Evaluation of an In Vitro Blood-Based Assay to Detect Production of Interferon-γ By Mycobacterium Bovis–Infected White-Tailed Deer (Odocoileus Virginianus)

Tuberculosis due to Mycobacterium bovis in captive Cervidae was identified as an important disease in the United States in 1990 and prompted the addition of captive Cervidae to the USDA Uniform Methods and Rules for eradication of bovine tuberculosis. As well, M. bovis infection was identified in free-ranging white-tailed deer in northeast Michigan in 1995. Tuberculosis in both captive and free-ranging Cervidae represents a serious challenge to the eradication of M. bovis infection from the United States. Currently, the only approved antemortem tests for tuberculosis in Cervidae are the intradermal tuberculin skin test and the blood tuberculosis test (BTB). At present, the BTB is not available in North America. Tuberculin skin testing of Cervidae is time-consuming and involves repeated animal handling and risk of injury to animals and humans. This study evaluated the potential of a new blood-based assay for tuberculosis in Cervidae that would decrease animal handling, stress, and losses due to injury. In addition, a blood-based assay could provide a more rapid diagnosis. Twenty 6–9-month-old white-tailed deer, male and female, were experimentally inoculated by instillation of 300 colony-forming units of M. bovis in the tonsillar crypts. Seven, age-matched uninfected deer served as controls. Blood was collected on days 90, 126, 158, 180, 210, 238, 263, and 307 after inoculation and was analyzed for the production of interferon-γ (IFN-γ) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-g in response to PPDb was significantly greater (P < 0.05) at all time points in samples from M. bovis–infected deer as compared with uninfected control deer, whereas IFN-γ production to PWM did not differ significantly between infected and control deer. Measurement of IFN-γ production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae.

In vitro evaluation, in vivo quantification, and microbial diversity studies of nutritional strategies for reducing enteric methane production

The main objective of the present work was to study nutritive strategies for lessening the CH4 formation associated to ruminant tropical diets. In vitro gas production technique was used for evaluating the effect of tannin-rich plants, essential oils, and biodiesel co-products on CH4 formation in three individual studies and a small chamber system to measure CH4 released by sheep for in vivo studies was developed. Microbial rumen population diversity from in vitro assays was studied using qPCR. In vitro studies with tanniniferous plants, herbal plant essential oils derived from thyme, fennel, ginger, black seed, and Eucalyptus oil (EuO) added to the basal diet and cakes of oleaginous plants (cotton, palm, castor plant, turnip, and lupine), which were included in the basal diet to replace soybean meal, presented significant differences regarding fermentation gas production and CH4 formation. In vivo assays were performed according to the results of the in vitro assays. , when supplemented to a basal diet (Tifton-85 hay sp, corn grain, soybean meal, cotton seed meal, and mineral mixture) fed to adult Santa Ines sheep reduced enteric CH4 emission but the supplementation of the basal diet with EuO did not affect ( > 0.05) methane released. Regarding the microbial studies of rumen population diversity using qPCR with DNA samples collected from the in vitro trials, the results showed shifts in microbial communities of the tannin-rich plants in relation to control plant. This research demonstrated that tannin-rich , essential oil from eucalyptus, and biodiesel co-products either in vitro or in vivo assays showed potential to mitigate CH4 emission in ruminants. The microbial community study suggested that the reduction in CH4 production may be attributed to a decrease in fermentable substrate rather than to a direct effect on methanogenesis.

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro?

Brossi P.M., Baccarin R.Y.A. & Massoco C.O. 2012 Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro? Pesquisa Veterinaria Brasileira 32(12):1355-1360. Departamento de Clinica Medica, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Butanta, Sao Paulo, SP 5508-210, Brazil. E-mail: baccarin@ usp.br Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)(4) - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (P<0.05). There was no difference between UBP and CBP effects. In conclusion, treatment of stimulated equine synovial cells with either UBP or CBP efficiently restored their redox equilibrium.

CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 – 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.

Effect of colchicine and amiprophos-methyl on the production of in vitro doubled haploid onion plants and correlation assessment between ploidy level and stomatal size

Doubled haploid onion (Allium cepa L.) plants allow the production of completely homozygous lines for a later production of hybrids. The haploid plants are normally produced using in vitro gynogenesis. The obtained haploid plantlets must be treated with different agents for doubling chromosomes. It is necessary to adjust the concentration and the length of treatment of the doubling agent. In this case, the effect of 250 and 500 mg.L-1 colchicine and 15.2; 30 and 60 mg.L- 1 amiprophos-methyl during 24 and 48 h was assessed over the rate of onion haploid plantlets chromosome doubling. The best duplication treatment was 250 mg.L-1 colchicine for 48 h, which yielded 100% of doubled haploid plants. On the other hand, a positive correlation resulted from the ploidy level and stomatal size, and a negative correlation between the level of ploidy and stomatal density. Significant differences between the stomatal length, width and density in haploid and doubled haploid plantlets were observed. An economical and quick method to test ploidy level in onion plantlets is proposed through the measurement of stomatal size and density.

The influence of diet on the effectiveness of garlic oil and cinnamaldehyde to manipulate in vitro ruminal fermentation and methane production.

The objective of this study was to evaluate the effects of increasing doses [0 (control: CON), 20, 60, 180 and 540 mg/L incubation medium] of garlic oil (GO) and cinnamaldehyde (CIN) on in vitro ruminal fermentation of two diets. Batch cultures of mixed ruminal microorganisms were inoculated with ruminal fluid from four sheep fed a medium-concentrate diet (MC; 50 : 50 alfalfa hay : concentrate) or four sheep fed a high-concentrate diet (HC; 15 : 85 barley straw : concentrate). Diets MC and HC were representative of those fed to dairy and fattening ruminants, respectively. Samples of each diet were used as incubation substrates for the corresponding inoculum, and the incubation was repeated on 4 different days (four replicates per experimental treatment). There were GO × diet-type and CIN × diet-type interactions (P < 0.001–0.05) for many of the parameters determined, indicating different effects of both oils depending on the diet type. In general, effects of GO were more pronounced for MC compared with HC diet. Supplementation of GO did not affect (P > 0.05) total volatile fatty acid (VFA) production at any dose. For MC diet, GO at 60, 180 and 540 mg/L decreased (P < 0.05) molar proportion of acetate (608, 569 and 547 mmol/mol total VFA, respectively), and increased (P < 0.05) propionate proportion (233, 256 and 268 mmol/mol total VFA, respectively), compared with CON values (629 and 215 mmol/mol total VFA for acetate and propionate, respectively). A minimum dose of 180 mg of GO/L was required to produce similar modifications in acetate and propionate proportions with HC diet, but no effects (P > 0.05) on butyrate proportion were detected. Methane/VFA ratio was reduced (P < 0.05) by GO at 60, 180 and 540 mg/L for MC diet (0.23, 0.16 and 0.10 mol/mol, respectively), and by GO at 20, 60, 180 and 540 mg/L for HC diet (0.19, 0.19, 0.16 and 0.08 mol/mol, respectively), compared with CON (0.26 and 0.21 mol/mol for MC and HC diets, respectively). No effects (P = 0.16–0.85) of GO on final pH and concentrations of NH3-N and lactate were detected. For both diet types, the highest CIN dose decreased (P < 0.05) production of total VFA, gas and methane, which would indicate an inhibition of fermentation. Compared with CON, CIN at 180 mg/L increased (P < 0.05) acetate proportion for the MC (629 and 644 mmol/mol total VFA for CON and CIN, respectively) and HC (525 and 540 mmol/mol total VFA, respectively) diets, without affecting the proportions of any other VFA or total VFA production. Whereas for MC diet CIN at 60 and 180 mg/L decreased (P < 0.05) NH3-N concentrations compared with CON, only a trend (P < 0.10) was observed for CIN at 180 mg/L with the HC diet. Supplementation of CIN up to 180 mg/L did not affect (P = 0.18–0.99) lactate concentrations and production of gas and methane for any diet. The results show that effectiveness of GO and CIN to modify ruminal fermentation may depend on diet type, which would have practical implications if they are confirmed in vivo.

Role of p38 mitogen-activated protein kinase in HIV type 1 production in vitro

The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) promote HIV type 1 viral replication in vitro. In the present studies, HIV production was increased in the macrophagic U1 cell line expressing the HIV genome after exposure to IL-1β, osmotic stress, or surface adhesion, suggesting a confluence of signaling pathways for proinflammatory cytokines and cell stressors. The p38 mitogen-activated protein kinase (MAPK) mediates both cytokine and stress responses; thus the role of this kinase in HIV production was investigated. HIV production as measured by p24 antigen correlated with changes in the expression of a specific (non-alpha) isoform of p38 MAPK. In the presence of a specific p38 MAPK inhibitor (p38 inh), IL-1β-induced HIV production was suppressed by more than 90% and IL-1β-induced IL-8 production was suppressed completely, both with IC50 of 0.01 μM. p38 inhibition blocked cell-associated p24 antigen and secreted virus to a similar extent. The p38 inh also decreased constitutive HIV production in freshly infected peripheral blood mononuclear cells by up to 50% (P < 0.05). Interruption of p38 MAPK activity represents a viable target for inhibition of HIV.

Constitutive production of granulocyte/macrophage colony-stimulating factor by hypodense mononuclear eosinophils developed in vitro from hybrid eosinophil/basophil granulocytes.

We recently described the development in vitro of cells with granules characteristic of eosinophils and basophils (hybrid granulocytes) from normal human cord blood mononuclear cells cultured for 14 days with recombinant human (rh) interleukin (IL)-3, rhIL-5, and a soluble basement membrane, Matrigel. Hybrid granulocytes constitutively produced granulocyte/macrophage colony-stimulating factor (GM-CSF) and rapidly developed into eosinophils after the exogenous cytokines and Matrigel were removed. To characterize the developmental progression of hybrid granulocytes, cells were maintained for an additional 14 days in medium containing rhIL-3, rhIL-5, and Matrigel. After 28 days, 73% +/- 1% (mean +/- SEM; n = 6) of the nonadherent cells were mononuclear eosinophils, 13% +/- 3% were eosinophils with two or more nuclear lobes, 13% +/- 4% were hybrid granulocytes, and 0.2% +/- 0.1% were basophils. More than 90% of the mononuclear eosinophils were hypodense as determined by centrifugation through metrizamide gradients. After an additional 5 days of culture in medium without exogenous cytokines, 65% +/- 3% (n = 5) of the 28-day cells excluded trypan blue. In contrast, 2% +/- 1% of freshly isolated peripheral blood eosinophils survived 5 days of culture without exogenous cytokines (n = 5). Fifty percent conditioned medium from in vitro derived 28-day mononuclear eosinophils and 14-day hybrid granulocytes maintained the survival of 60% +/- 7% and 77% +/- 7%, respectively, of freshly isolated peripheral blood eosinophils for 72 h, compared with 20% +/- 8% survival in medium alone (n = 3). The eosinophil viability-sustaining activity of 50% mononuclear eosinophil-conditioned medium was neutralized with a GM-CSF antibody. A total of 88% of the 28-day cells exhibited immunochemical staining for GM-CSF. Thus, during eosinophilopoiesis, both hybrid eosinophil/basophil intermediates and immature mononuclear eosinophils exhibit autocrine regulation of viability due to constitutive production of GM-CSF.

Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.

An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p-hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides.

Regulated production of a pleiotropic cytokine-platelet-derived growth factor--by differentiating erythroid cells in vitro and in vivo.

Erythroid progenitor growth in vitro is stimulated by exogenous platelet-derived growth factor (PDGF). We now report that both normal and transformed erythroid progenitor cells produce authentic PDGF in vitro and in vivo. Importantly, this production is highly regulated during erythropoiesis. Addition of soluble lysates from Rauscher murine erythroleukemia cells--an erythropoietin-responsive model progenitor cell line--to quiescent BALB/c 3T3 fibroblasts resulted in a mitogenic response identical to that observed with the addition of authentic recombinant PDGF. Polyclonal and monoclonal anti-PDGF antibodies immunoabsorbed 50-100% of this activity. Induction of Rauscher cell differentiation in vitro with dimethyl sulfoxide or erythropoietin for 48-72 hr markedly upregulated PDGF production by 17- to 18-fold and 14- to 38-fold, respectively. Importantly, stimulation of normal erythropoiesis in vivo in mice treated either with phenylhydrazine or with erythropoietin increased PDGF levels in the spleen by 11- to 48-fold and 20- to 34-fold, respectively. These results strongly suggest a role for erythroid cell-derived PDGF in normal erythropoiesis and provide documentation of the regulated production of a pleiotropic cytokine by erythroid cells.