Chromogranin A is an acidic protein costored and coreleased with catecholamines from storage vesicles. Its serum concentration is elevated in patients with peptide-producing endocrine neoplasia. We measured serum chromogranin A at the time of diagnosis in 34 children with all stages of neuroblastoma. With a sensitivity of 91% and specificity of 100%, serum chromogranin A emerged as a useful diagnostic tool for neuroblastoma, comparable to or better than other measurements such as neuron-specific enolase, ferritin, or dopamine-beta-hydroxylase. Mean serum chromogranin A correlated with disease stage (r = 0.76, P less than 0.01). The relationship of prognosis (progression-free survival) to baseline serum chromogranin A, age, and disease stage was determined in 34 patients at risk for relapse, with a median followup period of 18 mo (range, 1-48 mo). The survival rate for patients with lower serum chromogranin A levels (less than 190 ng/ml at the time of diagnosis) was 69%, whereas it was 30% for those with higher chromogranin A levels (P less than 0.05). Furthermore, when subjects were additionally stratified by either age or stage, chromogranin A was an effective prognostic tool in patients who either were older than 1 yr (P less than 0.005) or had more advanced disease (stage III or IV; P less than 0.05). We conclude that serum chromogranin A in neuroblastoma is (a) a valuable (sensitive and specific) diagnostic tool, (b) a correlate of tumor burden, and (c) a useful predictor of survival.
R J Hsiao, R C Seeger, A L Yu, D T O'Connor
Interleukin-3 (IL-3) is a hematopoietic growth factor that supports the growth of early hematopoietic progenitors in vitro. In vivo administration of recombinant human interleukin-3 (rhIL-3) to normal primates results in a modest and delayed leukocytosis secondary to increases in neutrophils, basophils, and eosinophils. We postulated that the effects of rhIL-3 might be more pronounced in hematologically stressed primates, and therefore administered rhIL-3 to primates after intensive myelosuppressive therapy. Primates received either cyclophosphamide (CPM) at 60 mg/kg or 5-fluorouracil (5-FU) at 75 mg/kg i.v. on two consecutive days. Subsequently, rhIL-3 was administered intravenously or subcutaneously at 20 micrograms/kg per d for 14 d. Compared to controls, all rhIL-3 treated primates experienced higher absolute neutrophil count (ANC) nadirs and dramatic decreases in the period of severe neutropenia (ANC less than 500) after myelosuppressive therapy. RhIL-3 administration resulted in a significant basophilia and eosinophilia, which resolved after discontinuation of the drug. RhIL-3 did not enhance erythroid recovery. Platelet recovery was earlier in rhIL-3-treated animals. However, variations in the platelet recovery observed in control animals, precluded accurate estimation of this effect or its significance. Our results indicate that the administration of rhIL-3 following intensive myelosuppressive therapy dramatically enhances myeloid recovery and ablates the predicted period of prolonged severe neutropenia.
A P Gillio, C Gasparetto, J Laver, M Abboud, M A Bonilla, M B Garnick, R J O'Reilly
To analyze the autoepitopes on the SS-B/La protein, a cDNA covering the entire region coding the protein was isolated from a human cDNA library. The cDNA was subcloned into an expression plasmid vector, pEX, to express its protein product as a fusion protein with cro-beta-galactosidase in Escherichia coli. A recombinant pEX plasmid expressing three-fourths of the protein (amino acid 112-408) was also constructed. The antigenicities of these recombinant proteins were confirmed with a patient's serum. Their various deletion mutants were produced with exonuclease III treatment from the 3' ends of the cDNAs without changing the proper translational frame. Immunoblot analysis and enzyme-linked immunosorbent assay were used to evaluate the reactivities of the recombinant proteins with patients' sera to determine the autoepitopes. A narrow segment (amino acid 88-101) and the region where several epitopes were located (amino acid 283-338) on the SS-B/La protein were universally recognized by all the sera with anti-SS-B/La antibodies examined. An additional epitope region (amino acid 179-220) was recognized by some patients' sera. Computer analysis revealed that the most distinct autoepitope, amino acid 88-101, had a striking homology to a retroviral gag polyprotein. These findings indicate that exogenous or endogenous retroviruses may play a role in initiation of the anti-SS-B/La autoimmunity.
H Kohsaka, K Yamamoto, H Fujii, H Miura, N Miyasaka, K Nishioka, T Miyamoto
Two distinct mutant alleles of the precursor (p) short chain acyl-CoA dehydrogenase (SCAD) gene were identified in a SCAD-deficient patient (YH2065) using the polymerase chain reaction to amplify cDNA synthesized from total RNA from her fibroblasts. Cells from this patient had previously been shown to synthesize a labile variant SCAD in contrast to the normal stability of variant SCADs in two other SCAD-deficient cell lines (Naito, E., Y. Indo, and K. Tanaka. 1989. J. Clin. Invest. 84:1671-1674). In the present study, both mutant alleles of YH2065 were found to contain a C----T transition, one at position 136 and the other at position 319 of the coding region of pSCAD cDNA. Clones of cDNA amplified from this region showed only one of the C----T transitions, indicating that each mutation was derived from different pSCAD alleles. Each of these mutations altered a known restriction endonuclease site, and restriction analysis of additional cDNA clones from amplified mutant cDNA and Southern blotting of mutant genomic DNA confirmed the presence of two unique mutant alleles in YH2065, indicating YH2065 is a compound heterozygote. These C----T transitions result in the substitution of Arg-22 and Arg-83 of the mature SCAD with Trp and Cys, respectively.
E Naito, Y Indo, K Tanaka
Most cases of cytosol-defective chronic granulomatous disease are due to the deficiency of a 47-kD protein (p47-phox) whose phosphorylation normally accompanies the activation of the respiratory burst oxidase. Recently, a form of chronic granulomatous disease was described in which the failure of O2- production was associated with the absence of a 67-kD polypeptide (p67-phox) from the cytosol of affected neutrophils. Using neutrophils obtained from a patient with this form of the disease, we examined the function of p67-phox in the activation of the oxidase. Our studies showed that in whole p67-phox-deficient neutrophils, p47-phox was phosphorylated in a normal fashion. In the cell-free oxidase-activating system, the ability of the p67-phox-deficient cytosol to support oxidase activation was partly restored by the addition of p47-phox-deficient cytosol; the p67-phox-deficient cytosol, however, was not complemented by cytosol inactivated with NADPH dialdehyde, an affinity label previously found to block the NADPH-binding component of the oxidase. Despite these differences, the kinetic properties of the p67-phox-deficient cytosol closely resembled those of the p47-phox-deficient cytosol. Taken together with earlier findings, these results suggest that (a) in the neutrophil cytosol, p67-phox is at least partly complexed to p47-phox; (b) it is in the form of this complex that p67-phox participates in oxidase activation; and (c) p47-phox appears to be translocated from the cytosol to the plasma membrane during oxidase activation, but complexation to p67-phox is not necessary for this translocation, nor for the accompanying extra protein phosphorylation.
N Okamura, B M Babior, L A Mayo, P Peveri, R M Smith, J T Curnutte
To compare extra-renal 1,25(OH)2D3 production in different types of granulomatous disease, and to identify the cell types responsible, we have evaluated the conversion of 25(OH)D3 in 1,25(OH)2D3 by uncultured cells recovered by bronchoalveolar lavage and blood mononuclear cells from normocalcemic patients with sarcoidosis and tuberculosis. 1,25(OH)2D3 was produced both by lavage cells (12/12 tuberculosis patients, 2/6 sarcoidosis patients) and blood mononuclear cells (3/5 tuberculosis patients, 0/3 sarcoidosis patients) from patients but not controls, but significantly greater amounts were produced by lavage cells from tuberculosis patients than those of sarcoidosis patients (P less than 0.001). 1,25(OH)2D3 production by lavage cells from tuberculosis patients correlated with the number of CD8+ T lymphocytes present but not other cell types. T lymphocytes appeared to be an important source of 1,25(OH)2D3 production, since purified T lymphocytes from all patients with tuberculosis produced 1,25(OH)2D3, and 1,25(OH)2D3 production by these cells correlated closely with that produced by unseparated lavage cells. Because 1,25(OH)2D3 can improve the capacity of macrophages to kill mycobacteria, our results support the conclusion that macrophage-lymphocyte interactions, mediated at least in part by 1,25(OH)2D3, may be an important component of a successful antituberculous immune response.
J Cadranel, M Garabedian, B Milleron, H Guillozo, G Akoun, A J Hance
Effective T cell vaccination against experimental autoimmune diseases involves treatment with activated, autoimmune T lymphocytes. The present study was undertaken to learn whether antigen-specific T cells present in low frequency could be selected in vitro without using the specific antigen. The rat models of adjuvant arthritis and experimental autoimmune encephalomyelitis were investigated using proliferation assays and limiting dilution techniques to quantify the changes in reactivity of a heterogenous population of lymphocytes to the relevant antigen. Stimulation with concanavalin A for 2 d and then culture in IL-2-containing medium led to a substantial increase in the activity and frequency of the specific autoimmune T cells. Enrichment of antigen-specific T cells could be demonstrated using lymph node, spleen, or peripheral blood lymphocytes, from rats late in the course of disease. The effect was not evident in lymphocytes from the thymus. These results are relevant to the clinical application of T cell vaccination and to investigation of self-antigens in autoimmune disease.
F Mor, A W Lohse, N Karin, I R Cohen
Experiments were performed in human working myocardium to investigate the relationship of intracellular calcium handling and availability to alterations in the strength of contraction produced by changes in stimulation rate and pattern. Both control and myopathic muscles exhibited potentiation of peak isometric force during the postextrasystolic contraction which was associated with an increase in the peak intracellular calcium transient. Frequency-related force potentiation was attenuated in myopathic muscles compared to controls. This occurred despite an increase in resting intracellular calcium and in the peak amplitude of the calcium transient as detected with aequorin. Therefore, abnormalities in contractile function of myopathic muscles during frequency-related force potentiation are not due to decreased availability of intracellular calcium, but more likely reflect differences in myofibrillar calcium responsiveness. Sarcolemmal calcium influx may also contribute to frequency-related changes in contractile force in myopathic muscles as suggested by a decrease in action potential duration with increasing stimulation frequency which is associated with fluctuations in peak calcium transient amplitude.
J K Gwathmey, M T Slawsky, R J Hajjar, G M Briggs, J P Morgan
Age-dependent alterations in the effects of catecholamines on lipolysis were investigated in 25 young (21-35 yr) and 10 elderly (58-72 yr) healthy, nonobese subjects using isolated adipocytes obtained from abdominal subcutaneous tissue. Basal lipolysis was not affected by aging, while the rate of catecholamine-stimulated lipolysis was reduced by 50% in the elderly subjects (P less than 0.005). To elucidate the mechanisms behind this phenomenon lipolysis was stimulated with agents that act at well-defined steps in the lipolytic cascade, from the receptor down to the final step: the activation of the protein kinase/hormone-sensitive lipase complex. All agents stimulated lipolysis at a 50% lower rate in elderly as compared with young subjects (P less than 0.05 or less). However, half-maximum effective concentrations of the lipolytic agents were similar in both groups. The antilipolytic effects of alpha 2-adrenoceptor agonists were also the same in young and old subjects. Moreover, the stoichiometric properties of the beta- and alpha 2-receptors did not change with increasing age. In vivo studies performed on the same individuals likewise demonstrated an impaired lipolytic responsiveness, with 50% lower plasma glycerol concentrations during exercise in the elderly subjects (P less than 0.05), in spite of a normal rise in plasma norepinephrine. The plasma glycerol levels correlated strongly to the glycerol release caused by catecholamine-stimulated lipolysis in vitro in both young and elderly subjects (r = 0.8-0.9, P less than 0.001). In conclusion, a decreased activation of the hormone-sensitive lipase complex appears to be the mechanism underlying a blunted lipolytic response of fat cells to catecholamine stimulation in elderly subjects. This finding may, explain the age-dependent decreased lipolytic response to exercise in vivo.
F Lönnqvist, B Nyberg, H Wahrenberg, P Arner
Adenosine has been proposed to act within the juxtaglomerular apparatus (JGA) as a mediator of the inhibition of renin secretion produced by a high NaCl concentration at the macula densa. To test this hypothesis, we studied the effects of the adenosine1 (A1)-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (CPX) on renin release from single isolated rabbit JGAs with macula densa perfused. The A1-receptor agonist, N6-cyclohexyladenosine (CHA), applied in the bathing solution at 10(-7) M, was found to inhibit renin secretion, an effect that was completely blocked by adding CPX (10(-5) M) to the bath. Applied to the lumen, 10(-5) M CPX produced a modest stimulation of renin secretion rates suppressed by a high NaCl concentration at the macula densa (P less than 0.05). The effect of changing luminal NaCl concentration on renin secretion rate was examined in the presence of CPX (10(-7) and 10(-5) M) in the bathing solution and in vehicle control experiments. The control response to increasing luminal NaCl concentration was a marked suppression of renin secretion, that was maintained as long as luminal NaCl concentration was high and was promptly reversible when concentration was lowered. CPX did not alter renin release when luminal NaCl was low, but diminished the reduction caused by high NaCl (P less than 0.01). It is concluded that A1-receptors are located within the JGA, and that A1-receptor activation inhibits renin release. A high NaCl concentration at the macula densa appears to influence A1-receptor activation, but a low NaCl concentration does not. The findings support participation of adenosine in macula densa control of renin secretion.
H Weihprecht, J N Lorenz, J Schnermann, O Skøtt, J P Briggs
No posts were found with this tag.