Diagnosis and monitoring of acute cytomegalovirus infection in peripheral blood of transplant recipients by nested reverse transcriptase polymerase chain reaction (RT-PCR).

The aim of this novel diagnostic approach is to monitor cytomegalovirus (CMV) infection in immunocompromised transplant recipients using early, sensitive, and specific predictors before and during antiviral therapy. The peripheral blood cells of 20 patients after transplantation (9 liver, 7 kidney, 4 simultaneous kidney-pancreas) were studied for an early diagnosis of acute infection. The mRNA and DNA of human CMV immediate-early antigen (IEA) were detected by nested-polymerase chain reaction (PCR) assay. Results of nested PCR were compared with the immunological detection of antigen pp65 and serological diagnosis of CMV infection. All data were correlated with clinical symptoms like leukopenia, thrombopenia, pneumonia, and allograft-rejection reaction. Of 20 transplant recipients, 12 were infected by CMV, and 9 suffered from a CMV-related disease. CMV mRNA were detected simultaneously with antigen pp65 and CMV DNA in all patients with symptomatic infection. Additionally, CMV mRNA was found over a longer period after ganciclovir treatment of infected recipients. Nested reverse transcriptase (RT)-PCR for CMV-IEA mRNA allows a sensitive and specific diagnosis of an acute CMV infection. CMV mRNA was found to be a good marker of acute viremia and could be a useful tool for CMV monitoring over the whole period of disease management, even during antiviral therapy.

Modulation of multidrug resistance in human leukemia cells with mdr1-targeted antisense oligonucleotides using variable treatment schedules.

OBJECTIVE: The purpose of the current study was to characterize the effect of chimeric AS-ODNs encapsulated with cationic lipids on MDR in human leukemia cells and to determine if this modification of the ODN alone or in combination with the cationic lipid might offer advantages over classical ODN treatment with free unmodulated or phosphorothiolated AS-ODNs. Furthermore, we extended the antisense method to the use of AS-ODNs in the parental drug-sensitive leukemia cells which express mdr1-mRNA at a relative low level and lack P170 expression to evaluate the effectiveness of prophylactic AS-ODN treatment. METHODS: The effect of a 4-day AS-ODN treatment in drug-resistant human leukemia cells which exhibit the classic MDR phenotype at a moderate level was examined. Twenty-four hours after the last ODN administration the cells were analyzed for mdr1-mRNA (quantitative RT-PCR) and P170 expression (FCM), for R123 accumulation/efflux capacity (FCM) and for sensitivity to vincristine (MTT). In the parental drug-sensitive CCRF-CEM cells the mdr1-mRNA expression was assessed 24, 48 and 72 h after AS-ODN treatment administered as free phosphorothioate or conjugated with DMRIE-C. RESULTS: Cationic lipids produced a clear increase in cellular ODN uptake but also caused an increase in variability of uptake rates (30% vs. 10% variability after free phosphorothioates). Both AS-ODNs inhibit P170 expression whereby the antisense effect of the chimeric ODN seems to be stronger compared to the phosphorothioate (30% vs. 22% MRK16 staining). Consistent with the inhibition of P170 expression, an increased sensitivity to vincristine was observed. In parental drug-sensitive cells, AS-ODN treatment caused nearly complete inhibition of mdr1-mRNA expression (5% of control). CONCLUSION: The data demonstrate that it is nearly impossible to achieve a complete reversal of the MDR phenotype in drug-resistant cells using AS-ODNs. A more promising approach seems to be the prophylactic treatment with AS-ODNs.

Absolute levels of MDR-1, MRP, and BCL-2 MRNA and tumor remission in acute leukemia.

Mononuclear cells prepared from peripheral blood or bone marrow of 119 AML and 28 ALL patients prior and following therapy were analyzed for absolute transcript levels of the chemoresistance genes mdr-1 and MRP, and the proto-oncogene bcl-2, by validated contamination-protected quantitative RT-PCR. In newly diagnosed AML mainly tumors of the granulocytic lineage (FAB M1-M2) expressed increased mdr-1 mRNA amounts. The MRP gene was expressed in all investigated samples without relation to a particular FAB class. High initial expression of both genes did not confer a poor prognosis even at high number of CD34+ cells. Data compared prior to and after therapy start (paired samples) revealed that AML patients who did not respond to therapy (NR) expressed increased levels of mdr-1 mRNA, as well as MRP and bcl-2 cDNA normalized to GAPDH reference transcripts, when compared to patients achieving complete remission (CR; p = 0.003, 0.008 and 0.0005, respectively). In ALL-NR the mdr-1 and bcl-2 genes were entirely more active after induction chemotherapy. Arbitrary cut-off values were established in order to delimit pathological from non-pathological gene expression. 59% of studied AML and 33% of ALL-NR exceeded the arbitrary values (mdr-1: > 2 amol/microgram RNA, MRP: > 10 zmol/amol GAPDH, bcl-2: > 5 zmol/amol GAPDH) for one and 11% of AML-NR for two parameters. Only 17% of the AML-CR and none of the ALL-CR group were above these limits. The results indicate that high individual activity of usually one, rarely two of the investigated genes might be associated with poor clinical outcome in treated acute leukemia.

Genotyping of human apolipoprotein E alleles by the new qualitative, microplate-based CASSI-detection assay.

A new qualitative PCR product detection assay called competitive amplified single mutation detection by selective probe hybridization immunoassay (CASSI) was developed for genotyping the most common apolipoprotein E (apoE) polymorphisms. Single target DNA strands immobilized using biotin on streptavidin-coated microplates were hybridized in separate wells with two distinct, 5'-fluorescein isothiocyanate (FITC)-labeled oligonucleotides, complementary to either the 112Arg or 158Arg encoding site. With this assay, only correctly matched hybrids that form between probe and target DNA can be cleaved with the HhaI restriction endonuclease, leading to loss of probe label in corresponding wells. However, allele-specific, probe-target mismatches due to G-->T exchanges in the HhaI recognition sequences are not cleaved. After digestion, the remaining microplate-adsorbed signal is measured colorimetrically by using anti-FITC, Fab-horseradish peroxidase conjugates. Our results show maximum intensity was detected when the respective probe hybridized incompletely to the target (i.e., no cleavage), and minimum signal was obtained when the probe matched the target completely (complete cleavage); whereas, an intermediate signal was recorded at 50% complementarity (i.e., heterozygote alleles). With this assay, we could demonstrate a high prevalence of the apoE2 allele in patients suffering from coronary artery disease even though they displayed normal triglyceride and cholesterol levels. Corresponding results were obtained by CASSI compared with conventional restriction fragment-length polymorphism analysis.

Calibration and storage of DNA competitors used for contamination-protected competitive PCR.

DNA fragments used as standards in competitive PCR were precisely calibrated using HPLC and commercially available DNA molecular mass markers. The accuracy of calibration was reflected by data that differed by only 2% from the mean when two independently purified and calibrated competitor preparations were compared. Highly dilute competitor solutions were stable at -20 degrees C for up to 1 year in the presence of carrier HindIII-digested lambda DNA, but progressive loss of competitor DNA with increasing storage time was observed when carrier DNA was omitted from the solution. Applying 0.2 U uracil-DNA glycosylase (UDG) per assay of remaining temperature-stable activity did not effect the ratios of synthesized products. This study describes quality management in PCR quantitation that is useful for the measurement of multidrug resistance-associated protein (MRP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts.

[Obesity, malic enzyme and aging--an animal experiment study]. / Adipositas, Malatenzym und Altern--eine tierexperimentelle Studie.

In male Wistar rats the influences of age and experimental obesity on the activity of malic enzyme (EC 1.1.1.40) in different organs were studied. Obesity was induced in newborn rats by injection of Na(+)-L-glutamate (2 mg/g b.w. daily) subcutaneously in the first 5 days. The enzyme activity was measured at the ages of 2, 6 and 18 months. In control animals the highest enzyme activities were found in the heart muscle, liver, epididymal fat pad and skeletal muscle after 6 months. After 18 months the activities in these organs are considerably reduced. In the kidneys the activity between the 2nd and the 18th months tends to decrease continuously and only the brain shows an opposite trend. In comparison with the control animals, in glutamate treated rats the enzyme activity doubles nearly in the lipogenic organs liver and fat tissue in all age groups. In liver and fat tissue of 6-month-old rats, previously treated with clonidine to stimulate growth hormone secretion, the activities are lower than in glutamate obese rats without clonidine, but still higher than in normal control animals. The qualification of glutamate obese rats as a model for the study of age-associated diseases like obesity or diabetes mellitus type II needs further investigation.

Reduced, positive nitrogen balance and elevated plasma free fatty acid concentration in growing, glutamate-induced obese rats.

Glutamate-induced obesity of Wistar-rats is known to develop under normophagic and normoinsulinemic conditions, although hyperphagia and hyperinsulinemia are common to obese individuals. Rats of this obesity model show retarded growth, reduced mass of some organs, carcass and whole body as well as an extraordinary high fat content, whereas protein content is reduced. In this study, nitrogen (N) balance, urinary excretion of urea-N, ammonia-N, creatine-N and alpha-amino acid-N and plasma free fatty acid concentration of growing, glutamate-induced obese rats were determined. The main results were independent of frame of reference (mmol N/kg body mass; mmol N/kg0.75 metabolic body mass; N in % of nitrogen intake): Nitrogen intake, urinary excretion of alpha-amino acids and nitrogen excretion in faeces were equal between lean and obese rats. Nitrogen excretion in urine was elevated in obese rats, mainly resulting from increased amounts of urea and ammonia. Nitrogen balance was positive in both groups, but reduced in obese rats. These data point to normal digestion of food proteins, but an unusual high oxidative desamination rate of the absorbed amino acids in obese rats. Taking into account the various hormonal and nerval alterations in glutamate-induced obese rats, resulting e.g. in increased hepatic insulin concentration, the retained amino acid carbon should be channelled into hepatic fatty acid synthesis. Really, unfasted and overnight fasted obese rats showed elevated plasma free fatty acid concentrations. Channeling of amino acids into lipogenesis may explain the low muscle mass and striking fat accumulation--despite normophagia and peripheral normoinsulinemia--of growing, glutamate-induced obese Wistar-rats.

In vivo assessment of insulin binding in different organs of growing and adult glutamate-induced obese rats.

Injection of Na-L-glutamate into neonate Wistar-rats (2 mg/g body mass s.c.; day 1-5 of life) induces hypothalamic lesions, which are followed by hypoplastic-hypertrophic obesity despite normophagia. In contrast to other animal models of obesity, these rats develop obesity under peripheral normoinsulinemic conditions. However, beginning at an age of 2 months (growing rats), peripheral insulin concentration rises gradually and at an age of 6 months (adults rats) hyperinsulinemia becomes manifest. Surprisingly, adult rats show normoglycemia, pointing to alterations in insulin sensitivity. In continuation to previous work, insulin binding of different organs of growing and adult rats was investigated using the in vivo radioreceptor assay described by Whitcomb et al. in 1985. In contrast to in vitro methods, this assay works under real metabolic and hormonal conditions in plasma of lean and obese rats. Insulin binding of liver, pancreas, adrenals, stomach, duodenum, spleen, and heart muscles was found to be not statistically different between lean and obese rats of both age groups. Thus, liver insulin binding was 6323 +/- 458 pg/g wet organ in growing, and 7586 +/- 959 pg/g in adult lean rats. Corresponding values for obese rats were 5755 +/- 445 pg/g and 7830 +/- 526 pg/g, respectively. Organ specific down regulation of insulin binding in obese rats was not detected, suggesting unalterated insulin sensitivity. It is concluded that hyperinsulinemia of adult glutamate-induced obese rats cannot be explained by diminished insulin binding and reduced organ specific insulin clearance.

Changes of ATPase activities in erythrocytes of rats with hypothalamic obesity.

Na,K- and Ca,Mg-ATPase activities in the membrane of red blood cells (RBC) were determined in glutamate treated obese rats (GOR). Both activities are related oppositely. In the obese rats the Na,K-ATPase is higher but the efficiency in maintenance of Na/K-ion concentration gradients is diminished. Ca,Mg-ATPase is decreased in GOR. Under the hypermetabolic condition of cold adaption the Na,K-ATPase activity decreases and the Ca,Mg-ATPase activity rises in both animal groups. The Na,K-ATPase activity in RBC-membranes is positively related to fat accumulation and age.

[Hyperinsulinemia and B-cell proliferation in rats treated postnatally with L-glutamate]. / Hyperinsulinämie und B-Zellproliferation in postnatal mit L-Glutamat behandelten Ratten.

Development of hyperinsulinemia was investigated which appeared late in the obese state in rats postnatally treated with L-glutamate. Insulin concentrations were estimated in the blood plasma of the caval and portal vein, and morphometric and immunohistochemical measurements of cells in the islets of Langerhans were performed, and also glucose tolerance tests. Not earlier than at 3 months hyperinsulinemia is shown in glutamate obese rats (GOR) in the peripheral blood plasma. Also in the portal blood plasma the insulin concentration is higher (167%) in GOR relative to controls. The insulin concentrations in the portal vein rise further in both animal groups whereas insulin concentration in the peripheral blood remains at the different levels in both animal groups. Impaired glucose tolerance was observed for GOR only. Islets of Langerhans in the Pancreas show enlargement and increased proliferation of B-cells in GOR. In contrast the number of D-cells is diminished. The hyperplasia of islets differs remarkably to hypoplasia of other organs in GOR. We conclude that the peripheral hyperinsulinemia is caused by a permanent hypersecretion of insulin.

[Glucose utilization in adipose tissue of rats in chronic somatotropin deficiency]. / Glukoseverwertung im Fettgewebe von Ratten bei chronischem Somatotropinmangel.

Injections of monosodium glutamate to neonate rats induce chronic growth hormone deficiency by hypothalamic lesions in the regions of the nucleus arcuatus and the eminentia mediana. The glutamate treated rats develop massive obesity. By this type of obesity hyperinsulinemia in the dynamic phase is not evident. The adipose tissue of the GOR (glutamate obese rat) is characterized by hypertrophic adipocytes and diminished number of adipocytes. In the GOR glucose oxidation and glucose lipid conversion are increased, but insulin sensitivity of glucose metabolism is diminished. The enhanced glucose utilization in adipose tissue of the GOR is discussed as being the consequence of the chronic growth hormone deficiency.

Development of hypothalamic obesity in growing rats.

Administration of monosodium glutamate to neonate rats causes hypothalamic lesions in the region of the nucleus arcuatus and the eminentia mediana, followed by massive accumulation of triglycerides, diminished secretion of growth hormone, reduced body length and organ weights and diminished number of adipocytes (hypoplastic-hypertrophic obesity). Locomotor activity of obese animals is reduced by about 50%. Food intake is increased by about 10% during growth and development of obesity but decreased beneath the level of that in control animals in the stationary phase of obesity. Hyperinsulinemia coupled with insulin resistance develops in the stationary phase of obesity, i.e. when adipocyte diameter has reached approximately 100 microns. The effects of reduced secretion of growth hormone are considered to be a main factor of fat accumulation in this type of obesity.

Na dependence of monosaccharide absorption in isolated rabbit small intestine, perfused through lumen and vascular bed.

Na dependence of D-glucose and 3-O-methyl-D-glucose absorption was investigated using the isolated rabbit small intestine perfused through the lumen and the vascular bed, thus imitating in vivo conditions. No dependence of monosaccharide transport of luminal Na concentration was demonstrable if the lumen was perfused at low flow rate. Due to Na secretion, however, Na concentration in the lumen bulk phase, initially being zero, was raised to more than 20 mmol/l during the course of the experiments. Na dependence of sugar transport could be shown, however, if (1) Na secretion was decreased (by use of a vascular medium with low Na concentration) or if (2) unstirred layer thickness was reduced (by enhancement of luminal flow rate). Both conditions allowed the Na concentration near the brush border membrane to be controlled. The results provide an experimental explanation for the apparently low degree of Na dependence of monosaccharide absorption under in vivo conditions.

[Comparative studies of the preparation of alpha-gliadin]. / Vergleichende Untersuchungen zur präparativen Darstellung von alpha-Gliadin.

alpha-gliadin was prepared from wheat flour by two different methods. The products were compared electrophoretically and by double radial immuno-diffusion. The alpha-gliadin fraction proved to be identical in the immunological test. Only the alpha-gliadin preparation received by ion exchange chromatography is suitable for further purification by multiple gel filtrations.