Production of hybridomas secreting monoclonal antibodies against the lympholine osteoclast activating factor.

The human lympholine osteoclast activating factor (OAF) is thought to be involved in several bone-destroying diseases. The current studies were designed to produce monoclonal antibodies against OAF for use in the subsequent design of immunoassays for OAF in clinical samples. Spleen cells from mice immunized with purified human OAF were hybridized with mouse plasmacytoma cells in vitro to yield hybridomas. Several clones of these hybridomas secreted into the culture medium antibodies, which neutralized the biological activity of OAF at dilutions as high as 1:100,000 relative to the initial culture medium. These antibodies did not interfere with the activities of parathyroid hormone in the same systems. These results represent the first report of monoclonal antibodies against a human lympholine, and validate the concept that hybridoma production is a useful technique for developing antibodies against weak or scarce antigens.

Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin.

IL-8 (also known as neutrophil-activating peptide 1) is recognized as a potent effector of neutrophil functions. Several different cell types that contact blood, namely T lymphocytes, monocytes, and endothelial cells, secrete this polypeptide following stimulation by cytokines, or lipopolysaccharide. Here we show that when IL-8 is added to blood it rapidly partitions from the plasma fluid to the blood cells and that erythrocytes account for the vast majority of this binding. Analysis of 125I-IL-8 binding [( ala-IL-8]77 form) to human red cells indicates a single, 5 nM Kd affinity class of binding sites, present at approximately 2,000 per red cell representing approximately 15 nmol of red cell IL-8 binding sites per liter of blood. These sites are protease sensitive. Their binding of IL-8 is rapidly reversible and does not result in receptor internalization, although bound IL-8 is resistant to extraction by pH 3 buffer at 5 degrees C. 125I-IL-8 binding to red cells was not inhibited by epidermal growth factor or interleukin 1, but was inhibited by monocyte chemotactic peptide-1, which is not a neutrophil chemotaxin, but is a member of the same family of polypeptides as IL-8. FACS analysis of IL-8-mediated mobilization of Ca2+ in neutrophils indicates that the IL-8 bound to red cells is incapable of stimulating neutrophils. Thus, red cell absorption of IL-8 may function to limit stimulation of leukocytes by IL-8 released into blood.

D-Phe-Pro-Arg-chloromethylketone: its potential use in inhibiting the formation of in vitro artifacts in blood collected during tissue-type plasminogen activator thrombolytic therapy.

In vitro artifacts due to proteolysis may occur in blood samples containing recombinant tissue-type plasminogen activator (rt-PA) due to continued activation of plasminogen to plasmin by rt-PA. The aim of this study was to identify a rapid inhibitor of rt-PA that would not interfere in assays designed to monitor thrombolytic events. When rt-PA was added at 5 micrograms/ml to whole blood and incubated at 25 degrees C, fibrinogen decreased 50 percent, plasminogen levels decreased 90 percent and alpha 2-antiplasmin decreased below detectable levels. If D-Phe-Pro-Arg-chloromethylketone (PPACK) or aprotinin were added before the addition of rt-PA there was no significant loss of fibrinogen. Only PPACK completely inhibited changes in fibrin degradation products, plasminogen and alpha 2-antiplasmin. PPACK was also found to inhibit the binding of rt-PA to plasma protease inhibitors in vitro. Rhesus monkeys were infused with rt-PA and blood samples were taken with either PPACK or aprotinin in the collection syringe. There was a significant increase in the recovery of immunoreactive rt-PA and consistent measures of fibrinogen, FDPs, plasminogen, and alpha 2-antiplasmin in the PPACK group as compared to the aprotinin group which indicates that PPACK will prevent the in vitro formation of artifacts due to the presence of active rt-PA.

Pharmacokinetics and bioactivity of intact versus truncated IGF-I during a 24-h infusion into lactating goats.

The aim of this study was to compare the plasma concentration profile, mammary blood flow response and transfer into milk of intact IGF-I with that of its truncated analogue, des(1-3)IGF-I (des-IGF-I). Each peptide was infused for 24 h into the pudic artery supplying one mammary gland of lactating goats (n = 5). Concentrations of IGF-I in plasma (from the jugular vein) rose rapidly during infusion of IGF-I or des-IGF-I to reach 510 +/- 62 and 640 +/- 32 ng/ml (mean +/- S.E.M.) respectively, compared with 262 +/- 35 ng/ml after a similar infusion of saline. Ligand blotting analysis indicated a significant increase in the intensity of [125I]IGF-I binding to the 40-43 kDa doublet (binding protein-3 (BP-3), P < 0.01) and the band at 31 kDa (P < 0.05) during infusion of either IGF-I or des-IGF-I, as compared with saline. Furthermore des-IGF-I induced a significant increase in intensity of binding to the 35 and 24 kDa bands, but IGF-I did not. Whereas [125I]IGF-I was distributed between BP-3 and the other binding proteins, [125I]des-IGF-I bound exclusively to BP-3. Mammary blood flow (MBF) increased 48 +/- 6% after 12 h of infusion of des-IGF-I, compared with an increase of 22 +/- 6% during IGF-I. The difference in response was significant at P < 0.05. In addition, more IGF-I was secreted into the milk of the infused than the non-infused gland during either infusion of IGF-I or des-IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I in rats.

Incubation of 125I-labelled insulin-like growth factor-I (IGF-I) with rat plasma at 4 degrees C led to the transfer of approximately half the radioactivity to 150 kDa and smaller complexes with IGF-binding proteins. The extent of association was greater with labelled IGF-II and essentially absent with the truncated IGF-I analogue, des(1-3)IGF-I. A greater degree of binding of IGF peptides with binding proteins occurred after i.v. injection of the tracers into rats, but most of the des(1-3)IGF-I radioactivity remained free. Measurement of the total plasma clearances showed the rapid removal of des(1-3)IGF-I compared with IGF-I and IGF-II; the mean clearances were 4.59, 1.20 and 1.34 ml/min per kg respectively. The mean steady-state volume of distribution was larger for des(1-3)IGF-I than for IGF-I and IGF-II (461, 167 and 181 ml/kg respectively), probably because of the differences in plasma protein binding. With all tracers, radioactivity appeared in the kidneys to a greater extent than in other organs. The amount of radioactivity found in the adrenals, brain, skin, stomach, duodenum, ileum plus jejunum and colon was in rank order, des(1-3)IGF-I greater than IGF-I greater than IGF-II. Since this ranking is the opposite of the abilities of the three IGF peptides to form complexes with plasma binding proteins, we propose that the plasma binding proteins inhibit the transfer of the growth factors to their tissue sites of action. Moreover, we suggest that IGF analogues that are cleared rapidly from blood may have greater biological potencies in vivo.

Intraocular penetration of topical tissue plasminogen activator.

Fifty-eight eyes of 31 anesthetized rabbits received one drop of proparacaine hydrochloride, 0.05%, and two drops of tissue plasminogen activator (tPA) separated by 5 minutes. Four eyes of two additional rabbits had epithelial defects created before drug delivery. Tissue plasminogen activator in multiple doses was given to eight eyes of four other rabbits. We used this dosing regimen to investigate the effect of topical tPA on anterior chamber fibrin clots in three rabbits. A two-site enzyme-linked immunosorbent assay test was used to measure tPA levels in the aqueous samples, obtained by paracentesis in each eye. Of 53 eyes treated with the original dosing regimen, 21 (40%) had detectable tPA aqueous levels. Blood and aqueous from eyes of untreated control rabbits, contralateral control eyes of treated rabbits, and eyes with epithelial defects had nondetectable tPA. Multiple tPA drop dosing resulted in 75% of aqueous samples with detectable tPA and a higher average tPA concentration than the original dosing regimen. Eyes treated with tPA showed a significantly faster resolution of anterior chamber fibrin clots than did control eyes.

A review of nonclinical toxicology studies of becaplermin (rhPDGF-BB).

Becaplermin (recombinant human platelet-derived growth factor-BB [BB homodimer, rhPDGF-BB]) has demonstrated a favorable safety profile in a series of nonclinical studies designed to assess its systemic toxicity, sensitization, local irritation, and genotoxic potential. No significant local or systemic toxicity directly attributable to becaplermin was observed following single and multiple intravenous or subcutaneous administration at doses up to 3 mg/kg in monkeys. Administration of single large intravenous doses (up to 100 mg/kg) and repeated dosing at 1 or 3 mg/kg in mice resulted in rapidly reversible vasodilation and central nervous system depression. In a bone-toxicity study, becaplermin produced histomorphologic changes suggestive of accelerated bone remodeling, which were judged to be potentially reversible. Similar findings have not been observed in humans. Although becaplermin was not considered a dermal or ocular irritant, some skin-sensitizing effects were observed in animals; this finding was not unexpected for a recombinant human-derived protein. Becaplermin was not genotoxic in a variety of in vitro assays and in one in vivo assay.

Production of human/mouse hybridomas secreting a human lymphokine, osteoclast activating factor.

A human/mouse hybridoma was developed which has the property of secreting a human bone resorbing factor similar or identical to the osteoclast activating factor (OAF) isolated from human tonsil lymphocytes. Mouse plasmacytoma cells negative for OAF production were fused with an enriched subpopulation of human tonsil lymphocytes that had been activated with phytohemagglutinin (PHA) to produce OAF (G.E. Nedwin, M.A. Mohler, and R.A. Luben, submitted for publication). Culture supernatants from mixed hybridomas contained a bone resorbing protein shown to cause the release of 45Ca from previously labeled mouse calvaria. The bone resorbing activity from these hybridomas was inhibited by the presence of OAF-specific monoclonal antibodies. Several hybridomas retained OAF production following limited dilution cloning. One clone, CD6.20, showed a biphasic dose-response curve for bone resorption similar to that of purified OAF from PHA-activated human tonsil lymphocytes. OAF production in the CD6.20 cell line has been retained for over 100 passages. Karyotype analysis of this cell shows the presence of human chromosomes 10 and 18 and the X chromosome.

Intraocular tissue plasminogen activator concentrations after subconjunctival delivery.

BACKGROUND: Topical application of recombinant tissue plasminogen activator (tPA) results in detectable intraocular tPA levels. The authors sought to determine whether subconjunctival delivery of recombinant tPA results in enhanced intraocular drug levels. METHODS: One week after first performing a gas vitrectomy in each eye, the authors injected 0.4 ml of a tPA solution (1 or 10 mg/ml) subconjunctivally in one eye of 16 rabbits and 0.4 ml of sterile water in the fellow control eye. Vitreous taps were performed to obtain vitreous samples for measuring tPA concentrations. An anterior chamber paracentesis was then done for each concentration at 15, 30, and 60 minutes. Aqueous and vitreous tPA concentrations were determined using a two-site enzyme-linked immunosorbent assay (ELISA). RESULTS: Vitreous tPA levels were higher than aqueous levels in the treated eyes. Vitreous levels averaged 7 +/- 7 ng/ml for the 1-mg/ml group (n = 7/7) and 202 +/- 230 ng/ml for the 10-mg/ml group (n = 7/8). Aqueous levels were positive in 8 of 16 samples. The authors conclude that subconjunctival delivery of tPA results in both vitreous and aqueous tPA levels.

Increase in cyclic AMP levels by relaxin in newborn rhesus monkey uterus cell culture.

A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus (NRMU). NRMU cells respond to relaxin, in the presence of 1 microM forskolin, by producing intracellular adenosine 3', 5'-cyclic monophosphate (cAMP). The increase in cAMP levels is dose, time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1 X 10(5) cells/well. Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP. Three synthetic analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin. Natural relaxin purified from human corpora lutea tissue also generated a response similar to synthetic human relaxin. Porcine and rat relaxins also increased levels of cAMP. Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however, 200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin. Combinations of relaxin with insulin, IGF I or IGF II did not increase cAMP levels above levels obtained with relaxin alone. The effect on NRMU cells of other hormones, growth factors and drugs potentially present in cell culture systems or serum samples was evaluated. In combination with relaxin, oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin E2 resulted in additive increases in cAMP. These data suggest that the NRMU cell line is an appropriate target tissue for studying relaxin-mediated biological responses in vitro as well as functioning as the primary component of a relaxin in vitro bioassay.