Rapid infarct imaging with a technetium-99m-labeled antimyosin recombinant single-chain Fv: evaluation in a canine model of acute myocardial infarction.

Studies of monoclonal antibody-based imaging agents show that blood clearance is inversely proportional to molecular size, i.e., Fab or Fab' > F(ab')2 > IgG. Indium-111-antimyosin Fab-DTPA is a highly specific and sensitive marker for myocardial necrosis. An improvement on current antibody diagnostic imaging may result from the use of smaller labeled fragments. We report the first in vivo targeting of acute myocardial infarction with a novel recombinant single-chain Fv (sFv) antimyosin protein. The sFv (MW = 27,594) is approximately one-half the size of the Fab and is comprised of the heavy and light chain variable regions from the myosin-specific murine monoclonal antibody R11D10 which were joined by a 15-amino-acid linker and expressed as a fusion protein (sFv) in E. coli. The binding affinity of the sFv for cardiac myosin was similar to the affinity observed for the Fab fragment. Technetium-99m labeling of the sFv was accomplished by the attachment of a cleavable, ester-linked bifunctional chelator (RP-1). Comparative studies in mice showed 99mTc-sFv-RP-1 cleared significantly faster (p < 0.001) than 99mTc-Fab'-RP-1 and 111In-Fab-DTPA antimyosin fragments. Furthermore, measurement of 99mTc-sFv-RP-1 blood clearance in a canine model of acute myocardial infarction gave a mean T1/2 of 0.54 +/- 0.13 hr versus 2.80 +/- 0.57 and 2.58 +/- 0.64 hr for Fab-DTPA and Fab'-RP-1 (p < 0.05), respectively. Despite its comparatively rapid clearance, 99mTc sFv-RP-1 had similar uptake in the infarct compared to the Fab'-RP-1. In addition, infarct visualization was more rapid with the sFv. Thus, these data demonstrate antimyosin sFv possesses characteristics necessary for rapid imaging of myocardial necrosis.

Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro.

We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.

Fibrinolytic drugs prevent pericardial adhesions in the rabbit.

Epicardial adhesions are believed to form secondarily to impaired pericardial fibrinolytic activity. This activity was reconstituted in a rabbit pericardial adhesion model with single doses of the fibrinolytic agents tissue plasminogen activator (t-PA), t-PA analog (Fb-Fb-CF), and streptokinase (SK), resulting in reductions in the extent and tenacity of adhesion formation. Adhesions of the median strip of the anterior cardiac surface were reduced in area from 89% (n = 22) in controls, to 28% (n = 5) by treatment with Fb-Fb-CF (0.94 mg), and to 49% (n = 7) by treatment with SK (93,750 IU). A modified fabric of oxidized regenerated cellulose (mTC7) used to deliver the agent to the cardiac surface did not interfere with the activity of these agents (Fb-Fb-CF 19%, n = 14; SK 33%, n = 7). t-PA (0.94 mg) was also found to reduce adhesion formation in combination with mTC7 (4%, n = 4), although the appearance of significant postoperative bruising and bleeding resulted in a decision to terminate the treatment of further animals with t-PA with and without mTC7. Postoperative bruising, bleeding, and swelling, to a lesser extent, were associated with SK and Fb-Fb-CF. Despite the efficacy of the these fibrinolytic drugs further work is required to assess their safety before they are used clinically.

Delayed treatment with a t-PA analogue and streptokinase in a rabbit embolic stroke model.

Fibrinolytic therapy may be effective in the treatment of ischemic stroke, and clinical trials are under way. We evaluated two fibrinolytic agents, an analogue of tissue plasminogen activator (Fb-Fb-CF, the catalytic fragment of the tissue plasminogen activator molecule with a prolonged serum half-life, n = 10) and streptokinase (n = 7), in a rabbit model of embolic stroke. Both agents were given 3 hours after stroke onset, a time relevant to the clinical setting. Fb-Fb-CF was significantly better (p less than 0.04) than saline (n = 7) in restoring blood flow to previously occluded intracranial arteries, but streptokinase was ineffective. Neither fibrinolytic agent was associated with a substantial risk for intracerebral hemorrhagic side effects. Our study demonstrates that Fb-Fb-CF can safely and effectively reperfuse rabbit intracranial arteries 3 hours after occlusion, while streptokinase does not.

The effects of a new tissue plasminogen activator analogue, Fb-Fb-CF, on cerebral reperfusion in a rabbit embolic stroke model.

Early fibrinolytic therapy with full molecular tissue plasminogen activator (t-PA) has been observed to be both angiographically and clinically effective when employed in animal stroke models. Preliminary clinical trials with t-PA are in progress. It is possible to refine t-PA by developing fragments or analogues of the drug. Using recombinant DNA technology in the Escherichia coli system, a t-PA analogue consisting of the catalytic fragment of t-PA and a dimer of the B fragment of staphylococcal protein A (Fb-Fb-CF) has been produced. Because this analogue has a long serum half-life of 90 minutes, we employed Fb-Fb-CF in a rabbit cerebral embolic stroke model to assess its efficacy as a reperfusion agent. When given as a bolus to 10 animals 15 minutes after embolization, Fb-Fb-CF produced angiographic cerebral reperfusion in 48 +/- 21 minutes (+/- SD), while in 8 saline-treated controls, reperfusion was not observed at 180 minutes in any animal (p less than 0.01). In another experiment reperfusion was demonstrated at 66 +/- 32 minutes in 11 animals treated with Fb-Fb-CF 90 minutes after embolization as compared with 100 +/- 25 minutes in 12 saline-treated controls (p less than 0.01). A small macroscopic hemorrhage within an infarct was seen in 1 Fb-Fb-CF-treated animal in the 15-minute experiment and in none of the controls. In the 90-minute experiment, macroscopic hemorrhagic infarction was seen in 4 Fb-Fb-CF-treated animals and in 3 controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of unique mRNAs by human interferons.

Treatment of human fibroblast cells with human interferon (INF-alpha, IFN-beta, or IFN-gamma) resulted in the accumulation of at least four newly synthesized mRNAs. The mRNAs code for proteins having molecular weights of 56,000, 57,000, 62,000, and 68,000 when characterized in a wheat germ cell-free translation system. A direct relationship was observed between the amount of IFN used and the degree of both the accumulation of the induced mRNAs and the development of an antiviral state. In the case of IFN-alpha or IFN-beta, time course studies indicated that the induced mRNAs appeared as early as 40 min, accumulated for 2 h, then remained ribosome bound for up to 16 h. The ability of fibroblast cells to develop an antiviral state always coincided directly with both the appearance and the level of accumulation of the induced mRNAs. Further mRNA synthesis beyond 2 h had a minimal effect on the development of an antiviral state. Human IFN-gamma also induced the synthesis of the same four mRNAs but required higher interferon titers and a longer incubation time. In addition, IFN-gamma induced a disproportionate amount of the mRNA coding for the 68,000 molecular weight protein and three new mRNAs not detected in cells treated with IFN-alpha or IFN-beta. Mouse interferon induces the original four mRNAs in human cells but to a far lesser extent. This correlated with the inability of these cells to develop much resistance to viral infection.

Stimulation of human gamma interferon production by diterpene esters.

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.

Partial characterization of gamma (immune) interferon mRNA extracted from human lymphocytes.

gamma (immune) interferon (IFN-gamma) was induced in cultures of fresh human lymphocytes by combined treatment with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA). Compared to the IFN-gamma yields obtained with PHA induction alone, the inclusion of TPA caused a significant enhancement of IFN-gamma production. Poly(A)-containing mRNA was isolated from mononuclear cells induced with TPA and PHA. Injected into Xenopus laevis oocytes, this mRNA preparation gave rise to IFN activity with characteristic properties of human IFN-gamma. Sucrose density gradient centrifugation analysis showed that IFN-gamma mRNA sedimented at 15 S, suggesting that it contains a total of about 1400 nucleotides.

Partial purification and characterization of human gamma (immune) interferon.

Human gamma (immune) interferon (IFN-gamma) was produced in lymphocyte cultures stimulated with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) and purified phytohemagglutinin. Physicochemical analysis showed that human IFN-gamma is a glycoprotein with an isoelectric point around 8.6 and an apparent molecular weight of 58,000 +/- 3000. A purification process for IFN-gamma was developed consisting of sequential chromatographic separations on controlled-pore glass, concanavalin A-Sepharose, and Bio-Gel P-200. This purification process resulted in an increase in specific activity from about 10(4) (crude culture fluid) to an estimated 10(7) units per mg of protein with a cumulative recovery of about 40% of the IFN activity.

Le interferon mRNA from human fibroblasts.

Human F and Le interferon can be clearly distinguished on the basis of different antigenic properties and host range. After inoculation with Newcastle disease virus (NDV), GM-258 fibroblasts produced Le as well as F interferon; in contrast, only F interferon was detectable after stimulation with poly(I) . poly(C). Polyadenylylated mRNA isolated from fibroblasts induced with poly(I) . poly(C) or NDV was injected into Xenopus laevis oocytes and the interferon activities thus produced were analyzed. Only F interferon production was demonstrable in oocytes injected with mRNA from cells induced with poly(I) . poly(C), whereas both F and Le interferons were made in oocytes injected with mRNA from NDV-induced cultures. The time course of accumulation of F and Le interferon mRNAs in NDV-induced cells corresponded to the kinetics of F and Le interferon synthesis in intact cells. The ratio of F and Le interferons made in oocytes was similar to that observed in intact GM-258 cells. F and Le interferon mRNA activities isolated from GM-258 cells could not be separated by sucrose density gradient centrifugation. However, the profile of F mRNA activity was more heterogeneous and its peak sedimented somewhat more slowly than that of Le interferon mRNA. These results suggest that the varying ratios of F and Le interferon synthesis in different cells after different modes of stimulation are determined at the level of mRNA. The induction mechanisms of F and Le interferon mRNA synthesis appear to be closely related but not identical.

Poly(U)-agarose affinity chromatography: specific, sensitivity selectivity, and affinity of binding.

These studies were done to determine four basic intrinsic properties of poly(U)-agarose affinity columns. Specificity of binding studies demonstrated that binding to these columns is highly specific with greater than 90% complementary binding and less than or equal to 3% noncomplementary binding. Sensitivity of binding studies indicated that a minimum sequence of 10 adenylates is required for detectable complementary binding. Selectivity of binding studies revealed that nonsequential adenylates in native RNAs and randomly distributed adenylates in synthetic poly(A)-poly(C) co-polymers did not bind to poly(U)-agarose affinity columns. Whereas, affinity of binding studies demonstrated that A=U complementary base pairing is independent of chain-lengths of greater than or equal to 25 adenylates and dependent of chain-lengths of less than 25 adenylates. Thus the data demonstrates that poly(U)-agarose affinity chromatography is scientifically sound and expedient for the detection and isolation of poly(A)-containing cellular and viral RNAs.

Characterization of Gazdar murine sarcoma virus by nucleic acid hybridization and analysis of viral expression in cells.

Gazdar murine sarcoma virus (Gz-MSV) and Moloney murine sarcoma virus (M-MSV) are closely related. The complete M-MSV-specific nucleic acid sequences constituted a major portion of Gz-MSV-specific sequences. The MSV-specific sequences in both Gz-MSV and M-MSV genomes shared homology with hamster leukemia virus nucleic acid sequences. Both rat cells (S+L+) and hamster (S+L-) cells expressed two viral proteins of 68,000 and 70,000 daltons. These proteins were immunologically related to p60 purified from m1 virions of M-MSV.

Regulation of substrate uptake in Nocardia erythropolis.

The regulation of uptake of glucose (GLU), glycerol (GLY), mannitol (MTL), and succinate (SUC) has been examined in Nocardia erythropolis 305. The apparent K(m) values of the uptake activities of cells subcultured in a medium with the corresponding substrate as the sole carbon source were 205, 48, 8.7, and 36 muM for GLU, GLY, MTL, and SUC, respectively. GLU and GLY uptake activities were constitutive, although there was evidence for an additional inducible component in GLY uptake. Moreover, MTL and SUC uptake activities were inducible. MTL uptake activity was markedly induced by cultivation in MTL medium and partially induced by growth in GLU medium, whereas SUC uptake was induced only by cultivation in SUC medium. SUC added to MTL medium partially repressed the formation of, or inhibited the activity of, MTL uptake. When not induced, uptake of MTL and SUC was proportional to the substrate concentration. The induced uptake of MTL and SUC and the constitutive uptake of GLU were energy dependent and carrier mediated. Uptake of GLY, constitutively or when induced, was also carrier mediated.