The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus

The NRAMP1 gene encodes a divalent cation transporter, located in the phagolysosomal membrane of macrophages, that has been associated with resistance to intracellular pathogens. In cattle, natural resistance against brucellosis has been associated with polymorphisms at the 3 untranslated region (3UTR) of the NRAMP1 gene, which are detectable by single-strand conformational analysis (SSCA). This study aimed to evaluate the association between NRAMP1 3UTR polymorphisms and resistance against bovine brucellosis in experimental and natural infections. In experimentally infected pregnant cows, abortion occurred in 42.1% of cows with a resistant genotype (SSCA r ; n ‫؍‬ 19) and in 43.1% of those with a susceptible genotype (SSCA s ; n ‫؍‬ 23). Furthermore, no association between intensity of pathological changes and genotype was detected. In a farm with a very high prevalence of bovine brucellosis, the percentages of strains of the SSCA r genotype were 86 and 84% in serologically positive (n ‫؍‬ 64) and negative (n ‫؍‬ 36) cows, respectively. Therefore, no association was found between the NRAMP1-resistant allele and the resistant phenotype in either experimental or naturally occurring brucellosis. To further support these results, bacterial intracellular survival was assessed in bovine monocyte-derived macrophages from cattle with either the resistant or susceptible genotype. In agreement with our previous results, no difference was observed in the rates of intracellular survival of B. abortus within macrophages from cattle with susceptible or resistant genotypes. Taken together, these results indicate that these polymorphisms at the NRAMP1 3UTR do not affect resistance against B. abortus in cattle and that they are therefore not suitable markers of natural resistance against bovine brucellosis.

Section: Resultsmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 80%

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NRAMP13′ Untranslated Region Polymorphisms Are Not Associated with Natural Resistance toBrucella abortusin Cattle

Paixão¹,

Poester

Neta³

et al. 2007

“…PBMC were then removed from the interface between the plasma and Percoll solution, pooled, diluted in 50 ml of citrated PBS, and centrifuged at 500 ϫ g for 15 min. The cell pellets were then washed three times with citrated PBS at 500 ϫ g for 10 min, suspended in CRPMI containing 4% autologous serum to facilitate adherence, placed at 5 ϫ 10 6 PBMC in 50-ml Teflon flasks (6), and cultured 4 h at 37°C under 5% CO 2 . Nonadherent cells were then removed by three washes with prewarmed PBS, and adherent monocytes were cultured just as described previously in CRPMI plus 12% autologous serum for 10 to 12 days until they differentiated to macrophages.…”

Section: Bacteriamentioning

confidence: 99%

Apoptosis-Inducing Factor Participation in Bovine MacrophageMycobacterium bovis-Induced Caspase-Independent Cell Death

Vega-Manriquez¹,

López-Vidal

Morán

et al. 2007

Cell death (CD) is a very important process in the development and conservation of multicellular organisms maintaining the equilibrium between life and death (10). Disorders in CD lead to (i) embryogenic problems after exposure to a wide variety of teratogens, (ii) neurodegenerative diseases, and (iii) cancer (17,28). Apoptosis is a type of CD that is morphologically defined by cellular and nuclear shrinkage, chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies. It is generally accepted that execution of the apoptotic pathway relies on the activation of a family of cysteine proteases, also known as caspases (21). However, activation of caspases is not a hallmark of every cell undergoing a death process. Several authors have shown that cell death occurs even in the presence of caspase inhibitors, giving rise to the concept of caspase-independent cell death (CICD). Cells experiencing CICD feature morphological changes resembling apoptosis but do not engage caspases to disassemble the cell (1,5,21).In one of the first CICD reports, Xiang et al. (40) proved that N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-Vad-fmk), a general caspase inhibitor, blocked caspase proteolytic activity but did not prevent the suicide of Jurkat cells overexpressing BAX. In addition, data from a different study demonstrated that BH3 domain-only molecules kill cells deficient in the downstream effectors Apaf-1, caspase 9, and caspase 3 by a caspase-independent process of mitochondrial dysfunction (9). CICD has also been associated with hostpathogen interactions; for instance, it has been reported that soluble toxins of Escherichia coli activated CD via extracellular signal-regulated kinase (ERK) signaling in renal proximal tubular epithelial cells without caspase involvement (8). Additionally, Salmond et al. (36) demonstrated that the B subunit of E. coli heat-labile enterotoxin (EtxB) induced a rapid loss of mitochondrial membrane potential and cell viability, which were not affected by caspase inhibitors, in CD8 ϩ T cells. Moreover, Carrero et al. (7) proved that listeriolysin O induced loss of mitochondrial membrane potential and that exposure of phosphatidylserine on the cell surface was not abrogated by the incubation of a pan-caspase inhibitor with murine T cells. Finally, pneumococci and the major cytotoxins H 2 O 2 and pneumolysin have been shown to induce apoptosis-like programmed cell death of cerebral endothelial cells in the presence of rapid mitochondrial damage that was followed by translocation of mitochondrial apoptosis-inducing factor (AIF) into the cytoplasm (3).

“…Extensive similarities between bovine macrophage function and resistance to brucellosis and murine macrophage function and resistance to Salmonella, Mycobacterium, and Leishmania have been documented (9,33,49,57). Specifically, macrophages from cattle that are genetically resistant to a stringent in vivo challenge with virulent B. abortus S2308 are superior in their capacity to control the intracellular replication of not only B.…”

mentioning

confidence: 99%

Stable Transfection of the BovineNRAMP1Gene into Murine RAW264.7 Cells: Effect onBrucella abortusSurvival

Barthel¹,

Feng²,

Piedrahita³

et al. 2001

Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. Bovine NRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance-and susceptibility-associated alleles of bovine NRAMP1 as stable transgenes under the regulatory control of the bovine NRAMP1 promoter in the murine RAW264.