Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived- Dendritic Cells and Monocyte-Derived Macrophages

Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2), plasmacytoid DC (pDC), and monocyte derived DC (MoDC). DC of Artiodactyla (pigs and ruminants) can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC). Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb) to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ). Functional analysis revealed each of these populations can take up and process antigens (Ags), present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo

Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived- Dendritic Cells and Monocyte-Derived Macrophages

Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2), plasmacytoid DC (pDC), and monocyte derived DC (MoDC). DC of Artiodactyla (pigs and ruminants) can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC). Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb) to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ). Functional analysis revealed each of these populations can take up and process antigens (Ags), present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo

PCR-based detection and genetic characterization of porcine parvoviruses in South Korea in 2018

with the advantage of sequencing technology, many novel porcine parvoviruses (PPV) rather than PPV1 has been reported. This study ultilized specific PCR- based method and gene- based analysis to study the presence and genetic diversity of porcine parvoviruses in South Korea in 2018. The present study was conducted in 2018 and found PPV1 and PPV7 in nine out of 151 field samples (organs and semen) by the PCR method. Among these, the complete genome sequences of five strains (N2, N91, N108, N133, and N141) were recovered. Phylogenic analysis revealed that the strains N2, N91, and N108 belong to the PPV1 genotype, while N133 and N141 belong to PPV7 genotype. The PPV7 strains collected in this study had deletion mutations in the VP2 gene but differed from that of PPV7 strains collected in 2017. Among the PPV1 strains, the amino acid variations in the B cell epitopes of the VP2 protein were observed between three Korean PPV1 field strains (N2, N91, and N108) and the reference PPV1 strains. Those substitutions resulted in six out of 12 predicted epitopes having significant differences in antigenic index compared to the other PPV1 strains. This study confirmed the presence of different genotypes of porcine parvoviruses in South Korea. The PPVs circulating in South Korea were phylogenetically classified as PPV1 and PPV7 genotypes. Three Korean PPV1 strains collected in 2018 were predicted to have antigenic alteration in VP2 compared to several reference strains of PPV1.This study was supported by Boehringer Ingelheim Vetmedica Korea Ltd. (Grant no. 20180002). The funder had no role in this study design, data collection and analysis, decision to publish, or preparation of the manuscript

Distribution and antimicrobial resistance profiles of bacterial species in stray cats, hospital-admitted cats, and veterinary staff in South Korea

Antimicrobial resistance is becoming increasingly important in both human and veterinary medicine. According to the One Health concept, an important step is to monitor the resistance patterns of pathogenic bacteria. In this study, the antimicrobial susceptibility patterns and trends of bacteria isolated from stray cats, hospital-admitted cats, and veterinary staff in South Korea between 2017 and 2018 were investigated. The minimum inhibitory concentrations of different antibiotics for Staphylococcus spp., Enterobacteriaceae, and Enterococcus spp. were determined to establish representatives of different antibiotic classes relevant for treatment or surveillance. For Coagulase-positive and Coagulase-negative Staphylococci, resistance to fluoroquinolones was below 13%, but resistance to ampicillin and penicillin was high (20–88%). A total of 9.5, 12.1, and 40.3% of staphylococcal isolates from stray cats, hospital-admitted cats, and veterinary staff, respectively, were confirmed to be mecA positive. For Enterobacteriaceae, resistance to carbapenems, fluoroquinolones, and 3rd generation cephalosporins was low (0–11.1%). The Enterococcus spp. isolates showed no resistance to vancomycin. The antimicrobial resistance rates of the Staphylococcus spp. and Enterobacteriaceae isolates from stray cats were usually lower than those of isolates from hospital-admitted cats and veterinary staff, but the Enterococcus spp. isolates revealed the opposite. Thus, the antimicrobial resistance varied across bacterial species according to the source from which they were isolated. Resistance to critically important compounds were low. However, the presence of antimicrobial resistance in cat isolates is of both public health and animal health concern.This work was supported by a grant from the Animal and Plant Quarantine Agency, Ministry of Agriculture and Forestry (Z-1543081-2017-18-03) and the Cooperative Research Program of Center for Companion Animal Research Jung et al. BMC Veterinary Research (2020) 16:109 Page 12 of 14 (Project No. PJ013985022019) of Rural Development Administration, South Korea. The funding bodies played no role in the design of the study, the collection, analysis, and interpretation of data and in writing the manuscript

Evaluation of two mutants of \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e as candidates for a live attenuated vaccine for Johne’s disease

Control of Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis, has been difficult because of a lack of an effective vaccine. To address this problem we used targeted gene disruption to develop candidate mutants with impaired capacity to survive ex vivo and in vivo to test as a vaccine. We selected relA and pknG, genes known to be important virulence factors in Mycobacterium tuberculosis and Mycobacterium bovis, for initial studies. Deletion mutants were made in a wild type Map (K10) and its recombinant strain expressing the green fluorescent protein (K10-GFP). Comparison of survival in an ex vivo assay revealed deletion of either gene attenuated survival in monocyte-derived macrophages compared to survival of wild-type K10. In contrast, study in calves revealed survival in vivo was mainly affected by deletion of relA. Bacteria were detected in tissues from wild-type and the pknG mutant infected calves by bacterial culture and PCR at three months post infection. No bacteria were detected in tissues from calves infected with the relA mutant (P \u3c 0.05). Flow cytometric analysis of the immune response to the wild-type K10-GFP and the mutant strains showed deletion of either gene did not affect their capacity to elicit a strong proliferative response to soluble antigen extract or live Map. Quantitative RTPCR revealed genes encoding IFN-ƴ, IL-17, IL-22, T-bet, RORC, and granulysin were up-regulated in PBMC stimulated with live Map three months post infection compared to the response of PBMC pre-infection. A challenge study in kid goats showed deletion of pknG did not interfere with establishment of an infection. As in calves, deletion of relA attenuated survival in vivo. The mutant also elicited an immune response that limited colonization by challenge wild type Map. The findings show the relA mutant is a good candidate for development of a live attenuated vaccine for Johne’s disease

Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium subsp. paratuberculosis peptide vaccine

Studies in cattle show CD8 cytotoxic T cells (CTL), with the ability to kill intracellular bacteria, develop following stimulation of monocyte-depleted peripheral blood mononuclear cells (mdPBMC) with antigen-presenting cells (APC, i.e. conventional dendritic cells [cDC] and monocyte-derived DC [MoDC]) pulsed with MMP, a membrane protein from Mycobacterium avium subsp. paratuberculosis (Map) encoded byMAP2121c. CTL activity was diminished if CD4 T cells were depleted from mdPBMC before antigen (Ag) presentation by APC, suggesting simultaneous cognate recognition of MMP epitopes presented by MHC I and MHC II molecules to CD4 and CD8 T cells is essential for development of CTL activity. To explore this possibility, studies were conducted with mdPBMC cultures in the presence of monoclonal antibodies (mAbs) specific for MHC class I and MHC class II molecules. The CTL response of mdPBMC to MMP-pulsed APC was completely blocked in the presence of mAbs to both MHC I and II molecules and also blocked in the presence of mAbs to either MHC I or MHC II alone. The results demonstrate simultaneous cognate recognition of Ag by CD4 and CD8 T cells is essential for delivery of CD4 T cell help to CD8 T cells to elicit development of CTL

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/relA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb

Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells

Studies focused on development of an attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis (Ptb) in cattle and other species, revealed that deletion of relA, a global gene regulator, abrogates the ability of Map to establish a persistent infection. In the absence of relA, cattle develop CD8 cytotoxic T cells (CTL) with the ability to kill intracellular bacteria. Analysis of the recall response to a relA mutant, Map/relA, with cells from a vaccinated steer demonstrated that a 35-kDa membrane peptide (MMP) is one of the targets of the response. This observation suggested that it might be possible to develop a peptide-based vaccine. As reported here, the gene encoding the hypothetical MMP ORF, MAP2121c, was modified for expression in mammalian cells as a first step in developing an expression cassette for incorporation into a mammalian expression vector. The modified sequence of MMP, tPA-MMP, was mutated to generate two additional sequences for the study, one with substitutions to replace five potential residues that could be glycosylated, tPA-MMP-5mut, and one with substitutions to replace the first two potential residues that could be glycosylated, tPA-MMP-2mut. The sequences were placed in an expression cassette to produce peptides for analysis. An ex vivo platform was used with flow cytometry and a bacterium viability assay to determine if modifications in the gene encoding MMP for expression in mammalian cells altered its capacity to elicit development of CD8 CTL, essential for its use in a peptide-based vaccine. Monocyte-depleted PBMC (mdPBMC) were stimulated with antigen-presenting cells (APC) pulsed with different MMP constructs. CD4 and CD8 T cells proliferated in response to stimulation with MMP (control) expressed in Escherichia coli (eMMP), tPA-MMP, and tPA-MMP-2mut. CD8 T cells retained the capacity to kill intracellular bacteria. The tPA-MMP-5mut failed to elicit a proliferative response and was not included in further studies. The data show that the expression cassettes containing MMP and MMP-2mut can be used to screen and select a mammalian expression vector for the development of an efficacious peptide-based vaccine against Ptb

Comparison of the Efficacy of Glimepiride, Metformin, and Rosiglitazone Monotherapy in Korean Drug-Naïve Type 2 Diabetic Patients: The Practical Evidence of Antidiabetic Monotherapy Study

BackgroundAlthough many anti-diabetic drugs have been used to control hyperglycemia for decades, the efficacy of commonly-used oral glucose-lowering agents in Korean type 2 diabetic patients has yet to be clearly demonstrated.MethodsWe evaluated the efficacy of glimepiride, metformin, and rosiglitazone as initial treatment for drug-naïve type 2 diabetes mellitus patients in a 48-week, double-blind, randomized controlled study that included 349 Korean patients. Our primary goal was to determine the change in HbA1c levels from baseline to end point. Our secondary goal was to evaluate changes in fasting plasma glucose (FPG) levels, body weight, frequency of adverse events, and the proportion of participants achieving target HbA1c levels.ResultsHbA1c levels decreased from 7.8% to 6.9% in the glimepiride group (P<0.001), from 7.9% to 7.0% in the metformin group (P<0.001), and from 7.8% to 7.0% (P<0.001) in the rosiglitazone group. Glimepiride and rosiglitazone significantly increased body weight and metformin reduced body weight during the study period. Symptomatic hypoglycemia was more frequent in the glimepiride group and diarrhea was more frequent in the metformin group.ConclusionThe efficacy of glimepiride, metformin, and rosiglitazone as antidiabetic monotherapies in drug-naïve Korean type 2 diabetic patients was similar in the three groups, with no statistical difference. This study is the first randomized controlled trial to evaluate the efficacy of commonly-used oral hypoglycemic agents in Korean type 2 diabetic patients. An additional subgroup analysis is recommended to obtain more detailed information