[Diagnosis of minimal residual disease in autologous bone marrow transplantation]. / Diagnostik af minimal residual sygdom ved autolog knoglemarvstransplantation.

ABMT (autologous bone marrow transplantation) is being increasingly used in the treatment of malignant diseases, including the acute leukaemias. Although ABMT seems to be superior to conventional chemotherapy in terms of disease-free survival, an unacceptably high frequency of relapse after ABMT remains a major problem. As such relapse may be due to malignant cells in the graft or residual malignant cells surviving in the patient after preconditioning therapy, it is essential to be able to detect and eradicate residual malignant cells. The article presents a review of available methods for the detection of minimal residual disease in conjunction with ABMT, especially regarding their relative sensitivity and specificity.

Collectin in a non-mammalian species: isolation and characterization of mannan-binding protein (MBP) from chicken serum.

A chicken serum lectin was isolated by affinity chromatography on TSK-75 beads derivatized with the monosaccharide N-acetyl-D-mannosamine (ManNAc). Serum was applied to the column in a Ca(2+)-containing buffer and proteins were eluted with EDTA. After recalcification, the eluate was passed through a new ManNAc-derivatized column. Bound proteins were eluted with 50 mM ManNAc. Anti-carbohydrate antibodies present in the eluate were removed by passage through a rabbit anti-chicken immunoglobulin derivatized column, and the lectin was further purified by ion-exchange chromatography and gel-permeation chromatography. The purified chicken lectin shows an overall structure similar to mammalian mannan-binding protein (MBP). SDS-PAGE revealed two polypeptides of M(r) 33 and 34 kDa (reduced) with identical sequence for the first 30 NH2-terminal residues. The NH2-terminal sequence shows 43% identity with the human MBP. Like mammalian MBP, the polypeptides of the chicken lectin are degraded by treatment with collagenase. Residues 26-30 (G-L-P(OH)-G-D) are likely to represent the beginning of the collagenous region. Mobilities on SDS-PAGE of the COOH-terminal collagenase-resistant fragment under reduced and non-reduced conditions indicate the presence of intrachain disulphide bonds, as are also found in mammalian MBP. Gel chromatography showed an intact mol. wt of 750 kDa. Binding of the chicken MBP to mannan was inhibited by monosaccharides in the following order of potency: ManNAc > L-fucose > mannose > N-acetylglucosamine. Other monosaccharides inhibited poorly or not at all. Chicken MBP, bound to mannan, activated the classical complement pathway in human serum. Electron micrographs show structures and dimensions resembling human MBP. Overall, the results show that the purified lectin is the chicken homologue to mammalian MBP and indicate the presence of a MBP-like clearance system outside mammals.

Comparative study of the structural and functional properties of a bovine plasma C-type lectin, collectin-43, with other collectins.

Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.

Differential expression levels of the heat shock protein 27 isoforms in pediatric normal, nonleukemic and common acute lymphoblastic leukemia B-cell precursors.

Heat shock protein 27 (hsp27) may function as a regulator of microfilament dynamics and may participate in signal transduction pathways of different cell growth regulators, with the mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 being a major enzyme responsible for its phosphorylation. Using two-dimensional gel electrophoresis, we have compared the expression levels of two hsp27 isoelectric variants (hsp27 isoforms) M2 (molecular weight, 26 kD; isoelectric point, 6.02) and M3 (molecular weight, 26 kD; isoelectric point, 5.60) in pediatric bone marrow CD19+CD10+B-cell precursors (BCPs) purified from either common acute lymphoblastic leukemia (c-ALL) patients, normal donors, or non-c-ALL patients. Compared with normal BCPs, we found increased hsp27 expressions (M2 isoform) (by a factor 5 to 9 of mean level) in c-ALL as well as in non-c-ALL (nonleukemic) precursors. Though increased phosphorylation of hsp27 (M3 isoform) was observed in BCPs from c-ALL patients at relapse (by a factor 3 of mean level compared with normal BCPs and precursors from c-ALL at diagnosis), which might represent a differential enzymatic activity, this was not distinguishable from that of non-c-ALL patients. Therefore, our studies suggest constitutive differences of hsp27 isoforms between pediatric leukemic BCPs and their relatively low-expressing, immunophenotypically normal bone marrow counterparts. In light of the occasional and possibly transient increase of hsp27 expression during nonleukemic BCP differentiation and the possible role of hsp27 in signal transduction to microfilaments, these differences might be of considerable biologic interest and of importance in future studies of regulated normal or dysregulated leukemic hematopoietic cellular differentiation.

Induced and down-regulated proteins in the human cultured hairy cell leukemia line JOK-1 and the Burkitt's lymphoma cell line Daudi during incubation with interferon-alpha: a kinetic study.

To elucidate the mechanism of action of interferon-alpha (IFN-alpha), the effect on cell proliferation and protein synthesis in the human hairy cell leukemia line JOK-1 and the Burkitt's lymphoma cell line Daudi were investigated. While Daudi cells were inhibited in proliferation and in total protein synthesis, no effect was seen on JOK-1 cells. However, high-resolution two-dimensional gel electrophoresis showed that four polypeptides were induced in JOK-1 cells after IFN-alpha incubation, while an additional 11 were induced and two down-regulated in Daudi cells. Kinetic studies revealed that the changes in JOK-1 cells were only temporary (within 8-16 h) and small to moderate in magnitude (less than four-fold). In Daudi cells, the changes for two of these polypeptides were early (within 2 h), for most of them prolonged (at least 24 h), and for three of them of great magnitude (between 6- and 30-fold). Quantitative analytical assessments indicated that four IFN-alpha-inducible polypeptides, present in low amounts of untreated cells, were highly expressed only in sensitive Daudi cells upon IFN-alpha treatment. This observation might indicate a role for these polypeptides in the inhibition of cell proliferation in Daudi cells. Furthermore, six of the other IFN-alpha-modulated polypeptides were synthesized constitutively in JOK-1 cells at levels comparable to those achieved in IFN-alpha-treated Daudi cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-alpha-induced changes in surface antigens in a hairy-cell leukemia (JOK-1), and a Burkitt's lymphoma cell line (Daudi) during in vitro culture.

In further studying the mechanism of action of IFN-alpha in HCL, we cultured the HCL cell line JOK-1 and the IFN-sensitive Burkitt cell line Daudi with and without IFN-alpha and investigated the changes in density of a number of surface antigens by use of mAb and flow cytometry analyses. During culture with IFN-alpha, reproducible changes were induced in both cell lines, which were qualitatively similar but differed quantitatively with small and transient changes in JOK-1. Significant decreases in surface antigen expression were observed for CD 19, 23, 37, and for IgM on both cell lines. Moreover, decreases were seen for CD 10, 22, 45, and MHC class II on Daudi, and for CD 20, 21, 27, and 40 on JOK-1. By contrast, only a few antigens increased in density, including CD 39, A96/G8 and SC9, on both cell lines, CD 22 on JOK-1, and CD 21 on Daudi. The increase in CD 39, A96/G8 and SC9 was probably directly related to the mechanism of action of IFN-alpha, whereas the other changes were most consistent with an unspecific inhibition of protein synthesis, possibly due to an accumulation of cells in G0, even though a differentiating effect cannot be ruled out. Thus, the unique in vivo effect of IFN-alpha in HCL was not paralleled by a specific direct effect on JOK-1 in vitro. Our findings therefore do not support the theory that IFN's mechanism of action in vivo is a direct effect on HC, but suggest that indirect effects are involved.

[Cesarean section under epidural anesthesia. A questionnaire study]. / Kejsersnit i epidural anaestesi. En spørgeskemaundersøgelse.

Seventy-four women selected at random who had been subjected to Caesarean section replied to questions about their satisfaction with epidural anaesthesia at various stages during the intervention. General discomfort increased during the intervention and pain contributed most to this. 96% of the women would recommend the method to others.

Towards establishing comprehensive databases of cellular proteins from transformed human epithelial amnion cells (AMA) and normal peripheral blood mononuclear cells.

Databases of protein information derived from the analysis of two-dimensional gels have been established from transformed human amnion cells (AMA) and peripheral blood mononuclear cells (PBMCs). A total of 1781 [35S]methionine-labeled AMA proteins (1274 IEF, 537 NEPHGE) and a total of 1311 proteins from PBMC (948 IEF, 363 NEPHGE) were resolved and recorded using computerized (PDQ-SCAN and PDQUEST softwares) two-dimensional gel electrophoresis. AMA and PBMC proteins (total, 454: 301 IEF, 153 NEPHGE) were matched both manually and by the computer. Information entered in the AMA database (in most cases for some major proteins) includes: molecular weight, protein name, HeLa protein catalogue number, mouse protein catalogue number, nuclear proteins, phosphorylated proteins, distribution of proteins in Triton X-100 supernatants and cytoskeletons, proliferation- and transformation-sensitive proteins, cell cycle-specific proteins, mitochondrial proteins, proteins matched in normal human embryonal lung MRC-5 fibroblasts and PBMC cells, heat shock proteins, proteins affected by interferons, cytoskeletal proteins, and the presence of antibody against protein in human sera. Additional information has been entered for the cell cycle-regulated and DNA replication protein cyclin (PCNA). Information entered in the PBMC database includes molecular weight and potential markers for sorted populations of lymphocyte subtypes. For those proteins that have been matched to AMA proteins, information contained in some entries may be transferred from the AMA database.

Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis.

We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific MoAbs, and separated to a high degree of homogeneity by FACS into CD4+ helper T cells, CD8+ suppressor T cells, CD20+ B cells, and N901 (NHK-1)+ NK cells. The four lymphocyte subpopulations were labeled with [35S]methionine for 14 hr, solubilized in lysis buffer, and analyzed by two-dimensional gel electrophoresis (IEF). Of about 1000 proteins resolved in each case, most were found to be common to all subpopulations. However, eight putative markers for B1+ (proteins 5525, Mr = 63,700; 5621, Mr = 63,700; 8311, Mr = 36,900; 2202, Mr = 36,300; 6121, Mr = 30,300; 106, Mr = 29,300; 5009, Mr = 23,000; 8012, Mr = 11,600) and one for N901+ (protein 8129, Mr = 30,400) were identified. In contrast, no major protein markers were found that could differentiate T4+ and T8+ cells from each other or from B cells and NK cells. With the exception of two B1+ markers (proteins 5525 and 5621), lower but variable levels of the other markers were observed in all cell types. All the putative protein markers have been identified in the protein database of human peripheral blood mononuclear cells (PBMCs) (see accompanying article by Celis et al.). Comparison of the overall patterns of protein synthesis of the unsorted PBMCs with those of the four subpopulations showed that the synthesis of some major PBMC proteins decreased substantially in the sorted subsets. These proteins are most likely not of monocyte origin, as these cells constituted only about 15% of the total PBMCs. Also, the inhibition does not seem to be due to the addition of the single MoAbs or to cell cycle differences. Taken together, the data provide a background for further studies of protein profiles in normal (resting or activated) and malignant hematopoietic cells.

Major proteins induced and down-regulated by interferons in human cultured cells: identification of a unique set of proteins induced by interferon-alpha in epithelial, fibroblast, and lymphoid cells.

In all, 40 major polypeptides ranging in molecular weights from 14.5 to 83 kDa were shown to be induced by IFNs alpha (also by IFN-alpha 2b and beta in a few cases) and gamma in human cultured cells of epithelial (transformed amnion cells (AMA)), fibroblast (proliferating and quiescent MRC-5 fibroblasts), and lymphoid origin (Molt-4). With the exception of a heat shock protein (IEF14 or hs x 70) and two tropomyosins (IEFs 52x and 55), none of these proteins corresponded to polypeptides (proliferation-sensitive or others) previously identified and catalogued by us. IFN-alpha induced the highest number of polypeptides in lymphoid cells, while the response to IFN-gamma was more pronounced in cultured epithelial and fibroblast cells. Several of the polypeptides induced by IFNs alpha and gamma were synthesized (albeit at different rates) by the control untreated cells, and in some cell types such as normal human peripheral blood mononuclear cells many were expressed at high levels. Only IFN-alpha-induced a unique set of proteins (alpha 1, 51 kDa; alpha 2, 15 kDa; alpha 19, 78 kDa; and gamma 10, 83 kDa) in all cultured cell types studied, implying that response to this IFN involves a shared biochemical pathway(s). Both IFN-alpha (also IFN-alpha 2b) and beta induced an identical group of proteins in AMA cells in agreement with the fact that type I IFNs share common receptors. IFNs alpha and gamma induced a few common polypeptides, but only gamma 10 (83 kDa) showed increased synthesis in all cell types exposed to either of these IFNs. A total of 28 major cellular polypeptides were down-regulated by IFNs in the various cell type studied. Different sets of proteins were affected, however, in each system, emphasizing the complexity of the mechanisms underlying the action of these factors. Treatment of synchronized G1 AMA cells with IFNs alpha, beta, or gamma (500 IU/ml, final concentration) did not inhibit their progression from G1 to S-phase as determined by indirect immunofluorescence using PCNA autoantibodies specific for cyclin. These observations were in line with the fact that IFNs did not affect dividin or cyclin(PCNA) synthesis (S-phase specific proteins) at least within the first 17 hr after their addition.

The effects of naloxone on central hemodynamics and myocardial metabolism in experimental propoxyphene-induced circulatory shock.

The courses of the hemodynamic and cardiometabolic effects of naloxone were evaluated in propoxyphene-induced shock in eight pentobarbital-anesthetized pigs. Circulatory shock was induced by an infusion of propoxyphene chloride 15 mg . min-1 i.v. At shock, i.e. MAP less than 60 mmHg and/or CI less than 2.0 l . min-1 . m-2, naloxone was administered at 0.75, 1.5 and 3.0 mg . kg-1 with an interval between increments of 8 min. The propoxyphene infusion of 15 mg . min-1 was continued throughout the study. Following the injection of naloxone 0.75 mg . kg-1, increases were observed (% of baseline value) in MAP (41%), i.e. deficit to baseline 59%, HR (66%), CI (67%) and SVI (108%), whereas MPAP and MPAOP were unchanged. dP/dt increased (34%). In the coronary circulation naloxone initiated the following changes: CSF increased (69%) as did MVO2 (48%) with unchanged MO2-extraction, but CVR decreased further (36%). The maximum effects of naloxone were registered 2-3 min after 0.75 mg . kg-1. Following 1.5 and 3.0 mg . kg-1, no changes in hemodynamics were observed other than those caused by progressing propoxyphene intoxication.

The effects of cardiac pacing on central hemodynamics in experimental propoxyphene-induced cardiac failure.

Coronary sinus pacing was evaluated in 10 pigs during propoxyphene-induced cardiac failure. From baseline, propoxyphene chloride 15 mg . min-1 was infused until circulatory shock developed. Cardiac pacing was evaluated at different dose levels expressed as % of the shock dose of propoxyphene: at intoxication levels below 50% of the shock dose, cardiac pacing improved cardiac performance. At dose levels above 50% of the shock dose cardiac performance deteriorated further during pacing. The results are consistent with a severe negative inotropic effect of propoxyphene in overdose.

The effects of dopamine on central hemodynamics and myocardial metabolism in experimental propoxyphene-induced shock.

The hemodynamic and cardiometabolic effects of dopamine were evaluated in propoxyphene-induced circulatory shock in eight pentobarbital anesthetized pigs. Circulatory shock was induced by an infusion of propoxyphene chloride 15 mg . min-1 i.v. At shock, i.e. CI less than or equal to 2.0 l . min-1 . m-2 and/or MAP less than or equal to 60 mmHg, dopamine was infused at 10, 20, 40, 80 and 160 micrograms . kg-1 . min-1 with an interval between increments of 8 min. After 30 min at 160 micrograms . kg-1 . min-1, the infusion rate was reversibly decreased. The propoxyphene infusion of 15 mg . min-1 was continued throughout the study. Dopamine improved the circulation in seven animals; one animal died in refractory shock during dopamine infusion. Dopamine infusion at shock level resulted in an increase of the following variables (% of baseline value): MAP (69%), HR (109%), CI (138%) and SVI (129%). Normalisation was seen in MRAP (120%) and in MPAOP (100%). A profound decrease in systemic vascular resistance was unchanged. Increases were seen in left and right ventricular stroke work index, to 88% and 176% of baseline, respectively. Left ventricular dP/dt increased (170%). In the coronary circulation myocardial blood flow increased (133%) as did myocardial oxygen consumption (65%) concomitant with a decrease in myocardial oxygen uptake (41%), but coronary vascular resistance progressively decreased (38%). The myocardial propoxyphene extraction changed from +54% to -86% during peak dopamine infusion. In conclusion, dopamine reversed cardiac failure in propoxyphene overdose by a marked positive inotropic stimulation restoring contractility. A marked positive chronotropic stimulation maintained a sufficient cardiac index and a normal blood pressure in spite of a profound vasodilatation which was unresponsive to dopamine.

Association of the M blood group system with bovine mastitis.

Associations of the 11 bovine blood group systems with mastitis were examined in Red Danish dairy cattle. The mastitis status was followed during three lactational periods. A significant effect of the M blood group system on mastitis incidence was observed in the first and second lactation periods and a lower frequency of mastitis is found among animals lacking the M' factor as compared to those having the M' blood group factor. The significance of these results are discussed in view of the close relation between the M blood group system and the bovine lymphocyte antigens (BoLA), and the expected effect of eliminating the M' gene from the breed is estimated. Among the remaining 10 blood group systems, the T' system was the only system showing an overall effect on mastitis, and only in first and third lactation. However, the T' system was inconsistent with regard to the effect of the T' gene on the various mastitis diagnoses.

Cardiac pacing and central hemodynamics in experimental propoxyphene induced shock.

Coronary sinus pacing was evaluated in 10 pigs with propoxyphene induced cardiac failure. During the early phase of intoxication cardiac pacing improved cardiac function slightly but significantly worsened it in severely intoxicated animals. The results are consistent with marked negative inotropic action of propoxyphene in overdose.