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Biomedical subjects

J Wessels

Publications and source records attributed to J Wessels.

At least 37 records · Page 2Linked to original sources
PubMed1991

Differences in repair of radiation induced damage in two human tumor cell lines as measured by cell survival and alkaline DNA unwinding.

We studied the relationship between the repair of radiation induced DNA strand breaks and cellular repair kinetics in two human tumor cell lines, NB-100 (neuroblastoma) and HN-1 (squamous cell carcinoma). Damage was quantified using the fluorometric analysis of DNA unwiding (FADU) for DNA damage, and cell survival was assessed using a clonogenic assay. In plateau phase cells repair of sublethal damage was virtually absent in NB-100 after 4 Gy (recovery ratio 1.0), whereas HN-1 cells did show sublethal damage repair (recovery ratio 1.4). Repair of potentially lethal damage was more pronounced in NB-100 cells (recovery ratio 2.3) than in HN-1 cells (recovery ratio 1.7) after 4 Gy. Graded doses of X-rays induced comparable levels of DNA damage in both tumor cell lines. However, in HN-1 cells more DNA strand breaks were repaired after 4 Gy, leaving about 25% of the initial damage unrepaired, whereas in NB-100 about 50% was unrepaired. This higher fraction of unrepaired DNA damage correlated well with the degree of sublethal damage repair which was lower in NB-100 than in HN-1 cell, but it did not correlate with the repair of potentially lethal damage, which was higher in NB-100 than in HN-1. Since the level of damage remaining post-irradiation may be the critical variable for survival, the FADU technique can contribute in elucidating the relationship between radiosensitivity and DNA damage repair capacity.

Alkalies↗
PubMed1990

Allogeneic bone marrow transplantation for leukemia with marrow grafts depleted of lymphocytes by counterflow centrifugation.

Eighty consecutive patients were transplanted with human leukocyte antigen (HLA)-identical sibling marrow for acute myelogenous leukemia (AML, N = 29), acute lymphoid leukemia (ALL, N = 23), or chronic myelogenous leukemia (CML, N = 28). Donor marrow was depleted of lymphocytes using counterflow centrifugation. Median age of the recipients was 31 years. Pretransplant conditioning consisted of cyclophosphamide and fractionated total body irradiation (TBI) with a low (4.1 +/- 0.3 cGy/min) or high (13.1 +/- 1.6 cGy/min) midline average dose rate. In 43 patients, cytosine-arabinoside or anthracyclines were added to the conditioning regimen. Immunoprophylaxis posttransplant consisted of methotrexate (MTX) alone, cyclosporine A (CsA) in combination with MTX, or CsA alone; two patients received no immunoprophylaxis at all. Graft failure occurred in 4 of 77 evaluable patients (5%). The probability of acute graft-versus-host disease (GVHD) greater than or equal to grade 2 at day 100 after transplantation was 15%. The projected 3-year estimate of extensive chronic GVHD was 12%. Only three patients died of cytomegalovirus-interstitial pneumonitis. The projected 3-year probability of relapse was 30% (95% confidence interval [CI], range 8% to 53%) in transplants for AML in first complete remission (CR1), 35% (95% CI, 1% to 69%) after transplantation for ALL in CR1, and 38% (95% CI, 2% to 74%) after transplantation for CML in first chronic phase (CP1). The projected 3-year probability of leukemia-free survival (LFS) was 56% (95% CI, 35% to 77%) after transplantation for AML-CR1, 42% (95% CI, 16% to 69%) in patients transplanted for ALL-CR1, and 49% (95% CI, 18% to 80%) after transplantation for CML-CP1. After transplantation for AML-CR1, ALL-CR1, or CML-CP1, the median follow-up time for leukemia-free survivors was 31+, 30+, and 21+ months, respectively. Probabilities of relapse, survival, and LFS in AML-CR1 and ALL-CR1 transplants were comparable with those reported in recipients of untreated grafts. In patients transplanted for CML-CP1, probability of relapse was higher and probability of LFS was lower than in recipients of untreated grafts. In transplants for leukemia in CR1 and CP1, preparative regimen and immunoprophylaxis posttransplant were not associated significantly with the probability of acute GVHD greater than or equal to grade 2, extensive chronic GVHD, relapse, survival, or LFS. In bone marrow transplantation for leukemia, counterflow centrifugation is a useful technique for the prevention of GVHD.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1990

DNA strand breaks in human leukocytes induced by chemotherapy and total body irradiation.

The occurrence of DNA strand breaks and/or DNA alkali-labile sites in peripheral blood leucocytes was demonstrated ex vivo in three patients during and after bone marrow ablative chemotherapy and total body irradiation (TBI) with use of fluorometric analysis of the DNA unwinding rate in alkaline solution (FADU assay). DNA damage was apparent after cyclophosphamide administration and after TBI, related to the amount of the applied dose. In vivo repair occurred within 24 hours, although not to pretreatment values. Demethoxydaunorubicin and busulfan at the dosages used did not induce measurable DNA strand breaks. The experiences described may be developed further to study ex vivo the occurrence of DNA lesions in patients during and after anticancer treatment. Such studies may be of value in comparing the DNA damaging potential of different chemotherapeutic or radiotherapeutic regimens and as a biological assessment of DNA damage after nuclear casualties in cases where the dose is greater than 1-2 Gy and measurement can be made within due time after the ionizing exposure.

Adult↗
PubMed1988

GM-CSF enhances sensitivity of leukemic clonogenic cells to long-term low dose cytosine arabinoside with sparing of the normal clonogenic cells.

Leukemic clonogenic cells (CFU-L) and normal myeloid progenitor cells (CFU-GM) were exposed to Ara-C in the presence of crude CSF obtained from placentas (HPCM) or recombinant human GM-CSF for varying periods. The cytotoxicity of Ara-C to CFU-L increased considerably when the exposure time to Ara-C in the presence of HPCM was extended from 20 hours to 10 days. The ID50 of the CFU-L was 1.5 +/- 2.2 x 10(-8) M Ara-C compared to 5.5 +/- 2.9 x 10(-8) M Ara-C for the CFU-GM after an exposure to Ara-C for 10 days (p less than 0.05). Replacement of crude CSF from placenta conditioned medium by rh GM-CSF resulted in identical observations. Interesting was the observation that secondary leukemic colony forming cells were more or at least equally sensitive to Ara-C in the presence of GM-CSF when compared to the primary leukemic clonogenic cells. This contrasted the secondary normal CFU-GM, which were less sensitive to Ara-C than the primary CFU-GM. This indicates that GM-CSF induces leukemic clonogenic cells with selfrenewal capacity into proliferation, and in doing so, it may enhance the cytotoxicity of a cell cycle specific drug like Ara-C with sparing of the normal clonogenic cells.

Bone Marrow↗
PubMed1987

Infusion-rate independent cellular adriamycin concentrations and cytotoxicity to human bone marrow clonogenic cells (CFU-GM).

The effect of adriamycin (ADM) infusion-rate on cellular ADM concentrations and on clonogenicity of human haematopoietic cells was studied in vivo and in vitro. In patients an ADM dose of 30 mg m-2 was administered as a bolus injection, or as a 4 h or a 24 h infusion. In vitro the effect of ADM on clonogenic cell growth was determined after exposure during 5 min, 2 h and 24 h of human bone marrow cells to increasing ADM concentrations. ADM showed rapid intracellular accumulation, to levels 100-fold the plasma concentration in vivo or the incubation medium concentration in the in vitro experiments. After a bolus injection or 5 min exposure only approximately 10% of the cellular peak ADM was retained after elimination of the drug from the plasma or the incubation medium. Ninety percent of the ADM was apparently 'loosely' bound. After 4 h and 24 h constant-rate infusions and also after 2 h and 24 h incubations in vitro, the cells accumulated ADM gradually, and the subsequent washing-out of the cellular ADM was substantially less, most of the ADM being 'tightly' bound. Despite these different patterns of uptake and retention after in vivo short- and long-lasting infusion of the same total dose, the 'tightly-bound' cellular ADM concentrations were the same. Moreover, comparable cellular ADM concentrations, retained after efflux of the 'loosely-bound' cellular ADM fraction were equally cytotoxic to normal human clonogenic cells. Short-lasting cellular peak ADM concentrations which occur after a bolus injection or after short exposure to high ADM concentrations are not essential for the cytotoxic effect, in contrast to the retained, 'tightly-bound' cellular ADM levels.

Bone Marrow↗
PubMed1987

A simple method to obtain low density marrow cells for human marrow transplantation.

Removal of more than 99% of the erythrocytes and 74% of the nucleated cells from marrow grafts was achieved by density floatation separation in Percoll gradients with a density of 1.070 g/ml in eight 250-ml tubes, containing up to 3 X 10(9) nucleated cells per gradient. More than 90% of the myeloid and erythroid progenitor cells were recovered in the low density fraction. It appeared mandatory to use a centrifuge with the possibility of a gradual acceleration and deceleration. Twenty-five patients received a marrow graft from a histocompatible sibling after additional lymphocyte depletion by counterflow centrifugation, and 5 patients with T lymphoblastic malignancies received an autograft after in vitro purging with immunotoxins. All evaluable patients engrafted within normal limits, except 1 patient with an autoimmune pancytopenia who responded to steroids and 1 patient with a CMV infection. Four patients died too early for complete evaluation. The described separation method is easy, cheap and requires only 2 h for the complete processing of a marrow graft.

Bone Marrow Transplantation↗
PubMed1987

Influence of dose and duration of exposure on the cytotoxic effect of cytarabine toward human hematopoietic clonogenic cells.

The cytotoxic effect of cytarabine (ara-C) on the clonogenic potential of human normal and leukemic hematopoietic cells was investigated. Cells were exposed to ara-C at different concentrations and for periods of one to 120 hours and continuously. Colony growth (CFU-GM/CFU-E/BFU-E) in semisolid culture was assessed after seven and 14 days. The exposure time to ara-C appeared much more important in colony growth inhibition than the drug concentration. For instance, one-hour exposure to 10(-5) mol/L ara-C appeared not cytotoxic, while cocultivation in the presence of 10(-7) mol/L ara-C resulted in a total inhibition of colony growth. Cell fractionation with use of counterflow centrifugation allowed enrichment in subfractions for cells with different amounts of DNA. The inhibition of the clonogenic potential by ara-C was most pronounced in the cell fractions with the highest proliferation activity. Colony-forming cells, assessed after 14 days of culturing appeared less sensitive for ara-C cytotoxicity than seven-day CFUs. This means that ara-C is more cytotoxic to cycling than noncycling cells because compared to the seven-day colonies, 14-day colonies originate from the more resting, more primitive progenitor cells. The cytotoxicity of ara-C to clonogenic cells appears to be related to the proliferating and the cycling state of the progenitor cells and increases with the time of exposure. This phenomenon may be explained by the fact that ara-C is a cycle-specific drug and by the fact that the probability of cells entering the cell cycle is a time-dependent process. The results of this study indicate that development of more effective antileukemic therapy should not aim so much at very high drug levels but rather at prolonged administration of ara-C.

Bone Marrow Cells↗
PubMed1986

The relation of exposure time and drug concentration in their effect on cloning efficiency after incubation of human bone marrow with cytosine arabinoside.

The effect of different cytosine arabinoside (Ara-C) concentrations and exposure times upon clonogenicity of normal bone marrow cells was studied. One hour incubation, even at the pharmacologically high Ara-C concentration of 10(-5) M, was not cytotoxic. Prolonged incubation resulted in a very great increase in Ara-C cytotoxicity, provided that inactivation of Ara-C by deamination was limited. The results show that long-term exposure to Ara-C, even at pharmacologically very low concentrations, is deleterious to clonogenic cells and provide an experimental basis for the reported therapeutic efficacy of long-term low dose Ara-C administration in leukaemia and preleukaemic syndromes.

Bone Marrow↗
PubMed1986

Enrichment of human bone marrow aspirates for low-density mononuclear cells using a haemonetics discontinuous blood cell separator.

Isopycnic density floatation centrifugation has been proven to be a suitable technique to enrich bone marrow aspirates for clonogenic cells on a small scale. We have tested a Haemonetics semicontinuous blood cell separator in order to process large volumes of bone marrow with minimal bone marrow manipulation. The efficacy of isopycnic density floatation was tested in a one and a two-step procedure. Both procedures showed a recovery of about 20% of the nucleated cells and 1-2% of the erythrocytes. The enrichment of clonogenic cells in the one-step procedure appeared superior to the two-step enrichment, first separating buffy coat cells. The recovery of clonogenic cells was 70 and 50%, respectively. Repopulation capacity of the low-density cell fraction containing the clonogenic cells was excellent after autologous reinfusion (6 cases) and allogeneic bone marrow transplantation (3 cases). Fast enrichment of large volumes of bone marrow aspirates with low-density cells containing the clonogenic cells by isopycnic density floatation centrifugation can be done safely using a Haemonetics blood cell separator.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1986

Depletion of donor lymphocytes by counterflow centrifugation successfully prevents acute graft-versus-host disease in matched allogeneic marrow transplantation.

Bone marrow from 22 histocompatible siblings was depleted of 98% of the lymphocytes using a combination of density flotation centrifugation followed by counterflow elutriation. Even with the marrow suppressive influence of methotrexate (MTX), the viability of the hematopoietic stem cells was not affected, as indicated by the normal repopulation after grafting in the evaluable patients. One patient (UPN 9) showed a primary graft failure, possibly resulting from persisting septicemia and long-term antibiotic therapy. Two patients have persistent host lymphocytes, one of whom was examined during relapse; the other remains in remission. Two patients did not receive immunosuppression after bone marrow transplantation (BMT), and acute graft-v-host disease (GVHD) developed in both. Nine patients received MTX as immunosuppression following BMT. GVHD did not develop in any of them, but fatal infections in the immediate posttransplant period developed in five patients. Eleven patients received cyclosporine (CsA) after transplantation. Beginning in week 5 after BMT, CsA was gradually replaced by MTX. Acute GVHD, substantial chronic GVHD, or fatal infections did not develop in any of these patients. Removal of 98% of the lymphocytes by counterflow centrifugation prevents development of acute GVHD, provided that immunosuppression is administered after BMT. Graft rejection was not observed, but the number of evaluable patients is limited at present.

Adolescent↗
PubMed1985

Influence of peripheral blood admixture on the number of hematopoietic progenitor cells (CFU-GM and BFU-E) in human bone marrow aspirates.

Peripheral blood contamination in human bone marrow aspirates was calculated from the ratios between the erythrocyte and nucleated cell counts in both the bone marrow aspirate and a simultaneously obtained peripheral blood sample. This method is based upon experimental data which showed that the erythrocytes in bone marrow aspirates are mainly derived from the intravascular blood compartment. A strong negative correlation was found between the peripheral nucleated cell fraction (FBl) and the number of myeloid progenitor cells in 65 bone marrow samples (correlation coefficient r = -0.51; p less than 0.001). A similar correlation was found between erythroid progenitor cells and peripheral blood fraction (r = -0.55; p less than 0.01). The culture conditions were continuously monitored, using large batches of frozen marrow samples as controls. The correction for peripheral blood admixture permits a more reliable and reproducible interpretation of the quantitative results obtained from studies on the number of clonogenic cells in human bone marrow aspirates. Moreover, the method allows the interpretation of data obtained from bone marrow samples which are heavily contaminated by peripheral blood.

Biopsy, Needle↗
PubMed1985

Binding characteristics of three complement dependent assays for the detection of immune complexes in human serum.

The fluid phase C1q binding, the solid phase C1q binding and the Raji cell assay are the most widely employed methods for the detection of complement fixing immune complexes in human disease. However, their binding characteristics for complexes formed in native serum have not been compared and studied in detail. For this purpose, Tetanustoxoid (TT): anti-TT complexes were formed closely to in vivo conditions by incubating sera of immunized people with TT. Then the molecular characteristics of those complexes, which could be detected by the 3 immune complex assays, were defined. The methods used are precipitation curve, gel chromatography and measuring the complexed antibody with a sensitive Elisa method. All 3 assays detected only complexes near the equivalence zone; the Raji cell assay being the most sensitive detecting 1.5 micrograms complexed antibody/ml serum followed by the solid phase (6 micrograms/ml) and the fluid phase C1q binding assay (50 micrograms/ml). As estimated by gel chromatography, both the C1q binding assays measured most sensitively complexes with a molecular weight distinctly over 2,000,000 daltons, whereas the Raji cell assay detected preferentially complexes in the range of 2,000,000 daltons. Smaller TT: anti-TT complexes with a molecular weight less than 600,000 daltons were not detectable by the 3 assays. In conclusion, the 3 assays, while differing in sensitivity, showed similar binding patterns with preference for high molecular complexes near equivalence. Subsequently, they might be insensitive to small complexes persisting in the circulation.

Antigen-Antibody Complex↗
PubMed1984

Separation of immunoreactive lymphocytes from human pluripotent stem cells (CFU-GEMM) by means of counterflow centrifugation.

Counterflow centrifugation with continuous monitoring of the output for cell number and cell scatter was used to separate low density (d less than 1.070 g/ml) human bone marrow cells in two fractions: one containing the majority of small size lymphocytes and the other the majority of the larger sized committed progenitor cells. The recovery of the pluripotent stem cells (CFU-GEMM) in the large cell fraction was complete. The mitogenic reactivity of this putative stem cell fraction had decreased to 6% and 11%, of the original value as measured with phytohemagglutinin stimulation and one way mixed lymphocytic culture respectively. Counterflow centrifugation offers a physical separation technique, by which the majority of the immunoreactive cells can be separated from the pluripotent hematopoietic stem cells.

Bone Marrow Cells↗
PubMed1984

Separation of human bone marrow by counterflow centrifugation monitored by DNA-flowcytometry.

Human bone marrow was fractionated by counterflow centrifugation into 16 fractions with increasing cell size. Three distinct subpopulations could be recognized: small lymphocytic cells, medium-sized nucleated erythroid cells and large myeloid elements. DNA-flowcytometry and 3H-thymidine uptake showed that within the erythroid and myeloid cell populations counterflow centrifugation separates each population according to the cell cycle phase. Hypotonic treatment of bone marrow for removal of the erythroid nucleated cells resulted in a complete abrogation of the proliferating erythroid cell population. Counterflow centrifugation also separates the small non-proliferating myeloid and erythroid committed stem cells from the larger proliferating stem cells. It appeared feasible to separate the small lymphocytic cells from the majority of BFU-E and CFU-GM, due to the larger size of the proliferating normoblasts and the committed progenitor cells. Elimination of the mature lymphocytes from the haematopoietic stem cells by counterflow centrifugation may offer an alternative approach to the prevention of graft versus host disease (GvHD).

Bone Marrow↗