Culture of normal and leukemic bone marrow in interleukin-2: analysis of cell activation, cell proliferation, and cytokine production.

Transplantation of bone marrow autografts activated by culture in interleukin-2 (IL-2) followed by administration of IL-2 represents a novel approach in an attempt to combine ex vivo purging and post-transplant in vivo immunotherapy, and initial clinical results have suggested its feasibility. To further characterize the mechanism of the in vitro anti-leukemia effect, fresh bone marrow from normal donors and from patients with acute myelogenous leukemia (AML) in remission was cultured for 6 days in the absence or presence of IL-2 (1000 IU/ml). Proliferation of CD3, CD8, CD14, and CD56 cells was determined by direct immunofluorescence using flow cytometry. Predominantly T-lymphocytes (CD3+) and to a lesser extent CD56+ natural killer (NK) cells proliferate in 6-day marrow cultures in IL-2. Fresh bone marrow cells have no measurable NK activity when tested against K562 and Daudi target cell lines in a 4 h chromium-51 release assay, and it requires at least 6 days of culture in IL-2 to develop optimal cytotoxic activity. Cytokines released in the supernatants of these cultures were measured by immuno- and bioassays. Tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-6 were found to be produced in significant amounts by marrow mononuclear cells during culture in IL-2. Even without IL-2 present, concentrations of these cytokines were increased in 6-day marrow cultures. In contrast, IL-3, IL-7, granulocyte and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF) were below the level of detection of the immunoassay, a result that could be confirmed for GM-CSF and IL-3 by bioassay. The data suggest that culture of marrow from normal donors as well as from patients with AML obtained in remission can generate anti-leukemia effector mechanisms which are non-crossreactive with chemo- and radiotherapy and may contribute to effective ex vivo purging of residual leukemic cells. The transplantation of such IL-2 'primed' marrow may also contribute to an in vivo graft-versus-leukemia effect.

Isozyme patterns and protein profiles in neuromuscular disorders.

The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity.

Increased calcium-activated neutral protease activity in muscles of dystrophic hamsters and mice.

A Ca2+-activated neutral protease activity was examined in muscles of normal and dystrophic hamsters and mice. Light grey and golden brown strains of normal and B10 14.6 strain of dystrophic hamsters were used. Normal and dystrophic mice were of the Bar Harbor 129 ReJ strain. Enzyme activity was measured in the post myofibrillar fraction (homogenate) and in the 75,000 x g pellet (particulate fraction) and supernatant using purified myofibrils. In normal and dystrophic hamsters or mice, the Ca2+-activated neutral protease was most active in the supernatant followed by the homogenate and particulate fractions. As compared to fractions from normal muscle, enzyme activity was significantly elevated in all 3 fractions from dystrophic muscles of hamsters and mice. Both homogenate and supernatant fractions from muscles of normal hamsters had significantly higher enzyme activity than those of normal mice. Enzyme activity was similar in the particulate fraction. Similarly enzyme activity in the 3 fractions from dystrophic hamster and mouse muscles showed no significant difference. It is suggested that the Ca2+-activated neutral protease may be involved in muscle fibre necrosis in muscular dystrophy.

Hypoxanthine-guanine phosphoribosyltransferase activity of blood and muscle in Duchenne dystrophy.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity was measured in red cells and in skeletal muscles of normal and Duchenne subjects. [8-14C] hypoxanthine was used as substrate, and 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) was used as the ribose-5-phosphate donor. The [8-14C] inosine monophosphate (IMP) formed was separated by high-voltage electrophoresis, and radioactivity was measured by lipid scintillation counting. HGPRT activity in Duchenne and normal red-cell hemolysates was similar, but such activity was significantly higher in Duchenne than in normal muscle homogenates. Red cells of both normal and Duchenne subjects had significantly higher enzyme activity than did skeletal muscles. It is suggested that increased HGPRT activity may be involved in enhancing protein synthesis by increasing intracellular levels of purine ribonucleotides.

Hypoxanthine--guanine phosphoribosyltransferase activity in blood and skeletal muscles of normal and dystrophic mice.

1. Hypoxanthine--guanine phosphoribosyltransferase (HGPRT) activity was measured in erythrocyte haemolysates and quadriceps muscle extracts of normal and dystrophic 129 ReJ and C57 BL/6J mice with [8(-14)C]hypoxanthine as substrate and 5-phosphorylribose 1-pyrophosphate as a ribose 5-phosphate donor. [8(-14)C]Inosine monophosphate formed was separated by high-voltage electrophoresis and radioactivity was measured by liquid-scintillation counting. 2. In erythrocyte haemolysates, HGPRT activity was similar in normal and dystrophic C57 BL/6J mice but was significantly higher in dystrophic than in normal 129 ReJ mice. Elevated enzyme activity was observed only in mice that were clinically severely affected. 3. In muscle homogenates, HGPRT activity was significantly higher in dystrophic than in normal animals of both 129 ReJ and C57 BL/6J mice. Enzyme activity was not related to the severity of the disease. 4. It is suggested that changes in erythrocytes are secondary to the dystrophic process and that elevated HGPRT activity in skeletal muscle may be related to abnormal energy metabolism, possibly via the pentose monophosphate shunt.

Comparison of gamma-glutamyl transpeptidase activity in tissues of normal and dystrophic hamsters and mice.

The activity of gamma-glutamyl transpeptidase (gamma-GT), a membrane-bound enzyme, was assayed by a sensitive fluorometric method in the brain, heart, kidney, liver, skeletal muscle and serum of normal and dystrophic hamsters and mice. Normal and dystrophic hamsters were of the golden brown and BIO 14.6 line respectively. Both normal and dystrophic mice were of the C57 BL/6J -dy2j strain. Kidneys of both hamsters and mice contained the highest enzyme activity followed by the brain, skeletal muscle, liver and heart. Gamma-GT activity was elevated in dystrophic skeletal muscles but decreased in the dystrophic kidneys and sera. In the brain, heart and liver of dystrophic animals from both species enzyme activity was similar to that in normals.

Concomitance of basophilia, ribonucleic acid and acid phosphatase activity in regenerating muscle fibres.

Regenerating fibres from tibialis anterior muscles of mice and hamsters transplanted as minced fragments for 7 and 9 days respectively were compared for basophilia, ribonucleic acid (RNA) and acid phosphatase activity with fibres in muscles of patients with Duchenne muscular dystrophy, limb--girdle dystrophy and dermatomyositis. Normal muscles of mice, hamsters and humans were used as controls. In normal muscle fibres basophilia and RNA activity were restricted to the nuclei and acid phosphatase activity was occasionally observed in the nuclei, endomysial connective tissue and around muscle spindles. Diseased human muscle fibres were rich in acid phosphatase activity, the enzyme being most prominent in degenerating and basophilic fibres. When examined in serial sections, all basophilic fibres showed RNA and acid phosphatase activity. Similarly, all regenerating fibres in minced transplants which were basophilic to haematoxylin contained high RNA and acid phosphatase activity. It is suggested that basophilia in diseased human muscle fibres represents regeneration and that the lysosomes, as defined by acid phosphatase activity in these fibres, may be promoting their growth and differentiation by facilitating greater nucleocytoplasmic communication which ultimately could lead to protein synthesis.

Familial poliodystrophy, mitochondrial myopathy, and lactate acidemia.

We describe a 16-year-old boy who has a progressive dementia and seizures. On investigation, he was found to have a mitochondrial myopathy and elevated lactate levels in the blood and cerebrospinal fluid. His sister died at 18 years of age of a similar condition.

Effect of pentobarbitone sodium on serum creatine kinase of normal and dystrophic hamsters.

1. The dose of pentobarbitone required for anaesthesia was significantly greater for dystrophic hamsters than for normal animals. 2. Serum creatine kinase activity was significantly higher in dystrophic than in normal hamsters. 3. Brain, heart and tibialis anterior muscle from dystrophic animals contained significantly less creatine kinase than the normal tissues. 4. Creatine kinase in normal and dystrophic sera, as in skeletal muscles, consisted of MM isoenzyme. Heart creatine kinase consisted of both MM and MB types and brain contained only the BB isoenzyme. 5. Pentobarbitone raised serum creatine kinase activity of normal and dystrophic hamsters to the same extent, elevation of enzyme activity being dependent on the amount of pentobarbitone injected. 6. The sera of pentobarbitone-treated normal and dystrophic hamsters contained only the MM isoenzyme.