Roles of the tissue-type plasminogen activator in immune response.

The COVID-19 pandemic has once again brought to the forefront the existence of a tight link between the coagulation/fibrinolytic system and the immunologic processes. Tissue-type plasminogen activator (tPA) is a serine protease with a key role in fibrinolysis by converting plasminogen into plasmin that can finally degrade fibrin clots. tPA is released in the blood by endothelial cells and hepatocytes but is also produced by various types of immune cells including T cells and monocytes. Beyond its role on hemostasis, tPA is also a potent modulator of inflammation and is involved in the regulation of several inflammatory diseases. Here, after a brief description of tPA structure, we review its new functions in adaptive immunity focusing on T cells and antigen presenting cells. We intend to synthesize the recent knowledge on proteolysis- and receptor-mediated effects of tPA on immune response in physiological and pathological context.

Role of tissue plasminogen activator in clinical aggravation of experimental autoimmune encephalomyelitis and its therapeutic potential.

Tissue plasminogen activator (tPA), a component of the plasminogen activator (PA) system, is elevated in inflammatory neurological disorders. In the present study, we explored the immunomodulatory activity of tPA in experimental autoimmune encephalomyelitis (EAE). The EAE was treated with two catalytic inactive tPA variant proteins: S(481)A and S(481)A + KHRR(296-299)AAAA. EAE-induced tPA-/- mice presented with markedly more severe disease than wt EAE mice. Further, treatment with tPA variants, demonstrated a significant suppression of disease severity in tPA-/- and wt mice. Immunological evaluation showed that specific T-cell reactivity was markedly reduced in the tPA-/- animals, as indicated by decreased T-cell reactivity and reduction in T-regulatory cells. The current findings indicate that tPA plays a role in the pathogenesis of EAE. Moreover, successful amelioration of EAE was achieved by administration of tPA variant proteins. This might mean that these proteins have potential for the immunomodulation of neuroinflammation.

Efficacy of heat-labile enterotoxin B subunit-adjuvanted parenteral porcine epidemic diarrhea virus trimeric spike subunit vaccine in piglets.

Devastating outbreaks of porcine epidemic diarrhea (PED) started in China in late 2010 and rapidly spread to North America and Asia causing severe diarrhea and high mortality in neonatal piglets, indicating that a new generation of vaccine against porcine epidemic diarrhea virus (PEDV) is urgently needed. In the present study, to mimic the native spike (S) glycoprotein, a stable cell line producing the trimeric ectodomain of S glycoprotein of the PEDV Pintung-52 (PEDV-PT) strain was successfully established by incorporating T4 bacteriophage foldon sequence of fibritin trimerization domains at the C-terminal end and replacing the signal peptide of S protein with the tissue plasminogen activator signal peptide sequence at the N terminal end. The trimeric structure, bio-reactivity to PEDV-specific antibodies, and the N-glycosylation level of the recombinant S protein were characterized. To induce systemic and mucosal immunity, conventional 5-week-old piglets were immunized with the trimeric S glycoprotein combined with the B subunit of Escherichia coli heat-labile enterotoxin (LTB) by the intramuscular (IM) route. As compared with the control group, all piglets in the S protein-LTB immunized (IM PEDV S-LTB) group generated systemic PEDV S-specific IgG and neutralizing antibody in blood but a low level of fecal PEDV-specific IgA and limited protection against challenge of PEDV-PT strain. Our results suggest that the recombinant PEDV trimeric S glycoprotein could be a potential subunit vaccine candidate against PEDV, but IM immunization with LTB as the adjuvant provided insufficient protection. The development of a vaccine regimen for inducing mucosal immunity is an important task for generating a successful subunit vaccine against PEDVs.

The plasminogen activation system in neuroinflammation.

The plasminogen activation (PA) system consists in a group of proteases and protease inhibitors regulating the activation of the zymogen plasminogen into its proteolytically active form, plasmin. Here, we give an update of the current knowledge about the role of the PA system on different aspects of neuroinflammation. These include modification in blood-brain barrier integrity, leukocyte diapedesis, removal of fibrin deposits in nervous tissues, microglial activation and neutrophil functions. Furthermore, we focus on the molecular mechanisms (some of them independent of plasmin generation and even of proteolysis) and target receptors responsible for these effects. The description of these mechanisms of action may help designing new therapeutic strategies targeting the expression, activity and molecular mediators of the PA system in neurological disorders involving neuroinflammatory processes. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Antibodies preventing the interaction of tissue-type plasminogen activator with N-methyl-D-aspartate receptors reduce stroke damages and extend the therapeutic window of thrombolysis.

BACKGROUND AND PURPOSE: Tissue-type plasminogen activator (tPA) is the only drug approved for the acute treatment of ischemic stroke but with two faces in the disease: beneficial fibrinolysis in the vasculature and damaging effects on the neurovascular unit and brain parenchyma. To improve this profile, we developed a novel strategy, relying on antibodies targeting the proneurotoxic effects of tPA. METHODS: After production and characterization of antibodies (αATD-NR1) that specifically prevent the interaction of tPA with the ATD-NR1 of N-methyl-d-aspartate receptors, we have evaluated their efficacy in a model of murine thromboembolic stroke with or without recombinant tPA-induced reperfusion, coupled to MRI, near-infrared fluorescence imaging, and behavior assessments. RESULTS: In vitro, αATD-NR1 prevented the proexcitotoxic effect of tPA without altering N-methyl-d-aspartate-induced neurotransmission. In vivo, after a single administration alone or with late recombinant tPA-induced thrombolysis, antibodies dramatically reduced brain injuries and blood-brain barrier leakage, thus improving long-term neurological outcome. CONCLUSIONS: Our strategy limits ischemic damages and extends the therapeutic window of tPA-driven thrombolysis. Thus, the prospect of this immunotherapy is an extension of the range of treatable patients.

Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tPA signal sequence.

Ideal immunogenicity in antigens is a prerequisite to eliciting a sufficiently strong immune and memory response via either DNA or protein vaccines. To improve immunogenicity, efforts have focused on high-level expression of target proteins and on maintaining their natural conformations. In the present work, two trimer motifs (MTQ and MTI) were designed and introduced into a plasmid vector with the tissue plasminogen activator signal peptide (tPA-SP). Next, we examined the efficacy and the efficiency of the two motifs as well as the introduction of tPA-SP and its mutant forms, 22P/A and 22P/G, in facilitating the secretory expression of trimeric proteins in mammalian cells. We found that both trimer motifs could produce the target protein in a trimeric form at a high level. Introduction of tPA-SP 22P/A markedly increased the secretory expression level. The combination of the trimer motif, MTQ, and the signal peptide, 22P/A, may serve as a universal mammalian vector for producing trimeric proteins in vaccine development.

Masked hypertension unfavourably affects haemostasis parameters.

OBJECTIVE. Recent evidence demonstrates that masked hypertension (MH) is a significant predictor of cardiovascular disease. The aim of our study was to examine the impact of MH on haemostasis parameters and to compare the findings to those of healthy normotensives matched for age, sex, body mass index and the rest of risk factors. DESIGN AND METHOD. 130 (60 male, 70 female) healthy subjects mean age 45 ± 12 years who had clinic blood pressure < 140/90 mmHg were studied. The whole study population underwent 24-h ambulatory blood pressure monitoring (ABPM). According to the ABPM recordings, 24 individuals (eight males, 16 females) had MH (daytime systolic blood pressure ≥ 135 mmHg or daytime diastolic blood pressure ≥ 85 mmHg - group A) and the remaining 106 subjects (52 males, 54 females) had normal ABPM recordings - group B. Fibrinogen, thrombomodulin ™, the antigens of plasminogen activator inhibitor 1 (PAI-1Ag) and tissue plasminogen activator (tPA-Ag) were determined in the two groups. Results. The PAI-1 Ag, tPA-Ag, fibrinogen and TM levels were significantly higher in the masked hypertensive group than to normotensive control group. CONCLUSIONS. Our findings suggest that subjects with MH have significantly higher fibrinogen, TM, PAI-1Ag and tPA-Ag plasma levels compared with normotensives. This observation may have prognostic significance for future cardiovascular events in subjects with MH and needs further investigation.

Functional occurrence of the interaction of tissue plasminogen activator with the NR1 Subunit of N-methyl-D-aspartate receptors during stroke.

BACKGROUND AND PURPOSE: Despite its fibrinolytic effect, tissue-type plasminogen activator (tPA) displays deleterious effects in the brain, including proexcitotoxicity, that can reduce the overall benefit from thrombolysis during stroke. We have proposed that tPA potentiates excitotoxicity by interacting with and cleaving the aminoterminal end of the NR1 subunit of N-methyl-d-aspartate receptors, leading to an increased calcium influx, Erk1/2 activation, and neurotoxicity. Because this mechanism is debated, our aim was to demonstrate its in vivo occurrence and relevance. Because tPA is released under ischemic conditions, we hypothesized that if it indeed processes NR1, then the released fragment should reactivate the immune system in animals that had been immunized long before with recombinant aminoterminal end of the NR1. This effect should be exacerbated in ischemic animals thrombolysed with recombinant tPA. METHODS: At a time when specific antibodies could not be detected any longer, mice previously vaccinated with recombinant aminoterminal end of the NR1 were subjected to thromboembolic stroke induced by injecting thrombin in the middle cerebral artery alone or with intravenous thrombolysis. RESULTS: Stroke performed 1 year after active immunization induced the reappearance of antibodies against the aminoterminal end of the NR1 in the plasma, an effect significantly increased when ischemia was followed by recombinant tPA-induced reperfusion. Moreover, immunization preventing the interaction of tPA with aminoterminal end of the NR1 reduced ischemic brain damages and extended the therapeutic window of tPA-induced thrombolysis. CONCLUSIONS: We demonstrate that the tPA-dependent interaction and cleavage of the NR1 subunit of N-methyl-d-aspartate receptors occurs in vivo after stroke and that this interaction is a relevant therapeutic target for stroke treatment.

Anthrax protective antigen administered by DNA vaccination to distinct subcellular locations potentiates humoral and cellular immune responses.

Based on the hypothesis that immune outcome can be influenced by the form of antigen administered and its ability to access various antigen-processing pathways, we targeted the 63 kDa fragment of protective antigen (PA) of Bacillus anthracis to various subcellular locations by DNA chimeras bearing a set of signal sequences. These targeting signals, namely, lysosome-associated membrane protein 1 (LAMP1), tissue plasminogen activator (TPA) and ubiquitin, encoded various forms of PA viz. lysosomal, secreted and cytosolic, respectively. Examination of IgG subclass distribution arising as a result of DNA vaccination indicated a higher IgG1:IgG2a ratio whenever the groups were immunized with chimeras bearing TPA, LAMP1 signals alone or when combined together. Importantly, high end-point titers of IgG antibodies were maintained until 24 wk. It was paralleled by high avidity toxin neutralizing antibodies (TNA) and effective cellular adaptive immunity in the systemic compartment. Anti-PA and TNA titers of approximately 10(5) and approximately 10(3), respectively, provided protection to approximately 90% of vaccinated animals in the group pTPA-PA63-LAMP1. A significant correlation was found between survival percentage and post-challenge anti-PA titers and TNA titers. Overall, immune kinetics pointed that differential processing through various compartments gave rise to qualitative differences in the immune response generated by various chimeras.

The multifaceted role of plasminogen in inflammation.

A fine-tuned activation and deactivation of proteases and their inhibitors are involved in the execution of the inflammatory response. The zymogen/proenzyme plasminogen is converted to the serine protease plasmin, a key fibrinolytic factor by plasminogen activators including tissue-type plasminogen activator (tPA). Plasmin is part of an intricate protease network controlling proteins of initial hemostasis/coagulation, fibrinolytic and complement system. Activation of these protease cascades is required to mount a proper inflammatory response. Although best known for its ability to dissolve clots and cleave fibrin, recent studies point to the importance of fibrin-independent functions of plasmin during acute inflammation and inflammation resolution. In this review, we provide an up-to-date overview of the current knowledge of the enzymatic and cytokine-like effects of tPA and describe the role of tPA and plasminogen receptors in the regulation of the inflammatory response with emphasis on the cytokine storm syndrome such as observed during coronavirus disease 2019 or macrophage activation syndrome. We discuss tPA as a modulator of Toll like receptor signaling, plasmin as an activator of NFkB signaling, and summarize recent studies on the role of plasminogen receptors as controllers of the macrophage conversion into the M2 type and as mediators of efferocytosis during inflammation resolution.

Role of viral receptors TLR3, RIG-I and MDA5 in mesothelial tissue-type plasminogen activator and plasminogen activator inhibitor-1 synthesis.

Tissue-type plasminogen activator (t-PA) and its specific inhibitor plasminogen activator inhibitor-1 (PAI-1) are key mediators in the regulation of fibrin generation and fibrinolysis. Mesothelial cells (MC) line the inner surface of serosal cavities and are a source of procoagulant and fibrinolytic system components. Viral inflammation and infection of MC are a major problem in several organ systems including pleura, pericardium and peritoneum. MC express the viral receptors Toll-like receptor 3 (TLR3), RIG-I and MDA5. TLRs recognise molecular patterns associated with microbial pathogens and induce an immune response. TLR3 recognises dsRNA of viral origin as exemplified by poly (I:C) RNA, a synthetic analogue of viral dsRNA. The helicases RIG-I and MDA5 may also act as sensors of viral infections. Activation of these receptors by poly (I:C) RNA leads to an time- and dose-dependent increase of mesothelial PAI-1 synthesis and a decrease of t-PA expression. To show the specific effect of viral receptors knockdown experiments with siRNA specific for TLR3, RIG-I and MDA5 were performed. This finding of viral induced changes of t-PA and PAI-1 synthesis may indicate a novel link between viral infections and formation of mesothelial fibrin deposits and progression of viral associated disease of the pleura, peritoneum and pericardium.

t-PA Suppresses the Immune Response and Aggravates Neurological Deficit in a Murine Model of Ischemic Stroke.

Introduction: Acute ischemic stroke (AIS) is a potent trigger of immunosuppression, resulting in increased infection risk. While thrombolytic therapy with tissue-type plasminogen activator (t-PA) is still the only pharmacological treatment for AIS, plasmin, the effector protease, has been reported to suppress dendritic cells (DCs), known for their potent antigen-presenting capacity. Accordingly, in the major group of thrombolyzed AIS patients who fail to reanalyze (>60%), t-PA might trigger unintended and potentially harmful immunosuppressive consequences instead of beneficial reperfusion. To test this hypothesis, we performed an exploratory study to investigate the immunomodulatory properties of t-PA treatment in a mouse model of ischemic stroke. Methods: C57Bl/6J wild-type mice and plasminogen-deficient (plg-/-) mice were subjected to middle cerebral artery occlusion (MCAo) for 60 min followed by mouse t-PA treatment (0.9 mg/kg) at reperfusion. Behavioral testing was performed 23 h after occlusion, pursued by determination of blood counts and plasma cytokines at 24 h. Spleens and cervical lymph nodes (cLN) were also harvested and characterized by flow cytometry. Results: MCAo resulted in profound attenuation of immune activation, as anticipated. t-PA treatment not only worsened neurological deficit, but further reduced lymphocyte and monocyte counts in blood, enhanced plasma levels of both IL-10 and TNFα and decreased various conventional DC subsets in the spleen and cLN, consistent with enhanced immunosuppression and systemic inflammation after stroke. Many of these effects were abolished in plg-/- mice, suggesting plasmin as a key mediator of t-PA-induced immunosuppression. Conclusion: t-PA, via plasmin generation, may weaken the immune response post-stroke, potentially enhancing infection risk and impairing neurological recovery. Due to the large number of comparisons performed in this study, additional pre-clinical work is required to confirm these significant possibilities. Future studies will also need to ascertain the functional implications of t-PA-mediated immunosuppression for thrombolyzed AIS patients, particularly for those with failed recanalization.

Molecular and Antigenic Properties of Mammalian Cell-Expressed Theileria parva Antigen Tp9.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, is a leading cause of morbidity and mortality in cattle of sub-Saharan Africa. The infection and treatment method (ITM) is currently the only vaccine available to control T. parva. Although ITM elicits levels of protection, its widespread adoption is limited by costs, laborious production process, and antibiotic co-treatment requirement, necessitating the development of a more sustainable vaccine. To this end, efforts have been concentrated in the identification of new T. parva vaccine antigens and in the development of suitable platforms for antigen expression. In this study, we investigated the molecular and antigenic properties of T. parva antigen Tp9 expressed by mammalian cells. Data indicate that Tp9 contains a signal peptide that is weakly functional in mammalian cells. Thus, Tp9 secretion from mammalian cells increased 10-fold after the native signal peptide was replaced with the human tissue plasminogen activator signal peptide (tPA). Sera from all T. parva-immune cattle recognized this recombinant, secreted Tp9. Additionally, PBMC from ITM-immunized cattle produced significant (p < 0.05) amounts of IFNγ following ex vivo exposure to Tp9, but this response varied between cattle of different MHC class I and class II genotypes. In addition, depletion experiments demonstrated that IFNγ to Tp9 was primarily produced by CD4+ T cells. Molecular analysis demonstrated that Tp9 presents a signal peptide that is weakly functional in mammalian cells, suggesting that it remains within lymphocytes during infection. Tp9 secretion from mammalian cells was substantially increased when the tPA secretion signal sequence was substituted for the native secretion signal sequence. Using full-length, recombinant Tp9 secreted from mammalian cells, we demonstrated that T. parva-immune cattle develop both humoral and cellular immune responses to this antigen. Collectively, these results provide rationale for further evaluation of Tp9 as a component of a T. parva subunit vaccine.

Short-term effects of angiotensin receptor blockers on blood pressure control, and plasma inflammatory and fibrinolytic parameters in patients taking angiotensin-converting enzyme inhibitors.

INTRODUCTION: Angiotensin-converting enzyme (ACE) inhibitors reduce cardiovascular events in patients with established vascular disease and heart failure (HF). ACE-inhibitors have important effects on fibrinolytic balance, which may be the underlying mechanism for a reduction in cardiovascular events. Although angiotensin-receptor blockers (ARBs) offer greater tolerability than ACE-inhibitors, the major ARB trials have demonstrated a lack of reduction in myocardial infarction (MI) occurrence and mortality in contrast to ACE-inhibitors. In this study, we investigated the combined effects of ARBs and ACE-inhibitors on fibrinolytic and inflammatory parameters in patients with uncontrolled hypertension. METHODS: Twenty-four patients with uncontrolled hypertension despite taking adequate doses of ACE-inhibitor therapy were selected. Patients were started on Candesartan 16 mg once a day. Plasma plasminogen activator inhibitor (PAI-1) antigen (Ag), tissue plasminogen activator (t-PA) Ag, thrombin-activatable fibrinolysis inhibitor (TAFI) % activity and high sensitivity C-reactive protein (hsCRP) levels, were measured during low salt intake at baseline and two weeks after therapy with an ARB. RESULTS: Addition of ARB to the regimen reduced systolic (155+/-17 vs. 139+/-13, p<0.001), and diastolic (91+/-9 vs. 81+/-8, p<0.001) blood pressures (BP). No significant changes were observed in PAI-1 Ag (66+/-51 vs. 68+/-52, p=0.9), t-PA Ag (12.6+/-5.3 vs. 13.3+/-4.7, p=0.3), TAFI % activity (119+/-30 vs. 118+/-32, p=0.9) and hsCRP (3.9+/-3.4 vs. 3.6+/-3.6, p=0.7) levels after adding an ARB. CONCLUSIONS: Combined ARB and ACE-inhibitor use provide better BP control without any detrimental effect in plasma inflammatory and fibrinolytic parameters.

Fibrin targeting of echogenic liposomes with inactivated tissue plasminogen activator.

Fibrin-specific molecular targeting strategies are desirable for site-specific imaging and treatment of late stage atheroma, but fibrin-specific antibodies are difficult to produce and present immunogenicity problems. Tissue plasminogen activator (tPA) is an endogenous protein that has been shown to bind fibrin with high affinity and may circumvent antibody difficulties. Use of tPA-derived proteins or peptides, however, requires that the plasminogen-activating proteolytic activity be neutralized or removed. As an initial step in determining the feasibility of this targeting strategy, human recombinant tPA (Activase) was irreversibly inhibited with D-phe-L-pro-L-arg-chloromethyl ketone (PPACK) and conjugated to intrinsically echogenic liposomes (ELIP) by a thioether coupling protocol. Fibrin-binding affinities were assessed with a novel two-stage fibrin pad ELISA. We achieved 95-99% inactivation, while retaining both tPA fibrin-binding activities of K(D) approximately 2 nM and 33 nM. Thermodynamic analysis of the PPACK-inactivated tPA (tPA(P)) revealed highly exothermic interactions, indicative of ionic associations, especially for the higher affinity. The conjugation efficiency of tPA(P) to ELIP was within the range of that previously achieved for IgG and exhibited satisfactory fibrin targeting, characterized by striking increases of enthalpy and entropy increments. Evidence for coupling of noncovalent association energetics with the phosphatidylethanolamine major phase transition, observed in previous IgG antibody conjugations, was also evident in this case, but the nature of the transduction mechanism was different. These results demonstrate that tPA-derived components lacking proteolytic activity can be employed as fibrin-targeting agents for delivery of therapeutic and diagnostic formulations.

Amino-Fucoidan as a Vector for rtPA-Induced Fibrinolysis in Experimental Thrombotic Events.

Acute ischaemic stroke, myocardial infarction and pulmonary embolism are the main causes of mortality and morbidity worldwide. Thrombolysis by intravenous injection of recombinant tissue plasminogen activator (rtPA) remains the most common non-interventional treatment to recanalize occluded vessels. However, this procedure is limited by significant drawbacks, including high doses and bleeding complications. Recent studies showed that fucoidan targets the intraluminal thrombus in vivo. We have developed a chimaera covalently linking fucoidan, able to target platelets within the thrombus, to dilysine, able to non-covalently bind rtPA. We hypothesize that this construct should vectorize rtPA to the thrombus, thus increasing its fibrinolytic efficacy and avoiding its deleterious effects. In vitro, rtPA mixed to dilysine fucoidan (DLF) shows a greater fibrinolytic effect than rtPA alone, both on platelet-rich thrombus and in whole blood. In vivo, occluded mesenteric vessels, carotid artery and vena cava were more efficiently recanalized by DLF complexed to rtPA than by rtPA alone. This study thus provides evidence that DLF may be a promising therapeutic tool to fight against acute thrombosis by enhancing rtPA fibrinolytic efficiency.

Comparing Pathways of Bradykinin Formation in Whole Blood From Healthy Volunteers and Patients With Hereditary Angioedema Due to C1 Inhibitor Deficiency.

Multiple pathways have been proposed to generate bradykinin (BK)-related peptides from blood. We applied various forms of activation to fresh blood obtained from 10 healthy subjects or 10 patients with hereditary angioedema (HAE-1 or -2 only) to investigate kinin formation. An enzyme immunoassay for BK was applied to extracts of citrated blood incubated at 37°C under gentle agitation for 0-2 h in the presence of activators and/or inhibitory agents. Biologically active kinins in extracts were corroborated by c-Fos accumulation in HEK 293a cells that express either recombinant human B2 or B1 receptors (B2R, B1R). Biological evidence of HAE diagnostic and blood cell activation was also obtained. The angiotensin converting enzyme inhibitor enalaprilat, without any effect per se, increased immunoreactive BK (iBK) concentration under active stimulation of blood. Tissue kallikrein (KLK-1) and Kontact-APTT, a particulate material that activates the contact system, rapidly (5 min) and intensely (>100 ng/mL) induced similar iBK generation in the blood of control or HAE subjects. Tissue plasminogen activator (tPA) slowly (≥1 h) induced iBK generation in control blood, but more rapidly and intensely so in that of HAE patients. Effects of biotechnological inhibitors indicate that tPA recruits factor XIIa (FXIIa) and plasma kallikrein to generate iBK. KLK-1, independent of the contact system, is the only stimulus leading to an inconsistent B1R stimulation. Stimulating neutrophils or platelets did not generate iBK. In the HAE patients observed during remission, iBK formation capability coupled to B2R stimulation appears largely intact. However, a selective hypersensitivity to tPA in the blood of HAE patients suggests a role of plasmin-activated FXIIa in the development of attacks. Proposed pathways of kinin formation dependent on blood cell activation were not corroborated.

Induction of a protective response in ducks vaccinated with a DNA vaccine encoding engineered duck circovirus Capsid protein.

Duck circovirus (DuCV) is an immunosuppressive pathogen that causes a huge economic loss in the avian industry. Efficient vaccination has become a necessary strategy for preventing DuCV infection in the breeding industry. Three DNA vaccines encoding the Capsid (Cap) protein of DuCV were developed in this study, which were based on the eukaryotic vector pcDNA3.1 containing (i) the full length of Cap gene, pcDNA3.1-Cap, (ii) the Cap gene with a deletion of its nuclear localization signal (NLS) peptide encoding sequence, pcDNA3.1-CapΔNLS, and (iii) the Cap gene without NLS but harboring a fragment encoding the secretory signal peptide of tissue plasminogen activator (tPA), pcDNA3.1-tPA-CapΔNLS. Production of Cap protein-derived antigens from these three DNA vaccines was confirmed in vitro. The deletion of the NLS coding sequence of the Cap gene changed the subcellular location of the Capsid protein from the nucleus to the cytoplasm. Secretion of the Cap protein was observed in pcDNA3.1-tPA-CapΔNLS-transfected cells. The immunogenicity of these three DNA vaccines was assessed in vivo by measuring Cap-specific antibody and related cytokine levels. The results demonstrated that all these vaccines could induce a significant, specific immune response to protect ducks from DuCV challenge. Notably, higher titers of Cap-specific antibody were produced in ducks vaccinated with pcDNA3.1-tPA-CapΔNLS, which provided the highest protective efficacy at a rate of 90% in the challenge experiment. Taken together, DNA vaccines expressing the DuCV Cap protein show promising immunogenicity, which can be enhanced by replacing the NLS of the Cap protein with a secretory signal peptide of tPA.

Effect of a cholesterol-lowering diet during pregnancy on maternal and fetal Doppler velocimetry: the CARRDIP study.

OBJECTIVE: The purpose of this study was to evaluate the effect of a low-cholesterol low-saturated fat diet on Doppler indices in the fetus and mother. STUDY DESIGN: Two hundred ninety nonsmoking white women, aged 21-38 years, without previous pregnancy complications and carrying a single fetus were assigned randomly to continue their usual diet (control subjects; n = 149) or to adopt a low-cholesterol low-saturated fat diet (intervention group; n = 141) from gestational week 17-20 to birth. Doppler velocimetry of the umbilical artery and both uterine arteries were assessed at gestational weeks 24, 30, and 36. RESULTS: The physiologic gestational decrease in umbilical artery pulsatility index (PI) from week 24-30 was more pronounced in the intervention group, compared with the control group, with median values (interquartile range) of -0.17 (-0.29, -0.06) and -0.11 (-0.25, 0.01), respectively (P = .048). Assignment to the intervention diet did not influence the changes in mean PI value of the 2 uterine arteries (P = .3). The change in umbilical artery PI and mean PI value of the uterine arteries between weeks 24 and 36 were not significantly different between the 2 groups (P = 1.0 and .2 respectively). CONCLUSION: Our study shows that a cholesterol-lowering diet during pregnancy may modify fetoplacental circulation in mid pregnancy.