Alteration of the pharmacokinetics of small proteins by iodination.

The pharmacokinetics of several proteins were investigated using two different assays. A 23 kDa recombinant protein fragment of bactericidal/permeability-increasing protein (rBPI23) was radiolabeled with 125I using Iodo-beads and administered rats. Plasma samples were collected and assayed for 125I-rBPI23 by radioactivity. In a separate experiment, rBPI23 was administered to rats and plasma samples were assayed for rBPI23 by ELISA. The clearance determined from plasma concentrations of 125I-rBPI23 measured by radioactivity was about 2.5-fold lower than that of rBPI23 determined by ELISA. In addition, the steady state volumes of distribution and mean residence times of 125I-rBPI23 measured by radioactivity were four-fold and 10-fold greater, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were different when assayed by radioactivity or ELISA, but those of proteins with molecular weights of at least 80 kDA revealed only minor differences. To determine which assay method yielded the correct plasma pharmacokinetic profile, rBPI23 was metabolically labeled with 35S-methionine and administered to rats, and plasma samples were assayed by radioactivity. The concentration-time profile assessed by this method was very close to that determined by ELISA. Exposing rBPI23 to chloramine-T (the oxidant used in the iodination process) and measuring its plasma concentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeling rBPI23 using iodinated Bolton-Hunter reagent (which avoids exposing the protein to oxidant), and measuring 125I-rBPI23 by radioactivity, yielded pharmacokinetics that were similar, although not identical, to the pharmacokinetics of rBPI23 measured by ELISA. Thus, our data suggest that directly iodinating low-molecular-weight proteins by oxidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.

Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein.

Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

Competition between rBPI23, a recombinant fragment of bactericidal/permeability-increasing protein, and lipopolysaccharide (LPS)-binding protein for binding to LPS and gram-negative bacteria.

Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are two structurally related lipid A-binding proteins with divergent functional activities. LBP mediates activation of macrophage and other proinflammatory cells. In contrast, BPI has potent bactericidal and LPS-neutralizing activities. A recombinant fragment of BPI (rBPI23) retains the potent biological activities of the holo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis, and endotoxemia. For therapeutic effectiveness in many clinical situations, rBPI23 will have to successfully compete with high serum levels of LBP for binding to endotoxin and gram-negative bacteria. The relative binding affinities of rBPI23 and human recombinant LBP (rLBP) for lipid A and gram-negative bacteria were evaluated. The binding of both proteins to lipid A was specific and saturable with apparent Kds of 2.6 nM for rBPI23 and 58 nM for rLBP. rBPI23 was approximately 75-fold more potent than rLBP in inhibiting the binding of 125I-rLBP to lipid A. The binding affinity of rBPI23 (Kd = 70 nM) for Escherichia coli J5 bacteria was also significantly higher than that of rLBP (Kd = 1,050 nM). In addition, rBPI23 at 0.2 micrograms/ml was able to inhibit LPS-induced tumor necrosis factor release from monocytes in the presence of 20 micrograms of rLBP per ml. These results demonstrate that rBPI23 binds more avidly to endotoxin than does rLBP and that, even in the presence of a 100-fold weight excess of rLBP, rBPI23 effectively blocks the proinflammatory response of peripheral blood mononuclear cells to endotoxin.

An amino-terminal fragment of human lipopolysaccharide-binding protein retains lipid A binding but not CD14-stimulatory activity.

LPS-binding protein (LBP) mediates the pro-inflammatory effects of bacterial LPS by enhancing LPS-induced cytokine production by monocytic cells. LBP binds specifically to LPS to generate a complex that interacts with the CD14 receptor on the surface of responsive cells. To identify the biologically active regions of the protein responsible for mediating these activities, we cloned and expressed human rLBP (456 amino acids) as well as a truncated form encoding amino acids 1-197 (rLBP25). Both forms of LBP bound to LPS with the same affinity, and similarly inhibited LPS activity in the Limulus amebocyte lysate assay. These results demonstrate that the LPS-binding domain of LBP resides entirely within the N-terminal 197 amino acids of the protein. rLBP and rLBP25 were compared for their ability to mediate CD14-dependent LPS effects on cells. rLBP was effective in mediating uptake of LPS and stimulation of TNF production by human monocytic THP-1 cells, whereas rLBP25 had no significant activity in these assays. Similarly, rLBP was able to mediate LPS-induced TNF production by human PBMC whereas rLBP25 was essentially inactive. These results suggest that the structural features of LBP required for mediating LPS effects via CD14 are probably located in the C-terminal region of the protein. Thus, the LPS-binding activity of LBP can be separated from the CD14-stimulatory activity, suggesting these activities are mediated by structural elements residing in different regions of the protein.

Cysteine analogs of recombinant barley ribosome inactivating protein form antibody conjugates with enhanced stability and potency in vitro.

Antibody immunoconjugates were made with native and recombinant forms of the type-I ribosome inactivating protein from barley (BRIP) and with three recombinant BRIP (rBRIP) analogs engineered to contain a unique cysteine residue near the C terminus (at amino acid 256, 270, or 277). rBRIP and all three cysteine analogs (rBRIPc256, rBRIPc270, and rBRIPc277) were produced in E. coli, with yields of soluble protein as high as 1 g/L, and were as active as native BRIP in inhibiting protein synthesis in vitro. Interestingly, the position of the engineered cysteine influenced not only the efficiency of conjugation to antibody but also the efficacy and disulfide bond stability of the immunoconjugates. Anti-CD5 antibody conjugates prepared with native and rBRIP were relatively inactive against antigen-positive target cells, while the conjugate made with rBRIPc277 was 5-fold more cytotoxic. Anti-CD7 antibody conjugates made with rBRIPc277 or rBRIPc270 also exhibited improved potency and stability compared to the conjugate with native BRIP. These results indicate that engineering a cysteine residue into selected positions near the C-terminus of a type-IRIP such as BRIP can improve immunoconjugate yield, disulfide bond stability, and potency.

[Phagotest and Bursttest (Phagoburst), test kits for study of phagocyte functions]. / Phagotest und Bursttest (Phagoburst), Testkits zur Untersuchung von Phagozytenfunktionen.

Phagotest and Bursttest are complete test kits for the investigation of the phagocytic function of granulocytes and monocytes in whole blood using flow cytometry. Phagotest allows the quantitative determination of leukocyte phagocytosis (uptake of bacteria). It determines the percentage of phagocytes which ingest fluorescein-isothiocyanate (FITC) labelled opsonized E. coli bacteria and their activity (number of bacteria per cell). Reduced phagocytosis is observed in patients with sepsis, renal failure, and recurrent bacterial infections. Bursttest allows the quantitative determination of leukocyte oxidative burst. It determines the percentage of leukocytes which oxidize the fluorogenic substrate dihydrorhodamine (DHR) 123 and their enzymatic activity (amount of rhodamine 123). Reduced burst activity is observed in patients with chronic granulomatous disease (CGD).

Measurement of bactericidal/permeability-increasing protein in human body fluids by sandwich ELISA.

A sensitive sandwich ELISA has been developed to measure levels of native bactericidal/permeability-increasing protein (BPI) as well as two recombinant forms of BPI (rBPI and rBPI23) in human body fluids. The linear range for the rBPI and rBPI23 standard curves were 100-6000 pg/ml and 25-800 pg/ml respectively. Recovery of different concentrations of rBPI spiked into pooled human plasma samples averaged 83% and ranged from 65% at 300 ng/ml to 97% at 3 ng/ml. Recovery of rBPI23 averaged 56% and ranged from 30% at 0.5 ng/ml to 90% at 50,000 ng/ml. Because LBP is present in normal human plasma and shares sequence homology with BPI, the effects of rLBP on the BPI ELISA were also evaluated. Under standard assay conditions, rLBP caused minimal interference with BPI detection. At 100 micrograms/ml, rLBP generated a signal equivalent to 3 ng/ml of rBPI and 0.6 ng/ml of rBPI23. Matched serum and plasma samples were collected from 20 healthy adults to measure endogenous levels of BPI. The range of BPI concentrations was < 0.2-2.1 ng/ml in plasma and 4.9-72.1 ng/ml in serum. Western blot analysis indicated that the BPI ELISA immunoreactivity in plasma and serum correlated with the presence of a protein doublet (M(r) approximately 60,000), which comigrated with native BPI extracted from human neutrophils. These data demonstrate that low levels of holo-BPI are present in plasma, and suggest that additional quantities of BPI were released from neutrophils during the process of coagulation.

Ecdysone-dependent proteolysis of an apical surface glycoprotein may play a role in imaginal disc morphogenesis in Drosophila.

An apical surface glycoprotein, designated gp125 for its apparent molecular weight of 125,000, appears in Ca2(+)-free, ionic detergent extracts of imaginal discs of Drosophila melanogaster in response to the steroid hormone, 20-hydroxyecdysone (20-HE). Gp125 is not synthesized in response to 20-HE, but results from modification of an existing macromolecule. Treatment of discs or larval epidermis with serine protease (e.g., trypsin) results in hormone-independent production of gp125. Antiserum raised to electrophoretically purified gp125 recognizes, in addition to gp125, two closely related glycoproteins with higher apparent molecular weights, gp200 and gp180. This family of glycoproteins is localized at the apical surface of imaginal disc cells and of the epidermal epithelium in embryos, larvae and prepupae. Ca2+ affects both the solubility and the proteolytic products of this family of glycoproteins. We discuss the possibility that gp125 is generated through the action of a hormonally controlled serine protease in a process that is necessary for disc morphogenesis.

Detection and analysis of two serotypes of ammonia-oxidizing bacteria in sewage plants by flow cytometry.

Two different serotypes of the genus Nitrosomonas were isolated from samples of the sewage plant Heidelberg. These nitrifiers were enumerated in activated sludge of various other sewage plants after immunofluorescent labeling and staining with propidium iodide by flow cytometry. The concentrations of these serotypes of Nitrosomonas spp. were in the range of 0.1 to 2%. Also, a test for the determination of the activity of ammonia-oxidizing bacteria was developed. Nitrite-oxidizing bacteria were specifically inhibited with sodium chlorate, and the activity of ammonia-oxidizing bacteria could be calculated from the increase of nitrite. Concentrations and activities of ammonia oxidizers were measured for a period of 6 months in the sewage plant Heidelberg. With one exception, activities and concentrations of ammonia-oxidizing bacteria decreased and increased in parallel.

Virus neutralizing and enhancing epitopes characterized by synthetic oligopeptides derived from the feline leukaemia virus glycoprotein sequence.

Synthetic peptides that mimic antigenic determinants of viral proteins were used in vaccine studies of feline leukaemia virus (FeLV) infection. Immunoreactive epitopes on FeLV gp70 and p15E were predicted according to the criteria of their terminal position, hydrophilicity and the probability of them constituting helical structures. Nineteen peptides, consisting of seven to 19 amino acid residues, were synthesized, of which two peptides were derived from the FeLV subtype A, 16 from subtype B and one from subtype C. Rabbits were immunized with individual peptides coupled to keyhole limpet haemocyanin and the specificity and biological activities of these hyperimmune sera were determined by ELISA, Western blotting, virus neutralization and cytotoxicity assays. All sera reacted specifically with the immunizing peptide. Twelve of the 19 peptides induced antibodies against purified gp85 and antibodies to 11 peptides reacted with the whole virus. One peptide representing the carboxy terminus of the transmembrane protein p15E, and two peptides derived from the external glycoprotein gp70 elicited neutralizing antibodies, whereas antisera against four peptides enhanced virus infection in vitro. None of the peptide antisera mediated complement lysis of FeLV-infected cells.

Solid phase synthesis and biological activity of mast cell degranulating (MCD) peptide: a component of bee venom.

Mast cell degranulating (MCD) peptide, a 22 amino acid residue basic peptide from bee venom, was synthesized by stepwise solid phase synthesis on a benzhydrylamine resin support. N alpha-t-butyloxycarbonyl and benzyl type side chain protection was used. The two disulfide bridges were formed selectively by using S-acetamidomethyl protection for the cysteine residues in position 5 and 19 and S-methylbenzyl protection for the cysteine residues in positions 3 and 15. Crude synthetic MCD peptide was obtained following deprotection and cleavage from the resin by the low/high HF method. The peptide was isolated in pure form by ion exchange chromatography and gel filtration. The final product has physical, chemical, and biological properties identical with those reported for the natural product. The synthetic strategy utilized for MCD peptide will facilitate the availability of structurally similar analogs for evaluating antihistaminic and anti-inflammatory activities.

Flow-cytometric studies with eleutherococcus senticosus extract as an immunomodulatory agent.

A placebo-controlled study of the effect of an Eleutherococcus senticosus extract (Eleukokk) on the immune system was performed with 36 healthy volunteers utilising quantitative multi-parameter flow cytometry with monoclonal antibodies directed against specific surface markers of human lymphocyte subsets. Volunteers in the verum group received 10 ml of an ethanolic (vincamine free) eleutherococcus senticosus preparation, 3 times daily for 4 weeks. In the placebo, the eleutherococcus extract was substituted by additional wine, resulting in identical final concentrations of ethanol in both preparations. The purpose of the double-blind study was the demonstration of possible effects on the cellular immune status, as determined by quantitative flow cytometry. The most salient feature in the verum group was a drastic increase in the absolute number of immunocompetent cells, with an especially pronounced effect on T lymphocytes, predominantly of the helper/inducer type, but also on cytotoxic and natural killer cells. In addition, a general enhancement of the activation state of T lymphocytes was observed. No side effects were observed during the trial or afterwards (observation period 6 months).

Biochemical characterization and immunomodulatory action of thymic components as determined by flow cytometry on human lymphocytes.

A commercial preparation from calf thymus was separated into its components, then chemically characterized and tested for immunomodulatory efficiency. For the latter, a novel in vitro test system is described as a relatively simple screening procedure. It employs whole blood cultures and flow cytometry with fluorescent monoclonal antibodies defining human T-cell subsets. As a reference substance, thymosin fraction 5 was used. It could be shown that the effects of the entire thymic preparation as well as of one of its peptidic components resembled closely the ones obtained with thymosin fraction 5. In several cases, i.e. with donors relatively deficient in suppressor/cytotoxic T-lymphocytes, the effect of the former was even superior to that of thymosin fraction 5.

Differentiation changes in cord blood T lymphocytes induced by synthetic C-terminal peptides of thymosin alpha 1.

Thymosin fraction 5 and its component thymosin alpha 1 has been reported to play a regulatory role in the later stages of T lymphocyte differentiation. Previous studies from our group have also demonstrated that thymosin fraction 5 can induce changes in purine degradative enzymes and in surface antigenic markers of cord blood lymphocytes, which have been shown to be immature compared with normal adult lymphocytes. Birr et al., have shown that the C-terminal region of thymosin alpha 1 is essential for the biological activity. In this study, the effects of these synthetic peptide fragments on cord blood T lymphocytes were studied. After incubation with different peptide sequences, cord blood lymphocytes were analysed for surface antigenic markers (OKT4/OKT8) and for purine degradative enzymes (PNP, 5 NT). In the range of 1.3 X 10 to 1.3 X 10(-5)M, one of the eleven peptides examined (sequence 13-19) exhibited activity approximately equal to that of the parent peptide thymosin alpha 1: increase in activity of 5'NT (p less than 0.01) and reduction of cells positive for OKT4 (p less than 0.01). Another sequence (20-28) was able to induce an increase in 5'NT only and a third one (20-25) a reduction of cells positive for OKT4. The results obtained in this study confirm that the C-terminal region contains molecular signals for T cell differentiation.

The auricular myocardiocytes of the heart constitute an endocrine organ. Characterization of a porcine cardiac peptide hormone, cardiodilatin-126.

A peptide hormone was extracted from the porcine right atrium following a bioassay for differential vasorelaxant effects on smooth muscle strips from aorta and renal and inferior mesenteric arteries. The isolation procedure included several steps of gel-permeation and ion-exchange chromatography, and high performance liquid chromatography. During the isolation procedure, other peptides of smaller molecular weight were also found, which, in relation to cardiodilatin-126 (CDD-126), are shorter at their N-terminal. Among these, CDD-88 has also been isolated and characterized, and has been established as a prominent member of the cardiac hormone family. The N-terminal and C-terminal segments of the 126 amino acid-containing molecule were synthesized and used to raise region-specific antibodies. The natural peptide was then localized within myoendocrine cells of the right atrium where specific atrial granules are located. Renal effects of cardiodilation were studied in conscious dogs and showed strong diuretic and natriuretic activities. According to our functional studies, cardiodilatin-126 and cardiodilatin-88 possess qualities of a significant hormone family regarding the regulation of extracellular fluid volume and blood pressure.

Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src.

To study the function of pp60src, the transforming protein encoded for by Rous sarcoma virus, we have raised antibodies against synthetic oligopeptides corresponding to the primary structure of pp60src. All eight investigated peptides were immunogenic in rabbits, and four induced pp60src-specific antibodies. We screened tumor-bearing rabbit (TBR) sera for antibodies against the peptides; this revealed that five out of six of the peptides, chosen according to a high hydrophilicity plot, were related to epitopes of native pp60src, in contrast to two peptides of low hydrophilicity, which contained a cleavage site for protease. Antibodies against three of the peptides appeared to react with the kinase-active site of pp60src, as these antibodies were phosphorylated in their heavy chain upon immune precipitation. Antibodies against two of the peptides, in contrast to the others, did not precipitate pp60src when this molecule was complexed with two cellular proteins, pp50 and pp90. This observation allows speculation about the location of the pp60src site involved in the formation of this complex.

Carbonyldiimidazole/1-hydroxybenzotriazole activation in polymer phase synthesis of the arginine rich proalbumin hexapeptide extension.

The synthesis on different polymer phases of Arg-Gly-Val-Phe-Arg-Arg, the proalbumin extension, is reported. The peptide was prepared both on a 0.5% cross-linked polystyrene gel containing 2-oxoethyl bromide anchor functions and on a 1% cross-linked chloromethyl polystyrene. For temporary blocking of the amino groups we utilized the 2-(3,5-dimethoxyphenyl) propyl-(2)-oxycarbonyl (Ddz). The guanidino groups of the three arginine moieties were protected by the 4-toluenesulfonyl (Tos) group. As coupling procedure we used 1 equiv. carbonyldiimidazole (CDI)/2 equiv. 1-hydroxybenzotriazole (HOBt). The C-terminal activation of the Ddz-amino acids with CDI/HOBt made it possible to recover the excess Ddz-amino acids in 60-80% yield. We also investigated different procedures to cleave the 2-oxoethyl ester bond between the peptide and the polymer. This bond was completely stable against trifluoromethanesulfonic acid and was split by 1 N triethylamine in methanol/dioxane 1:1 (v/v) + 1 vol% 1 N NaOH. The optical rotation and HPLC properties of Arg-Gly-Val-Phe-Arg-Arg from this synthesis are identical to the product from a different synthesis published earlier.

Hexa- and pentapeptide extension of proalbumin: feedback inhibition of albumin synthesis by its propeptide in isolated hepatocytes and in the cell-free system.

Addition of the chemically synthesized proalbumin hexapeptide in a concentration of 110 micro M to the medium of isolated rat hepatocytes decreased net albumin synthesis by 12%. The synthesis of other secretory proteins was not altered. A weaker inhibitory effect on albumin synthesis was found for a tetrapeptide, a possible degradation product of the proalbumin hexapeptide. For the uptake of the hexa- and tetrapeptide into the cells, bovine serum albumin is required. In a reticulocyte and in a wheat germ cell-free system a propeptide concentration of 600 micro M inhibited albumin synthesis by 50%, whereas total protein synthesis was inhibited by 19% only, and the synthesis of alpha 1-antitrypsin was not inhibited. These results suggest that the synthesis of preproalbumin is regulated by a feedback mechanism with its propeptide as inhibitor.