Mode of action of chemotherapy in vivo on human acute leukemia. I. Daunomycin.

The leukocytes of 16 adult patients with acute myeloblastic leukemia were studied by autoradiographic methods to elucidate the mode of action of daunomycin. It was shown that daunomycin, at clinically useful doses, exhibits a cytolytic effect on all leukemic blasts whatever their cell-cycle phase. This cytolytic action affects, however, preferentially S-phase cells. It was shown also that blasts of patients less sensitive to daunomycin or receiving a lesser dose of the drug are temporarily blocked in G(2) phase (delayed mitosis) or in G(2) phase (prolonged generation time). Finally daunomycin appeared to hamper the passage of G(2)-blocked blasts from the bone marrow to the blood, while G(2)-phase cells crossed freely.

Four different mutations in codon 28 of alpha spectrin are associated with structurally and functionally abnormal spectrin alpha I/74 in hereditary elliptocytosis.

Hereditary elliptocytosis (HE) Sp alpha I/74 is a disorder associated with defective spectrin (Sp) heterodimer self-association and an abnormal tryptic cleavage of the 80-kD alpha I domain of Sp resulting in increased amounts of a 74-kD peptide. The molecular basis of this disorder is heterogeneous and mutations in codons 28, 46, 48, and 49 (codons 22, 40, 42, and 43 in the previous nomenclature which did not include the six NH2-terminal amino acids) have been reported. In this study we present data on seven unrelated HE Sp alpha I/74 kindred from diverse racial backgrounds in whom we identified four different mutations all occurring in exon 2 of alpha Sp at codon 28. Utilizing the polymerase chain reaction we established a CGT----CTT; Arg----Leu 28 mutation in one kindred of Arab/Druze origin. In two unrelated white kindred of English/European origin the substitution is CGT----AGT; Arg----Ser 28 and in two apparently unrelated white kindred from New Zealand, the mutation is CGT----TGT; Arg----Cys 28. Finally, in one American black kindred and in a black kindred from Ghana the mutation involves CGT----CAT; Arg----His 28. Allele specific oligonucleotide hybridization confirmed that the probands are heterozygous for the respective mutant alleles. All four point mutations abolished an Aha II restriction enzyme site which allowed verification of linkage of the mutation with HE Sp alpha I/74. Our results imply that codon 28 of alpha Sp is a "hot spot" for mutations and also indicate that Arg 28 is critical for the conformational stability and functional self association of Sp heterodimers.

Primary local orbital amyloidosis: biochemical identification of the immunoglobulin light chain kappaIII subtype in a small formalin fixed, paraffin wax embedded tissue sample.

BACKGROUND: Amyloidosis refers to a heterogeneous group of disorders associated with the deposition of chemically distinct amyloid fibril proteins. Precise determination of chemical amyloid type has diagnostic, therapeutic, and prognostic relevance. Although immunohistochemical techniques are used routinely to determine the amyloid type, the results can be negative or inconclusive, so that biochemical characterisation is often required. The development and application of new biochemical microtechniques suitable for examination of extremely small tissue samples is essential for precise identification of the deposited amyloid proteins. AIMS: To investigate biochemically the amyloid proteins present in a formalin fixed paraffin wax embedded orbital tissue from a patient with localised orbital amyloidosis in whom immunohistochemistry was not helpful in the determination of amyloid type. METHODS: Extraction of amyloid proteins from fixed tissue and their identification was carried out by a recently developed microtechnique. An extremely small tissue sample was dewaxed and extracted with formic acid. The extracted material was analysed using electrophoresis, western blotting, and amino acid sequencing. RESULTS: Biochemical examination of the extracted proteins showed the presence of immunoglobulin (Ig) derived amyloid proteins, which were composed of the N-terminal fragments of the Ig light chain kappaIII subtype (AL-kappaIII) (16, 8, and 3 kDa). CONCLUSIONS: This is the first chemically proved AL case reported in association with primary localised orbital amyloidosis. The biochemical microtechnique used was useful in achieving a precise diagnosis of amyloid disease, in a case where the results of routine immunohistochemical examination of amyloid were inconclusive.

Participation of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation in patients with essential hypertension.

Oxidative stress and inflammation have recently been linked to endothelial damage in essential hypertension (EH). Activated peripheral polymorphonuclear leukocytes (PMN) damage surrounding tissue by releasing reactive oxygen species (ROS) and proteolytic enzymes before self-necrosis. PMN necrosis further exacerbates inflammation and promotes chemotaxis and PMN recruitment. The number and properties of PMN from untreated EH patients is the focus of the present study. Oxidative stress was assessed by measuring the rate of superoxide anion release from separated, phorbol ester-stimulated PMN and the redox state of plasma glutathione. Inflammation was estimated indirectly by determining PMN number and their in vitro survival. PMN from EH patients (n = 37) released superoxide anion faster (P < .0001) than those of normotensives (NC, n = 37), 17.7 +/- 1.14 v 9.54 +/- 0.51 nmol/10 min/10(6) cells. The redox state of glutathione was twofold higher in EH plasma (P < .02) indicating systemic oxidative stress. PMN survival in vitro correlates linearly with the rate of superoxide release (r2 = 0.60, P < .02) and PMN count of EH patients, although in the normal range, were significantly higher (P < .0001), indicating necrosis and recruitment. Hypertensive plasma significantly reduced NC PMN viability, whereas normal plasma significantly increased EH PMN viability. What our studies show is that EH is accompanied by a primed state PMN that does not correlate with the levels of blood pressure. PMN priming in EH patients reflects an in vivo exposure to a constant stimulus ending in oxidative stress, increased self-necrosis, and cell recruitment. Oxidative stress and inflammation will result in endothelial damage and atherosclerosis in the long run.

Immunophenotypic characterization of myelopoiesis in early and late myelodysplastic syndromes: use of CD44 as an aid in early diagnosis.

We investigated the possibility that myeloid cells from the bone marrow (BM) of myelodysplastic patients differ in their expression of CD44 antigen compared with expression of the antigen in normal controls. In addition, two triple-surface marker assays incorporating, respectively, CD44/CD33/CD66 and CD33/CD34/HLA-DR were used to evaluate the degree of myeloid maturation and assess the number of blasts in BM by flow cytometry. Patients with early-stage myelodysplastic syndrome (MDS; RA [FAB classification]) have significantly decreased expression of CD44 on gated myeloid cells. In contrast, patients with late-stage MDS (RAEB and RAEB-T [FAB classification]) showed an elevated expression of CD44 and an increased number of CD34 blasts compared with early-stage MDS patients and normal controls. Late-stage MDS patients also had an increase in the immature myeloid compartment (CD66 weak expression) compared with early-stage MDS patients and normal controls. We have already included this assay as part of our MDS evaluation protocol alongside BM morphology and cytogenetics.

Red cell inclusion bodies in a case of preleukaemia.

A 26-year-old female patient is reported in whom red cell inclusion bodies (Hb-H) and thrombocytopenia appeared more than 5 years before the emergence of atypical chr. myeloid leukaemia. Attention is drawn to the fact that the recently uncovered mechanism of acquisition of Hb-H (absence of transcription of specific genes) has previously been implicated in other biochemical changes typical of leukaemia.

The possible role of fibronectin in multiple myeloma.

Fibronectin (FN) has an active role in the immune response, interacting with a number of different cells and components. It has been implicated in the formation of cryoprecipitates in rheumatic diseases and is present in tissues where under pathological conditions immune complexes are deposited. Under physiological conditions of pH and ionic strength both heavy and light chain of all multiple myeloma and normal IgG show affinity to FN. FN binds to both B and T cells and is shown to inhibit thrombin and collagen-induced platelet aggregation. We have found elevated levels of FN in the plasma of multiple myeloma patients tested compared to a group of normal subjects. Even though the level of FN did not correlate with the level of the paraprotein, our findings raise the possibility that FN might be implicated in some of the clinical symptoms of multiple myeloma.

Spontaneous immunoglobulin changes in human plasma-cell dyscrasia.

A series of spontaneous changes affecting the nature of the immunoglobulin secretion of plasma cels is described in a patient initially diagnosed as IgG lambda benign monoclonal gammopathy. After several years a slight increase in the amount of serum monoclonal immunoglobulin occurred; shortly thereafter an aggressive form of multiple myeloma was diagnosed. Unexpectedly a rapid spontaneous decrease of the monoclonal immunoglobulin, accompanied by the appearance in the serum of increasing quantities of a complex containing intact lambda light chains, then occurred. Concomitantly a fragment of the corresponding free light chain was was detected in the urine. A parallel is drawn between the facts observed in this patient and in an animal model recently proposed to explain the different types of structural immunoglobulin abnormalities in multiple myeloma.

High plasma fibronectin levels in multiple myeloma patients: possible mechanisms and clinical implications.

Fibronectin (FN) has been implicated in the formation of cryoprecipitates in rheumatic diseases and is present in tissues where, under pathological conditions, immune complexes are frequently deposited. We found elevated levels of FN in the plasma of 92% of multiple myeloma patients tested compared with a group of normal subjects, although the level of FN did not correlate with the level of the paraprotein. We then characterized the interacting fragments on both molecules and found that under physiological conditions of pH and ionic strength both heavy and light chain of all multiple myeloma and normal IgG showed affinity to FN; the active fragment on FN was found to be the aminoterminal heparin-binding domain. These findings raise the possibility that FN might be implicated in some of the clinical symptoms of multiple myeloma.

Viral-related information in oncornavirus-lik particles isolated from cultures of marrow cells from leukemic patients in relapse and remission.

Characterization of ribonucleic acid content of particles released from cultures of marrow cells of leukemic patients indicates the presence of RNA molecules of size and base sequence characteristic of oncornarviruses. Seventeen marrow samples obtained from leukemic patients in relapse or in a chronic phase of the disease yielded particles containing high-molecular-weight RNA with a sedimentation velocity (about 70 S) similar to that obtained for murine oncornavirus RNA. Eight of nine marrow samples from non-leukemic patients did not yield detectable high-molecular weight RNA. Among patients in firm hematological remission, three of three samples from patients with acute lymphoblastic leukemia and three of nine samples from patients with acute myeloblastic leukemia were positive for high-molecular-weight RNA. The base sequence of the RNA in particles was characterized by synthesizing complementary (3-H)DNA in an endogenous reaction and hybridizing to excess RNA from known oncornaviruses. Hybridization of 40-60% of input complementary DNA to simian sarcoma virus RNA was detected. No monology was detected with an avian oncornavirus (Rous sarcoma virus) while an intermediate level of homology (10-30%) was detected in hybridization to murine sarcoma virus (Kirsten) and murine leukemia viruses (Rauscher, Moloney, and Gross).

Malignant tumor masquerading as eosinophilic gastroenteritis.

A 49-year-old patient presented with urticaria, vomiting, diarrhea and peripheral eosinophilia. A histological diagnosis of eosinophilic gastroenteritis was made. Within 3 weeks of admission a highly papillary adenocarcinoma of the right ovary was diagnosed. The gastrointestinal symptoms and the eosinophilia disappeared after partial resection of the tumor and chemotherapy. A possible relationship between cancer, eosinophilia and eosinophilic gastroenteritis is discussed.

Prenatal diagnosis of congenital methemoglobinemia with mental retardation.

A case of congenital enzymopenic methemoglobinemia associated with severe mental retardation is described. The deficiency of cytochrome b5 reductase activity in the erythrocytes and the leukocytes of the propositus is demonstrated by kinetic measurement and by disc gel electrophoresis. Analysis of cultured amniotic fluid cells during a second pregnancy of the mother revealed an almost complete deficiency of the enzyme. The absence of cytochrome b5 reductase activity in the blood from the aborted fetus confirmed the prenatal diagnosis. The data presented support the opinion that in pregnancies at risk for the severe form of congenital enzymopenic methemoglobinemia, prenatal diagnosis is warranted.

Glucose-6-phosphate isomerase deficiency-Nahariya: extreme in vitro and in vivo lability of the mutant enzyme.

A glucose-6-phosphate isomerase deficiency is described in an Arab boy suffering from chronic hemolytic anemia. The patient was probably true homozygous for the defect. The residual enzyme activity in his red blood cells (RBC) was approximately 30% of normal. The most striking enzyme abnormality observed was an extreme heat lability: upon incubation at 45 C, greater than 90% of activity was lost within 15 min. Furthermore, an increased affinity for the substrate glucose-6-phosphate was shown. The lability of the enzyme was also shown to exist in vivo by separating the patient's RBC into four fractions of different cell age by centrifugation on a discontinuous density gradient. This in vivo lability of the enzyme is believed to be the main cause of the hemolytic diathesis. Remarkably, the residual activity of the enzyme in the RBC of obligate heterozygotes was comparable to that in the patient. However, their enzyme activity was only slightly more labile than that in normal RBC and consequently no signs of hemolysis were noticed.

Serum immunoglobulin-lipid complexes in plasma cell dyscrasia.

Immunoglobulin-lipid complexes (SILC) are found in the serum of all patients with monoclonal gammopathy. Fractionation by density gradient ultracentrifugation, Sephadex G-200 chromatography and Sepharose protein A affinity, followed by extensive immunoassaying for immunoglobulins and lipoproteins, does not confirm the accepted assumption that SILC are immune complexes between monoclonal immunoglobulins and lipoproteins. Lipid extraction of monoclonal fractions isolated on protein A columns followed by thin-layer chromatography shows a lipid pattern characteristic of the one found in cellular membranes. It is proposed that SILC are hydrophobic complexes between immunoglobulins and lipids, as described in membranes of B lymphocytes.

Remission maintenance of acute nonlymphoblastic leukemia with BCNU (NSC-409962) and cyclophosphamide (NSC-26271).

Thirteen of 29 patients with acute nonlymphoblastic leukemia achieved complete remission with cyclophosphamide, cytosine arabinoside, and vincristine, and remissions were maintained with a combination of BCNU and cyclophosphamide. The maintenance drugs (200 and 1000 mg/m respectively) were given at 8-week intervals intravenously. Only six of the 13 patients achieving a complete remission have relapsed. The projected median duration of complete remission is 65 weeks and of survival from diagnosis is 144 weeks. These remission and survival durations compare favorably with the results achieved using other forms of remission-maintenance therapy. The advantage of our form of maintenance therapy is that only overnight hospitalization is required at 8-week intervals.

Particles with characteristics of leukoviruses in cultures of marrow cells from leukemic patients in remission and relapse.

An enzyme activity with the characteristics of RNA-directed DNA polymerase (reverse transcriptase) was detected in marrow from patients with leukemia in relapse and in firm hematological remission. Material having the enzyme activity, when analyzed in sucrose gradients, appeared as two distinct homogeneous bands of particles with densities of about 1.17 and 1.23 g/ml. The enzyme activity was stimulated by exogenous template poly(rC).(dG)(12-18) but not by (dT)(12-18). The enzyme activities in these bands also increased (1.7- to 24-fold) after culture, and both bands with enzyme activity were obtained from the cultured cells and from the supernatant medium. Electron microscopic studies showed that the two bands contained particles resembling leukoviruses or their cores.

Acute tumor lysis syndrome induced by high-dose corticosteroids in a patient with chronic lymphatic leukemia.

Acute tumor lysis syndrome (TLS) has been reported in hematological malignancies, such as aggressive non-Hodgkin's lymphoma, acute lymphoblastic leukemia, and rarely, in other malignancies (solid tumors) in association with the administration of cytotoxic therapy. We report a case of a patient with chronic lymphatic leukemia (CLL) who developed autoimmune hemolytic anemia treated by high dose corticosteroids and, following this treatment, developed acute tumor lysis syndrome. Only one similar case has been reported recently. Clinicians should be aware that corticosteroids alone may produce this potentially life-threatening complication.

Transplantation of human neuroblastoma cells, catecholaminergic and non-catecholaminergic: effects on rotational behavior in Parkinson's rat model.

Cultured human catecholaminergic and non-catecholaminergic donor cells were used in neural transplantation experiments in a rat model of Parkinson's disease. Using two different human catecholaminergic neuroblastoma cell lines, one control non-catecholaminergic neuroblastoma cell line, and one sham control (tissue culture medium), transplants were made into the striatum using a modified Ungerstedt hemiparkinsonian rat model. Significant decreases in apomorphine-induced rotational behavior were produced by two of three catecholaminergic cell lines. Grafted cells staining positively for tyrosine hydroxylase (TH) and catecholamine fluorescence indicated viable catecholamine activity in the two cell lines which produced reductions in rotational behavior. Catecholamine fluorescence was not detected in either of the two controls. These data suggest a link between catecholamine secretion by transplanted cells and motor improvement using a rat rotational behavior model.