Biochemical and immunological properties of cytokines conjugated to dendritic polymers.

Here we describe a post-translational modification of SC-63032, a variant of the species restricted, multi-lineage hematopoeitic factor human interleukin-3 (hIL-3). We have made two new dendritic polymer (polyamidoamine or PAMAM dendrimers, generation 5)-SC-63032 bioconjugates. Using two distinct chemistries (one of which is novel to this work), we achieved site-specific conjugation with respect to the amino acid in the proteins ligated to the dendrimers. In both bioconjugates, conjugated cytokine maintains its ability to bind the hIL-3 alpha receptor subunit, but is significantly (about 10-fold) less potent in inducing hIL-3 dependent in vitro cell proliferation than is the free cytokine. In vivo data indicates that conjugation decreases the immunogenicity of the conjugated cytokine modestly. In the absence of pharmacokinetic or biodistribution effects associated with the bioconjugates that increase their potency in vivo (which can only be tested in a higher primate, due to the species restriction of hIL-3 and its derivatives), these immune mitigation effects may be too small to be therapeutically significant. Though unmodified PAMAM dendrimers fail to elicit an antibody response in mice, protein conjugation to dendrimers haptenizes them, and a dendrimer-specific antibody response is produced. In toto, the principal limitation of the dendrimer-cytokine bioconjugates herein is in their reduced receptor affinity and potency in vitro. Were the in vivo potency of the bioconjugates to parallel the in vitro potency of the conjugates reported here, it is likely that particular dendrimer bioconjugates could not justify their higher costs of goods relative to the parent SC-63032 molecule, though retention of SC-63032 biological activities in conjugates suggests that other cytokine-dendrimer bioconjugates may be bioactive. This is good news to the nanotechnology community, in as much as PAMAM dendrimers are among the monodisperse polymeric nanomaterials available, and these results show that they can be used successfully in conjugates to bioactive proteins.

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin.

Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor. Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very 'deep' mutagenesis at selected residues: >10(5) substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.

Phage presentation and affinity selection of a deletion mutant of human interleukin-3.

A deletion derivative of the cytokine human interleukin-3 (hIL-3(15-125), comprising amino acids 15-125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble-cytokine, phage-presented hIL-3(15-125) exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-3(15-125) species (designated hIL-3(15-125) epsilon) had 20 amino acids appended to its C-terminus; hIL-3(15-125) epsilon did not exhibit reduced bioactivity. hIL-3(15-125)-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.

Four new clerodane diterpenes from the leaves of Casearia guianensis which inhibit the interaction of leukocyte function antigen 1 with intercellular adhesion molecule 1.

Four new clerodane diterpenes, casearinols A and B (1 and 2) and casearinones A and B (3 and 4), were isolated from the leaves of Casearia guianensis. These immunomodulatory compounds have been structurally elucidated primarily on the basis of 2D NMR analysis and spectral data comparison with known compounds. These compounds inhibited the binding of T-cell leukocyte function antigen 1 to intercellular adhesion molecule 1.

High-level expression of biologically active, soluble forms of ICAM-1 in a novel mammalian-cell expression system.

LFA-1/ICAM-1 interaction is important in facilitating a number of cellular events including antigen-specific T-cell activation and leukocyte transendothelial migration. We are interested in defining residues and contact sites that mediate ICAM-1 interaction with the integrin receptor, LFA-1. To provide sufficient material to facilitate study of the interaction of this ligand-receptor pair, we have developed a new high-level mammalian-cell expression system based on the use of the herpes simplex virus (HSV) VP16 transactivator and the HSV IE175 promoter to direct expression of foreign genes in BHK cells. In this system, the gene of interest is expressed as a fusion protein with a carboxyl terminal decapeptide tail to aid in identification, quantitation, and affinity purification of recombinant protein. This system allowed rapid generation of cell lines producing high levels of levels of soluble proteins corresponding to the full-length extracellular (sICAM453) and the amino terminal two immunoglobulin domains (sICAM185) of ICAM-1. Both sICAM453 and sICAM185 were biologically active and were purified in a single step from conditioned media by antibody affinity chromatography.

Pharmacokinetic and galactopoietic response to recombinant variants of bovine growth hormone.

Two studies were designed to examine the pharmacokinetic and galactopoietic potency of three molecular variants of recombinant-derived bovine GH (rbGH): [Met1, Leu127]-bGH, [Ala1, Val127]-bGH and [Ala1, Val127, His133]-bGH. Histidine substitution for arginine at residue 133 of rbGH was shown to impart thrombin resistance. In a Latin square design, nine lactating Holstein cows received a 25 mg rbGH bolus infusion via the jugular vein followed by frequent blood sampling over the next 12 h. The serum GH concentration data were found to fit a two-compartment open model. Neither primary nor secondary kinetic parameter estimates differed significantly (P > 0.05) among the three rbGH variants. Thus, the disposition of GH concentration at time t was described by the equation C(t) = (1295.5 micrograms/l) (e-(0.11/min)(t)) + (317.3 micrograms/l)(e-(0.03/min)(t)). Overall averages were: area under the curve = 27.1 mg.min per l, clearance = 0.15 litres/min per 100 kg and volume of distribution of the central compartment = 2.59 litres/100 kg. The t 1/2 for the two compartments averaged 8.2 and 29.1 min. In the second study, 36 lactating Holstein cows received i.m. injections of one of four oil-based formulation treatments: control vehicle or 500 mg of one of the three rbGH variants every 14 days for 42 days. Average and maximum serum GH concentrations and area under the curve estimates were increased by approximately 3-6 micrograms/l, 5-15 micrograms/l and 40-90 micrograms.day per 1 respectively. Ala1, Val127 rbGH treatments elicited greater blood GH concentrations than [Met1, Leu127]-bGH when administered in an oil-based formulation. Blood GH responses did not directly translate into milk response differences, possibly due to differences in biopotency or receptor availability. Thrombin resistance resulting from substitution of histidine at position 127 of rbGH did not affect blood GH pharmacokinetic parameters or milk response over other rbGH variants.

Secretion of active bovine somatotropin in Escherichia coli.

We have expressed a chimeric protein, comprising the LamB secretion signal sequence fused to mature bovine somatotropin (bST), in Escherichia coli. Plasmid constructs with the recA promoter showed significant protein accumulation prior to induction and cell lysis occurred after induction. In contrast, the lacUV5 promoter was tightly regulated. With the lacUV5 promoter, temperature and inducer concentration had significant effects on the total amount of recombinant protein produced and the fraction processed to mature bST. Quantitation of bST from shake flask cultures showed that 1-2 micrograms/ml/OD550 could be released from the periplasm by osmotic shock. N-terminal sequence analysis of the purified protein indicated that the majority of the secreted bST was correctly processed. The bST present in the osmotic shock fraction was judged to be correctly folded by comigration with oxidized methionyl-bST standard on a non-reducing polyacrylamide gel and activity in a bovine liver radioreceptor assay. These results provide a rapid method to produce bST for use in structure-function studies.

Familial hypophosphatemic vitamin D-resistant rickets: review of the literature and report of case.

Familial hypophosphatemia, commonly known as vitamin D-resistant rickets, is inherited in an x-linked dominant manner. This condition is characterized by impairment of renal tubular reabsorption of inorganic phosphate and is inherited as a sex-linked dominant trait. Early clinical signs, usually detected about two years of age, included lateral bowing of the lower extremities, scoliosis, and frontal bossing. Characteristic dental findings are often the first clinically noticeable signs of the disease. This case report describes the typical dental findings in a 10-year old Hispanic female.

Purification and characterization of pituitary bovine somatotropin.

Bovine somatotropin (bST) has been isolated from pituitary glands and compared in a variety of chemical analyses and bioassays with somatotropin derived from recombinant Escherichia coli. Comparison of pituitary extracts and purified bST by Western blot analysis of two-dimensional gels suggested that the immunoreactive somatotropin species present in the extract were also present in the purified material, with no significant losses or degradation as a result of the purification method. NH2-terminal sequence analysis indicated the presence of equal quantities of Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser- and Phe-Pro-Ala-Met-Ser-Leu-Ser- sequences. The Met-Ser-Leu-Ser-NH2-terminal sequence, a degradation product observed in NIH standard lots, was not detected. Assay of bioactivity in a bovine liver receptor-binding assay and in a female rat growth assay showed pituitary bST and recombinant methionyl-bovine somatotropin to be equipotent. Tryptic maps and sequence analysis of pituitary-derived somatotropin suggest the presence of isoaspartate derivatization at Asp128.

Effect of rapid normalization of plasma glucose levels on microvascular dysfunction and polyol metabolism in diabetic rats.

Effects of rapid normalization of plasma glucose levels (by insulin infused via Alzet pumps implanted intraperitoneally) on plasma insulin-like growth factor I (IGF-I) levels, granulation tissue polyol levels, and vascular permeation by 125I-labeled albumin were examined in male Sprague-Dawley rats with streptozocin-induced (60-65 mg/kg) diabetes. Two days after implantation of pumps, plasma insulin levels were twice normal levels and remained elevated (1.4-2.5 times normal) throughout the remainder of the study. Plasma glucose levels and granulation tissue polyol levels were normalized within 2 days after initiation of insulin treatment. Plasma IGF-I levels were significantly increased (2 times) by 2 days, but were not normalized until 7 days. In contrast, 125I-albumin permeation normalized at a much slower relatively linear rate and was still not completely normal after 14 days of insulin treatment. In view of 1) previous studies demonstrating that diabetes-induced increases in 125I-albumin permeation in this tissue are linked to increased metabolism of glucose to sorbitol and 2) the rapid normalization of tissue polyol levels in this study, the relatively linear rate of normalization of vascular permeability over 14 days in these studies suggests that impaired vascular barrier functional integrity in this model is mediated by structural and/or functional vascular alterations associated with sustained increased polyol metabolism rather than by increased polyol levels per se and/or by readily reversible functional and metabolic alterations associated with acute increases in polyol metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic regions of bovine somatotropin as defined by monoclonal antibodies.

Three distinct antigenic regions of bovine somatotropin (bST) were identified on the basis of the ability of a set of monoclonal antibodies to bind to proteolytic fragments and deletion variants of recombinant bST (rbST) in Western blot analyses. One of the regions is further subdivided into two epitopes on the basis of the cross-reaction of somatotropins from several species with the same set of antibodies in solid-phase RIA. The RIA and Western blot results suggest that amino acids 134-150, 181-190 and the amino terminus may be involved in the binding specificity of antibodies to the bovine somatotropin molecule. The total of four antigenic regions on the bST molecule parallels results described for human somatotropin. Labeled antibody competition tests were used to show that the epitope involving amino acids 134-150 is spatially separated from the other three epitopes.

Islet transplants in diabetic Lewis rats prevent and reverse diabetes-induced increases in vascular permeability and prevent but do not reverse collagen solubility changes.

The effects of islet transplantation on diabetes-induced increases in vascular permeability and collagen solubility were examined in new granulation tissue vessels and collagen formed after induction of streptozotocin diabetes in male Lewis rats. Albumin permeation was increased by 50% (p less than 0.001) and collagen solubility was decreased by 50% (p less than 0.001) in granulation tissue from untreated diabetic animals as compared with controls. The islet transplants reversed diabetes-induced vascular permeability increases in tissues formed prior to islet transplantation (tissue to blood isotope ratio = 2.1 +/- 0.1 - SD for controls, 3.2 +/- 0.2 for diabetic rats and 2.0 +/- 0.2 for diabetic rats given islets) and prevented permeability increases in new tissues formed following transplantation (tissue to blood isotope ratio = 2.1 +/- 0.1 for controls, 3.3 +/- 0.8 for diabetic rats and 1.9 +/- 0.2 for diabetic rats given islets). In contrast, while islet transplants prevented diabetes-induced decreased collagen solubility in tissues formed after transplantation (controls = 24%, diabetic rats = 12%, and diabetic rats given islets = 24%), collagen solubility in tissues formed prior to islet transplantation was virtually unaffected. These findings indicate that collagen changes induced by the diabetic milieu are not nearly as readily reversed by normalization of the diabetic milieu as (diabetes-induced) alterations in vascular functional integrity.

Diabetes-induced increases in vascular permeability and changes in granulation tissue levels of sorbitol, myo-inositol, chiro-inositol, and scyllo-inositol are prevented by sorbinil.

In a recently developed animal model, we investigated the pathogenesis of diabetic vascular disease and demonstrated that 125I-albumin permeation is markedly increased in new "granulation tissue" vessels formed in subcutaneous tissue after the onset of diabetes. The studies described in this report were undertaken to examine the effects of an aldose reductase inhibitor on diabetes-induced increases in vascular permeability in this animal model. 125I-albumin permeation was assessed 3 weeks after the subcutaneous implantation of sterile preweighed polyester fabric (to stimulate angiogenesis) in diabetic male Sprague-Dawley rats, in controls, and in diabetic rats given sorbinil approximately 12 or approximately 25 mg/kg/d mixed in ground rat chow. Sorbinil administration prevented the diabetes-induced increase in vascular permeability by approximately 60% at the lower dose and by approximately 80% at the higher dose without affecting body weight or plasma glucose levels. Diabetes-induced changes in tissue levels of sorbitol, myo-inositol, scyllo-inositol, and chiro-inositol were also prevented by the high dose of sorbinil (data were not obtained for the lower dose). These observations are consistent with evidence linking diabetic cataracts and neuropathy to imbalances in sorbitol/inositol metabolism and support the hypothesis that diabetic vascular disease as well as neuropathy and cataracts are mediated by excess metabolism of glucose through the polyol pathway. Furthermore, these observations suggest that increased vascular permeability associated with diabetic microangiopathy in humans may be prevented by inhibitors of aldose reductase without the need to normalize blood glucose levels.

Sex steroid dependency of diabetes-induced changes in polyol metabolism, vascular permeability, and collagen cross-linking.

The effects of castration on diabetes-induced increases in collagen cross-linking and vascular permeability and on polyol levels in new granulation tissue formed after induction of streptozocin (STZ) diabetes were examined in male Sprague-Dawley rats. New granulation tissue formation was induced by implanting sterile polyester fabric subcutaneously (s.c.) at the time of STZ injection 3 wk before assessment of vascular permeability and collagen cross-linking. Castration was performed 10 days before implanting the fabric. The characteristic increases in collagen cross-linking (manifested by decreased solubility in 0.5 M acetic acid) and in albumin permeation of the vasculature seen in intact diabetic rats were completely prevented by castration. Net collagen accumulation was not affected by diabetes or castration. Castration also markedly diminished diabetes-induced increases in tissue levels of sorbitol and completely prevented the decreases in tissue levels of myo-inositol and scyllo-inositol observed in intact diabetic rats, but had no effect on serum glucose levels, nonenzymatic glycosylation of plasma and granulation tissue proteins, or plasma somatomedin-C levels. The demonstration that castration prevents diabetes-induced increases in vascular permeability and collagen cross-linking as well as imbalances in tissue levels of sorbitol, myo-inositol, and scyllo-inositol in this model indicates that all of these changes are sex steroid-dependent phenomena. While the pathogenesis of these vascular permeability and collagen cross-linking changes is clearly multifactorial, these new findings: indicate that the role of sex steroids in the development of late complications of diabetes may be far more important than hitherto suspected, and suggest an explanation for the clinical observation that diabetic complications are uncommon in prepubertal diabetic subjects regardless of duration of diabetes.

Purification of recA-based fusion proteins by immunoadsorbent chromatography. Characterization of a major antigenic determinant of Escherichia coli recA protein.

Monoclonal antibodies to Escherichia coli recA protein were prepared, characterized, and used as affinity reagents for the purification of recA and recA:somatostatin fusion proteins. The monoclonal antibodies recognize an antigenic determinant or determinants located between amino acids 260 and 330 of recA. Addition of a fragment of the recA gene coding for these amino acids to an unrelated gene (beta-galactosidase) allowed the resulting beta-galactosidase fusion protein to be recognized by the recA monoclonal antibodies.

Sorbinil prevents diabetes-induced increases in vascular permeability but does not alter collagen cross-linking.

In recent studies we have demonstrated a marked increase in albumin permeation of new vessels formed by angiogenesis (in subcutaneous tissue) in the diabetic milieu. Likewise, lysyl oxidase-mediated collagen cross-linking is markedly increased in the scar tissue associated with angiogenesis. The present studies were undertaken to determine whether sorbinil, a chemical inhibitor of aldose reductase that has been shown to prevent and reverse diabetic cataracts and neuropathy, also could prevent the vascular permeability and collagen cross-linking changes in this model. Vascular permeation by 125I-BSA, collagen cross-linking, and tissue levels of sorbitol, myo-inositol, and scyllo-inositol were assessed in male Sprague-Dawley rats 3 wk after injection of streptozocin and induction of angiogenesis and collagen synthesis in polyester fabric implanted subcutaneously. Sorbinil (approximately 25 mg/kg/day) added to the diet of diabetic rats reduced the diabetes-induced increases in albumin permeation by 80%, completely prevented diabetes-induced changes in tissue levels of sorbitol and myo-inositol, and markedly reduced diabetes-induced changes in tissue levels of scyllo-inositol. In contrast, sorbinil had no effect on plasma glucose levels or collagen solubility (an index of collagen cross-linking). These observations indicate that increased vascular permeability associated with diabetes is linked to imbalances in sorbitol/inositol metabolism. These findings also indicate that diabetes-induced increases in vascular permeability and in collagen cross-linking are independent phenomena and diabetes-induced increases in vascular permeability are largely preventable by treatment with an aldose reductase inhibitor in the face of high plasma glucose levels.

Albumin permeation of new vessels is increased in diabetic rats.

125I-bovine serum albumin (BSA) permeation of the vasculature of 3-wk-old granulation tissue (induced by subcutaneous implantation of polyester fabric) formed in the diabetic milieu was assessed in female BB/W, spontaneously diabetic rats and in male, Sprague-Dawley rats with streptozocin-induced diabetes as well as in corresponding nondiabetic controls. Albumin permeation of new granulation tissue vessels was markedly increased in both groups of diabetic animals relative to that of nondiabetic controls, while albumin permeation of vessels in most other tissues did not differ for controls and diabetics. These observations indicate that the functional integrity of new vessels formed in the diabetic milieu is impaired: (1) to a greater extent than that of older vessels formed before induction of diabetes and (2) relative to new vessels in nondiabetics. The implication of these observations is that molecular constituents of vessels synthesized in the diabetic milieu are quantitatively and/or qualitatively abnormal and/or their incorporation into vessels is defective.

Monoclonal antibodies to bovine somatotropin: immunoadsorbent reagents for mammalian somatotropins.

Twenty-nine stable hybridoma cell lines secreting monoclonal antibodies to bovine somatotropin (bST) have been produced and characterized. Five of the monoclonal antibodies bind porcine and human somatotropins as well as bST. One of these antibodies was used as a reagent in immunoadsorbent chromatography of recombinant bST or pituitary bST from cell extracts. Following chromatography, the bST preparations retained activity in a rabbit liver radioreceptor assay and in a radioimmunoassay. The immunoadsorbent reagent bound human and porcine somatotropins as well as bovine.

Muscle capillary basement membrane in identical twins discordant for insulin-dependent diabetes.

Although hereditary factors clearly modulate susceptibility to develop diabetes, their role as determinants of vascular complications associated with diabetes remains unclear. These studies were undertaken to further assess the extent to which capillary basement membrane thickening (CBMT) is governed by metabolic derangements associated with relative or absolute insulin deficiency versus genetic determinants of vascular disease closely linked to but independent of those modulating susceptibility to develop relative or absolute insulin deficiency. Quadriceps muscle capillary basement membranes obtained by needle biopsy were examined in eight pairs of identical twins discordant for insulin-dependent diabetes (IDD) for 11-29 yr. Biopsy material from one of the diabetic twins was technically unsuitable for study. The average CBM width of the IDD twins was found to be significantly thicker than that of their nondiabetic (ND) twin mates (t = 2.50, P less than 0.025). Three IDD, but none of the ND twins, had basement membrane width values in excess of 95% upper tolerance intervals for age- and sex-matched controls with no family history of diabetes. The absence of CBMT in all of the ND twins and in four of the IDD twins with diabetes of 15-24 yr duration argues against the existence, in this group of subjects, of hereditary determinants of diabetic vascular disease linked to those governing susceptibility to develop diabetes. In addition, the absence of CBMT in four subjects with IDD of 15-24 yr duration is consistent with evidence from other studies indicating that diabetic microangiopathy is not an inevitable consequence of the diabetic milieu.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosylation of human glomerular basement membrane collagen: increased content of hexose in ketoamine linkage and unaltered hydroxylysine-O-glycosides in patients with diabetes.

To study the glycosylation of glomerular basement membrane collagen (GBMC) in diabetes, kidneys were obtained at autopsy from 5 patients with insulin-requiring diabetes of long duration and diabetic complications, and from 5 control subjects. Glomeruli were prepared by sieving and collagen was isolated by limited pepsin proteolysis followed by salt precipitations. Amino acid analyses of the collagen preparations, after acid hydrolysis, indicated a composition consistent with that of type IV collagen. No differences in the relative contents of various amino acids, and in particular, 3-hydroxyproline, 4-hydroxyproline and hydroxylysine, were noted between diabetic and control samples. Non-enzymatic glucosylation was assessed by measuring hexose in ketoamine linkage with thiobarbituric acid after conversion to 5-hydroxymethylfurfural. In 4 of the 5 patients studied, glucosylation values exceeded the mean +2 S.D. of the controls; in the fifth subject glucosylation was in the high normal range. No correlation between the severity of diabetes and hexose content of GBMC was noted, however. In further studies, enzymatic glycosylation of GBMC was assayed after alkaline hydrolysis by separation of glucosylgalactosyl-O-hydroxylysine, galactosyl-O-hydroxylysine, and unsubstituted hydroxylysine in an amino acid analyzer. No differences in the relative contents of hydroxylysine-O-glycosides were evident between diabetic and control GBMC. The results suggest that non-enzymatic glucosylation, but not glycosylation catalyzed by collagen glucosyl and galactosyl transferases, is increased in diabetes. The increased carbohydrate content of collagen may lead to decreased turnover and/or excessive accumulations of basement membrane collagen thus contributing to the vascular complications of diabetes.