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Platelet-rich fibrin (PRF), which has been shown to promote wound and bone regeneration, has demonstrated antimicrobial properties against periodontal pathogens. However, in veterinary medicine, no study has determined the antimicrobial effects of canine platelet-rich fibrin (cPRF). Therefore, this study aimed to determine the antimicrobial effect of cPRF against E. coli and S. pseudintermedius found in dogs' wounds and against the standard strain S. aureus . Additionally, the mechanism of the existing antibacterial activity of cPRF, which involves the formation of reactive oxygen species (ROS), was tested. Blood samples from six dogs were processed for cPRF. The antimicrobial properties of three groups (growth control, cPRF, and drug control) were evaluated at 0.5, 4, 8, and 24 h using a time-kill assay. The killing mechanisms involving ROS were evaluated using horseradish peroxidase (HRP) to suppress ROS production in PRF (PRF-SR). Subsequently, tests for antimicrobial properties and ROS generation were compared to those of the growth control and cPRF groups. The results showed that cPRF had significant antimicrobial properties against E. coli but no antimicrobial properties against S. pseudintermedius . After the ROS suppression, PRF-SR did not show an antimicrobial property against E. coli . Moreover, cPRF-treated bacteria exhibited significantly greater intracellular ROS than PRF-SR. In conclusion, canine PRF showed an antimicrobial effect against E. coli , and its antibacterial mechanism was related to releasing ROS.

Advanced platelet-rich fibrin (A-PRF) induces more proliferation and migration of fibroblasts compared with standard PRF, but it being freshly prepared prior to it being applied is necessary. Therefore, this study aimed to determine the effect of lyophilized A-PRF on growth factor release and cell biological activity. Blood samples were collected from six dogs and processed for fresh and lyophilized A-PRF. The growth factors released included transforming growth factor beta-1 (TGF-β1), vascular endothelial growth factor-A (VEGFA), and platelet-derived growth factor-BB (PDGF-BB), and the fibroblast proliferation as well as wound closure enhancement of both products were compared. The results showed that TGF-β1, PDGF-BB, and VEGFA were continually released from lyophilized A-PRF for over 72 h. Lyophilized A-PRF released significantly more accumulated VEGEA and a tendency to release more TGF-β1 at 72 h as well as VEGFA at 24 h and 72 h than fresh A-PRF. Moreover, lyophilized A-PRF increased fibroblast proliferation and induced a significantly faster wound closure than the control, while no significant difference between fresh and lyophilized A-PRF was found. In conclusion, the lyophilization of canine A-PRF can preserve the release of growth factors and has similar biological activities to a fresh preparation. This encourages the substitution of lyophilized A-PRF instead of fresh A-PRF in regenerative treatments in which the stability of the product is concerned.

Background: Feline panleukopenia virus (FPV) is an etiologic pathogen of feline panleukopenia that infects all members of Felidae including tigers (Panthera tigris). Vaccinations against FPV among wild felid species have long been practiced in zoos worldwide. However, few studies have assessed the tiger immune response post-vaccination due to the absence of a serological diagnostic tool. To address these limitations, this study aimed to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the monitoring of tiger antibody levels against the feline panleukopenia vaccine by employing the synthesized subunit capsid protein VP2. An in-house horseradish peroxidase (HRP) conjugated rabbit anti-tiger immunoglobulin G (IgG) polyclonal antibody (HRP-anti-tiger IgG) was produced in this study and employed in the assay. It was then compared to a commercial HRP-conjugated goat anti-cat IgG (HRP-anti-cat IgG). Sensitivity and specificity were evaluated using the Bayesian model with preferential conditional dependence between HRP-conjugated antibody-based ELISAs and hemagglutination-inhibition (HI) tests., Results: The posterior estimates for sensitivity and specificity of two indirect ELISA HRP-conjugated antibodies were higher than those of the HI test. The sensitivity and specificity of the indirect ELISA for HRP-anti-tiger IgG and HRP-anti-cat IgG were 86.5, 57.2 and 86.7%, 64.6%, respectively, while the results of the HI test were 79.1 and 54.1%. In applications, 89.6% (198/221) and 89.1% (197/221) of the tiger serum samples were determined to be seropositive by indirect ELISA testing against HRP-anti-tiger and HRP-anti-cat, respectively., Conclusion: To the best of our knowledge, the specific serology assays for the detection of the tiger IgG antibody have not yet been established. The HRP-anti-tiger IgG has been produced for the purpose of developing the specific immunoassays for tigers. Remarkably, an in-house indirect ELISA based on VP2 subunit antigen has been successfully developed in this study, providing a potentially valuable serological tool for the effective detection of tiger antibodies.

This study investigated how camp management and tourist activities affect body condition, adrenocortical function, lipid profiles and metabolic status in female tourist elephants. We compared twice monthly serum insulin, glucose, fructosamine, total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), and fecal glucocorticoid metabolite (FGM) concentrations to body condition scores (BCS) at five camps with different management styles (e.g., tourist activities, work type, diet) between the High (November-February) and Low (March-October) tourist seasons. There were significant camp effects on health parameters, with BCS, TC, HDL, insulin and glucose being among the highest, and G:I being the lowest (less heathy) in elephants at an observation camp compared to those at camps where elephants received exercise by providing rides to tourists. Differences between High and Low tourist season months also were found for all measures, except TG and FGM concentrations. Both work time and walking distance were negatively correlated to glucose, fructosamine and insulin, while walking distance was negatively related to FGM concentrations. By contrast, positive associations were found between tourist number and BCS, TG, and insulin, perhaps related to tourists feeding elephants. Quantity of supplementary diet items (e.g., bananas, sugar cane, pumpkin) were positively correlated with FGM concentrations, glucose, fructosamine, and insulin. This study provides evidence that body condition, adrenal activity, metabolic markers, and lipid profiles in captive elephants may be affected by visitor numbers, work activities, and the amount of supplementary foods offered by tourists. Some activities appear to have negative (e.g., feeding), while others (e.g., exercise) may have positive effects on health and welfare. We conclude that camps adopting a more hands-off approach to tourism need to ensure elephants remain healthy by providing environments that encourage activity and rely on more natural diets or foraging., Competing Interests: The authors have declared that no competing interests exist.

We previously found relationships between body condition and physiological function affecting health and welfare of female tourist camp elephants in Thailand, and used that approach to conduct a similar study of bull elephants in the same camps (n = 13). A body condition score (BCS) was done every other month, and fecal glucocorticoid metabolite (FGM) concentrations were measured twice monthly for 1 year. Effects of season, camp management and tourist activity on lipid profiles [total cholesterol (TC), low density lipoproteins (LDL), high density lipoproteins (HDL), triglycerides (TG)] and metabolic factors [insulin, glucose, fructosamine, glucose to insulin ratio (G:I)] were determined and correlated to measures of body condition, testosterone and FGM. Positive correlations were found between BCS and TG, between FGM and TG, HDL and glucose, and between testosterone and HDL, whereas BCS and testosterone were negatively associated with the G:I. There was a significant positive relationship between FGM and testosterone. Elevated FGM concentrations were associated with altered lipid and metabolic profiles and were higher in winter compared to summer and rainy seasons. Insulin and glucose levels were higher, while the G:I was lowest in the winter season. Strong positive associations were found between TC and HDL, LDL and HDL and glucose, and glucose and insulin. By contrast, negative relationships were found between the G:I and HDL and glucose, and between insulin and G:I. Differences also were found between High and Low tourist season months for FGM, insulin, and G:I. Last, there was notable variation among the camps in measured parameters, which together with tourist season effects suggests camp management may affect physiological function and welfare; some negatively like feeding high calorie treats, others positively, like exercise. Last, compared to females, bull elephants appear to be in better physical health based on normal BCSs, lower insulin levels and higher G:I ratios., Competing Interests: The authors have declared that no competing interests exist.

Studies in western zoo elephants have found relationships between body condition and physiological function, and identified mitigating management strategies to optimize health and welfare. A similar methodological approach was used in this study, which evaluated a body condition score (BCS; 1 = thinnest, 5 = fattest) every other month and fecal glucocorticoid metabolite (FGM) concentrations twice monthly in 33 tourist camp elephants in Thailand for a 1-year period to assess seasonal variations, and determine how lipid profiles [total cholesterol (TC), low density lipoproteins (LDL), high density lipoproteins (HDL), triglycerides (TG)] and metabolic parameters [insulin, glucose, fructosamine, glucose to insulin ratio (G:I)] related to measures of body condition and adrenal function. The most prevalent BCS was 3-3.5 (60.6%), with 27.3% at BCS = 4 (overweight) and 12.1% at BCS = 4.5-5 (very overweight); no elephants had a BCS <2. BCSs were higher in rainy and winter seasons compared to summer, with FGM, TG, HDL, LDL, and insulin also higher in the rainy and/or winter seasons (p<0.05). By contrast, TC and glucose were lowest in the rainy season. FGM measures were negatively associated with two environmental factors: temperature and rainfall, but not humidity. Positive correlations were found between BCS and TC, LDL, and HDL, and between FGM and TC, HDL, glucose, and insulin (p<0.05), whereas BCS and FGM were both negatively associated with the G:I (p<0.05). However, there was no relationship between BCS and FGM among the camp elephants. Using BCS and FGM measures as outcome variables in separate regression models, this study found high BCS and elevated FGM concentrations were associated with altered lipid profiles and metabolic status in elephants. Furthermore, more work hours/day was associated with better body condition and health measures. Thus, being overweight and exposed to factors that increase adrenal activity could adversely affect health status, requiring alterations in management for some individuals, whereas exercise appears to have a protective effect., Competing Interests: The authors have declared that no competing interests exist.

Aim: To characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan (HSPG), clone 1E4-1D9., Methods: The antigen recognized by mAb 1E4-1D9 was immunoprecipitated and its amino acid sequence was analyzed LC/MS. The transmembrane domain, number of cysteine residues, and glycosylation sites were predicted from these entire sequences. Data from amino acid analysis was aligned with glypican-3 (https://www.ebi.ac.uk/Tools/msa/clustalo/). The competitive reaction of mAb 1E4-1D9 and anti-glypican-3 on HepG2 cells was demonstrated by indirect immunofluorescence and analyzed by flow cytometry. Moreover, co-immunoprecipitation of mAb 1E4-1D9 and anti-glypican-3 was performed in HepG2 cells by Western immunoblotting. The recognition by mAb 1E4-1D9 of a specific epitope on solid tumor and hematopoietic cell lines was studied using indirect immunofluorescence and analyzed by flow cytometry., Results: Monoclonal antibody 1E4-1D9 reacted with an HSPG isolated from human liver and a band of 67 kD was detected under both reducing and non-reducing conditions. The specific antigen pulled down by mAb 1E4-1D9, having a MW of 135 kD, was analyzed. The results showed two sequences of interest, gi30722350 (1478 amino acid) and gi60219551 (1378 amino acid). In both sequences no transmembrane regions were observed. Sequence number gi30722350 was 99.7% showed a match to FYCO1, a molecule involved in induction of autophagy. Sequence number gi60219551 contained 15 cysteines and 11 putative glycosylation sites with 6 predicted N-glycosylation sites. It was also matched with all PDZ domain proteins. Moreover, it showed an 85.7% match to glypican-3. Glypican-3 on HepG2 cells competitively reacted with both phycoerythrin-conjugated anti-glypican-3 and mAb 1E4-1C2 and resulted in an increase of double-stained cell population when higher concentration of mAb 1E4-1D9 was used. Moreover, antigens precipitated from HepG2 cell by anti-glypican-3 could be detected by mAb 1E4-1D9 and vice versa. The recognition of antigens, on other solid tumor cell lines, by mAb 1E4-1D9 was studied. The results demonstrated that mAb 1E4-1D9 reacted with Huh7, HepG2, HT29, MCF7, SW620, Caco2, B16F1, U937, K562 and Molt4 cells. It was also found to be weakly positive to SW1353 and HL60 and negative to H460 and Hela cell lines., Conclusion: All findings show that mAb 1E4-1D9 specifically recognizes glypican-3. Moreover, a new partner molecule of glypican-3, FYCO1 is proposed based on the results from co-precipitation studies., Competing Interests: Conflict-of-interest statement: The authors declare that there are no conflicts of interest.

Pasteurella multocida causes haemorrhagic septicemia in livestock and wild animals, including elephants. The disease has been reported in Asian elephants in India and Sri Lanka, but to date there have been no reported cases in Thailand. ELISA or indirect hemagglutination assays (IHA) have been demonstrated to be able to detect the antibody against the disease in cattle, but no data are available for elephants. The present study reports a novel in-house indirect ELISA for antibody detection of haemorrhagic septicemia in Asian elephants, and evaluates the sensitivity and specificity of the method using a Bayesian approach. The characteristics of ELISA and IHA were analyzed using a one population Bayesian model assuming conditional dependence between these two diagnostic tests. The IHA was performed as recommended by the World Organization for Animal Health (OIE) manual for haemorrhagic septicemia. An in-house indirect ELISA was developed with a heat extract antigen of P. multocida strain M-1404 (serovar B:2) as a coating antigen and rabbit anti-immunoglobulin G conjugated with horseradish peroxidase (eIgG-HRP). The checkerboard titration method was done using elephant sera immunized with P. multocida bacterin and negative sera from colostrum-deprived elephant calves. The concentrations of heat extract antigen (160μg/ml), sample serum (1:100), and eIgG-HRP (1:1000) were optimal for the assay. The calculated cut-off value was 0.103. Of the elephant sera, 50.59% (43/85) were considered seropositive by ELISA. The sensitivity of the ELISA test was higher than that of the IHA test [median=86.5%, 95% posterior probability interval (PPI)=52.5-98.9%] while the specificity was lower (median=54.1%, PPI=43.6-64.7%). The median sensitivity and specificity of IHA were 80.5% (PPI=43.8-98.0%) and 78.4% (PPI=69.0-87.0%), respectively. These findings suggest that our in-house indirect ELISA can be used as a tool to detect the antibody against haemorrhagic septicemia in Asian elephants., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Heparan sulfate proteoglycans (HSPGs) were isolated from normal human liver and α monoclonal antibody (MAb) was raised against them. Preliminary studies showed that MAb clone 1E4-1C2 was able to react with many cell lines tested, including hematopoietic cells and solid tumors. MAb1E4-1C2 was used to study whether HSPG was involved in growth and proliferation of human liver cancer using hepatocellular carcinoma (HCC) cell line (HepG2) as a model. Inhibition by MAb1E4-1C2 of HepG2 cell proliferation was studied in vitro by MTT assay. For in vivo assay, xenograft induction in athymic mice was performed. The results showed that MAb1E4-1C2 inhibited proliferation of HepG2 cells significantly, compared to isotype and medium control. MAb1E4-1C2 also suppressed the growth of tumor, resulting in smaller tumor size and weight. The investigation also showed that MAb1E4-1C2 inhibited proliferation and restricted tumor growth through the induction of apoptosis. The results suggest that HSPG might be involved in liver cancer cell proliferation. Therefore, a specific MAb that was raised against liver HSPG might be an alternative therapeutic agent for the treatment of human liver cancer.

N,N,N-Trimethyl chitosan chloride is capable of forming nanocomplexes with protein through ionotropic gelation. A monoclonal antibody, raised against human liver heparan sulfate proteoglycan and specifically inhibiting hepatocellular carcinoma in vitro, was prepared in nanocomplexes of this modified chitosan. The smallest nanocomplexes (59 ± 17 nm, zeta-potential 16.5 ± 0.5 mV) were obtained at polysaccharide:antibody ratios of 5:0.3. Spherical particles with a smooth surface and compact structure having a mean diameter of ~11.2 ± 0.09 nm were investigated by Atomic Force Microscopy. Cellular uptake of fluorescently labeled nanocomplexes was studied in mouse monocyte models of cancer and normal cells. External and internal fluorescence was analyzed by flow cytometry. The results demonstrate that the nanocomplexes could enter cells and were retained for a longer period of time in cancer cells where they exhibited greater toxicity. These nanocomplexes appear safe and could potentially enhance the half-life of added antibodies.

The novel low-molecular-weight chitosan polysulfate (MW 5120-26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, (13)C NMR, and (1)H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay, and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.

Platelet refractoriness is caused by HLA antibodies and platelet-specific antibodies. Current methods used to detect antiplatelet antibodies have limitations. Solid phase red cell adherence (SPRCA) lacks sensitivity and requires a second assay using chloroquine-treated intact platelets to specify the response due to anti-HLA. We modified SPRCA by using 2 types of antihuman platelet antibodies with different specificities toward platelet lysate and tested samples from 361 patients (69 with unexplained thrombocytopenia and 292 with poor response to platelet transfusions not explicable by alloimmunization or the clinical situation) and 50 from healthy volunteers. Our method compared favorably with platelet suspension direct immunofluorescence. All samples from healthy volunteers were negative; of the samples from the patient population, 240 were positive (147 samples had only antiplatelet and 3 samples had only anti-HLA antibodies). This modified technique had a sensitivity of 98% and a specificity of 91%.

Heparan sulfate proteoglycans (HSPGs) play important biological roles in cell-matrix adhesion processes and are essential regulators of growth actions. The expression of the different HSPGs in itself is tightly regulated providing strict controls on the activities of the bound ligands. Human liver is a target for a number of pathogens, and HSPGs have been demonstrated in several cases to play a pivotal role in infectivity. Despite HSPGs important biological functions, little is known about its cell-specific distribution patterns. Human liver HSPG was isolated, and a specific monoclonal antibody (mAb) 1E4-1C2 was produced. Distribution of HSPG reactive to this mAb was studied in normal blood cells, hematopoietic cell lines and blood cells isolated from patients with various hematologic disorders using indirect immunofluorescence. There was no expression of molecules recognized by this mAb on lymphoid (Daudi, Jurkat, SupT-1) and monocytoid (U937) cell lines. Peripheral blood cells, normal bone marrow, together with leukocytes isolated from patients with acute lymphoblastic leukemia, chronic myelocytic leukemia, Hodgkin's disease or Non-Hodgkin's lymphoma, were also negative. In contrast, 1E4-1C2 showed significant positive results on human myeloid cell lines HL-60 and K562. Moreover, it is interesting that this mAb also recognized epitopes on leukocytes isolated from acute myeloblastic leukemia. These results suggest that malignancies of cells in myeloid lineage may cause expression of HSPGs that are detected by this specific mAb, making it a potential co-marker for the diagnosis of acute myeloid leukemia.

Monoclonal antibody against P. mameffei yeast secreted antigen was produced in order to develop a serological test for penicilliosis marneffei. The yeast form of P. marneffei was cultured in brain heart infusion broth at 37 degrees C for 7 days. A secreted antigen was prepared, partially purified from culture supernatant and subsequently immunized in a BALB/c mouse. Mouse monoclonal antibody was produced from immune spleen cells by a standard hybridoma technique. Specificity of the obtained monoclonal antibody was assessed with yeast secreted antigens for P. mameffei, C. alblicans, C. neoformans, and H. capsulatum by an indirect ELISA. Three of 46 hybrid clones (1 F1, 2G5, and 3G4) reacted positively with P mameffei secreted antigen. 1 F1 and 3G4 were cloned by two rounds of limiting dilution. Partially purified monoclonal antibody and rabbit polyclonal antibody against P. marneffei yeast secreted antigen were used to develop a double antibody sandwich ELISA to detect P. marneffei antigen in plasma or serum samples of 7 patients with penicilliosis marneffei and 5 healthy controls. The sandwich ELISA developed using monoclonal antibody as a capture antibody and rabbit polyclonal antibody as a detector was able to detect P. marneffei antigen in all the plasma and serum samples of penicilliosis marneffei patients, while negative in all the healthy controls. Thus, the monoclonal antibody produced in the present study appeared to be highly specific for P. marneffei and the double antibody sandwich ELISA developed using monoclonal and polyclonal antibodies against the yeast secreted antigen of P. marneffei showed a strong potential for the diagnosis of penicilliosis marneffei.

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.

Chitin prepared from the shells of rice-field crabs (Somanniathelphusa dugasti) was converted into chitosan with a degree of acetylation of 0.21 and then sulfated with chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions to give 87% of water-soluble sulfated chitosan with degree of substitution (d.s) of 2.13. 1H NMR revealed the sulfate substitution at C-2, C-3 and C-6. Gel filtration on Sepharose CL-6B of the sulfated chitosan gave three fractions with average molecular weights of 7.1, 3.5, and 1.9 x 10(4). The three sulfated chitosan preparations showed strong anticoagulant activities, with the same mechanism of action observed for standard therapeutic heparin.