Kazuhiro Yoshihara

Porcine TLR2 and TLR6: identification and their involvement in Mycoplasma hyopneumoniae infection.

We successfully cloned and sequenced porcine toll-like receptor (TLR2) and TLR6 cDNA from porcine alveolar macrophages stimulated with 10 microg/ml lipopolysaccharide (LPS). The open reading frames (ORFs) of the porcine TLR2 and TLR6 cDNA were shown to be 2358 and 2391 bp in length and to encode 785 and 796 amino acids, respectively. The predicted amino acid sequence of porcine TLR2 was 72.3% homologous to human TLR2 and 61.0% homologous to murine TLR2. That of porcine TLR6 was 74.4% homologous to human TLR6 and 66.1% homologous to murine TLR6. Porcine TLR2 and TLR6 genes were both mapped to porcine chromosome 8 (TLR2: SSC8q21.1 --> 21.5; TLR6: SSC8p11.1 --> p21.1) by fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Western blot analysis confirmed that TLR2 and TLR6 proteins were both expressed in porcine alveolar macrophages. Further, antiporcine TLR2 and TLR6 antibodies synergistically blocked tumor necrosis factor-alpha (TNF-alpha) production by porcine alveolar macrophages stimulated with Mycoplasma hyopneumoniae. These results indicated that both TLR2 and TLR6 are important in the recognition of M. hyopneumoniae in porcine alveolar macrophages and will be useful in understanding innate immunity against M. hyopneumoniae.

Salivary gland extract of Rhipicephalus appendiculatus ticks inhibits in vitro transcription and secretion of cytokines and production of nitric oxide by LPS-stimulated JA-4 cells

There is increasing evidence that compounds in tick saliva and salivary gland extract (SGE) have a suppressive effect on host immunity and that tick-borne pathogens exploit this situation to their benefit thus causing diseases. We have demonstrated that SGE derived from Rhipicephalus appendiculatus ticks has a suppressive effect on a macrophage like cell line, JA-4, in terms of secretion as well as mRNA transcription of three cytokines. Percent suppression of cytokine secretion by JA-4 cells cultured in the presence of lipopolysaccharide (LPS) and SGE in comparison to JA-4 cells cultured in the presence of LPS alone was 67.8, 89.1 and 82.0% for IL-1alpha, TNF-alpha and IL-10, respectively (P<0.05). A similar pattern of results was demonstrated in terms of mRNA transcription where SGE-induced suppression was 36.9% for IL-1alpha, 25.0% for TNF-alpha and 31.5% for IL-10 (P<0.05). In addition, we have demonstrated that SGE partially inhibited nitric oxide production by JA-4 activated with LPS. The results of the present study suggest that tick salivary gland compounds may exert their effect in vivo by blocking the functions of macrophages in the transcription of cytokines and production of nitric oxide. This SGE-induced immunomodulation may comprise a major gateway in the facilitation of tick feeding and transmission of pathogens in hosts.

Expression Cloning of Gamma Interferon-Inducing Antigens of Mycobacterium avium subsp. paratuberculosis

ABSTRACT Three recombinant proteins, Map10, Map39, and Map41, produced based on nucleotide sequences obtained from the screening of Mycobacteriumavium subsp. paratuberculosis genomic library expressed in Escherichiacoli significantly elicited gamma interferon production in peripheral blood mononuclear cells from infected cattle. Two of these proteins were members of the PPE protein family.

Molecular cloning and expression of canine interleukin 8 cDNA

Molecular cloning of canine interleukin-8 (IL-8) was performed to establish a basis for its investigation in the canine immune system. From a cDNA pool constructed from LPS-stimulated popliteal lymph node cells, canine IL-8 cDNA covering the whole coding region was amplified by polymerase chain reaction. The nucleotide sequence of a canine IL-8 clone, designated pcIL-8#38, was highly similar to those of human, rabbit and porcine IL-8, and comprised 353 bp with an open reading frame that encoded 101 amino acids. Analysis of the deduced amino acid sequence of insert DNA in pcIL-8#38 showed 76.5, 80.2, and 87.0% similarities with human, rabbit and porcine IL-8 proteins, respectively. Insert DNA of pcIL-8#38 was transferred to a mammalian expression vector, pcDL-SR alpha 296, and transfected into Cos7 cells. The supernatant of the transfectant had neutrophil chemotactic activity when it was examined by the neutrophil migration assay, suggesting that our cloned cDNA was biologically active. The cloned canine IL-8 cDNA will be useful for canine inflammatory disease and comparative immunology research.

Cloning and sequencing of cDNA encoding bovine macrophage colony-stimulating factor (bM-CSF) and expression of recombinant bM-CSF using baculovirus

The cDNAs encoding bovine macrophage colony-stimulating factors alpha and beta (M-CSF alpha and M-CSF beta) were cloned and recombinant bovine M-CSF alpha (rbM-CSF beta) in its dimeric form was expressed by using a recombinant baculovirus/insect cell system. The predicted amino acid sequence of rbM-CSF alpha and rbM-CSF beta shared 83.3 and 75.9% (alpha), 75.3 and 65.9% (beta) similarity with the sequence for human and murine M-CSFs, respectively. The biological activity of rbM-CSF beta was confirmed by the colony-forming assay using mouse bone marrow cells. SDS-PAGE under a reducing condition showed that the molecular weight of rbM-CSF beta was approximately 34 kDa. On the other hand, Western blot analysis under a non-reducing condition revealed that this rbM-CSF beta was secreted in dimeric form into the cell supernatant.

Cloning, Expression Analyses, and Chromosomal Location of Porcine Interleukin-18 Receptor α Chain (IL-18Rα)

We cloned and sequenced a cDNA that contains the coding sequence of the porcine interleukin-18 receptor α chain (PoIL-18Rα). Based on the conserved nucleotide sequences between human (HuIL-18Rα) and murine IL-18Rα (MuIL-18Rα), we performed reverse transcription-polymerase chain reaction (RT-PCR) with total RNA prepared from porcine peripheral blood lymphocytes (PBLs) stimulated with PoIL-12 to clone the cDNA of PoIL-18Rα. The open reading frame (ORF) of the PoIL-18Rα cDNA is 1620 base pairs (bp) in length and encodes 539 amino acids. The predicted amino acid sequence showed 68.2% and 50.2% identity to the human and murine amino acid sequences, respectively. Stimulation with concanavalin A (ConA) and IL-12, but not with IL-4, was shown to upregulate the expression of IL-18Rα mRNA in pig PBLs by RT-PCR analysis. Flow cytometric analysis also demonstrated that IL-18Rα was constitutively expressed on PoPBLs, and this expression was augmented by ConA stimulation. Furthermore, the PoIL-18Rα gene was mapped by f...

Cloning, expression, and tissue distribution of bovine interleukin-21.

Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows.