Rabbit Serum Research Articles

Evidence of infection with Toxoplasma gondii and Neospora caninum in South Australia: using wild rabbits as a sentinel species.

The aim of this study was to utilise wild rabbits (Oryctolagus cuniculus) as a sentinel species to study levels of environmental contamination with N. caninum and T. gondii in South Australia, and to examine associations with rainfall, climate and land use. Toxoplasma gondii (T. gondii), an apicomplexan parasite, causes the clinical disease toxoplasmosis, which can affect sheep as well as humans and many other animals. Neosporosis, the clinical disease caused by closely related Neospora caninum (N. caninum), causes abortions in cattle, with large economic impacts to cattle industries. Aliquots of wild rabbit (Oryctolagus cuniculus) serum were obtained from twelve sites across South Australia over a period of eighteen years, with a total of 2114 samples. An in-house Modified Agglutination Test (MAT) was developed, and samples were screened for the specific antibodies against both T.gondii and N. caninum. Overall, 9.9% of samples screened for T. gondii tested positive and 6.1% of samples screened for N. caninum tested positive. There was no difference observed in seroprevalence of T.gondii specific antibodies between samples collected throughout summer, autumn, winter or spring. By contrast, a significantly higher (p=0.030) seroprevalence of N. caninum specific antibodies was observed in spring than any other season. T. gondii and N. caninum antibodies were discovered at sites across a broad area of South Australia, indicating these environments maybe infected with both parasites. Results provide evidence that suggests N. caninum oocysts may have different survival characteristics, such as varying tolerances to low relative humidity, than T. gondii oocysts.

Fluorescent Determination of Mercury (II) and Glutathione Using Amino-MIL-53(Al) Nanosheets

Amino-MIL-53(Al) nanosheets were synthesized by a hydrothermal reaction and were employed as a fluorescent probe because of their high quantum yield and excellent stability. The amino-MIL-53(Al) nanosheets have a maximum fluorescence emission peak at 435 nm using an excitation wavelength of 335 nm. The fluorescent intensity of luminophor at 435 nm was linearly quenched when mercury (II) (Hg2+) was added, which occurred due to the adsorption of Hg2+ onto the luminophor surface. Under these conditions, the fluorescent signal of the system is in the “turn-off” state. Following the addition of glutathione (GSH) into the system, the quenched fluorescence intensity may be partially restored based on the reaction between glutathione and Hg2+. In this condition, the fluorescent signal of the amino-MIL-53(Al)-Hg2+ system is in the “turn-on” state. Therefore, the amino-MIL-53(Al) nanosheets were successfully used as a “turn-off-on” fluorescent sensing system to determine the concentrations of Hg2+ in Tibetan medicine and glutathione in rabbit serum. A good correlation coefficient was obtained between the quenching of the fluorescence intensity of luminophor and the concentration of Hg2+ using the optimized conditions. A linear range was acquired from 1.96 nM to 38.27 μM with a detection limit of 0.23 nM of Hg2+ based upon a signal-to-noise ratio of three. For glutathione, the linear dynamic range was from 210 nM to 15.25 μM with a detection limit equal to 8.11 nM based upon a signal-to-noise ratio of three. This developed sensor system has the advantages of high selectivity, favorable sensitivity and simple operation for the determination of Hg2+ and glutathione in real samples.

A novel Thioredoxin-related protein 14 from Fasciola gigantica has an immunodiagnostic potential for fasciolosis

In the definitive host, a trematode parasite can survive and evade the damage by reactive oxygen species that are generated from its metabolism and the host immune cells. Several anti-oxidant proteins are found in Fasciola spp. which play essential roles in cellular redox balance. One of them is thioredoxin-related protein 14 (TRP14) that has a highly conserved WCPDC motif and serves as a disulfide reductase-like thioredoxin (Trx). In the present study, a cDNA encoding TRP14 from F. gigantica (FgTRP14) was selected and cloned by immunoscreening with a rabbit infected serum. Phylogenetic analysis was performed by MEGA X program showed that FgTRP14 was most highly related to the Fasciola hepatica. Immunoblotting analysis of the polyclonal antibody rabbit serum against recombinant FgTRP14 (rFgTRP14) revealed that the molecular weight of natural FgTRP14 was at 14 kDa from metacercariae, NEJ, 4-week old juvenile and adult stage. The native FgTRP14 was expressed in caecal epithelial cells and preferentially localized on the cells’ surface lamellae of adult stage. By sandwich ELISA assay, the circulating FgTRP14 could be recognized in sera of experimentally F. gigantica metacercariae infection in mice. The native FgTRP14 in the excretory–secretory (ES) and whole body (WB) of adult F. gigantica were detected at the concentrations 6.3 ng/ml, and 45 ng/ml, respectively. Therefore, it could be considered for immunodiagnostic candidate for fasciolosis.

Anesthesia of laboratory animals in manufacturing of diagnostic and preventive biomedicines

Preparations such as XilaVet, Zoletil 100 as well as Aeranne (Isoflurane) are successfully applied for animal anesthesia in veterinary practice. We assessed a possibility of using parenteral narcosis with Zoletil 100 in combination with muscle relaxant Xila for producer-rabbits involved in manufacturing of natural rabbit serum subsequently deployed for production of diagnostic serum and immunoglobulin preparations. Administration of preparations into auricular vein is easy to do, while animals are sedated immediately allowing for safe fixation on restraining table and causing no additional stress for biomodels. This type of narcosis provides for expected depth of anesthesia and its maintenance until the end of blood-letting procedure. Parameters characterizing the state of cardiovascular system due to anesthetic products remained within the permitted limits. These preparations do not reduce heart beat rate allowing for collecting sufficient blood volumes. Application of inhalation anesthesia with Aeranne in laboratory animals provides for the specified depth of anesthesia and its maintenance until the end of the whole procedure. However, it requires specialized equipment and highly trained personnel with appropriate skills. Usage of Xila as a mono narcosis is not recommended as exhibits weak analgesic effects and strong hypotensive activity by decreasing quantity of collected blood volume. It was found that anesthetics such as Xila, Zoletil 100, Aeranne did not affect specific activity of immune sera in case of total dehematizing procedure. Moreover, antibody titers were not declined throughout entire observation (12 months) period and complied with the requirements of regulatory documentation. In addition, a feasibility of replacing old-fashioned anesthesia method with diethyl ether for a combination of safer contemporary preparations of Zoletil 100 and Xila was demonstrated while manufacturing tableted chemical cholera vaccine in experimental series with suckling rabbits used at diverse stages of raw material verification during surgical interventions. Xila, Zoletil 100, and Aeranne examined by us had no impact on the amount of blood obtained from donor-animals, immunological properties of the sera and ready-to-use diagnostic preparations. Such drugs were safe for all-age animals that comply with the requirements to anesthesia of animal biomodels and producer-animals in manufacturing of immunobiological preparations. Thus, our study allowed to conduct experiments with laboratory animals in a more humane manner.

Prokaryotic expression, purification and identification of the N protein of middle east respiratory syndrome coronavirus

Objective To express the N protein of middle east respiratory syndrome coronavirus （ MERS-CoV） in prokaryotic expression system and evaluate the immunogenicity of the purified recombinant N protein.Methods The gene encoding N protein of MERS-CoV was synthesized and cloned into the vector pET30a to construct the recombinant expression plasmid pET30a-MERS-CoV-N.The transformed E.coli BL21 （ DE3） strains carrying expression plasmid were induced by IPTG to express N protein.The expressed protein was purified by using affinity chromatography.SDS-PAGE and Western blot assays were used to iden-tify the expressed N protein and evaluate its immunogenicity.Results The recombinant N protein was suc-cessfully expressed in soluble form with the size of 46×103 .The results of Western blot assay showed that the recombinant N protein could specifically react with rabbit serum samples positive for antibodies against N protein B-cell epitope peptide and mouse anti-His tag antibodies.No positive band against N protein was found when primary antibody was used with thirty adult serum samples from Beijing in 2008.Conclusion N protein of MERS-CoV was successfully expressed in prokaryotic expression system.The successful expres-sion of N protein laid the foundation for immunological diagnosis of N protein of MERS-CoV and researches on its immunogenicity. Key words: Middle east respiratory syndrome coronavirus; N protein; Prokaryotic expression; Purification ; Identification

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type oppF1 influences its growth characteristics

ABSTRACTThe Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF1 (oligopeptide permease transporter) which results in expression of a truncated OppF1. The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF1 gene in the MS-H genome. Wild-type oppF1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF1-tetM or with a similar vector lacking oppF1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF1-tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF1 detected bands of expected size for full-length OppF1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF1-tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF1-tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF1-tetM (passage 27) showed that the chromosomal copy of the mutated oppF1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species.RESEARCH HIGHLIGHTSThe full-length OppF1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF1 in MS-H was replaced with wild-type oppF1.

Pathogenetic features of formation of experimental preeclampsia-like condition

Objective. To study the pathogenesis of clinical and laboratory syndromes of preeclampsia-like condition and the possibility of influencing them through the introduction of specific antibodies to marinobufagenin.
 Materials and methods. 36 pregnant rats were used in the experiment. 24 rats from 12 to 19 days of pregnancy were prescribed a 1,8% NaCl solution to simulate a preeclampsia-like condition. Subsequently, this sample was divided into 2 groups. In one group (12 animals, the second group), preimmune rabbit serum was administered intraperitoneally as an immune agent. In another group (12 animals, the third group), a single intraperitoneal administration of polyclonal anti-marinobufagenin antibodies was performed. After the antibody injection, a comprehensive clinical and laboratory evaluation was performed.
 Results. Against the background of a hypersol diet, an increase in systolic blood pressure was found, as well as the concentration of marinobufagenin in blood plasma. The introduction of antibodies to marinobufagenin caused a decrease in blood pressure. In addition, the formation of a preeclampsia-like condition showed an increase in endoglin and sFlt1 in the placenta, as well as an increase in sFlt1, collagen-1 and a decrease in Fli-1 in the thoracic aorta. The appointment of antibodies to marinobufagenin led to an increase in the content of Fli-1 to the level in the control group.
 Summary. In the formation of a preeclampsia-like condition, an increase in the level of marinobufagenin in blood plasma leads to an increase in the content of antiangiogenic factors in the placenta and in the thoracic aorta. Intraperitoneal administration of antibodies to marinobufagenin contributes to a significant increase in the content of Fli-1.

A Mechanistic and Translational Pharmacokinetic-Pharmacodynamic Model of Abicipar Pegol and Vascular Endothelial Growth Factor Inhibition.

Abicipar pegol (abicipar) is a novel DARPin therapeutic and highly potent vascular endothelial growth factor (VEGF) inhibitor intended for the treatment of neovascular age-related macular degeneration (nAMD). Here we develop a translational pharmacokinetic/pharmacodynamic (PK/PD) model for abicipar to guide dosing regimens in the clinic. The model incorporated abicipar-VEGF binding kinetics, VEGF expression levels, and VEGF turnover rates to describe the ocular and systemic PK data collected from the vitreous, aqueous humor (AH), choroid, retina, and serum of rabbits after a 1-mg abicipar intravitreal (IVT) dose. The model was translated to humans using human-specific mechanistic parameters and refitted to human serum and AH concentrations from patients with diabetic macular edema and nAMD. The model was then used to simulate 8-, 12- (quarterly), and 16-week dosing intervals in the clinic. Simulations of 2 mg abicipar IVT at 8-week or quarterly dosing in humans indicates minimum steady-state vitreal concentrations are maintained above both in vitro IC50 and in vivo human IC50 values. The model predicted virtually complete VEGF inhibition for the 8-week and quarterly dosing schedule during the 52-week treatment period. In the 16-week schedule, clinically significant VEGF inhibition was maintained during the 52-week period. The model quantitatively described abicipar-VEGF target engagement leading to rapid reduction of VEGF and a long duration of VEGF inhibition demonstrating the clinical feasibility of up to a 16-week dosing interval. Abicipar is predicted to reduce IVT dosing compared with other anti-VEGF therapies with the potential to lessen patient treatment burden. SIGNIFICANCE STATEMENT: Current anti-VEGF treatments for neovascular age-related macular degeneration require frequent (monthly) intravitreal injections and monitoring, which increases patient burden. We developed a mechanistic pharmakinetic/pharmadynamic model to describe the interaction between abicipar (a novel VEGF inhibitor) and VEGF to evaluate the duration of action. The model demonstrates extended abicipar-VEGF target engagement leading to clinical feasibility of up to a 16-week dosing interval. Our model predicted that abicipar 8-week and quarterly dosing schedules maintain virtually complete VEGF inhibition during the 52-week period.

Recovery of immunoglobulin G from rabbit serum using κ-carrageenan-modified hybrid magnetic nanoparticles

Immunoglobulin G (IgG) has been used in the treatment of cancer, autoimmune diseases and neurological disorders, however, the current technologies to purify and recover IgG from biological media are of high-cost and time-consuming, resulting in high-cost products. In this sense, the search for cost-effective technologies to obtain highly pure and active IgG is highly required. The present work proposes a simple and efficient method for the purification and recovery of IgG from rabbit serum using magnetic iron oxide nanoparticles (magnetite, Fe3O4) coated with hybrid shells of a siliceous material modified with the anionic polysaccharide κ-carrageenan. Experimental parameters such as pH, contact time between the hybrid magnetic nanoparticles (HMNPs) and rabbit serum, and total protein concentration or dilution factor of serum were evaluated. The best results were achieved at pH 5.0, with a contact time of 60 min and using a rabbit serum with a total protein concentration of 4.8 mg·mL−1. Under these conditions, it was obtained an IgG purification factor and adsorption yield onto the HMNPs of 3.0 and 90%, respectively. The desorption of IgG from the HMNPs was evaluated using two strategies: a KCl aqueous solution and buffered aqueous solutions. Comparing to the initial rabbit serum, an IgG purification factor of 2.7 with a recovery yield of 74% were obtained using a buffered aqueous solution at pH 7.0. After desorption, the secondary structure of IgG and other proteins was evaluated by circular dichroism and no changes in the secondary structure were observed, meaning that the IgG integrity is kept after the adsorption and desorption steps. In summary, the application of HMNPs in the purification of IgG from serum samples has a high potential as a new downstream platform.

Strong Hydration at the Poly(ethylene glycol) Brush/Albumin Solution Interface

Albumin molecules are extensively used as biocompatible coatings, and poly(ethylene glycol) (PEG) materials are widely used for antifouling. PEG materials have excellent antifouling property because of their strong surface hydration. Our previous research indicates that hydration at the PEG/bovine serum albumin solution interface is stronger than that at the PEG/water interface. This research shows that this observation is general for different types of albumin molecules. Different albumins including bovine, porcine, rat, rabbit, and sheep serum albumins were studied in this research. It was found that the hydration at the PEG methacrylate (pOEGMA)/albumin solution interface is always stronger than that at the pOEGMA/water interface. Here, we define "strong interfacial hydration" as "ordered strongly hydrogen-bonded interfacial water". We believe that such a strong hydration is because of the strong hydration on the albumin surface, leading to its biocompatible property. All of the albumin molecules demonstrated stronger hydration on the pOEGMA surface compared to other protein molecules such as lysozyme and fibrinogen. The strong hydration on albumin molecules is related to the high surface coverage of glutamic acid and lysine with similar amounts.

Engineered protein containing crotoxin epitopes induces neutralizing antibodies in immunized rabbits

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

Detection of Nervous Necrosis Virus of Grouper Fish in Lombok Island, Indonesia Based on Serological and Molecular Studies

Cantang grouper fish (Epinephelus fuscoguttatus x Epinephelus lanceolatus) is one of the marine fisheries commodities that have opportunities both in the domestic and international markets. The cultivation of marine fish in Indonesian water has been largely harmed by the outbreak of viral diseases, for example, those caused by Nervous Necrosis Virus (NNV). The aim of study is to: produce primary antibody of NNV; and to detect disease based on immunohistochemical (IHC) and molecular methods. Two New Zealand rabbits (Oryctolagus cuniculus) of 8 months old males, ± 2 kgs of weight were vaccinated against NNV disease each week in a dose of 0.50 cc, 1.00 cc, 2.00 cc, and 3.00 cc by intra-peritoneal injection. The rabbit serum was harvested in the fifth week activated, and filtered by membrane dialysis three times to obtain immunoglobulin as the primary antibody. The brain, eyes, and spleen of infected grouper, were processed for histological tissue and stained with Hematoxylin and Eosin and immunohistochemical methods. Positive results were supported by the molecular test of Polymerase Chain Reaction (PCR) with Nishizawa's primers: R3: 5 '-CGA GTC AAC ACG GGT GAA GA-3' and F2: 5 '-CGT GTC AGT CAT GTG TCG CT-3'. The NNV primary antibody was sensitive for the detection. The positive results of IHC stains were supported by the PCR test in grouper fish from Lombok.

Polyclonal antibodies production from porcine gelatin and its preliminary study for immunosensor applications

Gelatin was extracted from porcine skin and used as an antigen to produce the polyclonal antibodies of porcine gelatin using New Zealand white rabbits for application in the development of an ELISA-based immunosensor for porcine gelatin recognition. The extraction of porcine gelatin from porcine skin was successfully performed with 20.52 % of yield. The characterization performed by using UV and IR spectroscopy indicated that the purified gelatin has a comparable quality against the commercial standard of porcine gelatin. Further analysis of the gelatin with SDS-PAGE method observed five separation lines with molecular weight bands of 57.06; 49.50; 42.94; 33.89; and 29.40 KDa, indicating that the gelatin can be used as an antigen to produce the polyclonal antibodies of porcine gelatin. After five injections of gelatin into the rabbit, ELISA characterization of the purified serum of the blood rabbit showed that the antibody with a concentration of 0.289 mg/mL could be produced.

Construction of Polymeric Antigenic Diagnosticum Based on <i>Vibrio cholera</i> О1 Lipopolysaccharide

Representatives of the genus Vibrio cholerae differ in the structure of lipopolysaccharide, in particular, its O-polysaccharide chains (O-antigen), which determines the serological specificity of vibrios. Currently, the water-phenolic method is used to obtain the lipopolysaccharide preparation. However, this technique relates to harsh chemical methods, leads to a change in original molecular organization of biopolymer, violating its structure and biological properties. Modern technologies in the development of diagnostic kits for the immunosuspension reaction of volume agglomeration allow for obtaining synthetic carriers with different reaction groups on the particle surface capable to bind antigens/antibodies. The aim of this study was to construct cholera antigenic polymeric diagnostic kit based on the lipopolysaccharide of Vibrio cholerae O1 serogroup. Materials and methods . The lipopolysaccharide was used as a sensitizer obtained through the author's modification of enzymatic purification from the cell membranes of Vibrio cholerae using ultrasonic disintegration. Results and discussion . The resulting sensitin contains small impurities of protein (1.5 %) and nucleic acids (0.1 %). Diagnosticum is characterized by high analytical sensitivity in agglomeration reaction with commercial and experimental rabbit serum to Vibrio cholerae O1 serogroup (1:640 - 1:5120) and analytical specificity (the diagnosticum does not interact with heterologous sera, with serums to pathogens of acute intestinal infections, as well as with sera from healthy donors). A polymeric antigenic cholera diagnosticum designed to detect antibodies to lipopolysaccharide of Vibrio cholerae in the blood serum of patients who were ill, suspected of the disease or vaccinated people has been constructed.

Structural characterization of a safflower polysaccharide and its promotion effect on steroid-induced osteonecrosis in vivo

A water-soluble polysaccharide (SPAW) was purified from Safflower and it was identified to be (1→3)-linked β-d-Glucan. The therapeutic effect and underlying mechanism of SPAW on steroid-induced avascular necrosis of the femoral head (SANFH) in a rabbit model was performed here. The abnormal histopathologic changes and apoptosis of femoral head in model group were significantly reverted after SPAW (25, 100 and 200 mg/kg) administration for 60 days, as evidenced by the a decline of empty lacunae rate, the average bone marrow fat cell size and the proportion of apoptotic cells. Furthermore, administration of SPAW significantly decreased the Bax and caspase-3 protein expression, but increased the protein expression of Bcl-2 when compared these in model rabbits. Meanwhile, increased hydroxyproline (HOP) and decreased serum hexosamine (HOM) concentration in rabbit serum were turned to the opposite way. The present study suggested that SPAW may provide an alternative treatment for the treatment of SANFH.

Immunization with the Gly1127-Cys1140 amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies.

Background: The LRP1 (CR9) domain and, in particular, the sequence Gly1127-Cys1140 (P3) plays a critical role in the binding and internalization of aggregated LDL (agLDL). We aimed to evaluate whether immunization with P3 reduces high-fat diet (HFD)-induced atherosclerosis.Methods: Female New Zealand White (NZW) rabbits were immunized with a primary injection and four reminder doses (R1-R4) of IrP (irrelevant peptide) or P3 conjugated to the carrier. IrP and P3-immunized rabbits were randomly divided into a normal diet group and a HFD-fed group. Anti-P3 antibody levels were determined by ELISA. Lipoprotein profile, circulating and tissue lipids, and vascular pro-inflammatory mediators were determined using standardized methods while atherosclerosis was determined by confocal microscopy studies and non-invasive imaging (PET/CT and Doppler ultrasonography). Studies treating human macrophages (hMΦ) and coronary vascular smooth muscle cells (hcVSMC) with rabbit serums were performed to ascertain the potential impact of anti-P3 Abs on the functionality of these crucial cells.Results: P3 immunization specifically induced the production of anti-P3 antibodies (Abs) and did not alter the lipoprotein profile. HFD strongly induced cholesteryl ester (CE) accumulation in the aorta of both the control and IrP groups, and their serum dose-dependently raised the intracellular CE of hMΦ and hcVSMC, promoting TNFR1 and phospho-NF-kB (p65) overexpression. These HFD pro-inflammatory effects were dramatically decreased in the aorta of P3-immunized rabbits and in hMΦ and hcVSMC exposed to the P3 rabbit serums. Microscopy studies revealed that P3 immunization reduced the percentage of lipids, macrophages, and SMCs in the arterial intima, as well as the atherosclerotic extent and lesion area in the aorta. PET/CT and Doppler ultrasonography studies showed that the average standardized uptake value (SUVmean) of the aorta and the arterial resistance index (ARI) of the carotids were more upregulated by HFD in the control and IrP groups than the P3 group.Conclusions: P3 immunization counteracts HFD-induced fatty streak formation in rabbits. The specific blockade of the LRP1 (CR9) domain with Anti-P3 Abs dramatically reduces HFD-induced intracellular CE loading and harmful coupling to pro-inflammatory signaling in the vasculature.

Особливості мінерального обміну у кролів за умови корекції біологічно активними кормовими добавками на тлі імплантування PLA імплантатів

Humic substances are able to influence mineral metabolism due to their properties to form chelate complexes. Directly, the state of bone tissue is closely related to general processes that ensure the exchange of minerals in the body as a whole. Polylactic acid (PLA) is a biopolymer which, due to its high biocompatibility and osseointegration properties, is increasingly used for the manufacture of implants. Among the methods of improving the osseointegration of implants, gaining popularity, the use of pharmacological drugs and dietary supplements that have a direct effect on bone tissue or act indirectly by affecting the body’s systems and metabolism. For the experiment 32 rabbits were divided into groups of 8 rabbits each In two model groups, 16 rabbits were implanted with PLA implants in the bilateral defect of the parietal bone, one of which was immersed in a 1% humilid solution and the other in a 0.9% NaCl solution before administration. From the operated rabbits, 8 received humilid during treatment, and 8 others were intact. 16 rabbits who did not undergo surgery were divided into two groups, one of which drinked humilid and the other clean water. The aim of our work was to determine the mineral metabolism in rabbits during implantation of PLA implants. The ability of humic substances to influence the redistribution of calcium ionized and copper in the blood serum and femur in rabbits of the model group in comparison with intact animals was proved.. There was also an increase in the content of iron and calcium ionized in the serum, and calcium, iron, copper and a decrease in the content of zinc in the femur of rabbits treated with humilide. It has been proved that the introduction of humilid into the diet caused an increase in newly formed bone tissue in PLA implants of rabbits of the model group. Therefore, an increase in macro- microelements in blood serum and their redistribution in the bone tissue of rabbits receiving humilides suggests the possibility of using humilid to correct mineral metabolism and improve the condition of bone tissue. Leveling reduction macro- microelements in the blood and bone tissue, together with an increase in newly formed bone tissue in PLA implants of a model group of rabbits receiving humilide indicate a systemic effect of humilid on the osseointegration of PLA implants.

Spectroscopic demonstration of sinapic acid methyl ester complexes with serum albumins.

The methyl ester of sinapic acid (MESA) is a molecule with confirmed antioxidant properties. It is important to establish whether it can be transported across humans and animals. Therefore, we investigated MESA interactions with serum albumins, namely, human serum albumin (HSA), bovine serum albumin (BSA), rabbit serum albumin (RSA), and sheep serum albumin (SSA). Experiments were performed in a pH range from 5.9 to 10.7 using absorption and fluorescence techniques. It was found that MESA formed complexes with every albumin in the entire pH range under examination, which was confirmed by the appearances of new absorption and fluorescence complex bands. Fluorescence intensities were much higher (up to 20 times) and lifetimes were up to 340 times as compared to those for unbound MESA. The quenching experiments at pH 7.4 showed that the stoichiometry for every albumin was 1 : 1; the binding constant was the highest for HSA, which reached 52 000 M−1. The obtained results suggested that MESA preferred the hydrophobic binding sites in albumins. The analysis of the fluorescence spectra and fluorescence lifetimes showed two possibly different binding sites in BSA, RSA, and SSA as well as three binding sites in HSA.

Rapid immunochemical method for the detection of orthopoxviruses (Orthopoxvirus, Chordopoxvirinae, Poxviridae).

The abolition of smallpox vaccination has led to the disappearance of population immunity to pox viruses. However, the threat of infection by pathogenic orthopoxviruses persists and determines the need to develop sensitive and operational methods for indicating pathogens. Development of a sensitive, fast and easy-to-use immunochemical test for the detection of orthopoxviruses in the «point of care» format. We used preparations of cultural vaccinia virus (VV) with varying degrees of purification, polyclonal antibodies from hyperimmune rabbit serum, and equipment from a previously developed autonomous kit for dot-immunoassay on flat protein arrays. It has been established that rabbit polyclonal antibodies can be used in a single-stage dotanalysis, both as a capture agent immobilized on a substrate and as a detection reagent bound with colloidal gold particles. It is shown that the effectiveness of the detection of VV is inversely related to the degree of purification of viruses from sub-viral structures. The sensitivity of the rapid detection of viruses in a crude preparation was about 30 times higher than in pure viral material. The increase in sensitivity, presumably, occurs due to binding to the capture antibodies of subviral structures, which form large aggregates of sensitized gold particles. The test does not detect cross-reactions with heterogeneous viruses (measles, rubella and chickenpox) that cause exantematous diseases. The one-stage variant of the dot-immunoassay reduces the analysis time to 40 minutes and improves the detection sensitivity of orthopoxviruses in crude viral preparations to the range of 105-104 PFU / ml. Full makeup, ease of analysis and the ability to visually accounting for results allow the test to be used outside of laboratories.

Stanazolol derived ELISA as a sensitive forensic tool for the detection of multiple 17α-methylated anabolics

Two valuable forensic tools based on enzyme-linked immunoassays (ELISAs) for the analysis of 17α-methylated steroids were developed using haptens of stanazolol and its conjugates with biotin. Haptens containing terminal carboxylic group were conjugated to bovine serum albumin (BSA), rabbit serum albumin (RSA) or ovalbumin (OVA). Eight batches of antisera (RAbs) obtained by immunization of rabbits were tested in an indirect competitive ELISA system using immobilization of RSA conjugate (RSA/hapten) and competitor immobilization of the biotinylated conjugate (AB-ELISA) to avidin (avidin/hapten). The best results were achieved with the RAb 212 antibodies in RSA/ST-3 and avidin/ST-10 assembled variants. For the RSA/ST-3 system, an IC50 of 0.3 ng/mL and a detection limit of 0.02 ng/mL were measured. In case of avidin/ST-10 variant, IC50 was of 3.9 ng/mL and a detection limit of 0.57 ng/mL were obtained. The effect of solvent was tested as well as the stability of coated microtiter plates over four-month period. The cross-reactivity of the developed assays with other anabolic steroids was tested and high sensitivity towards 17α-methylated steroids was observed. RSA/ST-3 assay showed significant cross-reactivity with 17α-methyltestosterone (81.2%), oxymetholone (30.4%), methandienone (10.0%) and methyl dihydrotestosterone (7.7%). Similarly, in the avidin/ST-10 assay, 17α-methyltestosterone (34.5%), mestanolone (32.1%), oxymetholone (22.7%), methandienone (14.2%), 9-dehydromethyltestosterone (12.5%) and oxandrolone (1.2%) exhibited high cross-reactivity. The functionality of the developed systems was verified by the successful identification of a series of 17α-methylated anabolic steroids in a set of real samples including pharmaceutical preparations seized by the Police of the Czech Republic on the black market.