Bovine tuberculosis at a cattle-small ruminant-human interface in Meskan, Gurage region, Central Ethiopia

ABSTRACT: BACKGROUND: Bovine tuberculosis (BTB) is endemic in Ethiopian cattle. The aim of this study was to assess BTB prevalence at an intensive contact interface in Meskan Woreda (district) in cattle, small ruminants and suspected TB-lymphadenitis (TBLN) human patients. METHODS: The comparative intradermal test (CIDT) was carried out for all animals involved in the cross-sectional study and results interpreted using a < 4 mm and a < 2 mm cut-off. One PPD positive goat was slaughtered and lymph nodes subjected to culture and molecular typing. In the same villages, people with lymphadenitis were subjected to clinical examination. Fine needle aspirates (FNA) were taken from suspected TBLN and analyzed by smear microscopy and molecular typing. RESULTS: A total of 1214 cattle and 406 small ruminants were tested for BTB. In cattle, overall individual prevalence (< 2 mm cut-off) was 6.8% (CI: 5.4-8.5%) with 100% herd prevalence. Only three small ruminants (2 sheep and 1 goat) were reactors. The overall individual prevalence in small ruminants (< 2 mm cut-off) was 0.4% (CI: 0.03-5.1%) with 25% herd prevalence. Cattle from owners with PPD positive small ruminants were all PPD negative. 83% of the owners kept their sheep and goats inside their house at night and 5% drank regularly goat milk.FNAs were taken from 33 TBLN suspected cases out of a total of 127 screened individuals with lymph node swellings. Based on cytology results, 12 were confirmed TBLN cases. Nine out of 33 cultures were AFB positive. Culture positive samples were subjected to molecular typing and they all yielded M. tuberculosis. M. tuberculosis was also isolated from the goat that was slaughtered. CONCLUSIONS: This study highlighted a low BTB prevalence in sheep and goats despite intensive contact with cattle reactors. TBLN in humans was caused entirely by M. tuberculosis, the human pathogen. M. tuberculosis seems to circulate also in livestock but their role at the interface is unknow

Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes

Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

Breast Tumor Cells with PI3K Mutation or HER2 Amplification Are Selectively Addicted to Akt Signaling

Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin

CEACAM1 Negatively Regulates IL-1β Production in LPS Activated Neutrophils by Recruiting SHP-1 to a SYK-TLR4-CEACAM1 Complex

LPS-activated neutrophils secrete IL-1β by activation of TLR-4. Based on studies in macrophages, it is likely that ROS and lysosomal destabilization regulated by Syk activation may also be involved. Since neutrophils have abundant expression of the ITIM-containing co-receptor CEACAM1 and Gram-negative bacteria such as Neisseria utilize CEACAM1 as a receptor that inhibits inflammation, we hypothesized that the overall production of IL-1β in LPS treated neutrophils may be negatively regulated by CEACAM1. We found that LPS treated neutrophils induced phosphorylation of Syk resulting in the formation of a complex including TLR4, p-Syk, and p-CEACAM1, which in turn, recruited the inhibitory phosphatase SHP-1. LPS treatment leads to ROS production, lysosomal damage, caspase-1 activation and IL-1β secretion in neutrophils. The absence of this regulation in Ceacam1−/− neutrophils led to hyper production of IL-1β in response to LPS. The hyper production of IL-1β was abrogated by in vivo reconstitution of wild type but not ITIM-mutated CEACAM1 bone marrow stem cells. Blocking Syk activation by kinase inhibitors or RNAi reduced Syk phosphorylation, lysosomal destabilization, ROS production, and caspase-1 activation in Ceacam1−/− neutrophils. We conclude that LPS treatment of neutrophils triggers formation of a complex of TLR4 with pSyk and pCEACAM1, which upon recruitment of SHP-1 to the ITIMs of pCEACAM1, inhibits IL-1β production by the inflammasome. Thus, CEACAM1 fine-tunes IL-1β production in LPS treated neutrophils, explaining why the additional utilization of CEACAM1 as a pathogen receptor would further inhibit inflammation

Suppression of Ribosomal Function Triggers Innate Immune Signaling through Activation of the NLRP3 Inflammasome

Some inflammatory stimuli trigger activation of the NLRP3 inflammasome by inducing efflux of cellular potassium. Loss of cellular potassium is known to potently suppress protein synthesis, leading us to test whether the inhibition of protein synthesis itself serves as an activating signal for the NLRP3 inflammasome. Murine bone marrow-derived macrophages, either primed by LPS or unprimed, were exposed to a panel of inhibitors of ribosomal function: ricin, cycloheximide, puromycin, pactamycin, and anisomycin. Macrophages were also exposed to nigericin, ATP, monosodium urate (MSU), and poly I:C. Synthesis of pro-IL-ß and release of IL-1ß from cells in response to these agents was detected by immunoblotting and ELISA. Release of intracellular potassium was measured by mass spectrometry. Inhibition of translation by each of the tested translation inhibitors led to processing of IL-1ß, which was released from cells. Processing and release of IL-1ß was reduced or absent from cells deficient in NLRP3, ASC, or caspase-1, demonstrating the role of the NLRP3 inflammasome. Despite the inability of these inhibitors to trigger efflux of intracellular potassium, the addition of high extracellular potassium suppressed activation of the NLRP3 inflammasome. MSU and double-stranded RNA, which are known to activate the NLRP3 inflammasome, also substantially inhibited protein translation, supporting a close association between inhibition of translation and inflammasome activation. These data demonstrate that translational inhibition itself constitutes a heretofore-unrecognized mechanism underlying IL-1ß dependent inflammatory signaling and that other physical, chemical, or pathogen-associated agents that impair translation may lead to IL-1ß-dependent inflammation through activation of the NLRP3 inflammasome. For agents that inhibit translation through decreased cellular potassium, the application of high extracellular potassium restores protein translation and suppresses activation of the NLRP inflammasome. For agents that inhibit translation through mechanisms that do not involve loss of potassium, high extracellular potassium suppresses IL-1ß processing through a mechanism that remains undefined

Sublethal Doses of Anthrax Lethal Toxin on the Suppression of Macrophage Phagocytosis

BACKGROUND: Lethal toxin (LT), the major virulence factor produced by Bacillus anthracis, has been shown to suppress the immune system, which is beneficial to the establishment of B. anthracis infections. It has been suggested that the suppression of MEK/MAPK signaling pathways of leukocytes contributes to LT-mediated immunosuppressive effects. However, the involvement of MAPK independent pathways has not been clearly elucidated; nor has the crucial role played by LT in the early stages of infection. Determining whether LT exerts any pathological effects before being enriched to an MEK inhibitory level is an important next step in the furtherance of this field. METHODOLOGY/PRINCIPAL FINDINGS: Using a cell culture model, we determined that low doses of LT inhibited phagocytosis of macrophages, without influencing MAPK pathways. Consistent low doses of LT significantly suppressed bacterial clearance and enhanced the mortality of mice with bacteremia, without suppressing the MEK1 of splenic and peripheral blood mononuclear cells. CONCLUSION/SIGNIFICANCE: These results suggest that LT suppresses the phagocytes in a dose range lower than that required to suppress MEK1 in the early stages of infection