Transient thyrotoxicosis from thyroiditis induced by sibutramine overdose: a case report.

Sibutramine is an antiobesity drug that inhibits the reuptake of serotonin and noradrenalin in the hypothalamus. A 37-year-old Korean man presented to the emergency room for the oral intake of 280 mg of sibutramine. The patient was in thyrotoxic state. The (99m)Technetium-pertechnetate thyroid scan showed irregular uptake of radioisotope and thyroid-stimulating hormone receptor antibody and thyroperoxidase antibody were negative. Thyroid function normalized after that. The patient had transient thyrotoxicosis with thyroiditis. We report a case of thyrotoxicosis accompanied by thyroiditis resulting from the intentional overdose of sibutramine.

Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy.

In order to investigate the role of host factors in Helicobacter pylori infection and immunity, two different strains of inbred mice, C57BL/6 and BALB/c, were infected with a standard H. pylori strain, SS1. A month later, infected mice were immunized orally with whole-cell lysates of H. pylori SS1 and cholera toxin on days 1, 3, 6, 30, and 54. Ten days after the last immunization, mice were sacrificed and the stomach was collected to assess H. pylori colonization density by quantitative culture. H. pylori SS1 colonization was significantly greater in C57BL/6 than in BALB/c (P<0.02 and P<0.003 at 2 and 13 weeks post-inoculation, respectively). Colonization in C57BL/6 persisted at equivalent levels for 13 weeks but the colonization density in BALB/c decreased significantly during this period. In contrast to the pattern of bacterial colonization, antibody responses following H. pylori SS1 infection were greater in BALB/c than in C57BL/6, suggesting that host factors may modulate the immune responses to H. pylori infection. Following therapeutic immunization, H. pylori colonization in BALB/c mice was also significantly reduced (P<0.03), while no significant differences in bacterial density were observed in C57BL/6. These observations collectively demonstrate the great importance of host factors in H. pylori infection and the development of effective immune responses.

Depletion of plasma factor XIII prevents disseminated intravascular coagulation-induced organ damage.

The impact of clot stability affecting the vasculopathy and tissue necrosis in Shwartzman reaction was investigated using plasma Factor XIII A2-depleted rabbit (FXIII-DR). Plasma Factor XIIIA2 (FXIIIA2) was depleted by infusion of the mono-specific goat anti-rabbit FXIIIA2 IgG. Generalized Shwartzman reaction (GSR) was induced by priming and challenged by i.v. injection of LPS and local Shwartzman reaction (LSR) was primed by intradermal injection of LPS and challenged by i.v. injection of LPS. Histological examination of the GSR animals showed, extensive thrombi accumulation in renal tubules and bilateral cortical necrosis of kidney in 8 out of 10 rabbits but none in the FXIII-DR. Fibrinogen levels were elevated to 3 approximately 4 fold at 24 h and lowered at 48 h whereas a steady rise was seen in the FXIII-DR. FDP levels in GSR animals were significantly elevated at 24 h and further increased at 48 h but only slightly elevated in the FXIII-DR. Examination of the LSR tissues after 48 h showed an acute onset of progressive cutaneous vascular thrombosis, purpura, and secondary hemorrhagic necrosis whereas neither fibrin deposit nor necrosis of tissue were detected in FXIII-DR despite of an early edema formation. Fibrinogen levels were also increased two fold at 24 h but returned to basal levels at 48 h in control LSR animals but not affected at all in FXIII-DR. These results suggest that during the severe inflammatory conditions such as sepsis, the fibrinolytic system is functionally sufficient to dissipate the pathogenic accumulation of disseminated intravascular clots and exudated fibrin clots if those clots were prevented from getting crosslinked in plasma.

Attachment characteristics of normal human cells and virus-infected cells on microcarriers.

The attachment kinetics of normal and virus-infected LuMA cells were studied to improve the production of live attenuated varicella viruses in human embryonic lung (LuMA) cells. Normal LuMA cells and LuMA cells infected by varicella virus at various cytopathic effects (CPE) were grown on microcarriers. Ninety-three percent of suspended LuMA cells attached to the solid surface microcarriers within fifteen minutes and cell viability was greater than 95% when the cell suspension was stirred. Low serum levels did not affect the attachment rate of virus-infected cells in the microcarrier culture system. Kinetic studies showed that varicella infected cells had a lower attachment rate than normal LuMA cells. Virus inoculum (= infected cells) at low CPE showed a relatively better attachment rate on cell-laden microcarriers than virus inoculum at a higher CPE. Maximum titers were obtained at 2 days post-infection. Based on cell densities, the use of viral inoculum showing a 40% CPE led to an approximately 2- and 1.2-fold increase in the cell associated and in cell free viruses, respectively, than a virus inoculum with a CPE of 10%.However, the ratio of cell-free to cell-associated virus in a microcarrier culture was very low, approximately0.04-0.06. These studies demonstrate that the virus inoculum resulting in a high CPE yielded a high production of cell-associated and cell-free virus in microcarrier cultures because of the high cellular affinity of the varicella virus.

Production and characterization of recombinant guamerin, an elastase-specific inhibitor, in the methylotrophic yeast Pichia pastoris.

The elastase-specific inhibitor, guamerin, was expressed and secreted into a culture medium using the methylotrophic yeast Pichia pastoris, and the resulting recombinant guamerin was purified from the culture media using a two-step procedure composed of a hydrophobic interaction and reverse-phase chromatography. Up to 90 g/L of dry cell weight, the guamerin-producing recombinant P. pastoris was cultivated and guamerin was secreted into the culture medium at a level of 0.69 g/L. The recombinant guamerin was highly purified (>98%) with a recovery yield of 68%. Analyses of the purified guamerin revealed the same N-terminal amino acid sequence, amino acid composition, and molecular mass as found in the native leech protein. The recombinant guamerin exhibited the tight binding to porcine pancreatic elastase. Furthermore, the recombinant guamerin did not produce a humoral immune response in mice.

Transient expression of transglutaminase C during prenatal development of human muscles.

Tissue transglutaminase (TGase C, TGase II) is known to participate in cellular processes during morphogenesis, differentiation, and development of various prenatal tissues and organs. The expression of TGase C during myoblast proliferation and attachment to external laminae was examined by immunohistochemical (IH) localization at 5-12 weeks of developmental stages of prenatal human muscle in 23 embryos. IH detection using a monospecific antibody to TGase C showed a prominent expression of TGase C in muscle cells as stage- and spatial-specific patterns during an early embryonal period. The myoblasts of intervertebral, tongue, and limb muscles, attached to adjacent cartilaginous skeletons or fibrous fascia, showed a pronounced expression of TGase C at 5-6, 6-7, and 7-8 weeks after fertilization, respectively. The most intense activity of TGase C was observed in some cardiac myoblasts infiltrating into endocardial mesenchyme at 6-7 weeks after fertilization. Although weak staining was detected until 14 weeks after fertilization, the level of TGase C expression in all muscles was significantly decreased after 6-7 weeks, with the exception that the smooth muscle cells of blood vessels and gastrointestinal tract showed diffusely intense staining of TGase C between 5 and 12 weeks after fertilization. Western blotting analysis of the cellular extracts of pooled samples showed a single strong band at 80 kD at 6 weeks after fertilization. This band became weaker after 8-10 weeks of prenatal development. These findings of transient expression of TGase C, which coincides with the development of myoblast anchoring and differentiation, suggest that TGase C plays a role in myoblast attachment to the extracellular laminae during the early embryonal period.

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes.

Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.

Recombinant kringle 1-3 of plasminogen inhibits rabbit corneal angiogenesis induced by angiogenin.

PURPOSE: Angiostatin is a potent angiogenesis inhibitor that has been identified as a cryptic fragment of plasminogen molecule containing the first four kringle domain. Angiogenin, a 14-kDa monomeric protein, a potent blood vessel inducer, is expressed in tumors and present in mammalian plasma. The purpose of this study was to determine whether recombinant kringle 1-3 (rKI-3) of human plasminogen could interfere with angiogenesis induced by angiogenin and to evaluate the role of angiogenin in corneal angiogenesis in rabbit. METHODS: A Hydron polymer pellet containing 2.0 microg of angiogenin was implanted intrastromally into the superior cornea of each of 44 rabbit eyes. All eyes received an intrastromal pellet and were randomized into either one group treated with 12.5 microg of rKI-3 (n = 25) or the other group treated with phosphate-buffered saline (PBS; n = 19). Both pellets were positioned in parallel at the site 1.2 mm from the superior limbus. Two masked observers kept the angiogenesis score daily, based on the number and the length of new vessels. The corneas with induced angiogenesis also were examined histologically. RESULTS: On the third day of the angiogenin pellets implantation, the eye treated with rKI-3 had less angiogenesis (mean score, 4.2 +/- 6.6) than eye treated with PBS (mean score, 16.1 +/- 17.1; p < 0.05, Mann-Whitney U test). The cornea treated with PBS also showed much more leukocyte adhesion than the cornea treated with rKI-3. CONCLUSION: Recombinant kringle 1-3 appears to inhibit angiogenin-induced angiogenesis in a rabbit corneal pocket assay. Recombinant kringle 1-3 may have therapeutic potential as an antiangiogenic agent.

Disruption of interkringle disulfide bond of plasminogen kringle 1-3 changes the lysine binding capability of kringle 2, but not its antiangiogenic activity.

Kringle 1-3 of human plasminogen is a potent inhibitor of endothelial cell proliferation. To understand a possible role for the unique cystine bridge between kringle 2 and kringle 3, we disrupted the interkringle disulfide bond by mutating Cys(169) and Cys(297) to serine residues. The yield of the mutant during the refolding process was decreased significantly. Anti-endothelial cell proliferative activity of the mutant was similar to that of the wild type. There was no significant difference in in vivo antiangiogenic activity between the wild type and the mutant in chorioallantoic membrane assay. However, in the mutant, the weak lysine binding capability of kringle 2 was not detected and its mobility in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is different from that of the wild type. These results support the notion that the overall antiangiogenic function of angiostatin is mediated by individual kringles, and suggest that the lysine binding capability of kringle 2 is likely not important for the antiangiogenic activity of kringle 1-3.

Production characteristics of interferon-alpha using an L-arabinose promoter system in a high-cell-density culture.

Using high-cell-density culture of Escherichia coli under the control of an L-arabinose promoter (ParaB), several factors affecting the production of recombinant protein and the formation of inclusion bodies were studied. The inducer, L-arabinose, showed a maximal induction level above 10.7 mM in the final concentration. The concentration of inducer also affected the partition of interferon-alpha (IFN-alpha) into the soluble form and inclusion bodies. Induction kinetics of the rate of accumulation of IFN-alpha on the ParaB promoter showed a slower rate than those of other promoter systems, for example T7, lac or tac. These innate characteristics of ParaB enabled cells to grow continuously in spite of the metabolic burden induced by the expression of foreign protein. The duration time of induction could control the expression of both soluble and insoluble protein. The ratio of yeast extract to glycerol (N/C ratio) in feeding media significantly affected both the production level of recombinant protein and inclusion body formation. The reason for decreasing specific bioactivity during induction can be explained by the increased proportion of inclusion bodies in the total expressed IFN-alpha.

Cloning and characterization of a 22 kDa outer-membrane protein (Omp22) from Helicobacter pylori.

Helicobacter pylori is a causative agent of gastritis and peptic ulceration in humans. As the first step towards development of a vaccine against H. pylori infection, we have attempted to identify protective antigens. A potential target of vaccine development would be a H. pylori specific protein, which is surface-exposed and highly antigenic. We identified a 22 kDa outer-membrane protein (Omp22) from H. pylori, which was highly immunoreactive. By screening a H. pylori genomic DNA library with rabbit anti-H. pylori outer-membrane protein antibodies, the omp22 gene was cloned and 1.4 kb of the nucleotide sequence was determined. One open reading frame, encoding a 179-residue polypeptide, was identified and the amino acid sequence deduced showed homology with peptidoglycan-associated lipoproteins. The sequence was conserved among other H. pylori strains. Omp22 protein is expressed as a precursor polypeptide of 179 residues and undergoes lipid modification and cleavage of an 18 amino acid signal peptide to yield a mature protein. Omp22 protein in H. pylori as well as recombinant Omp22 protein expressed in E. coli was localized into the outer membrane and exposed on the cell surface. Omp22 may have the potential as a target antigen for the development of a H. pylori vaccine.

Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility.

Helicobacter pylori colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of H. pylori. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The fliD operon of H. pylori consists of flaG, fliD, and fliS genes, in the order stated, under the control of a sigma(28)-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the fliD gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the fliD gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the fliD mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The fliD mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic fliD mutant of H. pylori SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that H. pylori FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.

Inhibition of human malignant glioma growth in vivo by human recombinant plasminogen kringles 1-3.

Human malignant gliomas are highly vascularized and aggressive tumors. Angiogenesis inhibitors have been shown to induce regression of a variety of primary and metastatic tumors in vivo. However, their usefulness in treating brain tumors is not well understood. Angiostatin, a multiple kringle (1-4 of 5)-containing fragment of plasminogen, is one of the highly effective natural cryptic angiogenesis inhibitors. In our study, the therapeutic efficacy of non-glycosylated and small molecular size recombinant kringles 1-3 (rPK1-3) was examined in the treatment of brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days post-implant were treated daily with rPK1-3 (100 mg/kg) s.c. for 21 days. Treated animals showed suppressed brain tumor growth by greater than 71.2% along with a 3-fold increase of apoptotic index and suppressed vascularization by 78.9%, without any observable signs of toxicity. Analysis of bFGF and VEGF expression in the tumors of treated animals using immuno-histochemical methods showed near complete absence of growth factors. Our results indicate that the non-glycosylated, small molecular size rPK1-3 is an efficient tumoristatic agent for the treatment of intracranial human glioma xenografts in mice and might provide new strategies for the treatment of brain tumors.

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.

Oral immunization with Helicobacter pylori-loaded poly(D, L-lactide-co-glycolide) nanoparticles.

BACKGROUND: Helicobacter pylori is a major cause of chronic antral gastritis and peptic ulcer diseases. Many researchers have examined the possibility of immunologically-mediated prevention of H. pylori infection using an oral vaccine. The purpose of this study is to investigate whether mucosal and systemic immune responses are induced by oral immunization with H. pylori lysate-loaded poly(D, L-lactide-coglycolide)[PLG] nanoparticles, and if so, how the distribution of serum IgG subclasses are produced. METHODS: PLG nanoparticles (H. pylori-PLG) with encapsulated H. pylori lysates were prepared by the solvent evaporation method, and the physical properties of the nanoparticles were investigated. Following the oral immunization of the H. pylori-PLG nanoparticles into mice, antibody induction was assayed in serum and gut washings, and the pattern of serum IgG subclasses was determined by ELISA. RESULTS: The prepared H. pylori-PLG nanoparticles were spherical, nonporous particles with a mean diameter of less than 1 microm. The multiple oral immunization with H. pylori-PLG nanoparticles induced significantly H. pylori-specific mucosal IgA response as well as serum IgG responses. The serum antibody subclasses elicited were predominantly IgG1 and IgG2b. CONCLUSION: Our results suggested that oral immunization of H. pylori-PLG nanoparticles induced the H. pylori-specific mucosal and systemic responses in mice and enhanced Th2-type responses.

Quantitative analysis of polyvinyl alcohol on the surface of poly(D, L-lactide-co-glycolide) microparticles prepared by solvent evaporation method: effect of particle size and PVA concentration.

Polyvinyl alcohol (PVA) is an emulsion stabilizer that is used in the solvent evaporation method for poly(D,L-lactide-co-glycolide) (PLG) microparticles preparation. In this study, the surface binding of PVA on PLG microparticles was quantitatively examined by employing gel permeation chromatography. The PVA binding can affect hydrophobicity and digestibility of the microparticle surface. GPC analysis detected the presence of PVA bound on the microparticle surfaces in an irreversible manner. The PVA content per weight of microparticle increased with the specific surface area as the particle size decreased. The surface PVA density, which is the PVA content per unit surface area of microparticle, changed with particle size in a biphasic manner. In the size range larger than 1 microm, the surface PVA density increased from 0.8 mg/m2 to 2.2 mg/m2 as the particle size decreased. But below 1 microm, the surface PVA density remained unchanged at about 1.8 mg/m2. The surface PVA density of PLG microparticles prepared in different PVA concentrations (0.1, 1, 5, 10% w/v) showed similar values and size dependency, indicating that the PVA concentration of the continuous phase did not affect the surface PVA density of PLG microparticles. These data suggest that the influence of the surface PVA layer would be larger in the smaller particles, but be independent of the PVA concentration of the continuous phase in the manufacturing process.

Induction of mucosal and systemic immune response by oral immunization with H. pylori lysates encapsulated in poly(D,L-lactide-co-glycolide) microparticles.

Helicobacter pylori is a major cause of chronic antral gastritis and peptic ulcer diseases. Several kinds of poly(D,L-lactide-coglycolide) microparticles containing H. pylori whole-cell lysate (PLG-HP) were prepared by the solvent evaporation method using double emulsion. Physical properties, such as particle size, protein content, and morphology were investigated. All prepared microparticles showed a smooth surface morphology from 0.5-0.86 microm in diameter and high degree of encapsulation efficiency from 62-75%. SDS-PAGE and immunoblotting of extracted antigen confirmed that the molecular weight and antigenicity of the antigen remained unaltered by the encapsulation procedure. Following the oral immunization of the microparticles to mice, antibody production was assayed in serum and gut washings by ELISA and antibody secreting cells were determined in intestinal lamina propria lymphocytes (LPL) by ELISPOT. Multiple oral immunizations induced significant H. pylori-specific intestinal IgA response as well as serum IgG response than those detected with soluble antigen (P < 0.001). The presence of antibody-secreting cell in intestinal lamina propria lymphocytes (LPL) was correlated with IgA level in gut washing fluids. After boosting at week-8, the antibody induction levels were highly increased irrespective of microparticles prepared with different PLG molecular weights. These data suggested that PLG-HP could stimulate the H. pylori-specific mucosal and systemic response in vivo and might be useful adjuvant in future H. pylori vaccine development.

Purification and characterization of recombinant murine endostatin in E. coli.

Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent.

Characterization of yeast deoxyhypusine synthase: PKC-dependent phosphorylation in vitro and functional domain identification.

The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions.

The inhibitory effects of recombinant plasminogen kringle 1-3 on the neovascularization of rabbit cornea induced by angiogenin, bFGF, and VEGF.

Angiostatin is a potent angiogenesis inhibitor that is composed of the first four kringles of plasminogen fragment. Angiostatin with one less kringle molecule (kringle 1 to 3) was recently demonstrated to be an effective angiogenic inhibitor. To determine whether recombinant plasminogen kringle 1-3 (rPK1-3) can inhibit the corneal neovascularization induced by potent angiogenic factors; angiogenin, bFGF, or VEGF, hydron polymer discs each containing 2.0 microg of angiogenin, 500 ng of bFGF, or 500 ng of VEGF respectively were implanted into the corneal stroma of 138 rabbit eyes, and then discs each containing 10 microg, 12.5 microg, 20 microg or 30 microg of rPK1-3 were implanted randomly. Discs containing phosphate buffered saline were also implanted as a control. The angiogenesis score on number and length of newly formed vessels on the each of the rabbit's cornea were recorded daily by two observers (blinded). The treated corneas were also examined histologically. Recombinant PK1-3 treated corneas showed less neovascularization induced by all angiogenic factors (p < 0.05). and the extent of inhibition of neovascularization was proportional to the concentration of rPK1-3 (p < 0.05). Histologic examination showed leukocyte infiltration into the corneal stroma on the PBS treated eyes whereas rPK1-3 treated eyes showed only traces of leukocytes. These results of the effective rPK1-3 inhibition of corneal neovascularization induced by angiogenin, bFGF, or VEGF suggest that this angiostatin related fragment, rPK1-3, may be useful in the treatment of various neovascular diseases.