Brucellosis is a clinically and economically important disease. of apoptosis-related genes was not altered except TLR4. These results suggest that the cellular antigens of induce both humoral and cellular immunity via the production of IL-6, IL-12p40, and IFN- in bPBMC without exerting any adverse effects on the cells. Introduction Brucellosis is a highly contagious zoonosis caused by buy 116649-85-5 Gram-negative bacteria of the genus This disease affects livestock, wild animals, and humans. Ten species of the genus have been buy 116649-85-5 identified based on antigen variation buy 116649-85-5 and primary hosts [1, 2]. Brucellosis causes significant economic losses not only because it affects animal production (reduced milk production, abortion, delayed conception, and impaired fertility) but also because detection of the disease in a region or country causes enactment of international veterinary regulations as well as restrictions on animal movements and trade [1, 3]. In addition, brucellosis in human can be severely debilitating and remains an important public health concern [1, 4]. Most serological diagnostic methods for detecting infection use antibodies against common antigens . O-polysaccharide (OPS), a well-known immunodominant epitope in smooth lipopolysaccharide (SLPS), is commonly used in serological tests for diagnosing brucellosis [6C9]. Recently, several cellular proteins of have been considered new diagnostic antigens because traditional diagnostic methods using LPS have low specificity due to cross-reactivity with other relevant bacteria such as O:9 [10, 11]. is a facultative intracellular bacterial pathogen that can survive intracellular defenses and hamper the induction of host humoral immune responses . These properties help preventing the serological diagnosis of infection. Infection with potently activates both the innate and adaptive immune system, leading to Sele a proinflammatory response that favors the T-helper 1 (Th1) responses [13, 14]. Although both antibody- and cell-mediated immune responses can influence the course of infection, the latter is primarily responsible for the clearance of intracellular bacteria . spp. have mechanism that prevent activation of the host innate immune system . Invasion through the digestive tract does not elicit any inflammatory response including cytokine production from buy 116649-85-5 the host . Therefore, spp. invade silently or unnoticed by the innate immune system of the host . Cytokines are important for responses to infection. Much attention has thus been given to research on cytokine-mediated inflammatory reactions in cases of brucellosis. Previous studies have revealed that can induce the production of proinflammatory cytokines such as tumor necrosis factor- (TNF-), interleukin (IL)-6, IL-12 and IL-1 by a variety of cell types and in mice [19C23]. Understanding immune mechanisms is an important step for the development of new control measures including diagnostic antigen(s) since most of the antigens have been selected based on reaction with antibodies without considering the immune responses in the host [24C27]. However, the precious mechanisms of infection including the possible apoptotic activities have not been revealed in the expression of cytokines and apoptosis-related buy 116649-85-5 genes, yet. Therefore, to understand the mechanism underlying the immune responses to cellular proteins. Cytokine production and the expression of genes associated with apoptosis were then analyzed as the first step of understanding in the induction of immune responses. Materials and methods Cloning and expression of genes Genes encoding outer membrane protein 28 (OMP28), malate dehydrogenase (mdh), elongation factor Ts (tsf), arginase (rocF), and metal-dependent hydrolase (0628) of 544 were amplified by PCR (Table?1), cloned, and expressed with a cold shock expression vector (pColdTM TF DNA; Takara, Japan) in DH5. Sequences of the primers used for this procedure are shown in Table?1. Nucleotide sequences of the genes were.