Cleverson D. Souza

Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium.

Pathogenic mycobacterial organisms have the capacity to inhibit macrophage activation and phagosome maturation. Although the mechanism is complex, several studies have incriminated signaling through TLR2 receptors with subsequent activation of the MAPK pathway p38 (MAPKp38) and overproduction of IL‐10 in the survival of pathogenic mycobacterial organisms. In the present study, we compared the response of bovine monocytes with infection by Mycobacterium avium subspecies paratuberculosis (MAP), the cause of paratuberculosis in ruminants, with the closely related organism M. avium subspecies avium (Maa), which usually does not cause disease in ruminants. Both MAP and Maa induced phosphorylation of MAPKp38 by bovine monocytes; however, addition of a blocking anti‐TLR2 antibody partially prevented MAPKp38 phosphorylation of MAP‐infected monocytes but not Maa‐infected monocytes. Addition of anti‐TLR2 antibody enhanced phagosome acidification and phagosome‐lysosome fusion in MAP‐containing phagosomes and enabled monocytes to kill MAP organisms. These changes were not observed in Maa‐infected monocytes. The effect on phagosome maturation appears to occur independently from the previously described inhibitory effects of IL‐10 on phagosome acidification and organism killing, as IL‐10 production was not affected by addition of anti‐TLR2 antibody to monocyte cultures. Therefore, signaling through the TLR2 receptor appears to play a role in phagosome trafficking and antimicrobial responses in MAP‐infected bovine mononuclear phagocytes.

Mannosylated lipoarabinomannans from Mycobacterium avium subsp. paratuberculosis alters the inflammatory response by bovine macrophages and suppresses killing of Mycobacterium avium subsp. avium organisms.

Analysis of the mechanisms through which pathogenic mycobacteria interfere with macrophage activation and phagosome maturation have shown that engagement of specific membrane receptors with bacterial ligands is the initiating event. Mannosylated lipoarabinomannan (Man-LAM) has been identified as one of the ligands that modulates macrophage function. We evaluated the effects of Man-LAM derived from Mycobacterium avium subsp. paratuberculosis (MAP) on bovine macrophages. Man-LAM induced a rapid and prolonged expression of IL-10 message as well as transient expression of TNF-α. Preincubation with Man-LAM for up to 16 h did not suppress expression of IL-12 in response to interferon-γ. Evaluation of the effect of Man-LAM on phagosome acidification, phagosome maturation, and killing of Mycobacterium avium subsp. avium (MAA) showed that preincubation of macrophages with Man-LAM before addition of MAA inhibited phagosome acidification, phagolysosome fusion, and reduced killing. Analysis of signaling pathways provided indirect evidence that inhibition of killing was associated with activation of the MAPK-p38 signaling pathway but not the pathway involved in regulation of expression of IL-10. These results support the hypothesis that MAP Man-LAM is one of the virulence factors facilitating survival of MAP in macrophages.

Reference values for chinchilla (Chinchilla laniger) blood cells and serum biochemical parameters

Raising chinchilla (Chinchilla laniger) for commercial purpose has increased significantly; however, hematological and serum biochemical reference values have not yet been determined for chinchillas raised in south Brazil. Establishing blood cells and serum biochemistry reference values might be helpful to evaluate health status of chinchillas and might be used as a tool by clinicians. The purpose of this study was to determine the reference values for blood cells and serum biochemistry of Chinchilla laniger. Blood samples were collected by cardiac puncture from 16 adult males, at the time they were killed to remove the fur coat, and from 8 adult males anesthetized with ketamine and xylazine. Blood cell counts and serum biochemistry analysis were performed using standard techniques and the results were expressed as mean ± SEM. Analysis of blood parameters from post-mortem cardiac punctured and from anesthetized chinchillas indicated that blood samples from anesthetized chinchillas had higher PCV, Hemoglobin, MCHC and WBC (P < .05); in contrast, had lower levels of monocytes, basophils and eosinophils (P < .05). Serum biochemical parameters were less affected by sampling method: anesthetized chinchillas had lower levels of urea, glucose and triglycerids (P < .05). The data obtained might be useful as a parameter to monitor the health status of chinchillas raised in south Brazil.

Effects of nuclear factor-κB on regulation of cytokine expression and apoptosis in bovine monocytes exposed to Mycobacterium avium subsp paratuberculosis

OBJECTIVE To evaluate the role of the nuclear factor-kappaB (NF-kappaB) in the response of bovine monocytes to exposure to Mycobacterium avium subsp paratuberculosis (MAP). SAMPLE POPULATION Monocytes from healthy adult Holstein cows that were known to be negative for MAP infection. PROCEDURES Monocytes were incubated with MAP organisms with or without a specific inhibitor of the NF-kappaB pathway (pyrrolidine dithiocarbamate), and activation of the NF-kappaB pathway was detected by use of an electrophorectic mobility shift assay. The capacities of monocytes to express tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, and IL-12; to acidify phagosomes; to phagocytize and kill MAP organisms; and to undergo apoptosis were evaluated. RESULTS Addition of MAP organisms to monocytes activated the NF-kappaB pathway as indicated by increased NF-kappaB-DNA binding. Addition of pyrrolidine dithiocarbamate prevented nuclear translocation of NF-kappaB, decreased expression of TNF-alpha and IL-10, and increased IL-12 expression. Treatment of MAP-exposed monocytes with pyrrolidine dithiocarbamate increased the rate of apoptosis but failed to alter phagosome acidification, organism uptake, or organism killing by those cells. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that NF-kappaB rapidly translocated to the nucleus after exposure of bovine monocytes to MAP organisms. These data suggest that NF-kappaB is involved in initiation of inflammatory cytokine transcription and inhibition of apoptosis but that it is not directly involved in phagosome acidification or organism killing.

Mycobacterium avium Subspecies paratuberculosis Recombinant Proteins Modulate Antimycobacterial Functions of Bovine Macrophages.

It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinant proteins expressed from coding sequences annotated as lipoproteins were screened for their ability to induce IL-10 expression, an indicator of MAPKp38 activation, in bovine monocyte-derived macrophages. A recombinant lipoprotein, designated as MAP3837c, was among a group of 6 proteins that strongly induced IL-10 gene transcription in bovine macrophages, averaging a 3.1-fold increase compared to non-stimulated macrophages. However, a parallel increase in expression of IL-12 and TNF-α was only observed in macrophages exposed to a subset of these 6 proteins. Selected recombinant proteins were further analyzed for their ability to enhance survival of M. avium within bovine macrophages as measured by recovered viable bacteria and nitrite production. All 6 IL-10 inducing MAP recombinant proteins along with M. paratuberculosis cells significantly enhanced phosphorylation of MAPK-p38 in bovine macrophages. Although these proteins are likely not post translationally lipidated in E. coli and thus is a limitation in this study, these results form the foundation of how the protein component of the lipoprotein interacts with the immune system. Collectively, these data reveal M. paratuberculosis proteins that might play a role in MAPK-p38 pathway activation and hence in survival of this organism within bovine macrophages.

In Vitro Evaluation of the Biological Responses of Canine Macrophages Challenged with PLGA Nanoparticles Containing Monophosphoryl Lipid A.

Poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been considerably studied as a promising biodegradable delivery system to induce effective immune responses and to improve stability, safety, and cost effectiveness of vaccines. The study aimed at evaluating early inflammatory effects and cellular safety of PLGA NPs, co-encapsulating ovalbumin (PLGA/OVA NPs), as a model antigen and the adjuvant monophosphoryl lipid A (PLGA/MPLA NPs) as an adjuvant, on primary canine macrophages. The PLGA NPs constructs were prepared following the emulsion-solvent evaporation technique and further physic-chemically characterized. Peripheral blood mononuclear cells were isolated from canine whole blood by magnetic sorting and further cultured to generate macrophages. The uptake of PLGA NP constructs by macrophages was demonstrated by flow cytometry, transmission electron microscopy and confocal microscopy. Macrophage viability and morphology were evaluated by trypan blue exclusion and light microscopy. Macrophages were immunophenotyped for the expression of MHC-I and MHC-II and gene expression of Interleukin-10 (IL-10), Interleukin-12 (IL-12p40), and tumor necrosis factor alpha (TNF-α) were measured. The results showed that incubation of PLGA NP constructs with macrophages revealed effective early uptake of the PLGA NPs without altering the viability of macrophages. PLGA/OVA/MPLA NPs strongly induced TNF-α and IL-12p40 expression by macrophages as well as increase relative expression of MHC-I but not MHC-II molecules. Taken together, these results indicated that PLGA NPs with addition of MPLA represent a good model, when used as antigen carrier, for further, in vivo, work aiming to evaluate their potential to induce strong, specific, immune responses in dogs.

Blocking the mitogen activated protein kinase-p38 pathway is associated with increase expression of nitric oxide synthase and higher production of nitric oxide by bovine macrophages infected with Mycobacterium avium subsp paratuberculosis.

This study evaluated the role of the mitogen-activated protein kinase (MAPK)-p38 pathway in the nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by bovine monocyte-derived macrophages ingesting Mycobacterium avium subsp. paratuberculosis (MAP) organisms in vitro. Bovine monocyte-derived macrophages were incubated with MAP organisms with or without a specific inhibitor of the MAPKp38 pathway and activation of the MAPKp38, interleukin - (IL) IL-10, IL-12, iNOS mRNA expression and NO production were evaluated. Incubation of macrophages with MAP organisms activates the MAPKp38 pathway at early time points post infection. Chemically inhibition of MAPKp38 before incubation of bovine macrophages with MAP resulted in increased expression of IL-12 mRNA at 2, 6 and 24h, decreased expression of IL-10 mRNA at 2, 6 and 24h and increased expression of iNOS mRNA at 2 and 6h. Nitric oxide was evaluated to indirectly determine the effects of MAPKp38 pathway on the anti-microbial activity of bovine macrophages. Incubation of bovine macrophages with MAP resulted in modest increased production of NO at 4 and 6h post infection. Pretreatment of bovine macrophages with the MAPKp38 inhibitor SB203580 before addition of MAP organisms resulted in increased production of NO at 2, 4, 6 and 24h post infection. This study expanded our knowledge of the importance of the MAPKp38 pathway in limiting an appropriate macrophage response to MAP and suggested how activation of MAPKp38 pathway may be a target of this organism to disrupt earlier antimicrobial mechanisms of macrophages. These findings raises the interesting possibility that the cellular manipulation of MAPKp38 may be useful in designing novel vaccines against MAP.

Comparative sequences of the canine and feline vasopressin prohormones

Vasopressin (VP, antidiuretic hormone) is synthesized as a prohormone consisting of four domains: a signal sequence, the VP nonapeptide, neurophysin II (NP II), and a C-terminal glycopeptide. It plays a key role in cardiovascular homeostasis by regulating renal tubular water reabsorption and urine production. As part of our ongoing studies of the hormonal effects of heart failure in dogs and cats, we have been determining, when not known, the nucleotide and amino acid sequences of relevant peptide hormones. This information will be used to assist in the development and selection of regions of the prohormone for further study and the selection of molecular and antibody probes. To determine the comparative nucleotide and amino acid sequences of canine and feline VP, total RNA was extracted from feline and canine hypothalamus to obtain complementary DNA (cDNA) by reverse transcriptase polymerase chain reaction (RT-PCR) using oligo-T primers. Consensus primers were designed and used for PCR of the resulting cDNA, and the PCR products were then sequenced and analyzed. Each VP sequence was compared to the VP from several mammalian species by alignment with sequences obtained from the GenBank database at the National Center for Biotechnology Information (NCBI). The complete sequence of the canine prohormone was consequently derived from raw data available from the canine Whole Genome Shotgun sequencing site at NCBI. Canine and feline VP prohormones share a high degree of homology with that of other known mammalian species in the VP hormone and NP II domains. The nine amino acid code of canine and feline VP (CYFQNCPRG) is identical to that found in human beings, cows, mice, and rats. The sequence differs by one amino acid from that of swine. The results of this study should be of help in the design and use of antibodies and molecular probes for future studies.

A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages

We evaluated the potential of a nanoparticle (NP) delivery system to improve methods of delivery of candidate peptide‐based vaccines for Paratuberculosis in cattle.