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It is known that lipopolysaccharides (endotoxin) prime neutrophils for oxygen radical production. Monophosphoryl lipid A is a nontoxic derivative of lipid A that protects against lethal endotoxemia. We examined the effects of Salmonella minnesota monophosphoryl lipid A on S. minnesota lipopolysaccharide-induced priming of neutrophil superoxide anion generation. Human neutrophils were preincubated with and without either lipopolysaccharide or monophosphoryl lipid A before stimulation with 10(-5) formyl-norleucyl-leucyl-phenylalanine. Neutrophil priming reached a plateau at a concentration of 100 ng/ml of lipopolysaccharide, where superoxide anion generation increased from 10.1 +/- 0.8 to 25.2 +/- 1.7 nmol superoxide anions/10(6) neutrophils/10 min (p less than 0.01). In contrast, monophosphoryl lipid A did not exhibit any priming activity. Monophosphoryl lipid A also exhibited a time-dependent inhibitory effect on lipopolysaccharide-induced priming of neutrophils, which was maximal when monophosphoryl lipid A was added 15 minutes before lipopolysaccharide. Preincubation with monophosphoryl lipid A induced a dose-dependent inhibition of neutrophil priming by 1000 ng/ml lipopolysaccharide. Neutrophil superoxide anion generation decreased by 47% from 19.0 +/- 0.6 to 10.0 +/- 0.7 nmol superoxide anions/10(6) neutrophils/10 min by 2000 ng/ml monophosphoryl lipid A (p less than 0.01). These data indicate that monophosphoryl lipid A does not enhance neutrophil superoxide generation in response to formyl-norleucyl-leucyl-phenylalanine. Monophosphoryl lipid A also inhibits lipopolysaccharide-induced priming in a dose-dependent manner that may reflect blocking of lipopolysaccharide by monophosphoryl lipid A at cellular binding sites.

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Swainsonine, a Golgi mannosidase II inhibitor, causes a 15,000 dalton decrease in the apparent molecular weight of formyl peptide chemotactic receptor (FPCR) synthesized by three types of human phagocytic cells (differentiated HL-60 cells, differentiated U-937 cells, and cultured human macrophages derived from peripheral blood monocytes). This indicates that some of the asparagine-linked (N-linked) oligosaccharide of FPCR is of the complex type. Studies with endoglycosidase F and endoglycosidase H show that unaltered FPCR contains both complex and high mannose content N-linked oligosaccharide chains, and that FPCR synthesized in the presence of swainsonine contains only high mannose N-linked oligosaccharide chains. Cycloheximide prevents the swainsonine effect on FPCR molecular weight. This shows that swainsonine only affects the carbohydrate structure of FPCR which is newly synthesized in the presence of swainsonine. Since swainsonine decreases the molecular weight of FPCR from human peripheral blood monocytes in culture, these cells must be synthesizing new FPCR. The abnormally low molecular weight FPCR synthesized in the presence of swainsonine is converted to the normal, higher molecular weight form of FPCR when swainsonine is removed from the culture. This occurs even when new protein synthesis is inhibited by cycloheximide. This requires that previously synthesized, swainsonine-affected FPCR at the cell surface must come in contact with Golgi enzymes which complete the processing of the abnormal, high mannose oligosaccharide. This implies a potential Golgi-related repair mechanism for altered or "damaged" cell surface receptors.

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We previously showed that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL-60 cells induced to differentiate by N6,O2-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of 5 micrograms/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt2cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (formyl 125I-hexapeptide) and ethylene glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (Mr 32,000), and no fully glycosylated FPCR (Mr 62,000 to 85,000) was detectable. Scatchard analysis of formyl 125I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells (control cells, 82,000 +/- 32,000 sites/cell with Kd 10.0 +/- 4.3 nM and 520,000 +/- 40,000 sites/cell with Kd 250 +/- 80 nM; tunicamycin-treated cells, 11,000 +/- 5000 sites/cell with Kd 3.0 +/- 1.9 nM and 470,000 +/- 70,000 sites/cell with Kd of 500 +/- 140 nM). Both control and tunicamycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca2+ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin-treated cells were less than that of the control cells. The present studies demonstrate that N-glycosylation of FPCR is not essential for cell surface expression or for several FPCR-mediated cell responses.

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In consultation the authors were requested to evaluate a middle-aged diabetic woman for an apparent episode of biliary sepsis. The patient had been admitted to the dermatology service with a four-day history of rash and pruritus. This was initially thought to represent an allergic reaction to dicloxacillin in someone with a previous history of penicillin hypersensitivity. Persistent right upper quadrant pain, fevers, elevations of serum alkaline phosphatase, and a radionuclide scan which did not demonstrate a functioning gall bladder led to a cholecystectomy for acute cholecystitis and possible biliary sepsis. This diagnosis was not confirmed. Ultimately, this case illustrated the need to review carefully recent changes in any patient's drug regimen. Reactions to commonly prescribed agents may cause syndromes which are difficult to distinguish from episodes of apparent sepsis.

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Formyl peptide chemotactic receptors affinity-labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (where Nle represents norleucine) and ethylene glycol bis(succinimidyl succinate) consist of two isoelectric forms with cell type differences in both apparent size and charge (neutrophils: 55-70 kDa, pI 5.8, and 6.2.; monocytes: 60-75 kDa, pI 5.6 and 6.0; differentiated HL-60 cells: 62-85 kDa, pI 5.6 and 6.0). Endo-beta-N-acetylglucosaminidase F (endo F) cleavage of N-linked oligosaccharides from formyl peptide receptor generates 40-50- and 33-kDa products that can be affinity-labeled. Whereas both pI forms of this receptor from neutrophils are cleaved by endo F to 33-kDa final products, this cleavage does not eliminate pI differences. Tunicamycin decreases expression of formyl peptide receptor on differentiating HL-60 and causes a dose-dependent decrease in size of the major product seen after affinity labeling (0.5 micrograms/ml: 38-48 kDa; 2 micrograms/ml: 32 kDa). Thus, the formyl peptide receptor polypeptide backbone from all three cell types contains at least two N-linked oligosaccharide side chains which contribute to the cell type differences in Mr and are not required for ligand binding. Papain treatment of intact cells generates a membrane-bound formyl peptide receptor fragment that can be affinity-labeled and is of similar size (29-31 kDa) in all three cell types. Endo F treatment of the affinity-labeled papain fragment of formyl peptide receptor does not alter its size, suggesting that this fragment does not contain the N-linked oligosaccharide cleaved by endo F from intact receptor. The results indicate that at least two N-linked oligosaccharide chains are located on the distal 1-3-kDa portion of the receptor polypeptide backbone.

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Four fluorine- 18-labeled estrogens--16 alpha-[18F]fluoro-estradiol-17 beta (1), 16 beta-[18F]fluoro-estradiol-17 beta (2), (2R, 3S)-1-[18F]fluoro-2,3-bis(4-hydroxyphenyl)-pentane (1-[18F]fluoropentestrol) (3), and (3R, 4S)-1-[18F]fluoro-3,4-bis(4-hydroxyphenyl)hexane (1-[18F]fluorohexestrol) (4)--have been prepared by simple displacement reactions utilizing reactive trifluoromethane sulfonate (triflate) precursors and F-18 fluoride ion. All four fluoroestrogens have high affinity for the estrogen receptor. In immature female rats, they are taken up by target tissues, such as the uterus, with very high selectivity: uterus-to-blood ratios at 1 hr are: Compound 1, 39; Compound 2, 12; Compound 3, 13; and Compound 4, 19. Average uterus-to-blood ratios exceed 80 for Compound 1 at 2 hr. That the uptake process involves an estrogen-specific binder of limited capacity is demonstrated by the suppressive effect of coadministered unlabeled estradiol on target tissue uptake.

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Some of the problems which we see on the infectious disease consultation service can be quite frustrating. This is one such case. A middle-aged man presented to our medical service with fever and dyspnea. His fulminant downhill course was characterized by anemia, jaundice, hypercalcemia, pulmonary abnormalities, and a lack of responsiveness to conventional antimicrobial therapy. At autopsy, malignant-appearing histiocytes were present in several organs including spleen, lymph nodes, and lung. Histopathological examination of tissues obtained at autopsy confirmed the presence of phagocytized erythrocytes within such histiocytes. This case aptly illustrates the hazy dividing line which sometimes exists between infectious and/or malignant processes which are, at present, still of undetermined etiology.

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16 alpha-[77 Br]-Bromoestradiol-17 beta (3a) has been synthesized from estrone enol diacetate (1) by bromination with Na77Br and hydrogen peroxide-acetic acid, followed by reduction with lithium aluminum hydride to give a mixture of 16 alpha-[77 Br]-bromoestradiol-17 beta (3a) and 16 alpha-[77Br]-bromoestradiol-17 alpha (3b) from which the desired epimer (3a) can be obtained in 50% overall radiochemical yield (from Na77Br) by HPLC. Analogous procedures can be used in the preparation of 16 alpha-[77Br]-bromo-11 beta-methoxyestradiol-17 beta. (4a) The effective specific activities of these radiopharmaceuticals, determined by binding to the uterine estrogen receptor, are 900-1500 Ci/mmol. Both have affinity and good binding selectivity for the estrogen receptor and are useful as imaging agents for mammary tumors.

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Steroidal and nonsteroidal estrogens substituted with halogens ortho to the phenolic hydroxyl group in the D ring at C-16 have been prepared as potential estrogen receptor-based imaging agents for human breast tumors. Estrogens bearing an aromatic fluorine ortho to a phenolic hydroxyl group were prepared by the Schiemann reaction on the corresponding methyl esters; other ortho-halogenated estrogens were prepared by direct halogenation. Steroidal estrogens substituted at the 16 alpha position were prepared by halogenation of estrone 3-acetate (17-enol acetate) followed by hydride reduction, and those substituted at the 16 beta position were prepared by epimerization prior to reduction. The binding affinity of these halogenated estrogens to the uterine estrogen receptor was measured relative to that of [3H]estradiol by a competitive binding assay. All of the monosubstituted ortho-fluorinated estrogens show very high binding affinity for the receptor (64--250% that of estradiol). The monosubstituted and symmetrically disubstituted bromo- and iodohexestrols and 2- and 4-substituted estradiols have binding affinities considerably lower than those of the fluoro compounds, the 4-substituted estradiols have affinities greater than the corresponding 2-substituted isomers. Introduction of a halogen (Cl, Br, I) at the 16 alpha position of 17 beta-estradiol results in compounds with receptor affinities comparable to that of 17 beta-estradiol itself; the 16 beta-epimers and the estrone derivatives are bound less well. Thus, provided that they can be labeled with suitable gamma-emitting radioisotopes at sufficiently high specific activity, it appears that the A-ring fluoroestrogens and 16 alpha-bromo- and 16 alpha-iodoestradiol-17 beta are excellent candidates for receptor-based imaging of human breast tumors.

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We have synthesized as potential imaging agents for human breast tumors a series of hexestrol analogues bearing the halogens fluorine, chlorine, bromine, and iodine at the terminus of the hexane chain. The binding affinity of these compounds for the estrogen receptor from uterine tissues forms a monotonically decreasing series, starting at 129% of that of estradiol for the fluoro analogue and decreasing to 60% for the iodo analogue. Such a modest decrease in binding affinity is thought to reflect the preference of the receptor for lipophilic groups and for substituents of moderate steric size at this site, parameters which change in opposite directions in the halogen sequence going from fluorine to iodine. Three estrogenic bis(trifluoromethyl)diphenylethylenes, prepared by DuPont, also showed substantial binding affinities for the estrogen receptor. In terms of ease of radiolabeling and high receptor binding selectivity, the compound that appears to be the most promising candidate for a breast tumor imaging agent in these series is the chain terminal fluorohexestrol.

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Four halogenated estrogen analogs--o-fluorohexestrol, and 1-fluoro-, 1-bromo-, and 1-iodohexestrol--have been prepared and tritium-labeled in high specific activity, to investigate their potential as estrogen-receptor-based agents for imaging breast tumors. These compounds bind with high affinity in vitro to the cytoplasmic uterine estrogen receptor from rat and lamb and sediment as 8S receptor complexes on sucrose gradients. After 1 hr in immature rats, these compounds show high uptake into the uterus, but low uptakes (10--25% of the uterine levels) into most nontarget tissues. The uterine uptake is estrogen specific since it is depressed by excess nonradioactive estradiol. Uptake selectivity is greatest for the fluorohexestrols and decreases for the bromo and iodo compounds. In mature rats bearing DMBA-induced mammary tumors, selective uptake by the uterus and tumors is seen with 1-fluoro[3H4]hexestrol and o-fluoro[3H3]hexestrol. The studies indicate that these four halogenated hexestrols are promising candidates as estrogen-receptor-based agents for the imaging of human breast tumors.

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