PubMed Health⌕ Search

Biomedical subjects

J M Wessels

Publications and source records attributed to J M Wessels.

At least 37 records · Page 2Linked to original sources
PubMed1988

Relationship between internalization and cytotoxicity of ricin A-chain immunotoxins.

Immunotoxins (ITs) appear to vary considerably in their killing efficiency towards antigen positive cells. In order to unravel the mechanisms underlying these differences, the parameters responsible for these variations were studied. The efficacy of the monoclonal antibodies (MoAbs) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7) and WT82 (CD8) conjugated to ricin A-chain was expressed by the extent of protein synthesis inhibition of four leukaemic T cell lines (CEM, GH1, Jurkat and HPB-ALL). Large differences in cytotoxicity were observed. Efficient inhibition of protein synthesis was seen with anti-CD3 IT, anti-CD5 IT and anti-CD7 IT. In these cases the cytotoxicity was related to the antigen density on the target cell membrane. Anti-CD4 IT inhibited poorly and anti-CD8 IT was ineffective, even on cell lines with a high expression of the corresponding antigen. When antigen density and cytotoxicity were plotted for all CD antigens, no correlation could be found. Subsequently, internalization was studied with 125Iodine-labelled antibodies. Anti-CD7 showed the fastest internalization rate, followed by anti-CD3 and anti-CD5. Anti-CD4 and anti-CD8 antibodies were respectively moderately and hardly internalized. When the absolute amount of internalized MoAb was calculated, a highly significant correlation with cytotoxicity was found. We conclude that the degree of antigen expression is not so important as the absolute amount of antibody internalized in predicting the efficacy of ITs.

Antibodies, Monoclonal↗
PubMed1988

Different susceptibilities of normal T cells and T cell lines to immunotoxins.

In the context of ex vivo T cell elimination from bone marrow, the anti-T cell cytotoxic potential of immunotoxins (IT) prepared by conjugation of the monoclonal antibodies (MoAb) WT32 (CD3), T101 (CD5), and WT1 (CD7) to ricin A chain was evaluated. The cytotoxicity of IT was based on protein synthesis inhibition in human T cell lines: GH1, CEM, HPB-ALL, and Jurkat, and appeared closely related to the antigen density and internalization rate of and appeared closely related to the antigen density and internalization rate of the IT. Normal unstimulated T cells appeared to be rather insensitive to IT not due to a low antigen density or decreased internalization. The cytotoxicity of IT to T cells could be enhanced considerably by NH4Cl. Treatment of T cells with a cocktail of IT (10(-8) M) and 20 mM NH4Cl resulted in a 5000-fold cytoreduction as measured by clonogenic assays of limiting T cell dilutions, whereas the haematopoietic progenitor cells remained unaltered. Stimulation of T cells with phytohaemagglutinin (PHA) prior to incubation with IT considerably increased the sensitivity to IT treatment. Thus, normal T cells are less sensitive to anti-T cell IT than T cell lines and activated T cells. This suggests that a low protein synthesis is responsible for the resistance to IT. However, a high specific cytotoxicity of IT to normal T cells can be achieved in the presence of 20 mM ammonium chloride.

Ammonium Chloride↗
PubMed1988

Human T lymphocyte differentiation antigens as target for immunotoxins or complement-mediated cytotoxicity.

Graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) is initiated by immunocompetent T cells present in the graft. Selective elimination of distinct T-cell subsets or a sufficient, but not complete T-cell depletion, might abolish severe GVHD without graft rejection and loss of the anti-tumour potential. In this study we analysed the efficacy of different monoclonal antibodies (MoAb) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7), and WT82 (CD8) with respect to their cytotoxicity to T cells either as immunotoxin (IT) or in combination with complement. The cytotoxic potential was assessed by protein synthesis inhibition and clonogenic assays. The ricin A conjugated MoAb exerted only a minor effect on blood or bone marrow T cells, although they were highly inhibitory to T-cell lines. However, in the presence of 20 mM ammonium chloride, IT directed against CD3, CD5, and CD7 were highly cytotoxic. IT directed against CD4 and CD8 were less effective, due to a low internalization. The complement-mediated cytotoxicity was efficient for all antigens used. The natural killer (NK) activity, as measured by cytotoxicity to K562, was hardly depressed by anti-CD3, anti-CD4, anti-CD5, and anti-CD8, but was eliminated by anti-CD7. All procedures used had only a minimal effect on haematopoietic progenitors as measured by CFU-GM and BFU-E assays. We concluded that, although the T-cell population can be eliminated with the combination of anti-CD3, anti-CD5, and anti-CD7 antibodies plus complement, IT with 20 mM NH4Cl appear to kill higher amounts of T cells. Selective elimination of CD4- and CD8-positive cells is effectively obtained by MoAb with complement.

Antibodies, Monoclonal↗
PubMed1987

Determination of amsacrine in human nucleated hematopoietic cells.

A new method has been developed for the determination of amsacrine (AMSA) in human nucleated hematopoietic cells. In order to prevent efflux during the cell separation procedure, white blood cells (WBCs) were separated from red blood cells by dextran sedimentation, leaving the WBCs in their natural environment. After cell counting, pelletting the cell suspension and correcting for the admixture of supernatant, AMSA was extracted from the WBCs and determined by high-performance liquid chromatography. Linearity of extraction was observed up to 40.10(6) cells. The inter-assay variation was 4.7%. Plasma and cellular concentrations were measured in five patients at the end of a 3-h infusion of 100 mg/m2 AMSA. A pharmacokinetic study of plasma and cellular AMSA concentrations up to 19 h after infusion was carried out. AMSA concentrations in WBCs correlated well with the plasma levels (n = 20, r = 0.967) with an accumulation factor compared to the plasma concentration of 2.6-9.8 in the patients studied. The method described is useful for studying cellular pharmacokinetics of AMSA in man.

Amsacrine↗
PubMed1987

Pharmacokinetics and toxicology of sparsomycin in beagle dogs.

Sparsomycin is a cytotoxic drug exhibiting a broad spectrum of in vitro activity against murine tumors and many tumor cell lines. It also appears to be a potent stimulator of the antitumor activity of cisplatin against L1210 leukemia in vivo. However, because of its toxicity, the antitumor activity of sparsomycin on murine tumors in vivo has been disappointing. The purpose of our study was to investigate the pharmacokinetics of this drug as well as the possible mechanisms that produce sparsomycin toxicity. Tests on beagle dogs revealed that about 60% of the drug is eliminated by metabolic clearance, while 40% is eliminated by the kidneys. After a single bolus injection of 0.1 mg/kg sparsomycin without narcosis, sparsomycin was eliminated with a t beta 1/2 of 0.6-0.7 h, the AUC being 0.32-0.38 mg.h.l-1, and the volume of distribution (Vd) 0.26 l/kg. In addition to being subject to glomerular filtration, sparsomycin is probably also actively excreted and actively reabsorbed by the renal tubuli. Sparsomycin itself may inhibit its active tubular excretion, thus resulting in a decrease in the drug's renal clearance and its accumulation in the plasma. Sparsomycin appeared to be toxic primarily in the liver, disturbing its function and the synthesis of plasma proteins. Two out of five dogs developed hemorrhagic diathesis due to hypofibrinogenemia and deficiency of other blood-coagulation factors. Sparsomycin was not toxic to the bone marrow.

Animals↗
PubMed1987

In vitro proliferation of primary human head and neck squamous cell carcinomas evaluated by flow cytometry.

In vitro proliferation of primary human head and neck squamous cell carcinomas was investigated using single cell suspensions and tissue explants of primary specimens and xenografts from 20 tumor specimens. The evaluations of the cells emerging in culture were performed with flow cytometry. Epithelial-like cells proliferated in serum-free medium, while no fibroblast-like cells were observed in culture. The epithelial-like cells could be subcultured several passages before senescence occurred. Conditioned medium or serum supplementation was necessary for a sustained outgrowth of malignant squamous cells as documented by flow cytometry. From a tumor line established in nude mice slowly proliferating tumor cells emerged. After 4-5 months in culture tumor cells seemed to be adapted to the culture conditions used. This resulted in a more consistent tumor cell proliferation. Early passage cultures from primary human head and neck squamous cell carcinomas are clearly difficult to obtain either from primary human specimens or from tumor lines established in nude mice.

Animals↗
PubMed1987

Modulation of placental alkaline phosphatase activity and cytokeratins in human HN-1 cells by butyrate, retinoic acid, catecholamines and histamine.

The effects of butyrate and retinoic acid in combination with catecholamines or histamine on the HN-1 human head and neck squamous carcinoma cell line were investigated analysing cell proliferation, placental alkaline phosphatase (PLAP) activity, and relative cytokeratin content. Butyrate inhibited cell proliferation in agar, whereas retinoic acid induced a small inhibitory effect. Butyrate enhanced PLAP activity in a time related manner in contrast to retinoic acid, which had no significant effect. However, retinoic acid inhibited the efficacy of butyrate to induce PLAP activity. A synergistic enhancement of PLAP activity was demonstrated after treatment of butyrate pretreated cells with catecholamines or histamine. The beta-adrenergic antagonist propranolol partly inhibited the aforementioned enhancement of PLAP activity, whereas the alpha-adrenergic antagonist phentolamine further enhanced PLAP activity. Indirect labeling of keratins with a polyclonal antibody showed that cytokeratin content was enhanced by butyrate but not by retinoic acid. Further analysis of cytokeratin content using four monoclonal antibodies showed that labeling of cytokeratins (5 + 8) was increased by butyrate. PLAP activity could be modulated by a concerted action of either butyrate plus retinoic acid or butyrate plus catecholamines or histamine, indicating a possible role for PLAP in tumour cell proliferation.

Alkaline Phosphatase↗
PubMed1987

In vitro modulation of cisplatin cytotoxicity by sparsomycin inhibition of protein synthesis.

Inhibition of protein synthesis can alter cellular responsiveness to the classical anticancer drugs. The in vitro response of Chinese hamster ovary (CHO) cells to cisplatin with or without sparsomycin (Sm) was studied with the use of [3H]leucine and [methyl-3H]thymidine incorporation and clonogenic assay. Pretreatment of exponentially growing CHO cells with 1 microgram Sm/ml for 3 or 5 hours decreased [3H]leucine incorporation by 20% and resulted in significant resistance to cisplatin (P = .005). Sm in a concentration of 10 micrograms/ml reduced [3H]leucine and [methyl-3H]thymidine incorporation after 3 hours by 92 and 84%, respectively, and resulted in potentiation of the cisplatin cytotoxicity (P = .004). This effect was the same in the case of nonproliferating cells (P = .005), while protection due to Sm (1 microgram/ml) was seen only during cell proliferation. Simultaneous incubation and postincubation with Sm proved to have much less or no potentiating effect on cisplatin. The mechanisms of both protection and potentiation are still not clear, but our data indicate that Sm is a promising drug for further studies on the modulation of the cancer cell response to classical anticancer drugs.

Animals↗
PubMed1986

Cell size, DNA, and cytokeratin analysis of human head and neck tumors by flow cytometry.

Cell subsets have been discriminated in cell suspensions derived from 37 human head and neck tumors by means of light scatter, DNA, and cytokeratin flow cytometry (FCM). Cell dispersion was performed overnight at 4 degrees C in two different enzyme mixtures, i.e., trypsin/dithioerythritol and collagenase/DNase, under slight agitation of sliced tumor tissue. Cells were examined before and after fractionation on a discontinuous low-density bovine serum albumin (BSA) gradient. Forward and right-angle light scatter FCM of 23 tumor specimens revealed four main subpopulations with different size and structure. Fractionation of primary cell suspensions on a BSA gradient at unit gravity separated debris, small cells and large cells. DNA FCM of the enriched populations demonstrated a relation between large cells and DNA aneuploidy. Epithelial cells, as recognized by cytokeratin antibodies, were also related with large cells. The results demonstrated the usefulness of light scatter, DNA, and cytokeratin analysis of crude and fractionated tumor cell suspensions for assessment of the efficacy of a particular dispersion technique and to obtain information of the cell subsets dispersed.

Cell Separation↗
PubMed1986

In vivo potentiation of cis-diamminedichloroplatinum (II) antitumor activity by pretreatment with sparsomycin.

The influence of protein synthesis inhibition by sparsomycin (Sm) on in vivo cisplatin activity has been studied on BALBc X DBA2: F1 mice bearing L1210 leukemia i.p. Sm alone at the dose range from 0.5 to 3.0 mg/kg did not significantly improve animal survival. Sm potentiated cisplatin activity only when given 3 or 6 h prior to cisplatin (P less than 0.001). Sm 0.5-1.5 mg/kg 3 h prior to cisplatin resulted in a significant prolongation of animal survival (P less than 0.001) and 66% cures in each group versus 0% due to cisplatin alone. Sm pretreatment decreased weight loss due to cisplatin suggesting that it probably is able to decrease cisplatin toxicity.

Animals↗
PubMed1985

Flow cytometric evaluation of cell dispersion from human head and neck tumors.

The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.

Adult↗
PubMed1984

Lymphocyte isolation from human spleen by counterflow centrifugation employing two different flow chambers on line.

Studies on splenic lymphocytes have hitherto been performed on single cell suspensions depleted of phagocytic cells by adherence to plastic or incubation with carbonyl iron. These techniques have the disadvantages of selective cell loss, suboptimal cell purification and cell activation. This paper describes purification of splenic lymphocytes by the use of counterflow centrifugation (CFC). The method was adapted to overcome pelleting of cells in the separation chamber to form a plug at the inlet and impede adequate flow. By combining 2 different separation chambers on line in 1 rotor this problem was overcome. Of all lymphocytes recovered after CFC 88.8 +/- 1.4% were collected in 2 pooled fractions with a purity of greater than or equal to 98% and a cell viability of 95%. After CFC, 80.8 +/- 12.1% of the viable cells loaded were recovered.

Blood Platelets↗
PubMed1984

S-adenosylhomocysteine hydrolase activity during differentiation of HL-60 cells.

Early changes in S-adenosylhomocysteine (SAH) hydrolase activity during DMSO-induced granulocytic differentiation of HL-60 cells were followed. Within 24 h a decrease of activity of SAH hydrolase could be detected in induced cultures but not in control cultures. This decrease could be shown to be associated with G1 phase of the cell cycle and was detected prior to phenotypic changes of the cells.

Adenosylhomocysteinase↗
PubMed1983

Flow cytometric determination of the S-phase compartment in adult acute leukemia.

Flow-cytometric analysis of bone marrow aspirates and blood samples was performed in 106 adult patients with acute leukemia in order to assess the size and the prognostic significance of the percentage of S-phase cells in the bone marrow (%Sbm). A correction procedure was applied for the fraction of contaminating peripheral nucleated cells in bone marrow aspirates (%Fpb). In 82 out of 106 patients studied, the %Sbm could be reliably determined, and was compared to the %Sbm in 25 healthy controls. The %Sbm in these healthy controls ranged from 8.4 to 14.6%. The median %Sbm in 31 patients with acute nonlymphocytic leukemia (ANLL) at diagnosis (11.3%) and in 14 patients with ANLL at relapse (11.8%) did not differ significantly from the median %Sbm in normal bone marrow (11.7%). On the other hand, in 12 out of 23 patients with acute lymphocytic leukemia (ALL) at diagnosis and in 6 out of 11 patients with ALL at relapse the %Sbm was much higher and ranged from 17.8 to 44.0%. The prognosis of patients with ALL with a high %Sbm (greater than 15%) was significantly worse. Blast cells with an abnormal DNA content (aneuploid cells) were noticed in 7.7% of the patients with acute leukemia at diagnosis. This incidence, however, was significantly higher in ALL patients at relapse (i.e. 42.1%).

Acute Disease↗
PubMed1983

DNA-flow cytometry of blood and bone marrow in chronic myelogenous leukemia.

Flow cytometric analysis of bone marrow aspirates and blood samples in 42 patients with chronic myelogenous leukemia (CML) at various disease stages was performed to determine the size of the S-phase compartment of bone marrow and blood. 25 healthy controls were studied for comparative information with both DNA-flow cytometry (DNA-FCM) and 3H-thymidine autoradiography. A correction procedure was applied for peripheral nucleated cell admixture in bone marrow aspirates. The fraction of peripheral nucleated cells in bone marrow aspirates (Fpb) in individual patients was considerable, especially in those with a very high white blood cell count (greater than 100 x 10(9)/l). The size of the S-phase compartments of bone marrow (% Sbm) in patients with CML at diagnosis and in patients at apparent hematological remission was of the same order of magnitude as in normal bone marrow. However, in 3 out of 4 patients at malignant metamorphosis in which the % Sbm could be reliably determined, this percentage was significantly higher than normal (p = 0.013). In 4 out of 11 patients at malignant metamorphosis aneuploidy was noticed. From these findings it is concluded that bone marrow cell proliferation in CML patients at diagnosis and during apparent remission is not essentially different from normal. However, at malignant metamorphosis changes occur in ploidy level and proliferative activity, which can be detected by DNA-FCM already in an early phase.

Adult↗
PubMed1982

Variations of the phosphorylation of 1-beta-D-arabinofuranosylcytosine (ARA-C) in human myeloid leukemic cells related to the cell cycle.

Bone marrow cells of five patients with acute myeloid leukemia were fractionated by means of counterflow centrifugation (elutriation). The different fractions were enriched with cells belonging to subsequent stages of the cell cycle. Cytokinetic evaluation of these cell fractions was performed by [3H]thymidine autoradiography, [3H]thymidine incorporation and DNA/RNA-flow cytometry. Phosphorylation of cytosine arabinoside (ara-C, 1-beta-D-arabinofuranosylcytosine) in the different fractions was measured by incubation of the cells for 30 min with 1.07 microM [3H]ara-C. Phosphorylation of ara-C in the whole bone marrow samples ranged from 5.9 to 33.2 pmol/10(6) cells. In the fractions containing only G1-phase cells, phosphorylation ranged from 1.2 to 19.5 pmol/10(6) cells. The phosphorylation seems to increase before DNA synthesis starts. Maximal activities were found in the fractions enriched with cells in late G1- or S-phase of the cell cycle. In these fractions the ara-C phosphorylating activity was 1.5-8 times higher compared to the fractions with the lowest activity. One may therefore assume that not only S-phase cells are killed by ara-C, but that G1-phase cells which can phosphorylate ara-C, may also be doomed when they enter S-phase, since the elimination of the intracellular cytosine arabinoside tri-phosphate (ara-CTP) is a relatively slow process. The fraction of G1-phase cells phosphorylating ara-C, may be an important determinant in the extent of the cell-killing effect of ara-C treatment in the different leukemias.

Cell Cycle↗