A rapid immunocytochemical assay for CMV detection in peripheral blood of organ-transplanted patients in clinical practice.

This study describes clinical experience with a rapid method for diagnosis of cytomegalovirus infection in organ-transplanted patients, based on the detection of CMV-specific antigens in peripheral polymorphonuclear cells with a mixture of monoclonal antibodies. This CMV-pp65 assay was formerly called the "CMV immediate early antigen assay." A group of 180 organ-transplanted patients were examined with this assay; 75 of them could be observed from the date of transplantation. These 75 patients consisted of two groups: 59 kidney transplant patients receiving no CMV hyperimmunoglobulin prophylaxis (group I), 13 heart-transplanted patients, and 3 liver transplanted patients receiving prophylaxis (group II). Group III consisted of 105 patients who had been transplanted ca. 2 years before starting this study. In group I, 26 (44%) were CMV-pp65-positive (13 primary and 13 secondary infections). Fifteen of these 26 (58%) positive patients showed clinical symptoms of CMV infection. Eleven of these 15 (73%) were primary infections. Symptomatic patients had significantly more CMV-pp65-positive cells than asymptomatic patients; 12 patients showed a high number of positive cells and 11 of them developed severe CMV illness. Thirty-three patients were CMV-pp-65-negative (22 CMV IgG-sero-positive, 11 CMV IgG-seronegative). None of them had symptoms of CMV infection. In all patients of group I there were 36 periods of graft dysfunction in which CMV infection had to be differentiated from transplant rejection. In 10 out of 36 there was a CMV-pp65-positive test result and subsequent seroconversion. Treatment of viral infection resulted in improvement of clinical problems. In the remaining 26 episodes no CMV-pp65-positive cells were detected: in 17 cases graft dysfunction was caused by rejection, in 9 cases by other complications. In group II, 13 of 16 patients (81%) were positive in the CMV-pp65 assay (6 primary infections, 7 secondary infections). However, none of them showed clinical signs of CMV infection, regardless of the number of positive cells. No CMV-related graft dysfunction was observed. In group III, CMV infections did not play an important role. The experiences described suggest that this test is a valuable tool in early CMV diagnosis and in differentiating CMV-dependent graft dysfunction from other graft dysfunctions. It allows prompt therapeutic intervention.

Troponin T as a marker for posttransplantation adaptational problems of the donor heart.

BACKGROUND: Troponin T is used as a marker for myocardial cell damage (e.g., in aiding diagnosis and follow-up of myocardial infarction). Elevated troponin T levels are also observed after heart transplantation, although until now no explanation could be found for this phenomenon. METHODS AND RESULTS: Serum samples of 15 patients who underwent orthotopic heart transplantation were tested for troponin T with a one-step enzyme immunoassay. The highest concentrations of troponin T were seen between day 3 and 14 after transplantation (3.05 +/- 1.30 micrograms/L) and remained elevated up to 3 months. A correlation (r = 0.61, p < 0.02) was found between pretransplantation systolic pulmonary artery pressure and the cumulative troponin T release after transplantation. No association was found with rejection, and no correlation was found with ischemic time of the donor heart. CONDITIONS: These findings support the hypothesis that the acute exposure of the donor heart to the preexisting elevated right ventricular afterload in the recipient represents a strong mechanical stress for the transplanted heart. Measurement of troponin T may therefore be helpful in the posttransplantation monitoring and management of ventricular function after orthotopic heart transplantation.

Age dependency of selenium and cadmium content in human liver, kidney, and thyroid.

Selenium and cadmium concentrations were investigated in 60 autopsy tissue samples obtained from fetal life up to adulthood (defined in this study as 25-87 y of age) in Styria, a moderately industrialized region in Austria that has a low selenium supply. During the first 2 y after birth, median liver selenium concentrations were slightly lower (i.e., 1.5 nmol/g wet weight) than concentrations found in fetal life (i.e., 2.9 nmol/g) and adulthood (2.1 nmol/g). Whereas in the fetal period median selenium content in the kidney cortex (2.1 nmol/g) and the thyroid gland (1.6 nmol/g) was lower than that found in the liver, the reverse was true for adults (i.e., kidney, 5.5 nmol/g; thyroid, 4.3 nmol/g). Tissue cadmium concentrations approached 0 during gestation. Accumulation in the kidney and liver commenced immediately after birth. In the thyroid gland of adults, significantly higher concentrations of cadmium were found. Median concentrations in adults showed no statistical significant age dependency (i.e., liver, 7.6 nmol/g; kidney, 59.8 nmol/g; thyroid, 11.2 nmol/g). In summary, the data revealed very low tissue selenium concentrations and low cadmium burdens for the Styrian population that was not exposed occupationally.

[Helicobacter pylori and cytomegalovirus infection and atherosclerosis: an assessment of the current status]. / Helicobacter pylori- und Zytomegalievirus-Infektion und Atherosklerose: eine Standortbestimmung.

An association between Helicobacter pylori infection or cytomegalovirus infection and atherosclerosis has been described. During the last four years, new evidence evolved from a number of studies, which allows to reevaluate the clinical impact of this association. Current knowledge suggests that the association between atherosclerosis and Helicobacter pylori may be accounted for by chance or by confounding from a secondary phenomenon. Regarding cytomegalovirus, a number of animal models and molecular studies propose several different mechanisms, by which the virus may interact with cells of the vessel wall, the immune system or with some of the established risk factors to cause atherosclerosis. Conclusive evidence for the clinical relevance of these experimental findings is still lacking. Some of the uncertainties may be resolved by larger studies, which have started during the last years.

Simple decomposition procedure for determination of selenium in whole blood, serum and urine by hydride generation atomic absorption spectroscopy.

A digestion procedure for selenium determination by hydride generation atomic absorption spectroscopy (AAS) in whole blood, serum and urine is described, it employs sulfuric acid, hydrogen peroxide and vanadium (V) sulfuric acid reagent solution. The method is rapid, uses no explosive reagents and can be performed at a constant temperature of 100 degrees C. Therefore, it is easily applicable in a routine clinical laboratory for a large amount of samples. The coefficient of intra-assay variation was 4.3-5.6%, the coefficient for inter-assay variation was 5-5.9% in the medium and high concentration range, and 5.8-8.6% in the low range. In analyzing several commercial reference materials our results showed good agreement with the target values. Analytical recovery by addition of sodium selenite and seleno-DL-methionine to samples ranged between 97 and 104%. The correlation between the described digestion procedure and the nitric, sulfuric and perchloric acid digestion procedure recommended by the International Union of Pure and Applied Chemistry showed good agreement for whole blood, serum and for urine. We determined selenium in serum (n = 58) and whole blood (n = 50) in a collective of healthy children from 1 to 5 years living in Styria, Austria. The low values in serum (35 +/- 11 micrograms/L) and whole blood (42 +/- 6 micrograms/L) at one year of life increased significantly to 48 +/- 13 mu/L (p = 0.033) and 55 +/- 6 micrograms/L (p = 0.004) at three years of life in serum and whole blood, respectively. The selenium concentration showed no further increase up to five years of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinogen values in patients with and without restenosis following percutaneous transluminal coronary angiography.

Fibrinogen has turned out to be an independent risk factor for coronary heart disease (CHD). It is not known whether or not this parameter could be a prognostic factor for restenosis following percutaneous transluminal coronary angioplasty (PTCA), which represents the main problem limiting the long-term efficacy of this procedure. Therefore, we studied fibrinogen concentrations in a series of 50 males (mean age: 55, range: 38-70 years) with CHD and successful PTCA. Follow-up coronary angiography was performed 12 months following PTCA. Twenty-two patients had restenosis, and 28 patients were without restenosis. Both groups did not differ significantly in medical history (smoking habits, hypertension, positive family history for cardiovascular diseases), in routine lipid profile (total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, apolipoproteins A1 and B). Fibrinogen values were 405 +/- 128 mg/dl (range: 202-725) in patients with restenosis and 352 +/- 94 mg/dl (range: 187-568) in patients without restenosis (not significant). Elevated fibrinogen levels of more than 400 mg/dl were found in 8 patients in each group. Although fibrinogen is a proven marker for CHD in men, fibrinogen is not a risk factor for restenosis following PTCA.

[Differential diagnostic considerations in eosinophilia with reference to a 19-year-old patients with Löffler endocarditis]. / Differentialdiagnostische Uberlegungen zur Eosinophilie anhand einer 19jährigen Patientin mit Löfflerscher Endokarditis.

In this case report a 19-year-old girl suffering from hypereosinophilia with 3500 cells/mm3 and involvement of the right lung, lymph nodes, skin, serosa and heart is described. Within 10 days of admission an infiltration of the right upper lung lobe disappeared spontaneously and was diagnosed as transient eosinophilic lung infiltration according to Löffler. Both lymph node needle biopsy and several skin biopsies revealed merely general reactive changes whereas a bronchial lavage produced a significant number of eosinophilic granulocytes. Autoimmunologic or infectious-toxic disorders were ruled out as etiologic causes. Within 4 weeks after admission severe mitral and tricuspidal insufficiency as well as AV-block second grade and protodiastolic galloping rhythm developed in addition to progressing polyserositis. The echocardiographic pattern was in accordance with restrictive endocarditis. Due to the greatly reduced left ventricular ejection fraction cortisone (125 mg prednison equivalent) was tentatively administered as therapy. Within 2 weeks heart-, serosa- and lymph-node-findings became normal as well as the eosinophilic count, the scaling skin rash being only partly improved. After 10 months of continuous cortisone therapy (10 mg prednison equivalent) the patient was without evidence of disease. However in conclusion, it may be said that such pattern of findings suggests a hypereosinophilic syndrome with Löffler endocarditis with an unknown future course of disease although generally survival prognosis may not be too high.

Platelets contain interleukin-1 alpha and beta which are detectable on the cell surface after activation.

Activated platelets have been shown previously to exhibit membrane-bound IL-1 bioactivity, which leads to the question of localization of the cytokine in platelets. Using immunocytological and flow cytometric techniques, we found IL-1 alpha and IL-1 beta in the cytoplasma of both resting and thrombin-activated platelets. Immunogold-silver staining of the cell surface of activated platelets as well as preembedding antibody treatment of platelets revealed the presence of IL-1 (alpha and beta) in low density on the surface of intact cells in contrast to distinct enrichment in the cytoplasma of damaged platelets. Fibrin fibres present between cells indicated adsorbance of IL-1. There was also weak binding of anti-IL-1 alpha to the surface of thrombin-activated platelets as shown by flow cytometry. Following activation there appears to be some transfer of IL-1 onto the cell surface of activated cells, the bulk of the cytokine, however, is probably not released prior to platelet disintegration. In summary, we present evidence for the presence of both IL-1 alpha and IL-1 beta in resting and activated platelets without being able to demonstrate localization of the cytokines to specific subcellular structures.

[Photometric determination of HbA1c levels in clinical practice]. / Photometrische Bestimmung des HbA1c-Wertes in der Praxis.

This study was undertaken to evaluate the routine use of a new immunological photometer to measure the concentration of HbA1c in whole blood from 155 patients. The basis of the measurement is a latex agglutination reaction in which a monoclonal antibody as epitope recognizes glucose bound to HbA1c. The result is available within 9 min. High-pressure liquid chromatography (HPLC) served as control method. The photometer proved to be very precise (all coefficients of variant < 2.5%), and the values obtained agreed well with those by HPLC (y = 0.952x-0.12; r = 0.986; P < 0.001). The reference ranges for the photometrically measured HbA1c values (4.4-5.9%), obtained for 40 patients, agreed well with those by HPLC (4.6-6.2%). Interference study discovered no effect on the measured value by anaemia, polycythaemia or high rheuma factor (n = 31). In 12 patients on dialysis the photometer recorded significantly lower values than HPLC (P < 0.0005). It is possible that in these cases the photometer values are more accurate because the method is not affected by carbamylated haemoglobin. False results were obtained by the photometer in two patients with leucocytosis (79,000 and 216,000/microliters, respectively) due to chronic lymphocytic leukaemia.

Immunoquantification of lipoprotein(a): comparison of nephelometry with electroimmunodiffusion.

A new fully automated nephelometric immunoassay for lipoprotein(a) quantification in human serum was evaluated using the Behring Nephelometer Analyzer. The assay exhibited a good linearity in the concentration range of 110-1,770 mg/l; at higher concentrations, samples were automatically diluted by a factor of 4. The method is simple, robust, and shows an excellent stability of the calibration curve over several weeks. Intra-assay and day-to-day coefficients of variation were 2% and 4.5%, respectively. The method correlated well with electroimmunodiffusion (r = 0.977; n = 123; P = 0.0001). Unspecific turbidity as expressed by an elevated blank value occurred in 3% of all freshly measured samples (n = 392). Storage of the samples for 1 week at 4 degrees C had no significant influence on the results. Frozen sera, on the other hand, cannot be assayed by this method. We believe that this assay is well suited for use in clinical routine work.

Comparison of direct versus indirect determination of zinc concentration in erythrocytes in euthyroid and hyperthyroid subjects.

Zinc concentration in erythrocytes is a suitable marker of peripheral tissue response to thyroid hormones. Therefore, the determination of erythrocyte zinc concentration has become an important marker for duration of preexisting hyperthyroidism in the clinical laboratory. We compared a new indirect erythrocyte zinc determination method with a commonly used direct method in 42 euthyroid and 14 hyperthyroid subjects. Zinc concentrations (median and range) obtained by direct and indirect methods were 172.8 (134.4-241.1) and 176.8 (143-243.9) mumol/l erythrocytes in the euthyroid group and 117.1 (71-141.9) and 118.5 (73-137) mumol/l erythrocytes in the hyperthyroid group. Values measured by the indirect method were slightly higher in both groups. Regression analysis showed a good correlation (r = 0.967, p = 0.0001). The influence of the anticoagulants, potassium EDTA and heparin, on erythrocyte and plasma zinc values was studied, and zinc concentrations in serum and plasma were compared. No statistically significant differences were found. Thus, the indirect determination of erythrocyte zinc concentration is a simple and rapid technique well suited for use in clinical laboratory work, yielding accurate and reliable results. For the indirect method reference concentrations were established in a collective of 102 thyroid healthy subjects. An erythrocyte zinc concentration of 176.3 +/- 23.9 mumol/l (mean +/- SD) was found with a cut-off limit of 138 mumol/l to hyperthyroidism.

[Iodine content of thyroid tissue in the Styrian population]. / Untersuchungen zum Jodgehalt von Schilddrüsengewebe in der steirischen Bevölkerung.

The iodine concentration was determined in 89 thyroid tissue samples of post-mortem examination material of thyroid healthy persons in dependency to the age. Before the 22nd week little iodine is stored in the fetal thyroid gland (median and range: 24 micrograms/g wet weight [0 to 27 micrograms/g]). From the 23rd week on the concentration is significantly higher (p = 0.004). There is a further increase of iodine concentration in the first 6 months of life, with a median concentration of 171 micrograms/g (75 to 808 micrograms/g). With a median of 599 micrograms/g (464 to 699 micrograms/g) the iodine concentration in the second year of life is comparable with that of adults (640 micrograms/g [218 to 2772 micrograms/g]). From the 60th year of life the median concentration is 462 micrograms/g (155 to 2100 micrograms/g), but this decrease is not statistically significant. Additionally the iodine concentration was determined in 7 topographically defined regions of 28 thyroid glands. The median iodine concentration did not differ in the 7 regions, but the iodine content in one and the same thyroid gland differed from region to region. Therefore in the healthy thyroid gland no regions exist, which have generally stored less iodine than others. However, no conclusion can be drawn from the iodine concentration of one biopsy to the whole iodine concentration of the organ.

Automated determination of glycated hemoglobin: comparative evaluation of five assay systems.

We evaluated four newly introduced assays for determination of glycated hemoglobin allowing the processing of large amounts of samples in a clinical routine laboratory. These methods were compared to the Bio-Rad Diamat system. The investigated methods were the Merck Hitachi L-9100, a fully automated HPLC analyser, the Abbott IMx glycated hemoglobin ion capture assay, the DAKO HbA1c enzyme linked immunosorbent assay (ELISA), and the Boehringer-Mannheim HbA1c Tinaquant turbidimetric assay. All methods showed generally acceptable precision and good accordance with the Diamat system. Interference study showed influence of anaemia, polycythemia, rheumatoid factor, and chronic hemodialysis on the values of the DAKO ELISA and of anaemia and polycythemia on the values of the Boehringer-Mannheim Tinaquant assay. All of the investigated methods allow referring of results either as measured or standardized HbA1c values, the latter obtained after calibration with reference to an ion exchange high-pressure liquid chromatography method. Our data confirm the feasibility of this kind of standardisation of glycated hemoglobin assays, allowing direct comparison of results from various determination methods.

The presence of immunosuppressive 'p15E-like' factors in the serum and urine of patients suffering from malign and benign breast tumours.

Certain types of tumours are capable of producing factors inhibiting mononuclear phagocyte chemotaxis which may contribute to defects in immunosurveillance. In head and neck cancer these factors are said to be related to the retroviral protein p15E. This study examines the presence of p15E-like factors in serum and urine of patients with malign and benign breast tumours. Thirty patients with breast cancer, 29 patients with benign breast masses, and 28 healthy controls were tested blindly with the monocyte polarization assay, using N-formyl-methionyl-leucylphenylalanine as chemo-attractant. The low molecular weight fractions prepared of sera of the malign tumour patients inhibited the monocyte polarization significantly (mean inhibition 34%, s.d. = 12) compared with those of benign tumour patients (15%, s.d. = 7) and of controls (14%, s.d. = 6). The observed inhibitory effects on the monocyte polarization could be compensated by MoAbs reactive to p15E-related antigens. The mean difference between the polarization inhibition with and without anti-p15E adsorption (the 'p15E-like factor-induced inhibition') was 25% (s.d. = 13) in the breast cancer group, compared with 7% (s.d. = 5) in the benign tumour patients and 5% (s.d. = 4) in the healthy control group. Surgical removal of the tumours resulted in a restoration of the monocyte polarization in 20/23 (87%) patients of the breast cancer group. Results testing preoperative urine samples correlated well with those of corresponding sera. These data give additional support to the concept that tumour-derived p15E-like factors are responsible for the inhibitory effect on monocyte chemotaxis in breast cancer patients, and that these factors can be found in serum as well as in urine.

Prolonged monitoring of troponin T for the detection of anthracycline cardiotoxicity in adults with hematological malignancies.

The study was performed to describe the time course of serum cardiac troponin T (cTnT) elevations for the early detection of anthracycline cardiotoxicity. cTnT was analyzed serially in 78 patients with hematological malignancies receiving 142 treatment cycles including various anthracyclines. cTnT positivity was defined as an increase in cTnT >or=0.03 ng/ml and was observed in 12 patients (15%) during 16 treatment cycles (11%). Peak cTnT levels were observed on day +21.5 (median, range: day +6 to day +35) after initiation of anthracycline therapy. cTnT positivity lasted >or=3 days in 63% of cycles and began to occur after a median of two anthracycline doses. Follow-up echocardiography in 28 patients showed a greater decrease in left ventricular ejection fraction (LVEF) in cTnT-positive patients compared to the cTnT-negative group (10% vs 2%, p=0.017). Age, gender, and pretreatment LVEF had no influence on the occurrence of cTnT positivity. Serial measurement of serum cTnT reveals delayed subclinical myocardial damage even after minor anthracycline exposure, may identify patients at risk for subsequent myocardial dysfunction, and suggests prolonged damage to the cardiac myofibrillar system.

Involvement of the immune system in iodine deficient goiter.

Iodine deficient goiters were studied by immunohistochemistry and showed extensive presence and typical arrangement of dendritic cells, known to have excellent antigen presenting capacity. These cells were positive for all MHC-class II epitopes and for ICAM-1. Epithelial follicle lining cells were also seen to be class II positive but lacked ICAM-1. Thyroglobulin seemed not to be iodinated at the C-terminal hormogenic site, as shown by reactions with monoclonal antibodies. Iodine therapy, as well as thyroxine therapy were effective in reducing thyroid size. Both forms of therapy were found to decrease the pretreatment levels of circulating thyroid growth stimulating immunoglobulins (TGI).

Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells.

In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell surface expression of the complex occurs in insect cells. Three recombinant baculoviruses, expressing gL1, gH1, and truncated gH1 (gH1t), which lacks the transmembrane region, were constructed. It was shown that recombinant gH1/gL1 and gH1t/gL1 heterooligomers were produced in insect cells. As in mammalian cells, gH1 and gH1t were not detected on the surfaces of insect cells in the absence of gL1. When coexpressed with gL1, recombinant gH1 was displayed on the surfaces of insect cells. Coexpression of gH1t and gL1 resulted in secretion of the gH1t/gL1 complex into the cell culture medium, indicating that gH1t is also transported to the surfaces of insect cells. Our results indicate that the process of folding and intracellular transport of gH1 and gL1 is comparable in insect cells and mammalian cells and that the baculovirus expression system can be used to examine the complex formation and the intracellular transport of gH1 and gL1. The availability of secreted gH1t/gL1 complex offers the opportunity to further investigate the immunological properties of this complex.

Recombinant proteinase 3 (Wegener's antigen) expressed in Pichia pastoris is functionally active and is recognized by patient sera.

The open reading frame of human proteinase 3 (PR3) without the prepro-peptide was cloned and expressed in Escherichia coli (rcPR3) and in Pichia pastoris (rpPR3). The 6-histidine tagged rpPR3 was efficiently secreted into culture supernatant from which it could be purified by immobilized metal chelate chromatography. Purified rpPR3 migrated as a single 32-kD band on SDS-PAGE and harboured protease activity that could be inhibited with inhibitors specific for serine-proteases. By indirect antigen-capture ELISA using rpPR3, 60% of sera from patients with Wegener's granulomatosis bound to the recombinant product, although it was not recognized in ELISA with directly coated rpPR3.

Monitoring of cardiac function by serum cardiac troponin T levels, ventricular repolarisation indices, and echocardiography after conditioning with fractionated total body irradiation and high-dose cyclophosphamide.

OBJECTIVES: Highly differing rates of cardiac complications associated with high-dose cyclophosphamide (CY) have been reported, and only one clinical study has been performed on the cardiotoxic effects of CY monotherapy following total body irradiation (TBI). PATIENTS AND METHODS: We prospectively evaluated the potential cardiotoxic effects of conditioning with fractionated total body irradiation and high-dose cyclophosphamide (TBI/CY) by serial measurement of serum cardiac troponin T (cTnT), assessment of systolic and diastolic echocardiographic parameters and analysis of ventricular repolarisation indices (QT-dispersion and corrected QT-dispersion) in 30 adult patients with haematological malignancies undergoing haematopoietic stem cell transplantation. RESULTS: There was no evidence of pretreatment cardiac dysfunction in any patient. Although cTnT was determined serially for a median of 14 d after completion of conditioning, no elevated levels were observed. Echocardiographic parameters did not show any significant change at a median follow-up of 5 months except for one patient with evidence of impaired diastolic filling. No significant differences for mean values before and after high-dose CY were noted for ventricular repolarisation indices. Two patients had a significant increase in corrected QT-dispersion after CY without any other signs of cardiotoxicity. Congestive heart failure or arrhythmias were not observed. CONCLUSIONS: These data suggest that TBI/CY is safe with respect to cardiotoxicity in patients without pre-existing cardiac dysfunction. Hitherto unknown synergistic cardiotoxic effects of CY with other cytostatic drugs may constitute the major pathogenic factor of myocardial dysfunction after high-dose chemotherapy.