Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFβ. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease

Bedrock geology of DFDP-2B, central Alpine Fault, New Zealand

<p>During the second phase of the Alpine Fault, Deep Fault Drilling Project (DFDP) in the Whataroa River, South Westland, New Zealand, bedrock was encountered in the DFDP-2B borehole from 238.5–893.2 m Measured Depth (MD). Continuous sampling and meso- to microscale characterisation of whole rock cuttings established that, in sequence, the borehole sampled amphibolite facies, Torlesse Composite Terrane-derived schists, protomylonites and mylonites, terminating 200–400 m above an Alpine Fault Principal Slip Zone (PSZ) with a maximum dip of 62°. The most diagnostic structural features of increasing PSZ proximity were the occurrence of shear bands and reduction in mean quartz grain sizes. A change in composition to greater mica:quartz + feldspar, most markedly below c. 700 m MD, is inferred to result from either heterogeneous sampling or a change in lithology related to alteration. Major oxide variations suggest the fault-proximal Alpine Fault alteration zone, as previously defined in DFDP-1 core, was not sampled.</p

Petrophysical, Geochemical, and Hydrological Evidence for Extensive Fracture-Mediated Fluid and Heat Transport in the Alpine Fault's Hanging-Wall Damage Zone

International audienceFault rock assemblages reflect interaction between deformation, stress, temperature, fluid, and chemical regimes on distinct spatial and temporal scales at various positions in the crust. Here we interpret measurements made in the hanging‐wall of the Alpine Fault during the second stage of the Deep Fault Drilling Project (DFDP‐2). We present observational evidence for extensive fracturing and high hanging‐wall hydraulic conductivity (∼10−9 to 10−7 m/s, corresponding to permeability of ∼10−16 to 10−14 m2) extending several hundred meters from the fault's principal slip zone. Mud losses, gas chemistry anomalies, and petrophysical data indicate that a subset of fractures intersected by the borehole are capable of transmitting fluid volumes of several cubic meters on time scales of hours. DFDP‐2 observations and other data suggest that this hydrogeologically active portion of the fault zone in the hanging‐wall is several kilometers wide in the uppermost crust. This finding is consistent with numerical models of earthquake rupture and off‐fault damage. We conclude that the mechanically and hydrogeologically active part of the Alpine Fault is a more dynamic and extensive feature than commonly described in models based on exhumed faults. We propose that the hydrogeologically active damage zone of the Alpine Fault and other large active faults in areas of high topographic relief can be subdivided into an inner zone in which damage is controlled principally by earthquake rupture processes and an outer zone in which damage reflects coseismic shaking, strain accumulation and release on interseismic timescales, and inherited fracturing related to exhumation

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

A window into thousands of earthquakes: Results from the Deep Fault Drilling Project (DFDP)

In 1941, two geologists, Harold Wellman and Richard Willett, traversed the length of Westland, mapping what would become one of the most influential continental faults in the world, New Zealand’s Alpine Fault (Wellman &amp; Willet 1942). The Alpine Fault strikes down the western edge of the Southern Alps, a youthful mountain range on New Zealand’s South Island (Fig 1). Here, collision between the Australian and Pacific Plates forms peaks over 3000 m in elevation which trap rain-laden clouds, resulting in 5–15 m of precipitation a year in central and south Westland. Driven by gravity, the rain and snow migrate into fractures and voids along the Alpine Fault, becoming heated and saturated with reactive ions along the way. Within the fault zone, these fluids play a fundamental role in the processes that drive earthquake nucleation and rupture propagation. Measurements made, and rocks recovered, from boreholes drilled during phases one and two of the Deep Fault Drilling Project (DFDP) have enabled scientists to document and quantify these processes for the first time

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne’s disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFb. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne’s disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne’s disease

Fluid flux in fractured rock of the Alpine fault hanging-wall determined from temperature logs in the DFDP-2B borehole, New Zealand

Sixteen temperature logs were acquired during breaks in drilling of the 893m-deep DFDP-2B borehole, which is in the Alpine Fault hanging-wall. The logs record various states of temperature recovery after thermal disturbances induced by mud circulation. The long-wavelength temperature signal in each log was estimated using a sixth-order polynomial, and residual (reduced) temperature logs were analyzed by fitting discrete template wavelets defined by depth, amplitude, and width parameters. Almost two hundred wavelets are correlated between multiple logs. Anomalies generally have amplitudes &lt;1°C, and downhole widths &lt;20m. The largest amplitudes are found in the first day after mud circulation stops, but many anomalies persist with similar amplitude for up to 15 days. Our models show that thermal and hydraulic diffusive processes are dominant during the first few days of re-equilibration after mud circulation stops, and fluid advection of heat in the surrounding rock produces temperature anomalies that may persist for several weeks. Models indicate that the fluid flux normal to the borehole within fractured zones is of order 10−7 to 10−6 m s−1, which is 2–3 orders of magnitude higher than the regional flux. Our approach could be applied more widely to boreholes, as it uses the thermal re-equilibration phase to derive useful information about the surrounding rock mass and its fluid flow regime.</p

The significance of heat transport by shallow fluid flow at an active plate boundary; the Southern Alps, New Zealand

Fluid flow can influence fault behavior. Here we quantify the role of groundwater heat advection in establishing the thermal structure of the Alpine Fault, a major tectonic boundary in southern New Zealand that accommodates most of the motion between the Australian and Pacific Plates. Convergence on the Alpine Fault has rapidly uplifted the Southern Alps, resulting in high geothermal gradients and a thin seismogenic zone. A new equilibrium temperature profile from the 818 m‐deep Deep Fault Drilling Project 2B (DFDP‐2B) borehole has been interrogated using one‐dimensional analytical models of fluid and rock advection. Models indicate a total heat flux of 720 mW·m‐2 results from groundwater flow with Darcy velocities approximating to 7.8×10‐10 m·s‐1. Groundwaters advect significantly more heat than rock advection in the shallow orogen (&lt;6 km depth) and are the major control on the subsurface temperature field