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Biomedical subjects

A Gruber

Publications and source records attributed to A Gruber.

At least 163 records · Page 9Linked to original sources
PubMed1994

Neopterin--its clinical use in urinalysis.

Increased amounts of neopterin are released during cellular immune response. Neopterin concentrations can be monitored in serum and urine of patients since neopterin is removed from the circulation by renal excretion. In allograft recipients, rising neopterin concentrations indicate rejection episodes early. Neopterin concentrations correlate with the extent and activity of viral infections, malignancies, and autoimmune diseases. We investigated excretion kinetics of neopterin in a rhesus monkey which received a high dose of neopterin intravenously. A sharp increase of urinary neopterin concentrations was observed, and from the data the half-life of neopterin in the circulation was estimated to be 90 minutes. By comparing urine and serum neopterin concentrations in HIV seropositive and seronegative human individuals, a strong correlation and similar diagnostic sensitivity between urine and serum values was observed. Thus, neopterin concentrations in serum or urine seem of equal value for diagnostic application as long as renal function is normal.

Animals↗
PubMed1994

A non-P-glycoprotein-mediated mechanism of vincristine transport which is affected by resistance modifiers and present in chemosensitive cells.

The accumulation and cytotoxicity of vincristine (Vcr), etoposide (VP16), and daunorubicin (Dau) and effect of the resistance modifiers (RM) verapamil (Ver; 10 microM) and cyclosporin A (CyA; 3 microM) were studied in isolated rat cardiac myocytes, peripheral lymphocytes from seven patients with chronic lymphocytic leukemia (CLL), in the human leukemic cell line K562 and its two Vcr resistant mdr1 gene expressing sublines, K562/Vcr30, K562/Vcr150. Both RMs increased the accumulation and cytotoxic effect of Vcr and Dau in the resistant sublines. In K562 cells, lymphocytes from patients with CLL and rat cardiac myocytes, which all were mdr1 RNA negative RMs increased the cellular accumulation and potentiated the cytotoxic effect of Vcr but not that of Dau. K562/Vcr30 and K562/Vcr150 were cross resistant to Dau but not to VP16 and RMs had no effect on the cytotoxicity of VP16 in any of cell lines. The results indicate that chemosensitive cells also have a transport mechanism, not mediated by P-glycoprotein, which transports Vcr but not Dau and VP16. This suggests that addition of RMs to Vcr-containing chemotherapy may enhance the antineoplastic effect also by inhibition of non-P-glycoprotein mediated transport mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Generation of activated protein C during thrombolysis.

The observation that urokinase infusion increases circulating levels of the anticoagulant activated protein C (APC) in baboons implies that APC might be elevated during thrombolytic therapy. Patients undergoing coronary thrombolysis showed an 11-fold increase (means from 6 to 69 micrograms/L) in APC during infusion of streptokinase. Thrombolytic therapy thus generates at least two potent antithrombotic factors in the circulation--the fibrinolytic enzyme, plasmin, and the anticoagulant enzyme, APC. APC may help prevent reocclusion during or after thrombolysis.

Aged↗
PubMed1993

Normalization of markers of coagulation activation with a purified protein C concentrate in adults with homozygous protein C deficiency.

Homozygous or double heterozygous protein-C deficiency can present at birth with purpura fulminans or later in life with venous thrombosis. Two homozygous patients who had previously sustained thrombotic episodes were investigated at a time when they were asymptomatic and not receiving antithrombotic therapy. The plasma levels of protein-C antigen and activity in both individuals were approximately 20% of normal. We administered a highly purified plasma-derived protein C concentrate to these individuals and monitored levels of several markers of in vivo coagulation activation. Assays for protein-C activation (activated protein C and protein C activation peptide) showed a sustained increase from reduced baseline levels, whereas thrombin generation (as measured by prothrombin fragment F1 + 2) gradually decreased over about 24 hours into the normal range. These investigations provide direct evidence that protein C is converted to activated protein C in vivo, and that the protein-C anticoagulant pathway is a tonically active mechanism in the regulation of hemostatic system activation in humans.

Adult↗
PubMed1993

Trypanosoma cruzi: characterization of two recombinant antigens with potential application in the diagnosis of Chagas' disease.

A genomic library of Trypanosoma cruzi, constructed in the vector lambda gt11, was screened with a hyperimmune rabbit antiserum against tissue culture trypomastigotes. Two clones, B12 and B13, containing inserts of 350 and 600 bp, respectively, were isolated. Sequencing data indicated that both clones present a pattern of tandemly repeated nucleotide units of 60 bp for B12 and 36 bp for B13. Southern blot analysis suggests that both corresponding genes exist as a single copy. The inserts of both recombinants were subcloned in the vector pMSgt11, in phase with the lacZ gene. Recombinant proteins were affinity purified on pAPTG-agarose columns and employed to immunize rabbits, as well as to immunoselect human chagasic antibodies. By Western blot, antibodies to B12 reacted with bands of 230 kDa in trypomastigotes and 200 kDa in epimastigotes, while those to B13 identified bands of 140 and 116 kDa in trypomastigotes and epimastigotes. Immunoprecipitation of radioiodinated parasites revealed that the 140-kDa antigen recognized by antibodies to B13 is located on the membrane of trypomastigotes but not epimastigotes. The potential application of either recombinant antigen in the serological diagnosis of Chagas' disease was evaluated initially by RIA. It was observed that B13 presents a very good performance with sensitivity of 97%. For B12, the corresponding value was 82%. The reactivity to B13 was also evaluated by ELISA tests run in parallel with conventional serological reactions for Chagas' disease. Analysis of 209 serum samples indicates that B13 presents similar or even better performance in relation to the use of total epimastigote antigens, making it a promising candidate for the diagnosis of Chagas' disease.

Amino Acid Sequence↗
PubMed1993

Severe aplastic anemia associated with seronegative community-acquired hepatitis C virus infection.

Aplastic anemia (AA) is a rare complication of viral hepatitis affecting mainly children or young adults. Most reported cases have been associated with community-acquired non-A, non-B hepatitis, but hepatitis A and B have also been implicated in a few patients. We report on a 43-year-old woman with severe AA, in association with acute hepatitis C virus (HCV) infection, diagnosed by detection of HCV RNA by the polymerase chain reaction technique. Her AA was successfully treated with anti-thymocyte globulin and cyclosporin A. The hepatitis C progressed to chronic disease but, despite a follow-up time of 2 years, the patient still has no detectable anti-HCV antibodies, as evaluated with a second-generation anti-HCV assay.

Adult↗
PubMed1993

Intracellular concentrations of mitoxantrone in leukemic cells in vitro vs in vivo.

The aim of this study was to determine the intracellular pharmacokinetics of mitoxantrone in vivo and to use these results to establish how leukemic cells should be incubated to perform clinically relevant in vitro studies of this drug. Blood samples were obtained from 11 patients with acute nonlymphoblastic leukemia at certain intervals up to 20 h after the infusion of mitoxantrone 12 mg/m2. Plasma and leukemic cells were separated and the drug concentrations were determined with HPLC. Before treatment, leukemic cells from 12 patients were incubated with 0.02, 0.05, 0.1, 0.2 and 1.0 microM mitoxantrone for 1-4 h and thereafter cultured in suspension culture for 20 h; during this time cell samples were taken at certain intervals for drug determination. In cells incubated with 0.05 and 0.2 microM mitoxantrone the cytotoxic effect was measured with the DiSC assay after cultivation for 4-5 days. In vivo, the intracellular levels exceeded the plasma concentrations already at the end of infusion and after 2 h the intracellular concentrations were 200-300 times higher than in plasma. In vitro, the intracellular steady state level of mitoxantrone was reached after 1-2 h and there was a pronounced intracellular retention even after 20 h culture in drug-free medium. Incubation with 0.05 microM during 1 h gave intracellular concentrations of mitoxantrone similar to those achieved in vivo. This incubation concentration gave a mean cytotoxic effect of 53% living cells measured with the DiSC assay, which gives good possibilities to discriminate between mitoxantrone-sensitive and unsensitive cells. We believe that exposing leukemic cells in vitro for in vivo mimicking mitoxantrone concentrations could increase the clinical relevance of predictive assays.

Adult↗
PubMed1993

Effect of verapamil on daunorubicin accumulation in human leukemic cells with different levels of MDR1 gene expression.

The purpose of the present study was to evaluate the distribution of the multiple drug resistance phenotype in leukemic cells with different levels of mdr1 gene expression. Peripheral leukemic cells from 8 patients (5 with detectable and 3 with undetectable mdr1 RNA levels) were incubated with daunorubicin (1 microM) in the absence and presence of verapamil (6.6 microM). Daunorubicin accumulation in individual cells was analysed by flow cytometry. Verapamil increased (16-45%) the mean daunorubicin accumulation in 7 of the 8 cell samples. In cells from 4 of these 7 patients, the fluorescence histograms showed a general increase of daunorubicin accumulation. The increase was limited to a small subpopulation of cells in one patient. In the remaining two patients an increase of daunorubicin accumulation was seen in the majority of cells, while cells with the highest accumulation were unaffected. In conclusion, verapamil can increase daunorubicin accumulation in leukemic cells with and without detectable mdr1 gene expression and the increase seems to affect the majority of the cell population.

Adult↗
PubMed1993

Late seroconversion and high chronicity rate of hepatitis C virus infection in patients with hematologic disorders.

BACKGROUND: Patients with hematologic disorders requiring repeated blood and platelet transfusions are at high risk for development of post-transfusion non-A, non-B hepatitis. PATIENTS AND METHODS: Fifty-five patients with hematologic diseases and post-transfusion non-A, non-B hepatitis were studied. Sera were assayed for hepatitis C virus (HCV) antibodies with a second-generation enzyme-linked immunoassay. Sera from 40 patients were examined for the presence of HCV RNA with a nested PCR method. RESULTS: The clinical picture of acute non-A, non-B hepatitis did not differ from that described in other patient groups: however, progression to chronic hepatitis was very common (95%). Thirty-eight (95%) of 40 patients, whose sera were analysed both serologically and for the presence of HCV RNA had verified HCV infections. In some patients time to seroconversion was prolonged, up to more than 14 months. Seventeen patients with resistant or relapsed acute leukemia were treated with combination chemotherapy during the acute or chronic phase of hepatitis. Suppression of the inflammatory activity as reflected by a decrease of serum aminotransferase levels was recorded during the subsequent pancytopenic period. CONCLUSIONS: Hepatitis C has a high chronicity rate in patients with hematologic disorders which parallels the situation of hepatitis B in the immunocompromised host. Furthermore, like the situation in hepatitis B, the hosts' immune response to infection seems to be involved in the pathogenesis of liver injury. Time to seroconversion may be prolonged and detection of HCV RNA is therefore important for diagnosis.

Adolescent↗
PubMed1993

Reactivation of chronic hepatitis C after withdrawal of immunosuppressive therapy.

A patient with immune-mediated thrombocytopenia (ITP) and chronic hepatitis C virus (HCV) infection for 11 years was given immunosuppressive treatment because of an activation of his ITP. After 6 weeks of treatment with cyclophosphamide, cyclosporin A and cortisone the patient decided not to continue taking his medication. One month later he was readmitted to hospital due to fever, cough and jaundice. Clinical investigation revealed his condition to be caused by an activation of his HCV infection. It is concluded that, in parallel to the situation in hepatitis B, immunosuppressive treatment of patients with HCV infection may lead to increased viral replication, resulting in severe liver damage when immunocompetence is regained.

Adult↗
PubMed1993

On the interaction between cytosine arabinoside and etoposide in vivo and in vitro.

Cytosine arabinoside (ara-C) and etoposide are often used in combination in the treatment of acute myelocytic leukemia (AML). The intracellular phosphorylation of ara-C to its 5'-triphosphate (ara-CTP) is a prerequisite for its cytotoxic effects. It has been shown in vitro that etoposide can impair the formation of ara-CTP in leukemia cells. The present study was undertaken in order to elucidate whether this interaction may be of clinical importance. Leukemia cells were isolated from 3 patients with acute myelocytic leukemia and incubated in medium (RPMI-1640) with or without 10% fetal calf serum or in human plasma. When the cells were incubated in RPMI-1640 with ara-C (10 mumol/l) and etoposide during 2 h, the formation of ara-CTP was decreased to 71 +/- 18 (mean +/- S.D.) and 30 +/- 15% of control at 1 and 10 micrograms/ml etoposide, respectively. When the cells were incubated in human plasma, the formation of ara-CTP was not influenced by the presence of etoposide (101 +/- 6 and 103 +/- 20% at 1 and 10 micrograms/ml etoposide). When incubated in RPMI supplemented with 10% fetal calf serum, the corresponding figures were 81 +/- 8 and 70 +/- 20%. Six patients with AML were therefore treated with ara-C 0.5 or 1.0 g/m2 as a 2-h infusion every 12 h and, during 1 h before the second ara-C infusion, 100 or 200 mg/m2 etoposide was administered. The median change in the AUC of cellular ara-CTP between the first and second ara-C dose was 0% (-37 to +21%). The corresponding median change in rate of accumulation of ara-CTP in leukemia cells was 12% (-26 to +110%). The concentration of etoposide in plasma during the ara-C infusion was 18.7 +/- 5.1 micrograms/ml while the non-protein bound etoposide was 0.73 +/- 0.34 micrograms/ml. Thus, despite exposure to higher etoposide concentrations in vivo than in vitro, no impairment of ara-CTP formation was seen in the patients. This corresponds to the results obtained when leukemic cells were incubated in plasma. It is concluded that the inhibition of ara-CTP formation by etoposide seen in vitro is offset by the high protein binding of etoposide in plasma (96%) and that etoposide does not impair the formation of ara-CTP in leukemia cells in vivo during treatment with standard-dose etoposide.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates.

The effects on thrombosis and hemostasis of thrombin-induced activation of endogenous protein C (PC) were evaluated in baboons. Thrombosis was induced by placing into arteriovenous shunts a segment of Dacron vascular graft, which generated arterial platelet-rich thrombus, followed by an expansion region of low-shear blood flow, which in turn accumulated fibrin-rich venous-type thrombus. Thrombosis was quantified by 111In-platelet imaging and 125I-fibrinogen accumulation. Intravenous infusion of alpha-thrombin, 1-2 U/kg-min for 1 h, increased baseline activated PC levels (approximately 5 ng/ml) to 250-500 ng/ml (P < 0.01). The lower thrombin dose, which did not deplete circulating platelets, fibrinogen, or PC, reduced arterial graft platelet deposition by 48% (P < 0.05), and platelet and fibrin incorporation into venous-type thrombus by > 85% (P < 0.01). Thrombin infusion prolonged the activated partial thromboplastin clotting time, elevated fibrinopeptide A (FPA), thrombin-antithrombin III complex (T:AT III), and fibrin D-dimer plasma levels (P < 0.01), but did not affect bleeding times. Thrombin's antithrombotic effects were blocked by infusing a monoclonal antibody (HPC-4) which prevented PC activation in vivo, caused shunt occlusion, increased the consumption of platelets and fibrinogen, elevated plasma FPA and T:AT III levels, and reduced factor VIII (but not factor V) procoagulant activity (P < 0.05). We conclude that activated PC is a physiologic inhibitor of thrombosis, and that activation of endogenous PC may represent a novel and effective antithrombotic strategy.

Animals↗
PubMed1993

In vivo accumulation of etoposide in peripheral leukemic cells in patients treated for acute myeloblastic leukemia; relation to plasma concentrations and protein binding.

Since etoposide interacts with the nuclear enzyme topoisomerase II, the drug concentrations in the malignant cells during chemotherapy may have clinical correlates. Plasma protein binding of etoposide is extensive (94%) and alterations of the non-proteinbound fraction affect pharmacokinetic behavior of the drug. The pharmacokinetics of etoposide was therefore studied in plasma, total and non-proteinbound concentrations, and in leukemic cells isolated from peripheral blood samples from 22 patients after the first dose of the induction treatment for acute myelocytic leukemia. Fourteen patients received 100 mg/m2 and eight patients 200 mg/m2 as a 1 h infusion. The mean area under the concentration versus time curve AUC(0-infinity) in plasma was at the lower dose level 78.4 +/- 29.1 (mean +/- S.D.) micrograms/ml x h and 201.0 +/- 56.5 micrograms/ml x h at the higher dose level. The fraction of non-proteinbound etoposide in plasma was 5.2 +/- 3.4 and 5.4 +/- 2.1% in the two treatment groups. AUC(0-16h) in leukemic cells was 8.4 +/- 8.7 and 22.4 +/- 12.1 micrograms/ml x h at the two dose levels, respectively. The cellular etoposide concentration was 12.1 +/- 7.9 and 14.7 +/- 5.1% of the plasma concentration at the end of the infusion. The interpatient variability in cellular drug levels was considerable and exceeded the variability in plasma concentrations. Cellular accumulation of etoposide could be important for treatment outcome.

Adult↗
PubMed1993

Molecular approaches to diagnosis of Chagas' disease: use of defined antigens and a target ribosomal RNA sequence.

We evaluated the performance of two defined antigens in the serological diagnosis of Chagas' disease. One of them is a recombinant protein named B13 isolated from a genomic library of Trypanosoma cruzi in the expression vector lambda gtll. We show that the gene corresponding to B13 is conserved in the evolutive stages of the two "polar" strains of T. cruzi. The protein epitopes cloned in B13 are represented in 140 kDa, 116 kDa and 35 kDa polypeptides of trypomastigotes. The other antigen chosen for serodiagnosis is a lipopeptidophosphoglycan (LPPG). This glycoconjugate is also widely distributed in T. cruzi strains. The use of a rabbit serum to LPPG allowed the demonstration that this molecule bears epitopes in common to LPPG-like components and to 80-90 kDa glycoproteins of trypomastigotes. Both B13 and LPPG were evaluated in serodiagnosis by ELISA and RIA using a panel of normal human, Chagasic and Leishmaniasis sera. It was observed that B13 presents high sensitivity and specificity for Chagasic sera. For LPPG it was also concluded that this reagent discriminates between individuals infected and non-infected with T. cruzi. A heterogeneity in the level of antibodies to LPPG in Chagasic patients was detected. No correlation was found between the clinical form of Chagas' disease and the preferential reactivity to B13 or LPPG. We also report preliminary studies towards the characterization of a 100 bp sequence of the 24S alpha rRNA as a target for DNA-based detection systems for diagnosis. We show that polymerase chain reaction of total DNA of different trypanosomatids lead to the specific amplification of a 100 bp fragment only for T. cruzi. Northern blots confirmed the presence of the target region in the mature 24S alpha rRNA. Titration experiments based on the direct amplification of RNA with Taq DNA polymerase allowed the detection of 50 parasites. Studies are in progress to increase the sensitivity of the proposed system.

Animals↗
PubMed1992

Reevaluation of total, free, and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin.

The levels of total, free, and bound protein S and C4BP were determined using enzyme-linked immunosorbent assays (ELISAs) in plasma samples (8 males and 8 females) that were individually subjected to immunoadsorption studies in which "free protein S" was defined as that not adsorbed by anti-C4BP antibody-Sepharose column and "free C4BP" as that not adsorbed by anti-protein S antibody-Sepharose. Bound species were calculated as the difference between total and free species. Free protein S (131 nmol/L) averaged 38% of total protein S (346 nmol/L) and free C4BP (37 nmol/L) averaged 14% of total C4BP (264 nmol/L) in plasma. There was an excellent correlation between bound protein S and bound C4BP with 1:1 molar stoichiometry and a good correlation between free protein S antigen and protein S anticoagulant activity. It appears that free protein S is a necessary consequence of the molar excess of protein S over C4BP. C4BP beta chain antigen levels, measured using a new ELISA, averaged 218 nmol/L, a value indistinguishable from the molar concentrations of bound protein S (215 nmol/L) and bound C4BP (227 nmol/L). The free C4BP beta chain antigen was only 4 nmol/L compared with 131 nmol/L free protein S. These results suggest that free C4BP in plasma predominantly lacks the beta chain, that almost all C4BP capable of binding protein S is associated with protein S, and that the plasma levels of oligomeric forms of C4BP containing a beta chain (alpha 7 beta and alpha 6 beta) that bind protein S directly and stoichiometrically regulate free protein S levels.

Anticoagulants↗
PubMed1992

Direct detection of activated protein C in blood from human subjects.

The antithrombotic enzyme, activated protein C (APC) was measured in blood using an enzyme capture assay (ECA). The ECA involved (1) collection of blood into anticoagulant containing a reversible inhibitor of the enzyme, (2) specific affinity capture of the enzyme by an immobilized antibody that does not inhibit the enzyme, (3) removal of the reversible inhibitor by washing, and (4) direct assay of the captured enzyme's amidolytic activity. The ECA for APC used benzamidine for inhibition, anti-PC light-chain murine monoclonal antibody for capture, and the oligopeptide substrate S-2366 for enzyme assay. The sensitivity of this assay was 5 pmol/L (0.3 ng/mL) APC. The APC activity in normal pooled plasma corresponded to the amidolytic activity of 38 pmol/L (2.26 +/- 0.2 ng/mL) purified human plasma-derived APC in the ECA. APC levels in 41 normal donors ranged from 64% to 143%, averaging 104.9% +/- 19.6% (SD). Thus, APC is a measurable and normal component of circulating human blood, and this ECA may be useful for identifying APC deficiency. Moreover, similar ECAs for other enzymes in the circulation may be useful.

Antibodies, Monoclonal↗