Derivation of mixed phenotype cell lines with ras-myc- and raf-myc-containing retroviral vectors after infection of murine long-term bone marrow cultures.

Murine long-term bone marrow cultures were infected with retroviral vectors expressing the viral ras, raf, and myc oncogenes, alone and in combination. Stably transformed clonal cell lines were obtained after infection with ras-myc and raf-myc retroviruses but not by vectors expressing ras, myc, or raf alone. Northern blot analysis demonstrated that the two clonal cell lines expressed high levels of vector-specific transcripts. Phenotypic analysis of the cell lines by flow microfluorimetry and histochemical staining suggested that both cell lines expressed markers associated with cells of the megakaryocyte differentiation pathway. Histochemical staining demonstrated that these cell lines also expressed cytoplasmic enzymes associated with granulocytes and/or monocytes/macrophages. These cell lines, despite their clonal origin, are therefore of a mixed phenotype.

Interleukin 4 alone or in combination with interleukin 1 stimulates 3T3 fibroblasts to produce colony-stimulating factors.

Colony-stimulating activity (CSA) can be produced by fibroblasts when stimulated by interleukin 1 (IL-1). We show that like IL-1, interleukin 4 (IL-4) can stimulate 3T3 fibroblasts to produce CSA. Biological and molecular analyses show that a significant portion of the CSA is colony-stimulating factor 1 (CSF-1) and granulocyte colony-stimulating factor (G-CSF). CSF-1 production in cells stimulated with a combination of both IL-1 and IL-4 was greater than that observed when cells were stimulated with either cytokine alone. However, the data show a synergistic induction of the expression of high levels of G-CSF mRNA and protein in cells incubated in the presence of both IL-1 and IL-4. The concentration of G-CSF in supernatants from cells stimulated with both IL-1 and IL-4 was at least tenfold higher than that measured in supernatants harvested from cells stimulated with either IL-1 or IL-4 alone. Previous investigations have shown that IL-4 had direct effects on hematopoietic progenitor cell growth. The studies described herein indicate that IL-4 is also involved in the regulation of hematopoiesis in an indirect manner, that is, by playing a role in the regulation of hematopoietic growth factor production.

Poor premorbid adjustment and CT scan abnormalities in chronic schizophrenia.

The authors evaluated the premorbid adjustment of 51 chronic schizophrenic patients by retrospective chart analysis using a novel scale. Those patients with CT evidence suggestive of brain atrophy had significantly worse premorbid scores, particularly during childhood, than did the patients with normal scans. This finding provides further support for the clinical relevance of CT findings in chronic schizophrenic patients and implicates a neuropathological process that occurs early in development.

D-dopa and L-dopa similarly elevate brain dopamine and produce turning behavior in rats.

In the intact rat, intragastric administration of D-dihydroxyphenylalanine (D-DOPA) together with carbidopa (alpha-methyldopa hydrazine, a peripheral dopadecarboxylase inhibitor) increased striatal dopamine concentration to the same extent as a similar treatment with L-DOPA plus carbidopa. In rats with unilateral 6-hydroxydopamine-induced lesions of their substantia nigra, both stereoisomers of DOPA produced significant increases in dopamine and its metabolites in the intact striata. Although dopamine concentrations in the lesioned striata did not change, a significant increase in dopamine metabolites was observed, indicating some extraneuronal formation of dopamine. These results suggest that D-DOPA can be converted to dopamine in the normal striatum as well as in the striatum devoid of dopamine nerve terminals. D- and L-DOPA produced turning behavior in unilaterally lesioned rats with a similar efficacy. The onset of turning after D-DOPA was delayed compared with L-DOPA. Turning behavior elicited by these amino acids was attributed to stimulation of supersensitive dopamine receptors in the lesioned striata by the extraneuronally formed dopamine. Preliminary results suggest that D-DOPA is converted to dopamine via transamination and/or D-amino acid oxidation to 3,4-dihydroxyphenylpyruvic acid which upon further transamination gives rise to L-DOPA and hence dopamine. The relatively fast and slow onset of stimulation of dopamine receptors L-DOPA and D-DOPA respectively suggests that the use of the racemic mixture of DOPA combined with a peripheral dopadecarboxylase inhibitor may prove useful in the treatment of parkinsonism.

The Premorbid Adjustment Scale as a measure of developmental compromise in patients with schizophrenia and their healthy siblings.

Schizophrenia is associated with subtle developmental compromise, but the degree to which this is also associated with heritability and genetic risk is uncertain. The goal of the current study was to investigate the childhood, adolescent, and early adulthood social and academic function of patients with schizophrenia, their healthy siblings, and normal controls, using the Premorbid Adjustment Scale (PAS). Generalized Estimating Equations were conducted to account for nesting of subjects within families. Patients (N=286) scored significantly worse than their healthy siblings (N=315) at every age period; siblings scored significantly worse than controls (N=261) at every age period. In probands, PAS scores got worse after early adolescence while control and proband scores improved after late adolescence. Furthermore, patient PAS scores significantly predicted the scores of their own discordant siblings in childhood and late adolescence. This effect approached significance in early adolescence and in the general scale. Thus, the most premorbidly impaired patients tended to have non-ill siblings with worse premorbid adjustment scores than the siblings of less impaired probands. The results suggest that both patients and many of their siblings share poor adjustment in childhood and adolescence, possibly due to shared genetic or environmental risk factors.

T cell receptor V beta gene usage in the recognition of myelin basic protein by cerebrospinal fluid- and blood-derived T cells from patients with multiple sclerosis.

Because of its proximity to the central nervous system, the cerebrospinal fluid (CSF) represents an important source of T cells that potentially could mediate putative autoimmune diseases such as multiple sclerosis (MS). To overcome the low CSF cellularity, we evaluated culture conditions that could expand CSF T cells, with a focus on the expression of T-cell receptor V beta genes utilized by T cells specific for the potentially encephalitogenic autoantigen myelin basic protein (BP). Expansion of "activated" CSF cells with IL-2/IL-4 plus accessory cells optimally retained BP-responsive T cells that over-expressed V beta 1, V beta 2, V beta 5, or V beta 18, compared to expansion using supernatants from PHA-stimulated blood cells, or anti-CD3 antibody that led to different V gene bias and rare reactivity to BP. Sequential evaluation of paired CSF and blood samples from a relapsing remitting MS patient indicated that BP-reactive T cells were present in CSF during the period of clinical activity, and the pattern of BP recognition in CSF was partially reflected in blood, even after CSF reactivity had dissipated during remission. Over-expressed V beta genes were not always constant, however, since in three sequential evaluations of a chronic progressive MS patient, V beta genes over-expressed in the first BP-reactive CSF switched to a different V beta gene bias that was present in the second and third CSF samples. Blood samples reflected each pattern of CSF V beta gene bias, but retained the initial bias for at least 4 months after its disappearance from CSF. These data indicate that selective expansion of IL-2/IL-4-responsive CSF cells favors growth of the BP-reactive subpopulation, and, in a limited number of patients studied, reflected clinical disease activity. In comparison, blood T cells provided a partial but longer lasting reflection of the CSF BP reactivity and V beta gene bias.

Reactivity of monoclonal antibody E5 with endotoxin. I. Binding to lipid A and rough lipopolysaccharides.

The murine IgM monoclonal antibody (mAb) E5 was produced by a hybridoma derived from spleen cells of a mouse immunized with the J5 rough mutant of Escherichia coli O111:B4. In a multicenter randomized placebo-controlled clinical trial, E5 has been shown to reduce significantly the mortality and morbidity of patients with Gram-negative sepsis. The characteristics of E5 binding to endotoxin were studied in vitro. We report here the results of binding to an extensive panel of rough lipopolysaccharide (LPS) and lipid A preparations. Using standard immunologic techniques, including enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), as well as an antibody capture assay using immobilized antibody and a chromogenic Limulus amebocyte lysate (LAL) detection system, E5 was shown to bind to all rough LPS (chemotypes Ra through Re from Salmonella minnesota and E. coli J5) and lipid A preparations tested. E5 displayed a Kd for Ra LPS of approximately 6.5 nM. These results confirm and extend those reported previously and provide evidence that E5 binds specifically to lipid A and to the lipid A moiety of rough LPS.

6-[18F]fluoro-L-dopa and cerebral blood flow in unilaterally MPTP-treated monkeys.

Intravenous administration of 15O-labeled water and 6-[18F]-L-fluorodopa were used to assess abnormal striatal activity in monkeys after long-term recovery of unilateral lesions of the dopaminergic nigro-striatal system induced by the neurotoxin MPTP. PET data were examined in relation to behavioral and biological parameters. Cerebral blood flow and 6-[18F]-L-DOPA uptake were found to be significantly reduced in the lesioned striatum, compared to the unaffected side and to normal controls. There was no correlation between cerebral blood flow and any of the behavioral parameters. The uptake rate constant of 18F-DOPA from blood to striatum and the ratios of striatum to occipital areas were highly correlated to the concentrations of homovanillic acid in the cerebrospinal fluid of the same animals but not to the rotational behavior. This MPTP-induced model of striatal dopamine deficiency in primates presents similarities with idiopathic Parkinson's disease and may be used to evaluate the effects of dopaminergic lesions and transplants on brain function.