Biomedical subjects

M Hokland

Publications and source records attributed to M Hokland.

At least 37 records · Page 2Linked to original sources

PubMed1998

Modulatory function on autologous myeloid progenitor cells of clonal T-lymphocytes following autologous bone marrow transplantation.

We have studied the regulatory capacity of clonal T-lymphocytes from patients undergoing autologous bone marrow transplantation (ABMT) on the generation of CFU-GM from their harvest marrow preparations. To this end, T-lymphocytes from peripheral blood from 5 patients undergoing ABMT isolated 10 d before and 7, 14 and 28 d post-ABMT were placed in limiting dilution conditions (384 wells for each patient at each time point) and polyclonally stimulated. From more than 1600 wells with growth from the 5 patients, preparations from more than 900 wells could be expanded (range between patients 33-452) and identified by immunophenotyping (IP) and flow cytometry (FCM) by their exclusive expression of CD4 or CD8. This was significantly fewer than seen in normal donors, especially so at d 7 and 14 post-ABMT The ratio between CD4+ and CD8+ clones varied between 0.6 and 2.8 (median 1.3) and was significantly lower in the patients compared to normal donors (median 3.1; range 3.0-6.5). When the clonal T-cell preparations were co-cultured with autologous bone marrow cells obtained at the time of harvest and depleted for T-lymphocytes, the vast majority of both CD4+ and CD8+ clones exerted a clear enhancement on the CFU-GM growth with no relation to time of blood sampling in its the magnitude. Moreover, a trend seen in the normal donors towards CD4+ clones being more effective in this enhancement was not observed in ABMT patients. We conclude that clonal T-cells from ABMT patients, irrespective of their phenotype and time of isolation, exert an enhancement on the growth of autologous CFU-GM, which is equal to that seen in normal donors.

Adult↗

PubMed1997

High dose IL-2-activated murine natural killer (A-NK) cells accumulate glycogen and granules, lose cytotoxicity, and alter target cell interaction in vitro.

Activated natural killer (A-NK) cells, defined by immunophenotype and selected by adherence to the plastic, were cultured from murine splenocytes for up to 10 days with the addition of 1000 U/ml of recombinant human IL-2 at 48 h intervals. During culture days 2-4 with high DNA synthesis the initially non-granulated small cells established large granular lymphocyte (LGL) morphology and then differentiated further into giant hypergranulated cells with huge accumulations of glycogen. Timed EM observations indicated that specific dual-compartment (lytic) granules arose by a sequence of events starting with neo-synthesis of small progenitors with a dense core and a few membranous lamellae at one pole. Core and vesicular regions probably expanded independently to give the mature organization of the granule. Eventually, the vesicular region of granules contained large amounts of multi-lamellar material and probable debris, and the dense core could be multiplied. Intracellular proteoglycans, visualized with Cupromeronic Blue cytochemistry, were organized in a three-dimensional network within the dense cores. In contrast with earlier reports, and in spite of several-fold increased granularity, the in vitro cytotoxicity of the A-NK cells against YAC-1 and B16 cells decreased after the third day of culture. A-NK cells with glycogen accumulations caused focal clearing in melanoma monolayers whereas younger effectors adhered to the targets. It is concluded that high dose IL-2 stimulation causes more far-going progressive morphological and functional differentiations of the A-NK cells than has previously been observed with bearing for the use of these cells in experimental adoptive immunotherapy.

Animals↗

PubMed1997

Expression of adhesion and activation molecules on lymphocytes during open-heart surgery with cardiopulmonary bypass.

Open-heart surgery with cardiopulmonary bypass (CPB) and abdominal surgery are associated with lymphocytopenia. We measured a panel of adhesion and activation molecules on lymphocytes to clarify possible association of CPB with increased expression of these molecules. Eight patients undergoing open-heart surgery and eight with abdominal surgery were studied. The adhesion molecules CD11a/CD18 (LFA-1_, CD11c/CD18 and CD44 and the activation molecules CD25, CD69, CD71 and MHCII were measured, using monoclonal antibodies and flow cytometry. Lymphocytopenia was observed during CPB and for some hours after both open-heart and abdominal surgery. The proportion of CD11a/CD18-positive lymphocytes rose from 67.6 +/- 8% to 86.4 +/- 3% after aortic declamping (p < 0.05). The expression of activation molecules CD25, CD69 and CD71 was unchanged during and after open-heart as well as abdominal operations. Thus CPB was associated with increased expression of the adhesion molecule CD11a/CD18 on lymphocytes, while the expression of activation molecules on lymphocytes was unchanged.

CD18 Antigens↗

PubMed1996

A randomized controlled study of the effect of bedside leucocyte depletion on the immunosuppressive effect of whole blood transfusion in patients undergoing elective colorectal surgery.

In a randomized study the effect of whole blood transfusion versus bedside leucocyte-depleted blood transfusion on lymphocyte proliferation, CD4+:CD8+ ratio, and levels of soluble interleukin 2 receptor (sIL-2R) and interleukin (IL) 6, as well as on the development of postoperative wound infection and intra-abdominal abscess, was assessed in 60 patients undergoing elective colorectal surgery. Transfusion with whole blood induced a significant decrease in lymphocyte proliferation and CD4+ :CD8+ ratio (P < 0.01) as well as a significant increase in sIL-2R and IL-6 levels (P < 0.01). Furthermore, transfusion with whole blood was accompanied by a significant increase in postoperative infectious complications (P < 0.01). In patients transfused with leucocyte-depleted blood only slight and transient changes were observed, which were not significantly different from those observed in non-transfused patients.

Adult↗

PubMed1996

Myeloid progenitors in remission bone marrow in patients with malignant blood diseases: clues for slow hematopoietic regeneration after ABMT?

Slow hematopoietic recovery is a well-known feature in patients undergoing autologous bone marrow transplantation (ABMT), and here we demonstrate that compared with patients with other malignant blood diseases, the thrombocytopenia in acute myeloid leukemia (AML) patients stands out, with the median time to recovery of > 50 x 10(9) platelets being 101 days for AML patients and 22-37 days for other patient groups. We have consequently evaluated the content and fate of myeloid progenitor cells (BFU-E, CFU-GM, and CFU-GEMM) in bone marrow preparations from cancer patients in an attempt to uncover factors of importance for their slow hematopoietic recovery after ABMT using three experimental setups. First, we analyzed progenitors in marrow samples from 36 patients [18 AML, 5 acute lymphoblastic leukemia (ALL), and 13 non-Hodgkin's lymphoma (NHL)] exposed to multiple doses of severely myelotoxic cytoreductive regimens and observed that AML patients had suppressed numbers of CFU-GEMM and BFU-E (80% that of normal volunteers) but increased levels of CFU-GM. In contrast, the decrease in ALL patients was more pronounced (50% that of normal volunteers) and demonstrable for all progenitors. NHL patients exhibited normal progenitor frequencies of CFU-GM and CFU-GEMM, whereas their BFU-E counts were found to be increased nearly two-fold. Second, we followed the fate of progenitors through the laboratory manipulations of marrow grafts and found no evidence for selective losses in terms of disease (7 AML, 5 NHL, 1 ALL, and 2 testicular germ cell tumor patients) and progenitor type. Third, we retrospectively evaluated the cell yields of grafts from 121 cases [39 AML, 20 ALL, 22 NHL, 9 Burkitt's lymphoma (BL), 18 Hodgkin's disease (HD), and 13 testicular germ cell tumors] and found those of heavily treated AML, HD, and germinal tumors most affected, with a clear negative correlation in AML patients between age on the one hand and mononuclear cell yield and CFU-GM content on the other. We conclude that the amount of previous chemotherapy and age of patients appear to be among the determining factors on the number of myeloid progenitor cells in cancer patients and that thresholds for acceptability of grafts should take these variables into account.

Adolescent↗

PubMed1996

Distinct circulation patterns in peripheral blood of leukocyte subpopulations during the first 24 hours following autologous bone marrow transplantation.

We have studied the recirculation patterns of leukocyte subpopulations during the first 24 h at 5 min before and 5, 15, 180, and 1440 min after autologous bone marrow transplantation (ABMT) in 14 patients with cancer (6 with AML, 5 with malignant lymphomas, 2 with ALL, and 1 with Ewing's sarcoma) using multiparameter flow cytometry and measurements of myeloid progenitors (CFU-GM). Although the great majority of the injected cell populations were undetectable 5 min after graft infusion, the number of CD3+ T lymphocytes increased at 5 and 15 min and again at 24 h post-ABMT. In contrast, the number of CD56+ natural killer (NK) cells decreased rapidly after ABMT to remain low throughout the observation period. Mature myeloid and monocytic cells (identified by their expression of CD66 and CD14, respectively) were present before, as well as after ABMT in numbers indicating that they were probably of endogenous origin. Immature myeloid cells were identified in a three-color flow cytometric assay as CD13+ CD14- CD66- and tended to increase during the first 15 min after ABMT. Finally, when CFU-GM were followed longitudinally, they were found to be practically absent before ABMT but were clearly detectable in 12 of 14 patients throughout the observation period. We conclude that leukocyte subsets exhibit different recirculation patterns after ABMT, and in light of the increased knowledge about leukocyte-endothelial interactions, these data could provide a platform for attempts to control leukocyte recirculation during stem cell infusion.

Adolescent↗

PubMed1996

Little evidence for clonal evolution of malignant haematopoietic cells following relapse after autologous bone marrow transplantation.

By screening for immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements in bone marrow samples aspirated at different time points during the course of disease from 43 patients with acute leukaemia we have analysed the extent of clonal evolution after autologous bone marrow transplantation (ABMT) and addressed the issue of whether the Southern Blot method has the power to reveal clonal proliferations representing minimal residual disease (MRD) in the autologous bone marrow grafts. Our results show that no clonal proliferations were detectable in any of the 43 bone marrow grafts analysed, even after we analysed DNA preparations in 5 cases from cells highly enriched for cells of the original malignant immunophenotype. Moreover, as judged by the Ig- and TCR gene configurations in 11 patients, relapse arose from the original clone even though minor clonal variations did occur in about half of the relapsing patients. We conclude that while the Southern Blot method can detect gene receptor rearrangements in the majority of patients with acute leukaemias and high-grade non-Hodgkins lymphomas, it is not useful for predicting relapse after ABMT. On the other hand, it is possible-by employing it-to evaluate whether or not relapse after ABMT arises from the original malignant clone and to what extent clonal evolution has taken place.

Acute Disease↗

PubMed1996

Improved flow cytometric identification of myelopoiesis by the simultaneous labelling with CD13, CD14 and CD66 monoclonal antibodies.

The aim of the present study was to increase our knowledge of myelopoiesis evaluated by flow cytometry. We therefore designed a triple-marker assay employing monoclonal antibodies against the CD13 (immature), the CD14 (monocytic), and the CD66 (mature myeloid) antigens using three-colour immunofluorescence. In normal donor bone marrow the assay enables simultaneous identification of immature (CD13+, CD14-, CD66-), intermediate (CD13+, myelopoietic differentiation stages through the exclusion of CD14+ monocytic cells. In the diagnosis and longitudinal follow-up of AML patients the assay was of value in the fast determination of remission state. In MDS, the immature myeloid component could be distinguished in patients defined according to the FAB classification with the possibility of identifying aberrant phenotypes, the assay should also be of interest in other myeloproliferative disorders. Moreover, because it is easy to perform, time-saving, and yields comparable results to single antibody reactivity controls, it can replace more tedious and less-informative flow cytometric immunophenotyping procedures.

Acute Disease↗

PubMed1996

[Diagnosis of minimal residual disease in autologous bone marrow transplantation].

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.

Acute Disease↗

PubMed1995

DNA preparation from cryopreserved bone marrow cell samples.

1. Quickly thaw cryopreserved MNCs (1 ml) in 9 ml PBS supplemented with DNase I immediately before use (final concentration: 0.1 mg/ml). 2. Incubate at 37 degrees C for 10 min. 3. Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C. 4. Incubate with saturating amounts of selected MoAbs for 20 min at 4 degrees C. 5. Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C. 6. Immunomagnetic bead separation of viable cells as described by Lea et al. (1). 7. DNA extraction. 8. Measure quality and yield of DNA by spectrophotometry. In conclusion, the DNA extraction method presented here, based on DNase I pretreatment of the cryopreserved cells followed by an immunological selection for viable cells, provides cell suspensions with close to 100% viability; thus, high-quality DNA can be extracted even from cryopreserved cell samples of low initial viability. Furthermore, using this method DNA analyses can be performed on selected cellular subsets if desired. We thus recommend this method for DNA analyses in hematological research when the only materials available are cryopreserved bone marrow samples of low viability.

Bone Marrow↗

PubMed1995

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.

Adolescent↗

PubMed1995

Changes in lymphocyte subpopulations and adhesion/activation molecules following endotoxemia and major surgery.

Major surgery as well as endotoxin-induced sepsis is accompanied by lymphocytopenia in peripheral blood. The purpose of this study was to investigate the redistribution of lymphocyte subpopulations and adhesion/activation molecules on lymphocytes. Twenty-four rats were included in the investigation. Eight rats received an intraperitoneal injection of E. coli endotoxin (2 mg kg-1), eight rats had a sham operation performed while eight rats received isotonic saline and served as a control group. Blood samples were obtained by making an incision in the tail before and 2 and 5 h after surgery or administration of endotoxin or saline. After isolation of lymphocytes by gradient centrifugation, flow-cytometric immunophenotyping was performed using CD2, CD3, CD4, CD8, CD11/CD18, CD20, CD44 and MHC II monoclonal antibodies. Endotoxemia and surgery were both accompanied by increased serum cortisol, lymphocytopenia, and a decrease in CD2, CD3 and CD4 lymphocytes. Only endotoxemia was followed by a decrease in CD8, CD11/CD18 and CD44 lymphocytes in peripheral blood. Our results show that several of the changes in lymphocyte subpopulations following surgery and sepsis are associated with increased serum cortisol. Sepsis is, in addition, accompanied by an upregulation of adhesion receptors.

Animals↗

PubMed1995

Natural killer cells in peripheral blood after autologous bone marrow transplantation: a combined phenotypic and functional study.

The peripheral blood regeneration of natural killer (NK) cells was studied before and on 5 occasions during the first 6 weeks after autologous bone marrow transplantation (auto-BMT) in 10 patients with hematological malignancies and solid tumors. The number of NK cells (relative as well as absolute), enumerated by their lack of CD3 and their expression of CD56 recovered after a severe decline 1 week posttransplant to increase beyond pretransplantation levels during the next 2 weeks. Similar patterns were seen for the average NK activity against K562 as well as LAK cell lytic activity against Daudi but without the overshoot at weeks 2-3. Moreover, the fraction, but not the absolute numbers, of NK cells was found to correlate to the lytic NK activity. In contrast, no phenotypic marker was correlated to LAK activity. Immunophenotypic studies using three-color flow cytometry revealed that during the first 2 weeks after auto-BMT the phenotype of NK cells changed towards an immature phenotype (decreased CD45RA and CD11a) 1 week after transplant to an activated (increase in CD25, HLA-DR and CD11c) after 2-4 weeks. However, when the absolute number of selected phenotypically defined NK cell subsets(CD45RA, CD45RB, CD11a, CD11c, CD2, CD25, HLA-DR and fibronectin expressing CD56+,CD3- NK cells) were compared to the cytotoxic activity for each patient, we were not able to show any correlations except between the activation-related antigens CD25 and HLA-DR on the one hand, and NK lysis on the other, and only 3 weeks after auto-BMT. We conclude that NK cell function recovers quickly following auto-BMT concomitantly with the emergence of a transiently altered phenotype which increased expression of activation-related antigens.

Adult↗

PubMed1993

A quantitative evaluation of erythropoiesis in myelodysplastic syndromes using multiparameter flow cytometry.

By staining human bone marrow cells with a monoclonal antibody reacting with erythroid precursor cells (AS-E1) and propidium iodide, we have evaluated the proliferative capacity of erythropoiesis in patients with myelodysplastic syndromes (MDS) using flow cytometry. Comparing 36 patients (13 RA/RAS, 13 RAEB, 10 RAEB-t) with 7 normal controls, significant differences in both the percentage of AS-E1+ cells and the fraction of AS-E1+ cells in the S or S-G2M-phase between the four groups were found. Since neither the percentage of AS-E1+ cells nor their fraction in S or S-G2M alone was found to characterize their proliferative activity, we introduced the proliferative fractions of the erythroid cell, i.e. the number of the AS-E1+ cells in S or S-G2M related to all bone marrow cells in S or S-G2M. Applying these parameters, we found significantly increased proliferative AS-E1 fractions in the RA/RAS group compared to the normal controls (p = 0.03 and 0.002) respectively, as well as a highly significant decrease with disease progression.

Antibodies, Monoclonal↗

PubMed1993

Expression and regulation of an interferon-alpha-inducible membrane protein p106 on human hematopoietic cells.

p106 is a human membrane protein of 106 kD previously shown to be inducible by interferon-alpha (IFN-alpha) on Daudi cells. To investigate the role of p106 further, its distribution and inducibility within hematopoietic cells was studied. Multiparameter flow cytometry (FCM) analysis showed that p106 expression was restricted to B cells and monocytes, and in both cell lineages acquired at a late stage of differentiation. Thus, p106 was found on mature B lymphocytes and monocytes in peripheral blood and on a variety of freshly isolated leukemic cells of B and myeloid origin as well as on a variety of cultured B-cell lines. In contrast, no expression was found on T lymphocytes, natural killer (NK) cells or granulocytes. p106 expression could be further induced by IFN-alpha on monocytes and Daudi cells, and this capacity was shown to be selective for IFN-alpha, since no other cytokines tested induced p106. Moreover, IFN-alpha therapy of chronic myeloid leukemia (CML) and hairy cell leukemia (HCL) patients lead to a clearcut induction of p106 on such malignant cells. The distribution of p106 could suggest that it represents an activation antigen. Further studies, including cloning of p106 cDNA, are needed to determine the function of p106.

Cytokines↗

PubMed1992

Postoperative infection and natural killer cell function following blood transfusion in patients undergoing elective colorectal surgery.

The frequency of infection in 197 patients undergoing elective colorectal surgery and having either no blood transfusion, transfusion with whole blood, or filtered blood free from leucocytes and platelets was investigated in a prospective randomized trial. Natural killer cell function was measured before operation and 3, 7 and 30 days after surgery in 60 consecutive patients. Of the patients 104 required blood transfusion; 48 received filtered blood and 56 underwent whole blood transfusion. Postoperative infections developed in 13 patients transfused with whole blood (23 per cent, 95 per cent confidence interval 13-32 per cent), in one patient transfused with blood free from leucocytes and platelets (2 per cent, 95 per cent confidence interval 0.05-11 per cent) and in two non-transfused patients (2 per cent, 95 per cent confidence interval 0.3-8 per cent) (P less than 0.01). Natural killer cell function was significantly (P less than 0.001) impaired up to 30 days after surgery in patients transfused with whole blood. These data provide a strong case against the use of whole blood transfusion in patients undergoing elective colorectal surgery.

Adult↗

PubMed1992

Tissue distribution of adoptively transferred adherent lymphokine-activated killer cells assessed by different cell labels.

Assessment of the tissue distribution of adoptively transferred adherent lymphokine-activated killer A-LAK) cells by use of 51Cr indicated that these effector cells, after an initial phase in the lungs, distributed in high numbers to liver and spleen (30% and 10% of injected dose, respectively). However, when this experiment was repeated with 125IdUrd as cell label, fewer than 2% and 0.5% of the injected cells distributed into liver and spleen respectively. To analyse this discrepancy, we compared the tissue distribution of 51Cr- and 125IdUrd-labelled A-LAK cells with that indicated by alternative direct visual methods for identification of the injected cells, such as fluorescent dyes (rhodamine and H33342) or immunohistochemical staining of asialo-GM1-positive cells. The number of i.v. injected A-LAK cells found in the liver by all visual methods ranged from 1% to 5% of the injected dose, supporting the data obtained with 125IdUrd, whereas 25%-30% of the 51Cr label was consistently found in this organ. Autoradiography of the liver 24 h after i.v. injection of 51Cr-labelled cells revealed a background activity that was four- to fivefold higher than the control level, indicating substantial non-specific accumulation in the liver of 51Cr released from A-LAK cells. We conclude that 51Cr cannot be reliably used in investigations of cell traffic to the liver because of non-specific accumulation of the 51Cr label, particularly in this organ. In contrast, labelling with 125IdUrd or rhodamine and immunohistochemical staining of asialo-GM1-positive cells appear to be reliable and essentially equivalent methods for investigations of the fate of adoptively transferred A-LAK cells. Using these methods, we found that only few A-LAK cells redistribute to the liver upon i.v., i.e. systemic, injection, whereas 40%-50% of locally (intraportally) injected A-LAK cells remain in the liver for at least 24 h.

Animals↗

PubMed1992

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)

Burkitt Lymphoma↗