Use of polyclonal anti-myeloperoxidase antibody in myeloid lineage determination.

This study reports the production of a rabbit polyclonal antibody to myeloperoxidase (MPO) and its use in ascertaining the myeloid lineage of blasts in leukaemia. Comparison of the immunocytochemical stain using the anti-MPO antibody with the routine cytochemical methodology showed that the former was more sensitive. In all subtypes of acute myeloid leukaemia (AML; 72 patients, M1-M6) greater number of MPO positive blast cells were observed by immunocytochemistry, the highest being in the promyelocytic leukaemia. It was also extremely specific for cells of the myeloid lineage as it did not react with blasts from acute lymphoblastic (50 patients) and megakaryoblastic leukaemias (1 patient). In addition, it proved most useful for the lineage determination of blasts from patients with undifferentiated acute leukaemias (AUL) and those with chronic myeloid leukaemia in blast crisis (CML-BC). Out of 8 patients of AULs, 6 were classified as acute myeloblastic leukaemia due to their reactivity to the anti-MPO antibody. Similarly, out of 12 patients of chronic myeloid leukaemia in blast crisis, blasts from 8 showed reactivity to this antibody and thus could be identified as belonging to the myeloid lineage and/or of the mixed blast crisis type.

Characterization of ferritin from spleens of patients with Hodgkin's disease (HD).

Ferritin, an iron-storing protein, was isolated from disease-involved and -uninvolved regions of spleen biopsies obtained from patients with Hodgkin's disease (HD). Ferritin from all human spleen biopsies showed a major band of polypeptide of M(r) around 20 kDa in 1D-SDS-PAGE analysis. The corresponding bands for horse spleen ferritin and apoferritin (Sigma) were at a slightly lower M(r) level. In isoelectrofocusing (IEF) studies, the pI values of human spleen ferritin from the uninvolved and involved regions were 4.55 and 4.14, respectively. These were more acidic than that of horse spleen ferritin (4.79). Human spleen ferritin from the involved region also differed from that of the uninvolved region in the pattern of CNBr-generated peptide maps in 1D-SDS-PAGE. These results suggest that the presence of Hodgkin's disease in human spleen is associated with some physiochemical changes in the tissue ferritin.

Circulating immune complexes in Hodgkin's disease. Reactivity of IgG isolated from circulating immune complexes.

In our earlier report on circulating immune complexes (CIC) from sera of patients with Hodgkin's disease (HD), we demonstrated disease-associated increase in the intensity of a 40kD (pl 5.6) polypeptide in the CIC. In extension of these investigations, we now report that the 40kD moiety exists in CIC as a complex with IgG, and that IgG isolated from such CIC samples from sera of patients with HD shows preferential reactivity with the typical large binucleate Reed-Sternberg (R-S) cells in the sections of disease-involved lymph nodes from HD patients.

A major concanavalin-A-binding cell surface protein from normal and leukemic granulocytes: isolation and characterization.

This paper reports the isolation and biochemical characterization of a major concanavalin A (Con A)-binding cell surface protein (protein 2, M(r) 75-85 kD) from normal and chronic myeloid leukemic (CML) granulocytes. Our studies show that protein 2 has two differentially glycosylated forms, protein 2a (M(r) 75-85 kD), which does not bind the lectin RCA, and protein 2b (M(r) 80-90 kD), which does. Both molecules show identical retention times on reverse-phase HPLC, irrespective of the cell source. By the procedure used the amount of 2a obtained is about 2.4 times more than that of 2b in normal cells and about 2.6 times more in CML cells. Furthermore, both are approximately 2.4-fold more in CML granulocytes. A polyclonal antibody to protein 2a also immunostains protein 2b. The antibody to protein 2a does not prevent Con A binding but inhibits its internalization. Similarity of the molecules from both the cell types and their increased amounts in CML granulocytes suggest that factors/components other than its structure and amount are responsible for the known defective internalization of Con A by CML granulocytes.

Column isoelectric focusing in natural pH gradients generated by biological buffers.

Using 2 or 3 simple Good zwitterionic buffers at a 16 or 18 mmol/L final column concentration of the mixture, natural pH gradients of 4 to 8 and 3 to 9.5, respectively, were generated in a liquid LKB column. The pH gradients, stabilized by an anticonvective sucrose gradient, were linear, reproducible and stable in the electric field up to 5h. The pH gradients were used for isoelectric focusing of a number of impure proteins such as human hemoglobin, bovine serum albumin and chicken egg white lysozyme. The protein components could be well separated in the gradient, were easily recovered and appeared to be quite pure when analyzed by sodium dodecyl sulfate-gel electrophoresis. Furthermore, the pH gradient 4-8 was effectively used to isolate one of the acidic isozyme (pI 5.6) components of mouse liver alcohol dehydrogenase (EC 1.1.1.1) in an enzymatically active state, suggesting that the procedure does not denature proteins. The low cost, the ease with which the pH gradients are formed, their linearity, stability for a sufficient period to allow proteins to reach equilibrium and their subsequent recovery from buffer eluates should make the procedure interesting for electrofocusing of proteins.

The receptor for IgG on human normal and chronic myeloid leukemic granulocytes: functional and biochemical characterization.

Receptor mediated endocytosis of fluorescein isothiocyanate-conjugated heat-aggregated IgG (Fl-HAIgG) via the receptor for IgG (Fc gamma R) was studied using granulocytes from normal donors and patients suffering from chronic myeloid leukemia (CML). Within 15 min of incubation at 37 degrees C, 75% of the normal granulocytes internalized the ligand, while only 13% of the CML granulocytes could internalize the ligand in the same time. This functional defect was seen in all the CML patients analyzed. To elucidate the reason for this defective endocytosis, the Fc gamma R was isolated from normal and leukemic granulocytes and biochemically characterized, by gel electrophoresis, high pressure liquid chromatography, and one- and two-dimensional peptide mapping. Our results show that the molecule from the two cell types is very similar. The defective endocytosis must therefore be due to events which occur after ligand-receptor binding.

Circulating immune complexes (CIC) in Hodgkin's disease. I. Levels and two-dimensional analysis.

A polyethylene glycol 6000 (PEG) mediated precipitation procedure was used to estimate the levels of CIC in sera of healthy donors and patients with Hodgkin's disease (HD). In comparison with the normal serum samples, sera from untreated HD patients showed elevated CIC levels scattered over a wider range and a mean which differed significantly. Sera from treated patients who were in clinical remission exhibited decreased CIC levels. However, the mean level in this category was still above the mean found in the normal sera. The analysis of the data showed that the sera from HD patients in Stage I and II, and with LP and MC type histology showed preferential increase in CIC levels. The analysis of these CICs in 2D-SDS-PAGE and a careful scrutiny of the polypeptide patterns obtained revealed significantly elevated amounts of a component with a Mr of 40 kD and a pI of 5.6 in CICs from the untreated HD patients. Congruent peptide maps of this component and its decreased amounts in CICs from sera of patients in remission suggests its quantitative involvement in the disease.

Differential endocytosis of fluorescein iso-thiocyanate-concanavalin A by normal and chronic myeloid leukemic granulocytes.

Isolated granulocytes from normal individuals and patients suffering from chronic myeloid leukemia (CML) displayed different fluorescent patterns on treatment with fluorescein isothiocyanate concanavalin A (Fl-Con A). The ligand was internalized by 86% of the normal granulocytes, while 80% of the leukemic granulocytes exhibited Fl-Con A localized on the cell periphery. In further experiments, pretreatment of the normal granulocytes with cytochalasin B, iodoacetamide, 2-deoxyglucose and sodium fluoride (but not with sodium azide or dinitrophenol) was found to drastically inhibit internalization of the ligand. However, pretreatment of granulocytes from CML patients with cytochalasin B and 2-deoxyglucose, caused only a little alteration in the pattern of Fl-Con A labelling relative to untreated cells. These results indicate that CML granulocytes are defective in their ability to endocytose Fl-Con A. We suggest that this differential interaction between Fl-Con A and normal and leukemic granulocytes is a convenient system to study the initial steps in receptor mediated endocytosis of Concanavalin A.

Plasma membrane proteins from human normal and chronic myeloid leukemic granulocytes: identification and partial characterization of the concanavalin A-binding and detergent resistant proteins.

In this work granulocytes from normal human donors and patients suffering from chronic myeloid leukemia (CML) were externally labeled with 125Iodine, using the Iodogen method. 125Iodine labeled Concanavalin A binding proteins (CBP) and detergent-resistant proteins (DRP) were isolated from the cell lysates and characterized by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D- and 2D-SDS-PAGE). Autoradiographs of the 2D-gels of DRP show seven proteins with Mr 118,000 (spot 1 a), Mr 112,000 (spot 1b), Mr 78,000-85,000 (spot 2), Mr 85,000 (spot 4), Mr 52,000 (spot 3, 3 a and 3 b). Of this set, spot 1 b, 2 and 4 are also present in the autoradiographs of 2D-gels of CBP and, hence, may be considered to be transmembrane components. Spot 4 is expressed more intensely in the normal granulocytes while spots 3 a and 3 b are mainly expressed on the leukemic granulocytes.

Influence of splenectomy on the levels of circulating immune complexes (CIC) in Hodgkin's disease (HD).

A 3.75% polyethylene glycol 6000-precipitation method was used to estimate the levels of circulating immune complexes (CIC) in pre- and postsplenectomy serum samples obtained from 34 patients suffering from Hodgkin's disease (HD). In 26 out of the 34 patients (76.47%) the postsplenectomy samples showed a considerable decrease in CIC levels compared to the levels in the presplenectomy samples. Two patients showed a marginal change while the remaining 6 patients exhibited elevated levels of CICs after splenectomy. The mean levels (in terms of OD 450 nm) in the pre- and postsplenectomy sample sets were 0.66 +/- 0.07 and 0.42 +/- 0.05, respectively (p less than 0.01). There was no apparent relationship between the interval elapsed after splenectomy (6-22 days) and the magnitude of decrease (1.7-91.8%). Patients with LP-type histology and splenic involvement formed a category that preferentially showed a substantial decrease in CIC levels after splenectomy.

Terminal deoxynucleotidyl transferase in acute leukemias by direct immunofluorescence.

TdT (terminal deoxynucleotidyl transferase) can be detected by radio enzymatic assay, biochemical assay in cell extracts, serum or plasma, and intracellularly in the smear by indirect immunofluorescent methods. The IgG fraction of anti-TdT serum is conjugated with fluoresceinisothiocyanate and used directly on the cytospin smears of methanol fixed bone marrow/blood smears. The mice thymocytes and peripheral mononuclear cells of healthy donors were used as positive and negative controls, respectively, for TdT. 64% of our cases of ALL were found to be TdT+. The lymphoblasts of L1 morphology (FAB classification) were more frequently positive for TdT as compared to blasts with L2 morphology. 71% of our cALLa positive blasts in acute lymphoblastic leukemias were TdT+ve as compared to 58% of T-ALL blasts. 75% of PAS positive ALL cases were positive for TdT as well. Only 57% of the cases when acid phosphatase showed unipolar positivity (T type) were positive for TdT. 12% of cases with acute myeloid leukemia (6/47) were TdT+ve and 33% of CML in blastic crisis had TdT+ve blasts. Biochemical assay and IF assay for TdT were in good correlation in our study.

Peptide mapping of proteins in gel bands after partial cleavage with acidic cyanogen bromide vapors.

A procedure is described for obtaining peptide maps from microgram quantities of protein in gel bands, after cleavage at the methionyl peptide bonds with vapors of acidic cyanogen bromide (CNBr). Absence of direct contact of the gel pieces with CNBr eliminates the need for extensive equilibration of the gel piece to remove CNBr prior to electrophoresis. The milder conditions lead to partial cleavage of the proteins, yielding larger peptides and thereby reducing the risk of peptide loss during the postelectrophoresis procedures. The "fingerprints" obtained are reproducible and independent of an eightfold change in CNBr concentration.

Partial purification of cytochrome P-450 from human normal granulocytes.

Cytochrome P-450 was partially purified from human normal granulocytes, using four different purification procedures. PEG-6000 and ammonium sulfate fractionation of human granulocyte post-mitochondrial supernatant (S1) fraction resulted in 3.9 and 2.25 fold purification of cytochrome P-450 respectively. On the other hand, granulocyte S1 fractions subjected to noctylamino sepharose 4B and DEAE cellulose column chromatography separately revealed about 11 fold purification of cytochrome P-450. When these fractions were submitted to SDS-polyacrylamide gel electrophoresis, PEG-6000 and ammonium sulfate precipitated fractions showed multiple protein bands whereas n-octylamino sepharose 4B and DEAE eluates were relatively pure showing one or few bands. There was no indication of the existence of multiple P-450 species in the partially purified human granulocyte cytochrome P-450.

Depression of mouse liver microsomal mixed function oxidase enzymes by adriamycin.

Levels of cytochrome P-450 and cytochrome b5, and activities of NADPH cytochrome c reductase and 7-ethoxycoumarin O-deethylase, were found to be significantly decreased in hepatic microsomes prepared from mice killed 24 h after administration of a single intraperitoneal (i.p.) dose of adriamycin (ADR, 5 mg/kg). In contrast, both ascorbate-induced lipid peroxidation and conjugated dienes were increased in the same preparations. In vitro addition of ADR (5 micrograms/ml) to hepatic microsomal preparations (1 mg/ml protein) from the control mice also led to a substantial decrease in the mixed function oxidase (MFO) enzymes. A characteristic spectral change with an absorption peak at 408 nm and trough at 422 nm was associated with this in vitro interaction. It is suggested that the loss of cytochrome P-450 and related MFO enzymes due to ADR treatment is related to the generation of free radicals and subsequent lipid peroxidation in the liver.

Substrate-induced spectral changes in human normal and chronic myeloid leukemic granulocytes.

Several drugs/chemicals were allowed to interact with the cytochrome P-450 dependent mixed function oxidase system in the postmitochrondrial supernatant fractions of Ficoll-Hypaque-separated granulocytes from human normal subjects and patients with chronic myeloid leukemia. The substrate-induced spectral changes were followed by recording the difference spectra. Compounds conventionally classified as type I and type II substrates, on addition to S1 fractions of both normal and leukemic granulocytes, caused spectral changes that were reverse to those reported for the rat liver microsomes. Aminopyrine, phenobarbital, and Tween 80 evoked a reverse type I spectral change with a peak at 420-430 nm and a trough at 380-400 nm, whereas aniline and pyridine induced a modified type I (a reverse type II) spectral change characterized by a peak at 408 nm and a trough at 421 nm. These changes were found to be quantitatively proportional to the amounts of substrate added. However, the magnitude of the peaks and troughs was considerably less in the S1 fraction of the leukemic granulocytes. Correspondingly, total heme content was significantly decreased in S1 fractions of CML granulocytes as compared to similar fractions of normal granulocytes.

Plasma membranes from normal and chronic myeloid leukemic granulocytes: isolation and two-dimensional polyacrylamide gel electrophoretic analysis.

Normal human granulocytes obtained by Ficoll-Hypaque sedimentation were subjected to mild hypotonic shock and disruption by shear. The homogenate was fractionated by differential centrifugation and equilibrium ultracentrifugation to yield a plasma membrane preparation constituting 1% of the total cellular protein and enriched fifteen- and six-fold in alkaline phosphatase and Mg2+-adenosine triphosphatase activities, respectively. Granulocytes obtained from patients with chronic myeloid leukemia (CML) were identically processed. The protein constituents of both the normal and CML granulocyte plasma membranes were resolved by two-dimensional polyacrylamide gel electrophoresis. Comparison of the stained gels revealed CML-associated quantitative changes in four out of the fifteen protein spots examined. Thus, this analysis has permitted identification of those protein moieties that deserve attention for further isolation and purification.

Cyclic AMP metabolism in peripheral blood lymphocytes from patients with Hodgkin's disease.

Intracellular levels of cyclic AMP and cAMP phospodiesterase activity of peripheral blood mononuclear cells of patients with Hodgkin's disease (HD) were studied. The cAMP levels were elevated by 137% in HD patients as compared to normal subjects. The levels were reduced during clinical remission but remained significantly higher than normal controls. These high levels of cAMP in HD patients may be due to reduced degradation of cAMP as the cAMP phosphodiesterase activity was reduced to 50% of normal. This observed altered metabolism still persisted even after depletion of adherent monocytes, indicating that the defect was in the lymphocytes.

Pharmacokinetics of methotrexate in adult Indian patients and its relationship to nutritional status.

Since anticancer drugs have a low therapeutic index, any significant change in the pharmacokinetics of a drug would have a bearing on therapeutic efficacy and drug toxicity. Nutritional deficiencies have been shown to affect the four processes of pharmacokinetics, ie, absorption, distribution, biotransformation, and excretion. Malnutrition and undernutrition are widely prevalent in India and thus it was thought to be of practical importance to study the effect of initial nutritional status on the overall kinetics of methotrexate, a widely used anticancer drug. The results of the study reveal that relative weight correlates well with the anthropometric parameters, nutritional parameters, and dietary intake and may be used as a marker of nutritional status. When grouped on the basis of relative weights, the undernourished patients revealed a significant (P less than 0.01) prolongation of biological half-life and a significant (P less than 0.001) reduction in clearance. Based on these results, relative weight has been proposed as a basis for drug dosage determinations in place of the existing practice of administering antineoplastic drugs on the basis of body surface area or body weight alone.

Mixed function oxidase enzymes in human normal granulocytes and chronic myeloid leukemia cells. I. Detection and estimation.

Subcellular fractions prepared from human normal granulocytes and leukemic cells from patients with chronic myeloid leukemia (CML), were examined for 7-ethoxycoumarin O-deethylase activity as well as cytochrome P-450 and cytochrome b5 contents. The average 7-ethoxycoumarin O-deethylase activity was found to be significantly increased in all the fractions of CML cells when compared to the corresponding fractions of the normal granulocytes. The CML cells also differed from the normal cells by exhibiting decreased levels of both cytochrome P-450 and cytochrome b5. Examination of the microsomal and cytosolic fractions of both normal granulocytes and CML cells showed distribution of 7-ethoxycoumarin O-deethylase, cytochrome P-450, and cytochrome b5 in both the fractions. Such distribution seems to be unique for human leukocytes. Solubilization of protein in the postmitochondrial (S1) fractions of both the cell types with sodium cholate in the presence of 20% glycerol and further fractionation with 10% polyethylene glycol 6000 (PEG-6000) yielded a partially purified preparation with enriched 7-ethoxycoumarin O-deethylase activity and cytochrome P-450 as well as cytochrome b5 contents.