context. Cells withdrawn from the cell cycle permanently express p21. The fact that p2 1, in contrast to other cell cycle proteins, is not diminished when myotubes are reexposed to growth media, allocates this cyclin-dependent kinase inhibitor a special function. Here we report for the first time statistically increased p21 mRNA levels in dystrophin-deficient muscle tissue. Only 42% of conventional RT-PCRs from six muscle samples of human controls yielded positive results but almost all skeletal muscle biopsy samples (87%) from DMD patients (n=5). For p21 mRNA quantification in murine muscle samples we were able to use the exact real-time TaqMan PCR method due to generally higher p21 mRNA levels than in human muscles. In addition, contamination with fibroblasts can be excluded for the murine samples because they do not demonstrate fibrosis at the age of 350 days but start to lose their regenerative capacity. In accord with the results in humans, we observed p21 mRNA levels in mdx mice that were approx. four times as high as those in control mice. Elevated p21 mRNA level may indicate a shift in cell composition towards differentiated p21 expressing cells as a result of an exhausted pool of undifferentiated, non-p21-expressing satellite cells due to previous cycles of de- and regeneration. Alternatively, dystrophin-deficient cells per se may express higher p21 levels for unknown reasons. Although we cannot distinguish between these possibilities, the eventual transfec tion of a patient's own satellite cells with p21 antisense oligonucleotides may enable the dystrophic process to be influenced.
Actins↗
Cardiopulmonary bypass in patients with heparin-induced thrombocytopenia and thrombosis.
Heparin-induced thrombocytopenia and thrombosis (HITT) is an immunomediated disorder induced by the administration of heparin for therapeutic purposes. The presence of this condition in patients requiring full heparinization for cardiopulmonary bypass constitutes a formidable challenge for the cardiac surgeon. In this review, the clinical and experimental experience described in the literature are discussed in the perspective of the normal coagulation and the pathophysiology of HITT and in the light of a variety of old and new alternative anticoagulants.
Anticoagulants↗
Presence of human beta- and gamma-herpes virus DNA in Hodgkin's disease.
Herpes viruses have been implicated in the etiology of Hodgkin's disease (HD). We studied the prevalence of human cytomegalovirus (CMV), human herpes viruses type-6 (HHV-6), type-7 (HHV-7) and type 8 (HHV-8) DNA in up to 88 Hodgkin's disease biopsies in comparison to Epstein-Barr virus (EBV) DNA by polymerase chain reaction (PCR). Non-Hodgkin lymphomas (NHL) and reactive lesions served as controls. CMV and HHV-6 were found in 8/86 (9%) and 11/88 (13%) HD cases, respectively, by nested primer PCR. Except for three cases harbouring HHV-6 type-B, only HHV-6 type-A was detected in HD. HHV-7 was observed by nested PCR in 33/88 (38%) HD cases and was already detectable in 15/88 (17%) HD cases by a single-round PCR indicating elevated virus copy numbers. Seven of these cases showed co-infection with HHV-6, and 11 cases were found to contain EBV DNA. 7/8 CMV-positive HD cases also harboured EBV DNA. HHV-8 DNA was not detected by single round or nested PCR in any HD case investigated. Thus, CMV, HHV-6, and HHV-7 were present in small proportions of HD cases, with frequent co-infection of HHV-6 and HHV-7, and frequent association with EBV. In contrast to EBV, beta-herpes viruses are therefore unlikely to have a role in the aetiology of HD. Rather, the presence of these viruses seems to reflect impaired immunological surveillance.
Betaherpesvirinae↗
Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.
Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.
Base Sequence↗
Detection of human cytomegalovirus DNA by real-time quantitative PCR.
A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.
Blood Donors↗
Distinct expression patterns of the p53-homologue p73 in malignant and normal hematopoiesis assessed by a novel real-time reverse transcription-polymerase chain reaction assay and protein analysis.
The role of the recently identified first p53-homologue, p73, in neoplastic transformation is unknown. To elucidate p73 gene expression in hematopoiesis, we investigated samples from chronic myeloid leukemia (CML) and acute myeloid leukemia patients, leukemia cell lines, as well as mature and immature normal hematopoietic cells by real-time quantitative RT-PCR and Western blot analysis. We found a distinct p73 expression profile with highest p73 mRNA transcript levels in hematopoietic malignancies such as CML blast crisis and acute myelogenous leukemia versus CML chronic phase and normal controls. Mono- and biallelic p73 expression was found in both normal and malignant hematopoiesis. p73 protein was expressed at various levels in leukemia samples and cell lines but could not be detected in any normal controls tested. Our results point to a distinct yet undefined role of p73 in the pathogenesis of myeloid neoplasms.
Alleles↗
LightCycler technology for the quantitation of bcr/abl fusion transcripts.
Quantifying bcr/abl fusion transcripts in chronic myelogenous leukemia is thought to serve as a powerful parameter for monitoring the kinetic nature of this clonal disease in vivo and in vitro. Recently, we demonstrated the technical advantages as well as the clinical relevance of quantitating bcr/abl fusion mRNA using the 5-nuclease assay and a real-time fluorescence reverse transcriptase-PCR (RT-PCR) detection system (ABI PRISM 7700 SDS). Meanwhile, another technique was introduced (LightCycler technology) that may be used for the same purpose. To investigate whether this method may be an appropriate alternative to the described procedure, we have established bcr/abl LightCycler RT-PCR for major and minor bcr/abl fusion transcripts. We found that, with only minor modifications, TaqMan RT-PCR and fluorescent probe design can be used to obtain comparable results in the LightCycler system. The developed method could quantitate as little as 10 bcr/abl copies per 100 ng cDNA and was as safe and reproducible as the previously described technique. Because reaction efficiency was identical within different bcr/abl major fusions, one single RT-PCR could be established that simultaneously detects b2a3, b2a2, b3a2, and b3a3 fusion RNA with equal specificity and sensitivity. Compared to results generated by the ABI PRISM 7700 SDS, absolute amounts of bcr/abl did not differ significantly, and there was a linear correlation between the respective values. We conclude that TaqMan chemistry can be used in the LightCycler and that both real-time fluorescence PCR detection systems equally fulfill the criteria for the safe and reliable quantitation of bcr/abl fusion RNA in clinical samples. This may be of help for further standardization of quantitative bcr/abl RT-PCR, which, again, is necessary for the comparison of results generated by different investigators.
Calibration↗
[Recommendations for the diagnosis and therapy of cytomegalovirus infection after stem-cell transplantation. The Research Alliance on the Complications of Organ Transplantation due to Herpes Viruses].
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Antiviral Agents↗
Identification of human cytomegalovirus variants by analysis of single strand conformation polymorphism and DNA sequencing of the envelope glycoprotein B gene region-distribution frequency in liver transplant recipients.
Single strand conformation polymorphism analysis (SSCP) of PCR-amplified DNA and subsequent DNA sequencing of human cytomegalovirus (HCMV) glycoprotein B (gB) gene were applied to identify known HCMV strains and to detect new virus variants. 61 HCMV PCR positive patients were studied out of a cohort of 410 patients after liver transplantation (LTX). SSCP was able to distinguish between strains Davis, AD169, and Towne, and in addition could identify five new virus variants (Berlin B, C, E, F, and H). Their frequency, gB and gH types were determined. Simultaneous infections with two or three strains or variants, as well as a switch from one virus to another virus were observed during long-term follow-up. No correlation between the occurrence of certain virus strains or gB types and defined clinical manifestations of HCMV infection after LTX was drawn.
Base Sequence↗
Hepatocytes are permissive for human cytomegalovirus infection in human liver cell culture and In vivo.
The cytopathic potential of human cytomegalovirus (HCMV) in human liver cells was analyzed in cell culture and in tissue sections from patients with HCMV hepatitis. Liver cell cultures, consisting of hepatocytes, bile duct epithelial cells, and stromal cells were infected by various HCMV strains. Cytopathic effects, viral gene expression, and virus production were detected. Infected cell types were identified by immunocytochemical double labeling. Hepatocytes were the predominant target cells of HCMV infection in liver tissues and in cell culture. Late-stage infected cultured hepatocytes produced infectious progeny virus, and infectious virus was propagated from liver tissue specimens. HCMV infection in cultured liver cells closely resembled in vivo infection of the liver with regard to the target cell spectrum and the permissive course of infection. It is concluded that HCMV can cause direct liver parenchyma damage by efficient cytolytic infection of hepatocytes.
Antigens, Viral↗
Phase I trial of gemcitabine (Gemzar), 24 h infusion 5-fluorouracil and folinic acid in patients with inoperable pancreatic cancer.
Gemcitabine (Gemzar) has a significant impact upon survival and quality of life for patients with pancreatic cancer, compared with 5-fluorouracil (5-FU). This phase I study was initiated to define the recommended dose of 5-FU delivered as a 24 h infusion in combination with gemcitabine (1000 mg/m2) and folinic acid (200 mg/m2) in patients with inoperable pancreatic cancer, treated on an outpatient basis. Drugs were administered weekly for 4 weeks out of 6 weeks. Sixteen chemonaive patients (median age 59 years, range 51-66) were enrolled, 15 had stage IV and one stage III disease. The median Karnofsky performance score (KPS) was 70 (range 60-80). Six patients received 5-FU 750 mg/m2, eight received 5-FU 1000 mg/m2 and two received 5-FU 1250 mg/m2. The maximum tolerated dose of 5-FU was 1000 mg/m2. Hepatotoxicity was dose limiting. One patient who received 5-FU 1250 mg/m2 died as a result of hepatorenal failure. There was one partial response, nine patients had stable disease for more than 3 months and 13 patients had improved KPS. The median time to progressive disease was 31 weeks (range 5-50 weeks). A phase 11 trial is underway to further assess the activity of this combination at the recommended dose of 750 mg/m2 5-FU.
Adult↗
Different real-time PCR formats compared for the quantitative detection of human cytomegalovirus DNA.
BACKGROUND: The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. METHODS: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. RESULTS: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 10(1) to 10(7) HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. CONCLUSIONS: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.
Cytomegalovirus↗
Preemptive therapy in CMV-antigen positive patients after liver transplantation--a prospective trial.
OBJECTIVE: Preemptive therapy with intravenous ganciclovir and CMV-hyperimmunoglobulin in asymptomatic CMV pp65-antigen positive patients was compared with treatment of only symptomatic CMV-disease after liver transplantation in an open prospective study. PATIENTS AND METHODS: 48 out of 200 liver transplant recipients became positive during six weeks follow-up after transplantation. 17 out of these 48 patients who were already symptomatic at the time of positive antigen testing were successfully treated with ganciclovir and CMV-hyperimmunoglobulin. 31 asymptomatic antigen-positive patients were randomised to receive preemptive therapy or to receive therapy only at onset of clinical symptoms. RESULTS: Only two out of 15 patients in this latter group without preemptive therapy developed CMV-syndrome and were successfully treated with intravenous ganciclovir. 13 patients did not experience any clinical symptoms or disease and were therefore spared unneccessary toxicity and costs. The overall incidence of CMV-infection and -disease in the whole study population of 200 liver transplant recipients was 25% and 10%. As expected, CMV-negative patients who received an organ from a seropositive donor were at a higher risk of CMV-infection and -disease, but did not show more severe infections clinically. Patients with IL-2 receptor antibody induction therapy seemed to have a higher risk for CMV-infection and -disease.
Adult↗
EMS on the home front. Acute interventions for chronic-care patients.
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Chronic Disease↗