Isolation and structure of two novel muscarinic receptor antagonists.

The structures of two novel muscarinic receptor antagonists, 1 and 2, were determined by their spectral data and high-resolution mass measurements of their degradation products. Both are aliphatic long-chain compounds and contain amide and keto functionalities. The major microbial metabolite [1] contains three terminal guanidino groups and the minor compound [2] has two terminal guanidino groups.

Interleukin-6 production by Schwann cells and induction in sciatic nerve injury.

Interleukin-6 (IL-6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell-derived IL-6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL-6. Cocultured iSC transcribe IL-6 message as confirmed by northern analysis. Stimuli that induce IL-6 production in the hematopoietic lineage induced transcription and production of IL-6 by iSC cells. Lipopolysaccharide, tumor necrosis factor-alpha, IL-1 alpha, IL-6, and serum withdrawal induced iSC cell IL-6 mRNA. The kinetics of IL-6 production was confirmed in the mouse IL-6-dependent B9 bioassay and that activity could be neutralized with antisera to IL-6. Expression of both the IL-6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL-6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL-6 message is induced. IL-6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL-6 in response to PC12 cell coculture and to stimuli that induce IL-6 production in the hematopoietic lineage. The induction of IL-6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.

Elevated serum levels of interleukin-5 in patients with the syndrome of episodic angioedema and eosinophilia.

The syndrome of episodic angioedema and eosinophilia is characterized by cyclic edema, marked peripheral blood eosinophilia, and eosinophil degranulation in the dermis. Using a sensitive immunoenzymetric method, we measured serum interleukin (IL)-5 levels in four patients with this syndrome. We also determined the percentage of activated T cells in the peripheral blood of a new patient before and during an attack. In the patient presented, IL-5 levels peaked several days before maximal eosinophilia and then declined. This patient's lymphocytes showed an increased percentage, 28% (normal 2% to 3%), of activated T cells staining for both CD3 and HLA-DR 10 days before maximal eosinophilia, but no increase at the time of peak eosinophilia. In serum from three previously reported cases, elevated serum IL-5 levels were found during attacks. After glucocorticoid administration, IL-5 levels became undetectable in three of the four patients. Production of IL-5 is likely an important determinant of the pathophysiology of this syndrome.

Interleukin-5 and the posttreatment eosinophilia in patients with onchocerciasis.

To understand the role of the eosinophilopoietic cytokine IL-5 in humans, the posttreatment eosinophilic response in a group of microfilaria (mf)-positive patients with onchocerciasis (n = 10) was examined before and after treatment with diethylcarbamazine (6 mg/kg for 7 d). Sequential blood samples were assessed at 24 and 1 h before treatment (baseline values), then at frequent intervals over the next 14 d. Symptom scores, skin microfilariae (mf), and peripheral blood eosinophil counts were recorded as a function of time after treatment, and serum levels of IL-5 were quantitated by a highly sensitive (sensitivity greater than or equal to 20 pg/ml) monoclonal-based ELISA. Pretreatment eosinophil counts ranged from 240 to 1,186 eosinophils/microliter (geometric mean, 675), and the mf counts from 10 to 218 per mg skin (geometric mean, 79). After an initial decline in the peripheral eosinophil count to 28 +/- 8% of pretreatment levels at 8 h after beginning treatment, the eosinophil counts steadily increased over the next 2 wk, reaching a maximum at 14 d (257 +/- 38% of pretreatment levels). Serum levels of IL-5 rose sharply from pretreatment levels to a peak of 70.5 +/- 11 pg/ml by 24 h after treatment. Serum IL-5 remained elevated over the next 2-3 d and declined toward baseline by approximately 6 d after treatment, at which time the eosinophil levels were steadily increasing. IL-3 and granulocyte macrophage colony-stimulating factor, two other cytokines implicated in eosinophilopoeisis, were not detectable in the serum at any time before or after treatment. The rise in serum IL-5 before the posttreatment eosinophilia seen in this group of patients with onchocerciasis demonstrates a temporal relationship between IL-5 and the subsequent development of eosinophilia and implicates IL-5 as an important mediator of eosinophilia in humans.

Administration of interleukin-2 (IL-2) results in increased plasma concentrations of IL-5 and eosinophilia in patients with cancer.

Peripheral eosinophilia is almost invariably observed during the course of interleukin-2 (IL-2) therapy and is frequently accompanied by the development of a capillary leak syndrome characterized by edema, weight gain, and oliguria. We studied five patients with advanced malignancy treated with IL-2. Eosinophilia was not present initially but developed in all patients late in the course of therapy, with counts ranging from 2,328/mm3 to 15,958/mm3. In all patients, there was a temporal relationship between the infusion of IL-2 and the appearance of elevated plasma concentrations of IL-5, a growth factor for eosinophils. Granulocyte-macrophage colony-stimulating factor was not detectable in plasma. IL-4 and gamma-interferon plasma levels were variably elevated. Plasma concentrations of major basic protein, a toxic eosinophil granule protein, began increasing before eosinophil counts increased. By the time of the third IL-2 infusion, high concentrations of major basic protein were present in all five patients (up to 5,600 ng/mL) and skin biopsies showed major basic protein deposition in the dermis. Four patients developed significant capillary leak syndrome and all of these patients showed markedly elevated major basic protein levels. The lowest peak plasma concentration of major basic protein (1,751 ng/mL) was observed in the one patient who did not develop edema and weight gain. These results suggest that IL-2 induces IL-5 leading to marked peripheral eosinophilia and extravascular eosinophil degranulation. The release of toxic eosinophil products at extravascular sites and in the circulation may contribute to the pathogenesis of the capillary leak syndrome complicating IL-2 therapy.

Interleukin-5 is a human basophilopoietin: induction of histamine content and basophilic differentiation of HL-60 cells and of peripheral blood basophil-eosinophil progenitors.

Cytokine-induced differentiation of basophils may contribute to various inflammatory processes. We examined the effects of recombinant human interleukin-5 (IL-5) and other human cytokines in vitro on myeloid colony formation in methylcellulose and on alkaline passaged HL-60 basophilic cell differentiation. Myeloid colonies (CFU-C) at day 14, formed in the presence of either IL-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or G-CSF included peripheral blood-derived progenitors of the eosinophil/basophil lineage. IL-5 stimulated a greater proportion of basophil-containing, histamine-positive, eosinophil-type colonies compared with GM-CSF, IL-3, or G-CSF. IL-5 also stimulated dose-dependent increases in histamine content of alkaline-passaged, butyrate cotreated HL-60 cells. The concentration of IL-5 required for half-maximal induction of HL-60 histamine content was similar within twofold to that needed for half-maximal stimulation of the multifactor dependent TF-1 erythroleukemic cell line. Neutralizing rat monoclonal antibodies to human IL-5 were developed and used to demonstrate that each of these IL-5 bioactivities could be specifically blocked. We conclude that in addition to its previously described eosinophil differentiation activity, IL-5 may be considered a basophilopoietin.

Regulation of parasite-induced eosinophilia: selectively increased interleukin 5 production in helminth-infected patients.

Production of the eosinophilogenic cytokines interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and IL-5 by mitogen-stimulated peripheral blood mononuclear cells was compared between 11 noneosinophilic individuals and seven patients with helminth-induced eosinophilia. Both the kinetics and quantities of IL-3 and GM-CSF were similar in the two groups. In contrast, IL-5 production at both the protein and the mRNA level was markedly greater in the eosinophilic patients, an observation suggesting that IL-5 may be particularly important in mediating the selective eosinophilia seen in filarial and other helminth infections.

Effect of hydration on plasma volume and endocrine responses to water immersion.

To determine the effect of hydration on the early osmotic and intravascular volume and endocrine responses to water immersion the hematocrit, hemoglobin, plasma renin activity (PRA), and plasma electrolyte, aldosterone (PA), and vasopressin (PVP) concentrations were measured during immersion following 24-h dehydration; these were compared with corresponding values following rapid rehydration. Six men and one woman (age 23-46 yr) underwent 45 min of standing immersion to the neck preceded by 45-min standing without immersion, first dehydrated, and then 105 min later after rehydration with water. Immersion caused an isotonic expansion of the plasma volume (P less than 0.001), which occurred independently of hydration status. Suppression of PRA (P less than 0.001) and PA (P less than 0.001) during both immersions also occurred independently of hydration status. Suppression of plasma vasopressin was observed during dehydrated immersion (P less than 0.001) but not during rehydrated immersion. It is concluded that plasma tonicity is not a factor influencing PVP suppression during water immersion.

Effect of hand-arm exercise on venous blood constituents during leg exercise.

To test the hypothesis that ancillary arm and hand exercise would change the values of antecubital blood constituents during leg exercise, seven healthy men (19-27 yrs) performed static (10% of a maximal voluntary contraction) or dynamic (60 finger flexions/min) hand-arm exercise with one hand during submaximal leg exercise (50% V2 max) in the supine position. Venous blood was analyzed for serum Na+, K+, osmolality, albumin, total Ca2+, and glucose; blood hemoglobin, hematocrit, and lactic acid; and change in plasma volume. During leg exercise there were no significant differences in these blood constituents between right and left arms at rest. Only glucose and lactate were affected by additional arm exercise. Compared with resting arm values during leg exercise, glucose decreased from 4.7 to 4.5 mmol/l (delta = 4%, P less than 0.05) and lactate increased from 2.0 to 2.4 mmol/l (delta = 20%, P less than 0.05) during static arm exercise. With dynamic arm exercise, glucose decreased from a resting level of 4.8 to 4.7 mmol/l (delta = 2%, P less than 0.05). We conclude that additional static or dynamic hand-forearm exercise accompanying leg exercise could introduce significant errors in glucose (2%-4%) and lactic acid (6%-20%) concentrations measured in venous blood.

A 3' end fragment encompassing the transcriptional enhancers of nondefective Friend virus confers erythroleukemogenicity on Moloney leukemia virus.

Nondefective Friend helper murine leukemia virus (Fr-MuLV) induces primarily erythroleukemias in NFS mice, whereas Moloney murine leukemia virus (Mo-MuLV) induces T cell lymphomas. Using molecular clones of these two viruses, we constructed a recombinant in which a 0.62-kilobase fragment encompassing the U3 region at the 3' end of the Fr-MuLV genome replaced the corresponding region of Mo-MuLV. The recombinant virus obtained by transfection of this clone, whose genome is derived primarily from Mo-MuLV, induces almost exclusively erythroleukemias in NFS mice. This and the previous result of Chatis et al. (Proc. Natl. Acad. Sci. U.S.A. 80:4408-4411), showing that the reciprocal recombinant whose genome is primarily derived from Fr-MuLV induces almost exclusively lymphomas, argue that a strong determinant of the distinct disease specificities of Fr-MuLV and Mo-MuLV lies in this 3' end 0.62-kilobase fragment which contains the putative virus enhancers. To more precisely define this determinant, we have begun to construct recombinants in which smaller 3' end fragments of the Fr-MuLV and Mo-MuLV genomes are exchanged. Analysis of the first such recombinant showed that Fr-MuLV can be converted to a lymphoma-inducing virus in NFS mice by substitution of a 0.38-kilobase fragment encompassing the virus enhancers in U3 with the corresponding region of the Mo-MuLV genome.

Immersion diuresis without expected suppression of vasopressin.

To investigate fluid, electrolyte, and plasma vasopressin (PVP) and renin activity (PRA) responses, six men (20-35 yr) were immersed to the neck (NI) in water at 34.5 degrees C for six h after overnight food and fluid restriction. Diuresis was 1,061 +/- 160 (SE) ml/6 h during immersion and water balance was -1,285 +/- 104 ml/6 h. Preimmersion PVP was 0.7 +/- 0.2 pg/ml and increased to 3.0 +/- 0.6 pg/ml (P less than 0.05) at 6 h. PVP was unchanged at 1.2 +/- 0.1 pg/ml in the 6-h seated nonimmersion experiment at 25 degrees C. Plasma volume increased by 7.8 +/- 1.6% (P less than 0.05) at 60 min of NI and decreased thereafter. Serum osmolality was constant (292 +/- 1 mosmol/kg) throughout NI, whereas PRA decreased progressively from 1.9 to 0.5 ng angiotensin I X ml-1 X h-1 (P less than 0.05) at the end of immersion. In spite of moderate thirst just before NI, thirst sensations were attenuated and no water was consumed ad libitum during immersion. These data indicate that PVP is not suppressed when there is no fluid intake during immersion and suggest that the action of factors other than PVP suppression are necessary to explain the mechanism of immersion diuresis.

Susceptibility to Friend helper virus leukemias in CXB recombinant inbred mice.

The seven CXB recombinant inbred strains were tested for susceptibility to Friend helper virus (F-MuLV) hematopoietic neoplasms. BALB/c and CXB-H mice develop erythroblastosis after neonatal inoculation with F-MuLV, while C57BL/6 and the six other RI strains develop lymphoma and myelogenous leukemia. This strain distribution pattern is different from that for H-2, Gpd-1 (linked to Fv-1), Fv-2, Rfv-3, and Cv (linked to Rmcf) but the same as that for Bv, the endogenous ecotropic virus of C57BL/6. However, analysis of crosses segregating Bv show that resistance to F-MuLV erythroblastosis is not linked to Bv. Disease-free survival is shortest for BALB/c mice, intermediate for CXB-H and CXB-J, and longest for C57BL/6 and the other RI strains. We conclude: (a) the major C57BL/6 gene for resistance to F-MuLV erythroblastosis is different from previously identified Friend virus restriction loci; (b) latency for F-MuLV leukemias is controlled by more than one gene; and (c) latency and susceptibility to F-MuLV erythroblastosis are not inherited concordantly in the CXB-RI strains.

A new gene that controls the type of leukemia induced by Friend murine leukemia virus.

NB tropic Friend murine leukemia virus (F-MuLV) replicates equally well in BALB/c and C57BL mice inoculated as neonates but causes almost exclusively erythroblastosis in BALB/c mice and nonerythroid (lymphoid and myelogenous) leukemias in C57BL mice. The C57BL resistance to erythroblastosis appears to be controlled by a single dominant gene in first and second backcrosses to BALB/c. This resistance to erythroblastosis is distinct from other genes known to affect susceptibility to Friend virus including Fv-1, Fv-2, H-2, Rfv-3, Fv-4, and Rmcf. We suggest the name Fhe for the new gene controlling susceptibility to Friend helper virus erythroblastosis.

Cas spleen focus-forming virus. II. Further biological and biochemical characterization.

A new isolate of a murine erythroblastosis-inducing spleen focus-forming virus (Cas SFFV), derived from the wild mouse ecotropic murine leukemia virus Cas-Br-M, was further characterized after the production of a nonproducer cell line. When rescued from the nonproducer cells with a helper murine leukemia virus, the Cas SFFV induced rapid splenic enlargement and focus formation when inoculated into adult NFS/N mice. The Cas SFFV nonproducer cell line was also utilized to compare the envelope-related glycoprotein of Cas SFFV with gp52s from three strains of Friend SFFV. Cas SFFV was found to encode a 50,500-dalton glycoprotein (gp50) distinct in size to the envelope-related glycoproteins of the Friend SFFVs. The Cas SFFV was also compared in RNA blot hybridization studies. The genomic viral RNA of Cas SFFV was found to be slightly larger than two polycythemia-inducing strains of Friend SFFV and markedly larger than the anemia-inducing strain. Further comparisons between the SFFVs were made by examining their transforming capabilities in an in vitro erythroid burst assay. The erythroid bursts induced by Cas SFFV and the anemia-inducing strain of Friend SFFV showed similarities in their erythropoietin requirements. This study supports our recent observations that Cas SFFV is biologically similar to the anemia-inducing strain of Friend SFFV yet biochemically distinct from all Friend SFFVs.

Identification of a spleen focus-forming virus in erythroleukemic mice infected with a wild-mouse ecotropic murine leukemia virus.

An NFS/N mouse inoculated at birth with an ecotropic murine leukemia virus (MuLV) obtained from wild mice (Cas-Br-M MuLV) developed a lymphoma after 18 weeks. An extract prepared from the lymphomatous spleen was inoculated into newborn NFS/N mice, and these mice developed erythroleukemia within 9 weeks. Spleens from the erythroleukemic mice contained ecotropic and mink cell focus-inducing (MCF) MuLVs; however, when these viruses were biologically cloned and reinoculated into newborn NFS/N mice, no erythroleukemia was induced. In contrast, cell-free extracts prepared from the erythroleukemic spleens induced erythroleukemia within 5 weeks. Analysis of cell-free extracts prepared from the erythroleukemic spleens showed that they contained a viral species that induced splenomegaly and spleen focus formation in adult mice, with susceptibility controlled by alleles at the Fv-2 locus. The spleen focus-forming virus coded for a 50,000-dalton protein precipitated by antibodies specific to MCF virus gp70. RNA blot hybridization studies showed the genomic viral RNA to be 7.5 kilobases and to hybridize strongly to a xenotropic or MCF envelope-specific probe but not to hybridize with an ecotropic virus envelope-specific probe. The virus described here appears to be the fourth independent isolate of a MuLV with spleen focus-forming activity.

Polyoma T (tumor) antigen species in abortively and stably transformed cells.

Stable neoplastic transformation of cells by polyoma virus requires the particpation of two viral genes, designated ts-a and hr-t. The effects of mutations in these two genes on the patterns of T-antigen synthesis during productive infection have been previously described: ts-a mutants are affected in the "large" (100K) nuclear T antigen, and hr-t mutants are affected in the "middle" (36K, 56K, 63K) and "small" (22K) T antigens. The latter are associated predominantly with the plasma membrane (56K) and cytosol fractions, respectively. Here we examine the expression of the various forms of polyoma T antigen in nonproductive infection (abortive transformation) as well as in stably transformed cell lines of different species. The results on abortive transformation are essentially the same as those described above for productive infection. In stably transformed cells, the middle and small T antigens are seen to various extents. The large T antigen, however, is often absent or present below the level of detection. Clones lacking the large T antigen are found most often among mouse transformants, but are also seen among rat transformants. Retention of the 100K species in transformed cells therefore appears to be, at least in part, an inverse function of the level of permissivity of the host toward productive viral infection. These findings indicate that the induction of the transformed phenotype in both abortively and stably transformed cells generally does not require the large T antigen, but rather the products of the hr-t gene.

Tumor antigen(s) in cell productively infected by wild-type polyoma virus and mutant NG-18.

When isolated by means of an anti-polyoma tumor (T) antiserum, the major product from mouse cells productively infected by wild-type polyoma virus is a polypeptide of 100,000 apparent molecular weight as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In cells infected by NG-18, an hr-t mutant carrying a deletion of about 150 base pairs in the early region of the viral DNA, a T antigen species appears that comigrates with that of the wild-type virus. Comparisons of peptides after partial proteolysis reveal no differences between mutant and wild-type products. Both wild-type and mutant 100,000 products can be labeled in vivo with [(32)P]orthophosphate. An independent and more reliable estimate of the molecular weight of this protein using guanidine/Sepharose chromatography yields a value of 81,000 for both mutant and wild-type species. The apparent identity of wild-type and mutant products indicates that the deletion in NG-18 lies outside of the region encoding this major T antigen species. Immunoprecipitates from wild-type infected cells shows four bands in addition to the "100,000" band; these have apparent molecular weights of 63,000, 56,000, 36,000, and 22,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; the 56,000 and 36,000 species are phosphorylated. All four of these lower molecular weight bands are absent or drastically reduced in the immunoprecipitates from NG-18-infected cells.