David A. Loegering

An investigation of the cause of the eosinophilia–myalgia syndrome associated with tryptophan use

BACKGROUND The eosinophilia-myalgia syndrome is a newly recognized illness that has been associated with the consumption of tryptophan products. It is not known whether the cause is related to the tryptophan itself or to chemical constituents introduced by the manufacturing process. METHODS To describe the epidemiology of the eosinophilia-myalgia syndrome further and elucidate a possible association with the manufacturing process, we conducted surveillance for the syndrome in Minnesota, a community survey of tryptophan use in Minneapolis-St. Paul, and a case-control study to assess potential risk factors, including the use of tryptophan from different manufacturers. We performed high-performance liquid chromatography on tryptophan samples to identify other chemical constituents. RESULTS The prevalence of tryptophan use increased from 1980 to 1989 and was highest among women. Among the subjects for whom the source of the tryptophan was known, 29 of 30 case patients (97 percent) and 21 of 35 controls (60 percent) had consumed tryptophan manufactured by a single company (odds ratio, 19.3; 95 percent confidence interval, 2.5 to 844.9; P less than 0.001). This company used a fermentation process involving Bacillus amyloliquefaciens to manufacture tryptophan. Analysis of the manufacturing conditions according to the retail lot demonstrated an association between lots used by case patients and the use of reduced quantities of powdered carbon in a purification step (odds ratio, 9.0; 95 percent confidence interval, 1.1 to 84.6; P = 0.014), as well as the use of a new strain of B. amyloliquefaciens (Strain V) (odds ratio, 6.0; 95 percent confidence interval, 0.8 to 51.8; P = 0.04). There was a significant correlation (r = 0.78, P less than 0.001) between the reduced amount of powdered carbon used during manufacturing and the use of the new bacterial strain. High-performance liquid chromatography of this companys tryptophan demonstrated one absorbance peak (peak E) that was present in 9 of the 12 retail lots (75 percent) used by patients and 3 of 11 lots (27 percent) used by controls (odds ratio, 8.0; 95 percent confidence interval, 0.9 to 76.6; P = 0.022). CONCLUSIONS The outbreak of the eosinophilia-myalgia syndrome in 1989 resulted from the ingestion of a chemical constituent that was associated with specific tryptophan-manufacturing conditions at one company. The chemical constituent represented by peak E may contribute to the pathogenesis of the eosinophilia-myalgia syndrome, or it may be a surrogate for another chemical that induces the syndrome.

Elevated serum levels of the eosinophil granule major basic protein in patients with eosinophilia.

A radioimmunoassay was established for the human eosinophil granule major basic protein (MBP). The mean level of MBP in sera from 105 normal control patients was 454 ng/ml, whereas in a sample of 188 patients with various forms of diseases, including the hypereosinophilic syndrome, levels as high as 14,000 ng/ml were measured. Serum levels of MBP did not correlate with eosinophil counts in normal subjects, but a positive correlation was seen in patients with eosinophilia; the patients with eosinophil counts greater than 350/mm3 generally showed increased levels of MBP. Many patients with skin disease and normal eosinophil counts had elevated levels of serum MBP. Monomer MBP has a molecular weight of 9,300, but in sera of patients with eosinophilia, the MBP activity was of high molecular weight, greater than 50,000. Analyses of serum by Sephadex G-200 and by electrofocusing suggest that MBP is not simply polymerized, but rather is bound to a larger carrier molecule. Monomeric MBP can be isolated from serum by reduction of serum with dithiothreitol, alkylation with iodoacetamide, and acidification to pH 2 followed by fractionation on Sephadex G-50 at pH 2. Under these conditions, up to 80% of the MBP emerges in monomeric form. The results indicate that eosinophil granule proteins circulate in blood covalently bound to serum proteins, and that elevated concentrations of serum MBP are present in some diseases associated with eosinophilia.

Comparative properties of the Charcot-Leyden crystal protein and the major basic protein from human eosinophils.

Guinea pig eosinophil granules contain a protein, the major basic protein (MBP), which accounts for more than half of the total granule protein, has a high content of arginine, and displays a remarkable tendency to form disulfide-linked aggregates. In this study we have purified a similar protein from human eosinophil granules and have compared the human MBP to the protein comprising the Charcot-Leyden crystal (CLC). Eosinophils from patients with various diseases were purified and disrupted, and the granule fraction was obtained. Examination of the granule fraction by transmission electron microscopy showed numerous typical eosinophil granules. Analyses of granule lysates by gel filtration and by polyacrylamide gel electrophoresis revealed the presence of peroxidase and MBP with properties similar to that previously found in guinea pig eosinophil granules. The human MBP had a molecular weight of 9,200, contained less than 1% carbohydrate, was rich in arginine, and readily formed disulfide-bonded aggregates. CLC were prepared from eosinophil-rich cell suspensions by homogenization in hypotonic saline. The supernates following centrifugation of cell debris spontaneously formed CLC. Analysis of CLC revealed the presence of a protein with a molecular weight of 13,000 containing 1.2% carbohydrate. The protein displayed a remarkable tendency to aggregate even in the presence of 0.2 M acetic acid. Human MBP and CLC protein differed in their molecular weights, carbohydrate compositions, and amino acid analyses. Mixtures of the MBP and the CLC protein yielded two bands in polyacrylamide gel electrophoresis. Neither eosinophil protein increased vascular permeability in the guinea pig skin or contracted the guinea pig ileum. The results indicate that the human MBP and the CLC are distinct substances with properties such that one cannot be derived from the other.

In vitro and in vivo antitumor effects of the dual insulin-like growth factor-I/insulin receptor inhibitor, BMS-554417.

The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 micromol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo.

Eosinophil granule proteins in peripheral blood granulocytes.

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil‐derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in ly‐ sates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (X ± SEM) in ng/106 eosinophils (and in nM/106 eosinophils) were: MBP, 8,982 ± 611 (641.6); EDN, 3,283 ± 116 (178.4); ECP, 5,269 ± 283 (250.9); and EPO, 12,174 ± 859 (171.5). Basophils from a normal person contained (in ng/106 cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/106 cells) MBP, 3 ± 0.5; EDN, 72 ± 9; and ECP, 50 ± 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

Eosinophil- and eosinophil granule-mediated pneumocyte injury

The function of the eosinophil in eosinophilic pulmonary syndromes and asthma is uncertain. To determine if eosinophils might play a harmful role in these conditions, we cocultured purified human eosinophils, eosinophil major basic protein (MBP), and chromatographically eluted eosinophil granule fractions with human A549 and rat type II pneumocytes. Damage to these target cells was measured as cell lysis and nonlethal cell detachment. We found that unstimulated intact eosinophils affected minimal lysis or detachment of either pneumocyte target, but eosinophils stimulated with phorbol myristate acetate and other activators produced time- and dose-dependent nonlytic detachment of both targets. In contrast, supernatants from activated eosinophils did not produce significant injury, suggesting that close apposition of the effector and target cells was required. Catalase and superoxide dismutase did not inhibit the detaching activity of eosinophils, suggesting that hydrogen peroxide and superoxide anion were not activity of eosinophils, suggesting that hydrogen peroxide and superoxide anion were not responsible for mediating this form of injury. In contrast to our findings with intact eosinophils, we observed that the addition of purified eosinophil MBP to pneumocytes caused marked cytolysis with little detachment. When sequential fractions of eosinophil granules separated by Sephadex G-50 chromatography were added to A549 and rat type II pneumocyte targets, it was found that different fractions produced distinct forms of injury. Higher molecular weight fractions containing lysosomal enzymes and eosinophil peroxidase produced predominantly detachment, whereas fractions enriched in MBP produced lysis. These results indicate that intact eosinophils can produce nonlytic detachment of alveolar pneumocytes that is probably not dependent on the generation of toxic oxygen radicals but rather appears to be mediated by granule-associated products, possibly lysosomal enzymes. Furthermore, although intact eosinophils are not capable of lysing alveolar epithelial cells under the conditions of our assay, MBP has the potential to do so when the protein is released in high enough concentrations. The in vivo relevance of these findings in eosinophilic lung diseases may be that eosinophils, by producing both desquamation and death of alveolar epithelium cells, may increase the permeability of the alveolus to fluid and cells. Moreover, these forms of damage might also enhance the ingress of inhaled antigens across the pulmonary epithelial barrier, thus increasing immunologic sensitization.

MCL-1 as a Buffer for Proapoptotic BCL-2 Family Members during TRAIL-induced Apoptosis A MECHANISTIC BASIS FOR SORAFENIB (BAY 43-9006)-INDUCED TRAIL SENSITIZATION

Previous studies have suggested that Mcl-1, an antiapoptotic Bcl-2 homolog that does not exhibit appreciable affinity for the caspase 8-generated C-terminal Bid fragment (tBid), diminishes sensitivity to tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL). This study was performed to determine the mechanism by which Mcl-1 confers TRAIL resistance and to evaluate methods for overcoming this resistance. Affinity purification/immunoblotting assays using K562 human leukemia cells, which contain Mcl-1 and Bcl-xL as the predominant antiapoptotic Bcl-2 homologs, demonstrated that TRAIL treatment resulted in binding of tBid to Bcl-xL but not Mcl-1. In contrast, TRAIL caused increased binding between Mcl-1 and Bak that was diminished by treatment with the caspase 8 inhibitor N-(Nα-acetylisoleucylglutamylthreonyl) aspartic acid (O-methyl ester)-fluoromethyl ketone (IETD(OMe)-fmk) or the c-Jun N-terminal kinase inhibitor SP600125. In addition, TRAIL caused increased binding of Bim and Puma to Mcl-1 that was inhibited by IETD(OMe)-fmk but not SP600125. Further experiments demonstrated that down-regulation of Mcl-1 by short hairpin RNA or the kinase inhibitor sorafenib increased TRAIL-induced Bak activation and death ligand-induced apoptosis in a wide variety of neoplastic cell lines as well as clinical acute myelogenous leukemia specimens. Collectively, these observations not only suggest a model in which Mcl-1 confers TRAIL resistance by serving as a buffer for Bak, Bim, and Puma, but also identify sorafenib as a potential modulator of TRAIL sensitivity.

COMPARATIVE TOXICITY OF PURIFIED HUMAN EOSINOPHIL GRANULE PROTEINS FOR NEWBORN LARVAE OF TRICHINELLA SPIRALIS

Eosinophils have been implicated in both in vivo and in vitro destruction of helminths. One approach toward elucidating the role of the eosinophil in parasite killing has been to test the toxicity of purified eosinophil granule proteins for parasites in vitro. Previously, major basic protein (MBP) and eosinophil cationic protein (ECP) were shown to be toxic for schistosomules of Schistosoma mansoni, while eosinophil-derived neurotoxin (EDN) was only marginally so. We tested the toxicity of MBP, ECP, and EDN over a range of concentrations (0.006-5 X 10(-4) M) for newborn larvae of Trichinella spiralis. Our observations confirm previous reports of toxicity of mildly reduced and alkylated (R & A) MBP. At concentrations of 5 X 10(-5) M and above, R & A MBP killed 75% or more of the larvae within the first hour of culture. ECP was an effective toxin for these larvae after 3 hr of culture, and by 12 hr, dose-related toxicity was evident. After 24 hr, 100% of the larvae were killed at 5 X 10(-5) M ECP. EDN was much less toxic; after 12 hr, 90% of the larvae survived at concentrations of 1 X 10(4) M, while 5 X 10(-4) M EDN killed all the larvae. At the optimal toxic concentrations of 5 X 10(-5) M ECP and 5 X 10(-4) M EDN, kinetics of killing by these 2 proteins were essentially the same. Thus, on a molecular basis, both MBP and ECP appear to be potent helminthotoxins whereas EDN is much less so.

The injurious effect of eosinophil peroxidase, hydrogen peroxide, and halides on pneumocytes in vitro.

Recent data suggest that eosinophils may cause lung injury. To determine if the eosinophil peroxidase (EPO)-hydrogen peroxide (H2O2)-halide system could mediate this injury, we added human EPO, H2O2 (or glucose and glucose oxidase as a continuous source of H2O2), and various halides to monolayers of 51Cr-labeled human A549 and rat type II pneumocytes. Cell lysis was measured as soluble 51Cr release. In initial experiments, EPO in solution did not induce lysis under these conditions. Therefore, in subsequent experiments, pneumocytes were preincubated with EPO for 15 minutes, washed to remove unbound enzyme, and then glucose, glucose oxidase, and the halides were added. EPO alone was not injurious, nor was the addition of glucose and glucose oxidase in the absence of EPO. In contrast, the combined addition of EPO, glucose, glucose oxidase, and chloride produced marked target-cell lysis. This effect was time and EPO dose dependent and was enhanced by the addition of iodide. Catalase and azide substantially inhibited the lysis produced by the EPO-H2O2-halide system, suggesting that EPO-catalyzed products of halide oxidation mediated this form of injury. Finally, the addition of eosinophil major basic protein at 10(-5) mol/L to EPO-coated pneumocytes incubated with glucose, glucose oxidase, and halides failed to enhance or inhibit lysis. We hypothesize that the EPO-H2O2-halide system may injure the lung in asthma and eosinophilic pulmonary syndromes.

Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm

Phosphatidylserine (PS) exposure on the external leaflet of the plasma membrane is widely observed during apoptosis and forms the basis for the annexin V binding assay to detect apoptotic cell death. Current efforts to explain PS exposure focus on two potential mechanisms, activation of a phospholipid scramblase or calcium-mediated trafficking of lysosomes to the cell surface. Here, we provide evidence that apoptotic PS exposure instead reflects bidirectional trafficking of membrane between the cell surface and cytoplasm. Using a series of cell lines, some of which expose large amounts of PS during apoptosis and some of which do not, we demonstrate that accumulation of plasma membrane-derived cytoplasmic vesicles in a dynamin-, clathrin- and Cdc42-independent manner is a previously undescribed but widely occurring feature of apoptosis. The apoptotic exposure of PS occurs when these vesicles traffic back to cell surface in a calcium-dependent process that is deficient in a substantial fraction of human cancer cell lines. These observations provide a new model for PS externalization during apoptosis and simultaneously identify an altered step that accounts for the paucity of apoptotic PS exposure in many cell lines.