Novel assay for the detection of CRP protein in rabbit leukocytes using flow cytometry

C-reactive protein (CRP) is an acute phase serum protein secreted by liver hepatocytes. Besides its presence in the serum, CRP was also found on the surface of human leukocytes. However, the binding ability of CRP to rabbit leukocytes has never been previously investigated. The objective of our study was to optimize the detection of rabbit CRP binding leukocytes in order to observe the acute phase of immune response by flow cytometry after Complete Freund´s Adjuvant (CFA) administration. Blood samples were analysed using ELISA assay and flow cytometry immediately before and 2 days after the CFA administration. Significant (P<0.01) increase in the proportion of CRP+ leukocytes (up to 10%) and in their subsets (lymphocytes up to 14% and granulocytes up to 8%) were observed in samples after CFA immunization. ELISA also revealed significantly (P<0.01) higher CRP concentration in rabbit blood plasma after CFA immunization (about 3 mg/L) compared to control samples (about 1.5 mg/L) before immunization. In conclusion, the increase in level of CRP protein during the immune response in rabbits could be measured beside the ELISA also using flow cytometry via CRP binding leukocytes. This novel assay could be therefore successfully applied in further biomedical and veterinary research

Different MACS sorting strategies for the enrichment of Lin– (CD34+ CD45–) hematopoietic progenitor cells: preliminary study

Magnetic-activated cell sorting (MACS) has become a standard method for the isolation of hematopoietic stem/progenitor cells (HSC/HPC) in human or mouse model using CD34 antibodies. However, at the present there is no useable CD34 antibody that could be successfully used for the selection of rabbit HSC/HPC. Therefore, the aim of this preliminary study was to remove all mature cells (CD45+) from the heterogeneous mixture of rabbit peripheral blood and bone marrow mononuclear cells (PBMCs and BMMCs) in order to enrich these cell populations for the CD34+ cells. Briefly, cells were incubated with a CD45 antibody and proper magnetic microbeads. Three different MACS sorting strategies were used in the experiment that differed mainly in the sample loading rate and the number of used magnetic columns. Control (unsorted) and sorted cells were assessed for the sorting efficiency (% of double positive cells for CD45 and Labelling Check Reagent - LCR) by flow cytometry and for the relative expression of CD34 antigen by qPCR. According to flow cytometry, Depl025 mode showed the best sorting efficiency in terms of the lowest percentages of CD45+LCR+ cells for rabbit PBMCs as well as BMMCs. qPCR analysis confirmed this mode as the best in terms of the relative CD34 expression for rabbit PBMCs. However, higher relative expression of CD34 in BMMCs was obtained by other mode - Posselds. In conclusion, this study demonstrates a possible enrichment of rabbit (CD34+) HSC/HPC by the magnetic depletion of mature hematopoietic (CD45+) cells

Effect of GnRH (Lecirelinum) on some quality parameters of rabbit ejaculate

The aim of this study was to evaluate the effect of two concentrations of GnRH in insemination doses on selected quality parameters of rabbits ejaculate in vitro. Insemination doses (ID) were diluted to a concentration of 50 x 106 spermatozoa in ID (0.5 ml). Subsequently ID was divided into 3 samples (control - C, experiment 1, experiment 2). Implementor GnRH (Lecirelinum – commercial product Supergestran, Ferring Pharmaceuticals, the Czech Republic) was added to experimental insemination dose samples at concentrations as follows: experiment 1 to 0.2 ml (5 mg) GnRH / ID and experiment 2 to 0.3 ml (7.5 mg) GnRH / ID. Experimental samples were compared with the control sample. For the assessment of spermatozoa motility the CASA (Computer-Assistend Sperm Analysis) system SpermVision (MiniTüb, Tiefenbach, FRG) with a microscope Olympus BX 51 (Olympus, Japan) was used. Monitored spermatozoa parameters were motility (%), progressive motility (%), velocity (μm/s), curvilinear velocity of motility (μm/s) and beat cross frequency. In experimental samples (experiment 1, 2) increase of the spermatozoa motility values was detected in time periods of 1 and 3 hours (1 hour – C: 47.30 ± 7.99%, experiment 1: 86.39 ± 5.60%, experiment 2: 72.48 ± 3.80%, 3 hours – C: 57.09 ± 23.36%, experiment 1: 89.42 ± 2.41%, experiment 2: 63.92 ± 12.65%) and decrease over a period of 6 hours (C: 64.65 ± 8.60%, experiment 1: 35.26 ± 5.22%, experiment 2: 50.08 ± 8.27%). Progressive spermatozoa motility within time periods of 1 and 3 hours showed a similar trend as spermatozoa motility (1 hour – C: 30.50 ± 7.35%, experiment 1: 79.18 ± 6.58%, experiment 2: 59.85 ± 6.03%; 3 hours – C: 42.06 ± 22.69%, experiment 1: 82.31 ± 3.64%, experiment 2: 44.45 ± 12.01%) and decreased over a period of 6 hours (C: 56.34 ± 8.88%, experiment 1: 23.36 ± 5.95%, experiment 2: 39.07 ± 11.17%). Spermatozoa curvilinear velocity in experiment 1 reached after 1 hour 82.26 ± 4.47 μm/s, after 3 hours 68.40 ± 3.20 μm/s, after 6 hours 58.21 ± 3.89 μm/s; in experiment 2 was after 1 hour 62.00 ± 4.33 μm/s, after 3 hours 44.37 ± 9.19 μm/s and after 6 hours 52.73 ± 9.10 μm/s, in control group after 1 hour 71.86 ± 8.19 μm/s, after 3 hours 62.35 ± 7.89 μm/s and after 6 hours 73.93 ± 8.18 μm/s. Lower concentration of the implementor (1 to 0.2 ml GnRH / ID) ​​increased level of motility, progressive motility, velocity and curvilinear velocity of motility in the time period 1 and 3 hours after GnRH implementor application compared with the control sample. In 6 hours after application only lower changes of monitored parameters has occurred. The effect of GnRH under in vivo conditions may vary significantly comparing with results obtained in vivo

Secretome Analysis of Rabbit and Human Mesenchymal Stem and Endothelial Progenitor Cells: A Comparative Study

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) have been studied several years for their immunomodulatory effect through the paracrine mechanism and cytokine secretion. In combination with endothelial progenitor cells (EPCs), MSCs have great therapeutical potential for the repair of endothelium and wound healing. However, little is known about the cytokine profile of rabbit AT-MSCs or even EPCs. The aim of this study was to analyze the secretomes of these rabbit stem/progenitor cells. A large-scale human cytokine array (up to 80 cytokines) was used to identify and compare cytokines secreted into conditioned media of human and rabbit AT-MSCs as well as HUVECs and rabbit EPCs. Few cytokines were highly expressed by human AT-MSCs (TIMP-2, TIMP-1), HUVECs (MCP-1, TIMP-2, GRO, Angiogenin, IL-8, TIMP-1), or by rabbit EPCs (TIMP-2). Several cytokines have moderate expression by human (MCP-1, GRO, Angiogenin, TGF-&beta; 2, IL-8, LIF, IL-6, Osteopontin, Osteoprotegerin) and rabbit AT-MSCs (TIMP-2, TGF-&beta; 2, LIF, Osteopontin, IL-8, IL-5, IL-3) or by HUVECs (IL-6, MIF, TGF-&beta; 2, GCP-2, IGFBP-2, Osteoprotegerin, EGF, LIF, PDGF-BB, MCP-3, Osteopontin, Leptin, IL-5, ENA-78, TNF-&beta;) and rabbit EPCs (TGF-&beta; 2, Osteopontin, GRO, LIF, IL-8, IL-5, IL-3). In conclusion, the proposed method seems to be useful for the secretome analysis of rabbit stem/progenitor cells

Enrichment of Rabbit Primitive Hematopoietic Cells via MACS Depletion of CD45+ Bone Marrow Cells

Hematopoietic stem and progenitor cells (HSC/HPCs) of human or few animal species have been studied for over 30 years. However, there is no information about rabbit HSC/HPCs, although they might be a valuable animal model for studying human hematopoietic disorders or could serve as genetic resource for the preservation of animal biodiversity. CD34 marker is commonly used to isolate HSC/HPCs. Due to unavailability of specific anti-rabbit CD34 antibodies, a novel strategy for the isolation and enrichment of rabbit HSC/HPCs was used in this study. Briefly, rabbit bone marrow mononuclear cells (BMMCs) were sorted immunomagnetically in order to remove all mature (CD45+) cells. The cells were depleted with overall purity about 60&ndash;70% and then cultured in a special medium designed for the expansion of CD34+ cells. Quantitative Polymerase Chain Reaction (qPCR) analysis confirmed the enrichment of primitive hematopoietic cells, as the expression of CD34 and CD49f increased (p &lt; 0.05) and CD45 decreased (p &lt; 0.001) at the end of culture in comparison to fresh BMMCs. However, cell culture still exhibited the presence of CD45+ cells, as identified by flow cytometry. After gating on CD45&minus; cells the MHCI+MHCII&minus;CD38+CD49f+CD90&minus;CD117&minus; phenotype was observed. In conclusion, rabbit HSC/HPCs might be isolated and enriched by the presented method. However, further optimization is still required