Rabbit Peripheral Blood Research Articles

Effects of Tissue-Type Plasminogen Site-Specific Transgene in Gelatin-Coated Dacron on the Fibrinolysis Activity of Rabbit Left Atrium.

To investigate the effects of tissue-type plasminogen activator (tPA) gene transfer with left-atrium local positioning on the fibrinolytic activity of rabbit left atrial blood. A total of 48 rabbits were randomly divided into 3 groups (n = 16): gene therapy, vector control, and blank control groups. Each group was divided into 2 subgroups (8 rabbits in each subgroup) according to the sacrifice time on the postoperative 3(rd) and 14(th) days. The tPA mRNA transcriptional level and exogenous tPA protein expression within regional myocardial tissues of the left atrium were detected on the postoperative 3(rd) and 14(th) days. After excluding the animals that died, 6 samples of each subgroup were randomly selected for the statistics (n = 6). The tPA activities in rabbit left atrial blood and peripheral blood were also detected. The tPAmRNA and tPA protein expressions within regional myocardial tissues were detected on the postoperative 3(rd) and 14(th) days. The tPA activity in left atrial blood in the gene therapy group was higher than the tPA activity of other groups (p < 0.02). No significant differences were observed in the tPA activity of peripheral blood among the 3 groups before surgery. A gelatin-coated Dacron piece, which carried the tPA gene, was implanted in the left atrial appendage. The gelatin-coated Dacron piece could express and secrete tPA proteins in the region, thus enhancing the fibrinolytic activity of left atrial blood. Fibrinolytic activity; Gelatin coating; Gene; Left atrium; Tissue-type plasminogen activator.

Compare of different pretreatment methods for early endothelial progenitor cells detection from peripheral blood of rabbit

Objective In order to find out the most convenient,economic,accurate pretreatment methods for early endothelial progenitor cells (EPCs) detection from peripheral blood,this article compared the detection result of density gradient centrifugation method and two kinds of red cells lysis methods.Methods Arterial blood of New Zealand rabbits stored in vacuum heparin containing tubes was pretreated by 3 kinds of method respectively.The first pretreated method used lymphocyte separation liquid density gradient centrifugation,the second method used flow cytometry cell hemolysin and the third method used red cells lysis solution.After that,Anti-Mouse CD3-allophycocyanin (APC),Anti-Mouse CD45-phycoerythrin (PE),Anti-Mouse CD133 (Prominin-1)-fluorescein isothiocyanate (FITC) were labeled to detect early EPCs.Results There was no significant different of percentage of early endothelial progenitor cells pretreated by 3 kinds of method (P ＞ 0.05).Of the three methods,flow cytometry cell hemolysin method was the most convenient method for using the least blood,processing liquid,vitro and Instrument,not easy to be effected and repeating easily.Conclusion Flow cytometry cell hemolysin method was the most convenient pretreatment method for test endothelial progenitor cells percentage from peripheral blood. Key words: Pretreatment ; Early endothelial progenitor cells ; Lysis ; Density gradient centrifugation

187: West Nile virus (WNV) infection alters the cytokines and TLRs transcriptome profiles in rabbit PBL

The peripheral innate immune response to WNV is crucial for control of virus spread to the brain. Therefore, transcriptomes involved in the innate immune response against WNV were investigated in a model organism of rabbit peripheral blood mononuclear leukocytes (PBLs). PBLs were challenged with WNVNSW2011 in vitro and mRNA expressions were quantified at 2 h, 6 h, 12 h and 24 h post challenge (pc) using qRT-PCR. Compared to mock infected PBL, WNV infected PBLs expressed high levels of IFNα at 6 h pc. IFNγ expression increased markedly, peaking at 12 h pc with a 4-fold increase. IL6, 12 & 22 expression peaked at 12 h pc; while CXCL10 and PTX3 expression were higher at 2 h pc and then declined. TLR1, 2, 3, 4, 6 & 10 were up-regulated at 2 h pc with highest expression seen for TLR3, 4 & 6. TLRs-associated downstream genes (MyD88, STAT1, TRAF3, IRF7 & IRF9) were up-regulated between 6 and 24 h pc. Higher expression of TRAF3, STAT1, IRF7 & IRF9 suggest the activation of TLR signaling pathways in order to detect and clear the virus. Higher expression of TNFα, HO1, iNOS, Caspase3 & Caspase9 transcripts suggest the involvement of oxidative stress and apoptosis pathway in WNV infected rabbit PBLs. The expression dynamics of selected genes were validated in PBLs from rabbits experimentally infected with WNV in vivo. Higher expression of IFNα, IFNβ, IFNγ, TNFα, IL6, IL22, PTX3, TLR3 & TLR4, IRF7 & IRF9, STST1, TRAF3, Caspase3 & Caspase9 were found in PBL from WNV infected rabbits compared to PBLs from un-infected control rabbits, and coincided with expression patterns of these genes in vitro. WNVNSW2011 gene expression increased over time in PBLs challenged in vitro but remained undetected in PBLs from in vivo infected and control rabbits. This study highlights the array of cytokines and TLRs involved in the host innate immune response to WNV.

Effects of recombinant human interleukin-10 on Treg cells, IL-10 and TGF-β in transplantation of rabbit skin

The current study aimed to investigate the rejection and survival time of grafted skin, and the changes of Treg cells, interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) in peripheral blood following skin transplantation with recombinant human interleukin-10 (rhIL-10) or cyclosporin A (CsA), as well as the role of IL-10 in immunological rejection mechanisms. A total of 36 rabbits were divided into two groups. The skin of a donor rabbit was transplanted onto the back of one receptor rabbit. Receptors were randomly divided into six groups, including rhIL-10 low-dose (5 μg/kg/d), rhIL-10 high-dose (10 μg/kg/d), CsA low-dose (5 mg/kg/d), CsA high-dose (10 mg/kg/d), rhIL-10 (5 μg/kg/d) and CsA (5 mg/kg/d) and negative control normal saline (NS; 1 ml/d). All groups received intramuscular drug injection for ten days, beginning one day prior to skin transplantation surgery. Following transplantation, each rabbit’s peripheral blood was collected at different times. The changes of CD4+CD25+ regulatory T cells, IL-10 and TGF-β were determined by flow cytometry and enzyme-linked immunosorbent assay. When compared with the control group, the rejection and survival times of the experimental groups were longer following skin graft. Compared with the two CsA groups and the control group, the proportion of CD4+CD25+ regulatory T cells of rhIL-10 groups was significantly upregulated on the 4th and 7th days following surgery. However, TGF-β levels were not significantly different. Data suggested that the concentration of IL-10 was positively correlated with the proportion of CD4+CD25+ regulatory T cells. In addition, IL-10 may delay the rejection time of rabbit skin transplantation and prolong the survival time. Thus, the role of IL-10 in inhibited allograft rejection may be associated with CD4+CD25+ regulatory T cells and IL-10, and may be independent of TGF-β.

Transplantation of human thrombomodulin gene-modified endothelial progenitor cells to prevent vascular restenosis after balloon injury

Objective To explore the feasibility of transplantation of human thrombomodulin (hTM) gene-modified endothelial progenitor cells (EPCs) to rabbit carotid artery balloon injury model locally to prevent vascular restenosis.Methods Rabbit peripheral blood mononuclear cells were isolated and EPCs were modified with hTM gene using X-tremeGENE HP DNA Transfection Reagent.Genetically modified EPCs were incubated with Fe2O3-arginine.Sixteen adult New Zealand white rabbits were divided into two groups.hTM gene-modified EPCs,nom-gene modified EPCs and equal volume of normal saline were transplanted to the balloon injury local via Fogarty balloon catheter,respectively.Serial nuclear magnetic resonance (MRI) was performed on 1st,3rd,7th and 14th day after transplantation to assess the loss of MRI signal intensity at the injured sites.Four months later,the carotid arteries were isolated and cut for hematoxylin and eosin (HE) staining and elastic tissue staining to evaluate restenosis.Results The hTM gene could be successfully transfected into EPCs using Transfection Reagent and the efficiency rate was about 10％-15％.EPCs could be partly adhered to the injured site locally via Fogarty balloon catheter and result in the loss of magnetic resonance (MR) signal intensity at the injured sites.The neointimal/media ratio (N/M) in gene modified group,non-gene modified group and injured group was 0.30 ± 0.02,0.62 ± 0.05 and 1.58 ± 0.10,respectively (P ＜ 0.01).Conclusion hTM gene-modified EPCs could effectively prevent vascular restenosis after balloon injury. Key words: Thrombomodulin; Endothelial progenitor cells; Balloon injury; Restenosis

Verification of multipotential mesenchymal stem cell presence in peripheral blood of rabbits

The aim of this study was to verify the presence of multipotential mesenchymal stem cells in peripheral blood (PBMSC) of rabbits. For mobilization, granulocyte-colony-stimulating factor 30 µg/(kg·d) was injected into New Zealand White rabbits subcutaneously for 6 d, then the PBMSC were isolated from peripheral blood of rabbits by density gradient centrifugation and adhesive culture. The morphology of cell proliferation was observed by microscopy, the proliferative curve of cells was drawn. The phenotypes of PBMSC were detected by flow cytometry, the differential capability of PBMSC into osteocytes, chondrocytes and adipocytes was identified. The results showed that the morphology of subcultured PBMSC were spindle or polygonal shaped, and cell population doubling time was 37.4 h. The isolated PBMSC expressed mesenchymal marker CD29, but not expressed hematopoietic marker CD14. Under specific induction conditions, PBMSC demonstrated multipotency to differentiate into osteocytes, chondrocytes and adipocytes. It is concluded that PBMSC are successfully isolated from peripheral blood and cultured, and their multipotential capability of differentiation into osteocytes, chondrocytes and adipocytes are verified.

High throughput generation of antibody secreting B-cell clones from peripheral blood of rabbits

Event Abstract Back to Event High throughput generation of antibody secreting B-cell clones from peripheral blood of rabbits Sonja Offner1*, Stefan Seeber1, Francesca Ros1, Irmgard Thorey1, Georg Tiefenthaler1, Klaus Kaluza1, Valeria Lifke1, Jens Fischer1, Stefan Klostermann1, Josef Platzer1 and Brigitte Kaluza1 1 Roche Diagnostics GmbH, Germany We have developed a robust high throughput B-cell cultivation method using peripheral rabbit B cells. This rapid procedure generates a diverse set of antibodies using blood of immunized rabbits. The entire workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (v) the recombinant expression and purification of IgG antibodies. Rabbits immunized with the human IL1RL1 antigen (also known as ST2, DER4, FIT-1, IL33R, ST2L, ST2V, T1) were used for the proof-of-concept study. The IL33-IL1RL1 ligand-receptor system plays an important role in autoinflammatory diseases such as asthma, ulcerative colitis or arthritis. The fully integrated and to a large degree automated platform yielded clonal and very diverse IL1RL1-specific antibodies. Due to the high IgG productivity of the cultivated B-cell clones the IL1RL1-inhibiting IgGs from individual animals could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. In summary, this powerful B-cell cultivation method has proven to be an efficient and robust tool for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal. Acknowledgements We would like to express our sincere gratitude and thanks for expert technical assistance to Sabrina Kettenbach and Marco Friedrich (immunization of rabbits), Daniela Buntefuss (preparation of rbTSN), Basile Siewe and Ireneus Jonetzko (B-Cell Cloning), Manuela Paul and Sven Kroemmelbein (screening, biochemical assays), Dominique Ostler, Simone Hoege (B-Cell PCR), Peter Kern (in vitro NK-cell IL1RL1 inhibition assay), and Florian Lipsmeier (statistical analyses). Keywords: B-cell cultivation, antibody production, high throughput, rabbit, Peripheral Blood, B lymphocyte, costimulation, B-cell PCR, IL1RL1, peripheral B cell Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Offner S, Seeber S, Ros F, Thorey I, Tiefenthaler G, Kaluza K, Lifke V, Fischer J, Klostermann S, Platzer J and Kaluza B (2013). High throughput generation of antibody secreting B-cell clones from peripheral blood of rabbits. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01170 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Sonja Offner, Roche Diagnostics GmbH, Penzberg, Germany, sonja.offner@roche.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sonja Offner Stefan Seeber Francesca Ros Irmgard Thorey Georg Tiefenthaler Klaus Kaluza Valeria Lifke Jens Fischer Stefan Klostermann Josef Platzer Brigitte Kaluza Google Sonja Offner Stefan Seeber Francesca Ros Irmgard Thorey Georg Tiefenthaler Klaus Kaluza Valeria Lifke Jens Fischer Stefan Klostermann Josef Platzer Brigitte Kaluza Google Scholar Sonja Offner Stefan Seeber Francesca Ros Irmgard Thorey Georg Tiefenthaler Klaus Kaluza Valeria Lifke Jens Fischer Stefan Klostermann Josef Platzer Brigitte Kaluza PubMed Sonja Offner Stefan Seeber Francesca Ros Irmgard Thorey Georg Tiefenthaler Klaus Kaluza Valeria Lifke Jens Fischer Stefan Klostermann Josef Platzer Brigitte Kaluza Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

An in vitro study of pcDNA 3.0-hVEGF165 gene transfection in endothelial progenitor cells derived from peripheral blood of rabbits

The present study investigated the effect of the VEGF165 gene on adhesion, migration, and proliferation of endothelial progenitor cells (EPCs) derived from peripheral blood of rabbits. Peripheral blood mononuclear cells were isolated from rabbits by density gradient centrifugation with Ficoll-Plaque Plus. EPCs were characterized by immunofluorescence and immunostaining. A pcDNA 3.0-hVEGF165 expression vector was constructed and EPCs were transfected with the pcDNA 3.0-hVEGF165 gene. The EPCs derived from peripheral blood of rabbits were successfully transfected with pcDNA 3.0-hVEGF165. ELISA showed that the expression of VEGF165 increased significantly in the EPCs transfected with the hVEGF165 gene compared to control cells. Compared to control EPCs, EPCs transfected with the hVEGF165 gene had significantly enhanced adhesion, migration, and proliferative ability in vitro.

In vitro labeling of endothelial progenitor cells isolated from peripheral blood with superparamagnetic iron oxide nanoparticles.

The transplantation of endothelial progenitor cells (EPCs) provides a novel method for the treatment of human tumors or vascular diseases. Magnetic resonance imaging (MRI) has proven to be effective in tracking transplanted stem cells by labeling the cells with superparamagnetic iron oxide (SPIO) nanoparticles. The SPIO has been used to label and track the EPCs; however, the effect of SPIO upon EPCs remains unclear on a cellular level. In the present study, EPCs were labeled with home-synthesized SPIO nanoparticles in vitro and the biological characteristics of the labeled EPCs were evaluated. The EPCs were isolated from the peripheral blood of New Zealand rabbits and cultured in fibronectin-coated culture flasks. The EPCs were labeled with home-synthesized SPIO nanoparticles at a final iron concentration of 20 μg/ml. Labeled EPCs were confirmed with transmission electron microscopy and Prussian blue staining. The quantity of iron/cell was detected by atomic absorption spectrometry. The membranous antigens of EPCs were detected by cytofluorimetric analysis. Cell viability and proliferative capability between the labeled and unlabeled EPCs were compared. The rabbit EPCs were effectively labeled and the labeling efficiency was approximately 95%. The SPIO nanoparticles were localized in the endosomal vesicles of the EPCs, which were confirmed by transmission electron microscopy. No significant differences were found in cell viability and proliferative capability between labeled and unlabeled EPCs (P>0.05). In conclusion, rabbit peripheral blood EPCs were effectively labeled by home-synthesized SPIO nanoparticles, without influencing their main biological characteristics.

Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection

Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.

Differential analysis of peripheral blood‐ and bone marrow‐derived endothelial progenitor cells for enhanced vascularization in bone tissue engineering

For tissue engineering applications, effective bone regeneration requires rapid neo-vascularization of implanted grafts to ensure the survival of cells in the early post-implantation phase. Incorporation of autologous endothelial progenitor cells (EPCs) for the promotion of primitive vascular network formation ex vivo has offered great promise for improved graft survival, enhanced rate of vascularization and bone regeneration in vivo. For clinical usage, identification of an optimal EPC isolation source from the patient is critical. We have, for the first time, characterized and directly compared EPCs from rabbit peripheral blood and bone marrow (PB-EPCs and BM-EPCs, respectively). PB-EPCs outperformed BM-EPCs on all measures. PB-EPCs displayed typical endothelial cell markers, such as CD31, as well as high angiogenic potential in three-dimensional extracellular matrix in vitro. Furthermore, PB-EPCs cultured simultaneously with mesenchymal stem cells, displayed significantly enhanced expression levels of key osteogenic and vascular markers, including alkaline phosphatase, bone morphogenetic protein 2, and vascular endothelial growth factor. On the contrary, putative BM-EPCs did not express CD31, and instead, expressed key smooth muscle markers. BM-EPCs further failed to display vasculogenic activity. Hence, the highly angiogenic PB-derived EPCs may serve as an ideal cell population for enhanced vascularization and success of engineered bone tissue.

Effect of radiofrequency ablation on T lymphocyte subsets in peripheral blood of rabbits with renal VX-2 carcinoma

Objective To study the effect of radiofrequency ablation (RFA) on T lymphocyte subsets in peripheral blood of rabbits with renal VX-2 carcinoma.Methods Thirty New Zealand rabbits with VX-2 renal carcinoma were equally randomized into 3 groups.RFA and radical nephrectomy were applied in groups A and B respectively.In group C ( sham operation),animals were subjected to laparotomy and abdominal closure without any intervention.The cytokine profiles of T lymphocytes in peripheral blood such as CD3 + T,CD4 + T,CD8 + T cells were examined by flow cytometry before and 28 days after operation.Results After RFA treatment,the proportion of CD3 + T,CD4 + T and the ratio of CD4 +/CD8 + were significantly increased ( P ＜ 0.05 ) and CD8 + T significantly decreased in group A ( P ＜ 0.05 ) as compared with group B.In group B and group C,there was no significant difference before and after operation.However,there was significant difference in T lymphocyte subsets between groups B and C after operation.Conclusion RFA can result in remarkable changes of T-lymphocyte subsets in peripheral blood,which may also improve the anti-tumor cellular immunity. Key words: Radiofrequency ablation; Renal carcinoma; T-lymphocyte

Multimodality Imaging of Endothelial Progenitor Cells with a Novel Multifunctional Probe Featuring Positive Magnetic Resonance Contrast and Near-Infrared Fluorescence

The multimodal strategy incorporating T1-weighted magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence imaging can complement their strengths to provide images with high sensitivity and spatial resolution for noninvasively and dynamically monitoring endothelial progenitor cells (EPCs) in potential EPC-dominated therapies. Here we report the development of a protein-based imaging probe, bCD-PLL-Cy5.5 Conjugate 1, in which the bacterial cytosine deaminase (bCD) protein was modified with poly-l-lysine (PLL) that is labeled with imaging reporters, including T1-weighted MRI contrast chelator and NIR fluorophore. Conjugate 1 showed low cytotoxicity in EPCs isolated from the rabbit peripheral blood. The normalized cell viability was maintained above 90% after incubation for 1 to 5 days. Fluorescence microscopy of live cells indicated rapid cellular uptake of Conjugate 1 into EPCs in 15 minutes, and flow cytometry studies demonstrated the time-dependent internalization of Conjugate 1 with maximum uptake 48 hours after the treatment. MRI of phantoms demonstrated significant reduction of the T1 value of the EPC pellet that was pretreated with 2 μM of Conjugate 1 for 24 hours. Our preliminary data suggest that as a multimodal imaging contrast medium, Conjugate 1 offers a promising imaging probe for tracking the delivery and therapeutic response of EPCs in vivo.

The change and significance of CD4 + CD25 + regulatory T cells in peripheral blood in rabbit' s lung VX2 tumor resection model

Objective To analyze the change of CD4+ CD25 + regulatory T cells in peripheral blood in rabbit' s lung VX2 tumor resection model. Methods Thirty New Zealand rabbits were divided into 3 groups: group A, 10 rabbits with lung VX2 tumor receiving lobectomy; group B, 10 rabbits with lung VX2 tumor receiving no operation; group C, 10 rabbits without lung VX2 tumor receiving lobectomy only.The peripheral blood of rabbits was drawn. All the samples were analyzed by flow cytometry (FCM). Results The proportion of the CD4 + CD25 + regulatory T cells in peripheral blood of the rabbits was increased after the implantion of VX2 tumor ( P ＜ 0. 01 ), that in the rabbits with VX2 lung tumor receiving tumorectomy was obviously decreased at the 15th and 30th day after the operation (P ＜ 0. 05 ), but that in the rabbits with VX2 lung tumor receiving no operation did not have the same change ( P ＜ 0. 05 ). Conclusion The increase of the proportion of CD4 + CD25 + regulatory T cells in peripheral blood is related to immunosuppression. The functional disorder of immune system recovers in a certain extent after resection of the tumor. Key words: Lung tumor; Regulatory T cells; Operation; Flow cytometer

Strategic Approaches for Developing Alternative Tests for Safety and Potency of Vaccines

Safety control tests for vaccines need to be capable of quantifying in vivo toxicity of vaccines. Endotoxin contamination is a threat for vaccine safety and has traditionally been controlled using either an in vivo rabbit pyrogen test or the in vitro Limulus amebocyte lysate (LAL) assay. An in vitro pyrogen test was developed using prostaglandin E2 induction in rabbit peripheral blood. Responsiveness of LAL and rabbits to various endotoxins, however, differ from that of humans. A monocyte activation test was developed using human monocytes but is not applicable where the use of donated blood is limited to therapeutic purposes. Therefore, a clinically relevant in vitro pyrogen test was developed using a human monocytic cell line responsive to various pyrogens in a manner consistent with that of human peripheral blood. Residual pertussis toxin (PT) activity of acellular pertussis vaccines (aPs) is quantified by the mouse histamine sensitization (HS) test. The Chinese Hamster Ovary Cell clustering test can sensitively detect PT activity but is not adequate for testing aPs due to hampered interaction of aggregated PT molecules with cell surface receptors. An in vitro test system has been developed to estimate HS activity using the combined assay results of carbohydrate binding ELISA (B-subunit) and enzymatic-HPLC (A-subunit). Potency tests need to evaluate protective immunogenicity of vaccines. Protective potency of whole cell pertussis vaccine can be measured by the method whose results were shown to correlate with clinical protection. Identifying and quantifying the structures of antigen molecules relevant to the induction of protective immunity may be the ultimate goal.

Transfection of Endothelial Nitric Oxide Synthase Gene Improves Angiogenic Efficacy of Endothelial Progenitor Cells in Rabbits with Hindlimb Ischemia

Background: The present study explored the effect of endothelial nitric oxide synthase (eNOS) gene transfer on the angiogenic potential of ex vivo expanded endothelial progenitor cells (EPCs) in a rabbit model of hindlimb ischemia. Methods: Rabbit peripheral blood EPCs were cultured and transfected with mammalian expression vector pcDNA3.1-eNOS containing full-length human eNOS gene. Ischemia was induced in the right hind limb of three groups of rabbits by ligation of the distal external iliac artery and excision of the common and superficial femoral arteries. In one group of animals, ten days after the surgery, autologous eNOS-EPCs were transplanted intramuscularly in the ischemic limb. Two other groups received an equivalent number of unmodified EPCs or phosphate buffered saline (PBS) respectively. Results: Two weeks after cell transplantation, the in vivo expression of eNOS was detected in limb tissue sections of eNOS-EPCs treated animals. Animals treated with eNOS-EPCs had a significant reduction in ischemic muscle necrosis and inflammation, augmentation in the capillary density (P< 0.05) and angiographic scores demonstrating distal arterial reconstitution and enhanced angiogenesis in comparison to animals transplanted with EPCs or PBS (P< 0.05). Conclusion: We conclude that modification of EPCs by eNOS constitutes an effective strategy to improve the efficacy of EPCs for therapeutic angiogenesis.

Molecular cloning, in vitro expression and bioactivity of rabbit transforming growth factor-beta receptor type II (rTGF-βRII)

Transforming growth factor-beta receptor II (TGF-βRII) is an attractive target for anti-scarring therapy in wound healing because it attenuates excessive TGF-β which has pleiotropic effects on the immune system. In the present study, the cDNA of rabbit TGF-βRII (rTGF-βRII) was amplified from rabbit peripheral blood by RT-PCR. The open reading frame of rTGF-βRII encodes a protein consisting of 567 amino acids, which contains a predicted transmembrane domain and a Serine/Threonine protein kinase domain similar to other identified mammalian TGF-βRIIs. The amino acid sequences of the biologically active, soluble rTGF-βRII and mouse, rat, human and chicken counterparts are 81%, 81%, 89% and 61%, respectively, identical. Recombinant soluble rTGF-βRII (rsTGF-βRII) fused with His tag was efficiently expressed in Escherichia coli BL21 (DE3) expression host strain. This fusion protein's molecular weight of ∼19 kDa was identified by SDS-PAGE and Western blotting. In vitro, purified rsTGF-βRII was able to inhibit the proliferation of keloid rabbit fibroblasts and decrease the level of collagen. These findings indicate that rTGF-βRII plays an important role in inhibiting the proliferation of keloid rabbit fibroblasts and provides the basis for investigations on the role of TGF-βRII in this important domestic species.

HCl-induced inflammatory mediators in esophageal mucosa increase migration and production of H2O2by peripheral blood leukocytes

Exposure of esophageal mucosa to hydrochloric acid (HCl) is a crucial factor in the pathogenesis of reflux disease. We examined supernatant of HCl-exposed rabbit mucosa for inflammatory mediators enhancing migration of leukocytes and production of H(2)O(2) as an indicator of leukocyte activation. A tubular segment of rabbit esophageal mucosa was tied at both ends to form a sac, which was filled with HCl-acidified Krebs buffer at pH 5 (or plain Krebs buffer as control) and kept oxygenated at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Rabbit peripheral blood leukocytes (PBL) were isolated, and sac supernatant was used to investigate PBL migration and H(2)O(2) production. HCl-exposed esophageal mucosa released substance P (SP), CGRP, platelet-activating factor (PAF), and IL-8 into the supernatant. PBL migration increased in response to IL-8 or to supernatant of the HCl-filled mucosal sac. Supernatant-induced PBL migration was inhibited by IL-8 antibodies and by antagonists for PAF (CV3988) or neurokinin 1 (i.e., SP), but not by a CGRP antagonist. Supernatant of the HCl-filled mucosal sac increased H(2)O(2) release by PBL that was significantly reduced by CV3988 and by a SP antagonist but was not affected by IL-8 antibodies or by a CGRP antagonist. We conclude that IL-8, PAF, and SP are important inflammatory mediators released by esophageal mucosa in response to acid that promote PBL migration. In addition, PAF and SP induce production of H(2)O(2) by PBL. These findings provide a direct link between acid exposure and recruitment and activation of immune cells in esophageal mucosa.

Analysis of rabbit cell surface (CD) antigens by means of cross-reactive monoclonal antibodies with specificity for cattle CD antigens

Studies that involved testing monoclonal antibodies (mAbs) for cross-species reactivity proved to be efficient for the identification of previously unrecognized antigens in a number of different species. Twenty-six mAbs specific to different bovine CD (cluster defined) antigens (CD9, CD18, CD45R, CD41/61, CD62L, MHC class I and bovine IgG light chain molecule) were assayed for reactivity with rabbit peripheral blood leukocytes. Four of the mAbs recognizing CD9 and CD41/61 were reactive with rabbit platelets or granulocytes. These were investigated further by immunoblotting and immunohistochemical staining. The study identified CD9 and CD41/61 molecules on rabbit cells by mAbs IVA-50 and IVA-38. It showed that IVA-50 is a new valuable CD9 reagent for rabbit immunology which could be used for immunofluorescence staining or ELISA assay, immunohistological and molecular studies of rabbit CD9 antigen. IVA-38 recognizes the CD41/61 on rabbit platelets in indirect immunofluorescence and ELISA assay.

Growth of outgrowth endothelial cells on aligned PLLA nanofibrous scaffolds

Tissue engineering holds great promise in providing vascular grafts as substitutes for damaged small-diameter blood vessels. Two of the key factors in vascular tissue engineering are biocompatible scaffolds that mimic the effects of extracellular matrix and the source of seeding cells. Synthetic poly-L-lactic acid (PLLA) nanofibers has been shown to be excellent scaffolds for tissue engineering. Outgrowth endothelial cells (OECs) isolated from human peripheral blood could also be expanded in vitro and stably maintain the differentiated phenotypes and could be used as the seeding cells for engineering autologous vascular crafts. Here we tested the possibility of combining these two together. We found that PLLA nanofibers are not only biocompatible with OECs originally isolated from rabbit peripheral blood, the aligned PLLA fibers actually promoted and guided their sustained proliferation. These results suggest that aligned PLLA could be excellent both as the scaffolds and as a promoter of cell growth during vascular tissue engineering.