Complement evasion mechanisms of the systemic pathogens Yersiniae and Salmonellae.

Pathogens that colonize deep tissues and spread systemically encounter the innate host resistance mechanism of complement-mediated lysis and complement opsonization leading to engulfment and degradation by phagocytic cells. Yersinia and Salmonella species have developed numerous strategies to block the antimicrobial effects of complement. These include recruitment of complement regulatory proteins factor H, C4BP, and vitronectin (Vn) as well as interference in late maturation events such as assembly of C9 into the membrane attack complex that leads to bacterial lysis. This review will discuss the contributions of various surface structures (proteins, lipopolysaccharide, and capsules) to evasion of complement-mediated immune clearance of the systemic pathogens Yersiniae and Salmonellae. Bacterial proteins required for recruitment of complement regulatory proteins will be described, including the details of their interaction with host regulatory proteins, where known. The potential role of the surface proteases Pla (Yersinia pestis) and PgtE (Salmonella species) on the activity of complement regulatory proteins will also be addressed. Finally, the implications of complement inactivation on host cell interactions and host cell targeting for type 3 secretion will be discussed.

Induction of Protective Antiplague Immune Responses by Self-Adjuvanting Bionanoparticles Derived from Engineered Yersinia pestis.

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 × 105 CFU (80,000 50% lethal dose [LD50]) and intranasal challenge with 5 × 103 CFU (50 LD50) of virulent Y. pestis This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

Yersinia pestis Pla Protein Thwarts T Cell Defense against Plague.

Plague is a rapidly lethal human disease caused by the bacterium Yersinia pestis This study demonstrated that the Y. pestis plasminogen activator Pla, a protease that promotes fibrin degradation, thwarts T cell-mediated defense against fully virulent Y. pestis Introducing a single point mutation into the active site of Pla suffices to render fully virulent Y. pestis susceptible to primed T cells. Mechanistic studies revealed essential roles for fibrin during T cell-mediated defense against Pla-mutant Y. pestis Moreover, the efficacy of T cell-mediated protection against various Y. pestis strains displayed an inverse relationship with their levels of Pla activity. Together, these data indicate that Pla functions to thwart fibrin-dependent T cell-mediated defense against plague. Other important human bacterial pathogens, including staphylococci, streptococci, and borrelia, likewise produce virulence factors that promote fibrin degradation. The discovery that Y. pestis thwarts T cell defense by promoting fibrinolysis suggests novel therapeutic approaches to amplifying T cell responses against human pathogens.

Plasminogen activation is required for the development of radiation-induced dermatitis.

Skin damage caused by radiation therapy (radiodermatitis) is a severe side effect of radiotherapy in cancer patients, and there is currently a lack of effective strategies to prevent or treat such skin damage. In this work, we show with several lines of evidence that plasminogen, a pro-inflammatory factor, is key for the development of radiodermatitis. After skin irradiation in wild-type (plg+/+) mice, the plasminogen level increased in the irradiated area, leading to severe skin damage such as ulcer formation. However, plasminogen-deficient (plg-/-) mice and mice lacking plasminogen activators were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreased radiodermatitis in plg+/+ mice and prevented radiodermatitis in plg+/- mice. Together with studies at the molecular level, we report that plasmin is required for the induction of inflammation after irradiation that leads to radiodermatitis, and we propose that inhibition of plasminogen activation can be a novel treatment strategy to reduce and prevent the occurrence of radiodermatitis in patients.

Humoral and cellular immune responses to Yersinia pestis Pla antigen in humans immunized with live plague vaccine.

BACKGROUND: To establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of Y. pestis. This outer membrane protease is an essential virulence factor that is steadily expressed by Y. pestis. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination. CONCLUSIONS/SIGNIFICANCE: The Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen.

Bacterial pathogens activate plasminogen to breach tissue barriers and escape from innate immunity.

Both coagulation and fibrinolysis are tightly connected with the innate immune system. Infection and inflammation cause profound alterations in the otherwise well-controlled balance between coagulation and fibrinolysis. Many pathogenic bacteria directly exploit the host's hemostatic system to increase their virulence. Here, we review the capacity of bacteria to activate plasminogen. The resulting proteolytic activity allows them to breach tissue barriers and evade innate immune defense, thus promoting bacterial spreading. Yersinia pestis, streptococci of group A, C and G and Staphylococcus aureus produce a specific bacterial plasminogen activator. Moreover, surface plasminogen receptors play an established role in pneumococcal, borrelial and group B streptococcal infections. This review summarizes the mechanisms of bacterial activation of host plasminogen and the role of the fibrinolytic system in infections caused by these pathogens.

Impact of the Pla protease substrate α2-antiplasmin on the progression of primary pneumonic plague.

Many pathogens usurp the host hemostatic system during infection to promote pathogenesis. Yersinia pestis, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown in vitro to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor α2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP in vivo; the role of A2AP during respiratory Y. pestis infection is not known either. Here, we show that Y. pestis does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with Y. pestis, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection.

The plasminogen activator system: involvement in central nervous system inflammation and a potential site for therapeutic intervention.

BACKGROUND: Extracellular proteases such as plasminogen activators (PAs) and matrix metalloproteinases modulate cell-cell and cell-matrix interactions. Components of the PA/plasmin system have been shown to be increased in areas of inflammation, and have been suggested to play a role in inflammatory neurologic disorders such as epilepsy, stroke, brain trauma, Alzheimer's' disease and multiple sclerosis (MS). In the present study, we evaluated the involvement of the PA system in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced by myelin oligodendrocyte glycoprotein (MOG) in mice deficient for the urokinase PA (uPA-/-), or the urokinase PA receptor (uPAR-/-). Mice were evaluated for EAE clinical signs and histopathologic parameters, and compared with wild-type (WT) EAE mice. Lymphocytes from the knockout (KO) and WT mice were analyzed for ex vivo restimulation, cytokine secretion, and antigen presentation. Finally, WT EAE mice were treated with PAI-1dp, an 18 amino acid peptide derived from the PA inhibitor protein (PAI-1). RESULTS: EAE was aggravated in uPA-/- and uPAR-/- mice, and this was accompanied by more severe histopathologic features and microglial activation. By contrast, specific T- cell reactivity towards the encephalitogenic antigen MOG was markedly reduced in the KO animals, as shown by a marked reduction in proliferation and pro-inflammatory cytokine secretion in these mice. Antigen presentation was also reduced in all the KO animals, raising an immunologic paradox. When the mice were treated with PAI-1, a peptide derived from the PA system, a marked and significant improvement in EAE was seen. The clinical improvement was linked to reduced T-cell reactivity, further emphasizing the importance of the PA system in immunomodulation during neuroinflammation. CONCLUSIONS: Cumulatively, our results suggest a role for uPA and uPAR in EAE pathogenesis, as exacerbation of disease was seen in their absence. Furthermore, the successful amelioration of EAE by PAI-1 treatment suggests that the PA system can be considered a potential site for therapeutic intervention in the treatment of neuroimmune diseases.

Fast and simple detection of Yersinia pestis applicable to field investigation of plague foci.

Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37°C. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20°C and 37°C, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples.

Plasminogen activator inhibitor-1 and angiotensin converting enzyme gene polymorphisms in Turkish asthmatic children

Background: Polymorphisms of plasminogen activator inhibitor-1 (PAI-1) and angiotensin-converting enzyme (ACE) genes have been implicated in susceptibility to asthma. In this study, we aimed to investigate whether there was any association between childhood asthma and polymorphisms of the PAI-1 and ACE genes. Methods: Two hundred and three Turkish children aged 5–15 years, including 102 asthmatic patients and 101 healthy control subjects were included in this study. The asthma group was divided into two groups as follows: Group I: Asthmatic children with positive family history for atopy (n=53), Group II: Asthmatic children without any family history for atopy (n=49). One hundred and twenty-eight atopic family members were also included in the study. The insertion/deletion (I/D) polymorphism of the ACE and PAI-1 4G/5G gene polymorphisms was carried out by polymerase chain reaction. Results: The prevalence of the PAI-1 4G allele was significantly greater in asthmatic children compared to control group (p<0.05, OR: 1.64 (1.11–2.43)) but there was no significant relation between ACE I/D genotypes and childhood asthma. No significant difference was detected between Groups I and II in terms of these ACE and PAI-1 genotypes and allele frequencies. No significant relationship was found between both gene polymorphisms and total serum IgE and skin prick test results. Conclusion: It has been established that PAI-1 4G allele may be a genetic risk factor for childhood asthma but ACE gene I/D polymorphisms do not play a role in the development of asthma in the sample of Turkish children(AU)

Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

Inhibition of Mycobacterium tuberculosis-induced signalling by transforming growth factor-ß in human mononuclear phagocytes.

Tuberculosis (TB) is associated with excessive production and bioactivation of transforming growth factor bets (TGF-ß) in situ. Here, modification of expression of components of plasminogen/plasmin pathway in human monocytes (MN) by inhibitors of TGF-ß signalling was examined. Smad3 siRNA effectively inhibited TGF-ß-induced urokinase plasminogen activator receptor (uPAR). Agents known to interfere with TGF-ß signalling, including the Smad inhibitors SIS3 and erythromycin derivatives, and ALK5 receptor inhibitor (SB 431542) in inhibition of uPAR expression in response to Mycobacterium tuberculosis (MTB) were examined. Inhibition by SIS3 only inhibited uPAR mRNA significantly. SIS3 may prove to be an effective adjunct to TB therapy.

[Preparation and identification of monoclonal antibodies against Pla protein of Yersinia pestis].

AIM: To prepare the monoclonal antibody of Pla with recombinant Pla (rPla) by hybridoma cell technology, which will lay the foundation for related research work. METHODS: Purified rPla was collected by washing repeatedly with urea, and BALB/c mice were immunized by them. Hybridoma cells were achieved by Sp2/0 cell fusion with mouse spleen cells from successfully immunized mice. Monoclonal antibody was screened by indirect ELISA and Western blots with rPla, natural crude Pla and GST respectively. RESULTS: Three strains of hybridoma cells (named 15B8, 14H4 and 19A4 respectively) which secreted stably the monoclonal antibody of Pla were obtained. Their subclasses were IgG2a and IgG1 in heavy chains and κ chains in light chains. The ELISA titers of ascites were 10(6); respectively.Three of monoclonal antibody can react with natural crude Pla tested by western blots. CONCLUSION: Monoclonal antibody of natural Pla of Yersinia pestis were successfully got, which has laid the foundation for further study of the Pla protein and development diagnosis reagent.

Cpa, the outer membrane protease of Cronobacter sakazakii, activates plasminogen and mediates resistance to serum bactericidal activity.

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

Invited review: Breaking barriers--attack on innate immune defences by omptin surface proteases of enterobacterial pathogens.

The omptin family of Gram-negative bacterial transmembrane aspartic proteases comprises surface proteins with a highly conserved beta-barrel fold but differing biological functions. The omptins OmpT of Escherichia coli, PgtE of Salmonella enterica, and Pla of Yersinia pestis differ in their substrate specificity as well as in control of their expression. Their functional differences are in accordance with the differing pathogenesis of the infections caused by E. coli, Salmonella, and Y. pestis, which suggests that the omptins have adapted to the life-styles of their host species. The omptins Pla and PgtE attack on innate immunity by affecting the plasminogen/plasmin, complement, coagulation, fibrinolysis, and matrix metalloproteinase systems, by inactivating antimicrobial peptides, and by enhancing bacterial adhesiveness and invasiveness. Although the mechanistic details of the functions of Pla and PgtE differ, the outcome is the same: enhanced spread and multiplication of Y. pestis and S. enterica in the host. The omptin OmpT is basically a housekeeping protease but it also degrades cationic antimicrobial peptides and may enhance colonization of E. coli at uroepithelia. The catalytic residues in the omptin molecules are spatially conserved, and the differing polypeptide substrate specificities are dictated by minor sequence variations at regions surrounding the catalytic cleft. For enzymatic activity, omptins require association with lipopolysaccharide on the outer membrane. Modification of lipopolysaccharide by in vivo conditions or by bacterial gene loss has an impact on omptin function. Creation of bacterial surface proteolysis is thus a coordinated function involving several surface structures.

Relative immunogenicity and protection potential of candidate Yersinia Pestis antigens against lethal mucosal plague challenge in Balb/C mice.

Yersinia Pestis outer proteins, plasminogen activator protease and Yop secretion protein F are necessary for the full virulence of Yesinia pestis and have been proposed as potential protective antigens for vaccines against plague. In the current study, we used DNA immunization as a tool to study the relative protective immunity of these proteins with a standardized intranasal challenge system in mice. While the natural full-length gene sequences for most of these Y. pestis proteins did not display a good level of protein expression in vitro when delivered by a DNA vaccine vector, the overall immunogenicity of these wild type gene DNA vaccines was low in eliciting antigen-specific antibody responses and gene sequence modifications improved both of these parameters. However, even modified YopD, YopO and YscF antigens were only able to partially protect immunized mice at various levels against lethal challenge with Y. pestis KIM 1001 strain while no protection was observed with either the YopB or Pla antigens. These results demonstrate that DNA immunization is effective in screening, optimizing and comparing optimal antigen designs and immunogenicity of candidate antigens for the development of a subunit-based plague vaccine.

Immunomodulatory effects of plasminogen activators on hepatic fibrogenesis.

Tissue-type plasminogen activators (tPA) and urokinase-type plasminogen activators (uPA) are involved in liver repair. We examined the potential immunomodulatory actions of uPA, tPA and uPA-receptor (uPAR) in carbon-tetrachloride-induced hepatic fibrosis in wild-type (WT), tPA-/-, uPA-/- and uPAR-/- mice. Carbon-tetrachloride treatment increased fibrosis in four groups but significantly less in three knock-out models. Serum cytokines and intrahepatic T cells elevated significantly following fibrosis process in WT animals but not in the knock-out groups. In culture, uPA increased lymphocyte proliferation significantly in WT and uPA-/- but not uPAR-/- animals. Following uPA exposure in vivo, there was CD8 predominance. To isolate uPA's effect on lymphocytes, WT mice were irradiated sublethally and then reconstituted with WT or uPA-/- lymphocytes. In these animals fibrosis was decreased and T cells were reduced in the uPA-/- recipients. Based on these data we postulate that plasminogen activators affect fibrosis in part by liver-specific activation of CD8 subsets that govern the fibrogenic activity of hepatic stellate cells.

Effect of immunization with the mannose-induced Acanthamoeba protein and Acanthamoeba plasminogen activator in mitigating Acanthamoeba keratitis.

PURPOSE: The mannose-induced cytopathic protein (MIP-133) and Acanthamoeba plasminogen activator (aPA) play key roles in the pathogenesis of Acanthamoeba keratitis by inducing a cytopathic effect on the corneal epithelial and stromal cells and by production of proteolytic enzymes that facilitate the invasion of trophozoites through the basement membrane. The goal of the present study was to gain insight into the pathogenicity of Acanthamoeba infection as well as to determine whether oral immunization with aPA and MIP-133 produce an additive protection against Acanthamoeba keratitis. METHODS: MIP-133 and aPA were isolated by chromatography. The purity of the concentrated MIP-133 and aPA was confirmed by SDS-PAGE and fibrinolytic activity, respectively. aPA activity of Acanthamoeba cultures was quantitated by radial diffusion in fibrin-agarose gel. The capacity of aPA and MIP-133 to induce cytolysis of corneal epithelial cells was tested in vitro. Chinese hamsters were orally immunized with four weekly doses of aPA or MIP-133 conjugated with cholera toxin. The animals were immunized before infection to determine the prophylactic effect of oral immunization. The therapeutic effect of oral immunization with aPA and MIP-133 was determined after corneal infection had been established. The animals were then infected via Acanthamoeba castellanii-laden contact lenses. RESULTS: aPA was characterized in pathogenic and nonpathogenic strains of Acanthamoeba spp. Oral immunization with MIP-133 before and after infection with Acanthamoeba significantly reduced the severity of corneal infection which includes infiltration and ulceration (P < 0.05) and shortened the duration of the disease. Immunization with aPA alone did not significantly affect the course of disease (P > 0.05). CONCLUSIONS: These data suggest that once trophozoites invade the cornea, MIP-133 production is necessary to initiate corneal disease and plays an important role in the subsequent steps of the pathogenic cascade of Acanthamoeba keratitis.

Design and characterization of a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency.

To obtain a thrombus-targeted plasminogen activator with high affinity for activated platelets and enhanced thrombolytic and antithrombotic potency, we engineered a sequence encoding RGDS (Arg-Gly-Asp-Ser) peptide into the loop between domains II and III of the sequence-deleted mutant of annexin B1 and then constructed a chimaeric plasminogen activator gene mAnxB1-RGDS-ScuPA by fusing ScuPA32k [low-molecular-mass single-chain urokinase (32 kDa)] with the N-terminus. The chimaeric protein was expressed in inclusion bodies in Escherichia coli at 25% of the total cellular protein content. Ion-exchange and gel-filtration chromatographies were applied to purify the chimaeric protein, achieving purity greater than 98%. We demonstrated that this chimaera can be expressed and purified in an active form; in vitro testing indicated that the chimaera fully retained the thrombolytic activity, platelet membrane-binding activity and anti-platelet aggregation activity of the parent molecules. The plasma clearance of the chimaera was similar to that of urokinase and ScuPA32k. In vivo experiments in a canine system indicated that animals administered the chimaera presented a decreased time to reperfusion, higher reperfusion ratio and less bleeding effects than treatment with urokinase. These results show that the chimaera is a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency that may have advantages over currently available thrombolytic agents.

Contribution of somatic cell-associated activation of plasminogen to caseinolysis within the goat mammary gland.

Functional regression of the mammary gland is partly reflected by proteolysis of milk protein and tissue protein. The involvement of the plasminogen activation system in degradation of milk protein and mammary tissue damage has been demonstrated under inflammatory conditions. In this study, mammary secretion from 23 dairy goats primarily grouped as lactation (milking twice daily) or involution (milking once daily or less) was used to determine the ratio of gravity-precipitated casein to total milk protein (casein ratio) as an index of caseinolysis, and activities of components of plasminogen activation system as well as their expressions on somatic cells. Based on the casein ratio, lactation goats were subcategorized as very active (71.8 +/- 1.0%) or less active (29.9 +/- 1.0%) in mammary function; involution goats were subcategorized as gradual (21.7 +/- 1.0%) or acute (5.9 +/- 0.2%) involution. This result suggests that caseinolysis occurred during regular lactation as well as during involution. On the other hand, activities of components of the plasminogen activation system in mammary secretion were increased along with the decreasing casein ratio, in contrast to the similar activities of their counterparts in circulation throughout various mammary statuses. Correlation analysis between casein ratio and activities of plasminogen activation system of goat milk indicated a significant negative relationship for plasmin (r = -0.64), plasminogen (r = -0.69), and urokinase-type plasminogen activator (uPA; r = -0.78) during involution but not during lactation. As for the cellular components of plasminogen activation system, there was an increase in immunoreactivity on somatic cells toward both monoclonal antibodies of human uPA and human uPA receptor under involution conditions suggesting their upregulation relative to lactation condition. Collectively, these results suggest that plasminogen activation system within the mammary gland differentially contribute to milk caseinolysis along the various stages of goat lactation. Meanwhile, a somatic cell-mediated local elevation of plasmin activity may be committed to extensive caseinolysis during involution.