Mesenchymal Stromal Cell Secretome Is Affected by Tissue Source and Donor Age.

Variation in mesenchymal stromal cell (MSC) function depending on their origin is problematic, as it may confound clinical outcomes of MSC therapy. Current evidence suggests that the therapeutic benefits of MSCs are attributed to secretion of biologically active factors (secretome). However, the effect of donor characteristics on the MSC secretome remains largely unknown. Here, we examined the influence of donor age, sex, and tissue source, on the protein profile of the equine MSC secretome. We used dynamic metabolic labeling with stable isotopes combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify secreted proteins in MSC conditioned media (CM). Seventy proteins were classified as classically secreted based on the rate of label incorporation into newly synthesized proteins released into the extracellular space. Next, we analyzed CM of bone marrow- (n = 14) and adipose-derived MSCs (n = 16) with label-free LC-MS/MS. Clustering analysis of 314 proteins detected across all samples identified tissue source as the main factor driving variability in MSC CM proteomes. Linear modelling applied to the subset of 70 secreted proteins identified tissue-related difference in the abundance of 23 proteins. There was an age-related decrease in the abundance of CTHRC1 and LOX, further validated with orthogonal techniques. Due to the lack of flow cytometry characterization of MSC surface markers, the analysis could not account for the potential effect of cell population heterogeneity. This study provides evidence that tissue source and donor age contribute to differences in the protein composition of MSC secretomes which may influence the effects of MSC therapy.

Serial Interferon-Gamma Release Assay (IGRA) Testing to Monitor Treatment Responses in Cases of Feline Mycobacteriosis.

The interferon-gamma release assay (IGRA) is used to diagnose cases of feline mycobacteriosis, but the use of serial testing to monitor treatment responses has not been evaluated in this species. From a population of cats that underwent IGRA testing for diagnostic investigation, individuals were identified with a pre- and end-of-treatment IGRA that passed control thresholds. The number of cats which reverted to negative at the end-of-treatment IGRA, changes in paired antigen-specific optical density (OD) values and differences in the pre-treatment antigen-specific OD values for those which underwent reversion were compared. Factors to explain reversion or recurrence of disease post-treatment were explored. Four of 18 cats (22%) reverted to negativity at the point of clinical resolution (p = 0.33), there was no difference in paired antigen-specific OD values (p ≥ 0.12), and cats that reverted did not have a lower baseline OD value (p = 0.63). No statistically significant factors were identified to predict reversion (p ≥ 0.08). Remaining positive at the end of treatment IGRA was not associated with recurrence of disease post-treatment (p = 0.34). Overall, these data suggest there is limited value in the use of the IGRA to monitor treatment responses in cats.

Diagnostic accuracy of the interferon-gamma release assay (IGRA) for cases of feline mycobacteriosis.

The aim of this study was to evaluate the sensitivity and specificity of the interferon-gamma release assay (IGRA) for diagnosing infections with members of the Mycobacterium (M.) tuberculosis-complex (MTBC) and non-tuberculous mycobacteria (NTM) in domestic cats, and to generate defined feline-specific cut-off values using receiver operating characteristic (ROC) curve analysis to improve test performance. Records of 594 cats that had been tested by IGRA were explored to identify individuals that had a culture and/or polymerase chain reaction (PCR)-confirmed case of mycobacterial disease, and those that had a final diagnosis of non-mycobacterial disease. A total of 117 cats - 80 with mycobacterial disease and 37 diagnosed with a condition other than mycobacteriosis - were identified for further detailed analysis. This population was used to estimate test sensitivity and specificity, as well as likelihood ratios for the IGRA to correctly identify a cat with or without mycobacterial disease. Agreement between IGRA results and culture/PCR using current and proposed new cut-off values was also determined. ROC analysis of defined confirmed infected and non-mycobacterial disease control cats allowed an adjustment of current test cut-offs that increased the overall test sensitivity for MTBC infections from 83.1 % (95 % confidence interval [CI]: 71.5-90.5 %) to 90.2 % (95 % CI: 80.2-95.4%), and M. bovis infection from 43 % (95 % CI: 28.2-60.7%) to 68 % (95 % CI: 51.4-82.1%) while maintaining high test specificity (100 % in both cases). Overall agreement between IGRA results and culture/PCR, while recognising that neither culture nor PCR tests have perfect sensitivity, improved from weak (κ = 0.57) to moderate (κ = 0.71) using new proposed IGRA test cut-off values. Application of these results, based upon the statistical analysis of accumulated test data, can improve the diagnostic performance of the feline IGRA, particularly for identifying infections with M. bovis, without compromising specificity.

An outbreak of tuberculosis due to Mycobacterium bovis infection in a pack of English Foxhounds (2016-2017).

Mycobacterium bovis can cause tuberculosis (TB) in social mammals including lions, cattle and man, but canine infections are considered rare. In 2016/17 we investigated a M. bovis TB outbreak in a pack of approximately 180 Foxhounds within the bovine TB Edge Area of England. We employed a combination of immunological tests including an interferon gamma release assay (IGRA) and a serological assay (DPP VetTB, Chembio). Test-positive hounds were euthanased and subjected to post-mortem examination (PME). Overall 164 hounds were tested; 97 (59%) responded positively to at least one test. Eighty-five (52%) dogs responded to M. bovis antigens by IGRA while only 21 (12.9%) had detectable serological responses. At PME three hounds (3.1%) had visible lesions (VL) due to M. bovis infection, later confirmed by culture. Samples from 24 non-VL hounds were cultured and M. bovis infection was confirmed in a further three hounds (11%). This study is the first investigation and report of an outbreak of M. bovis TB in a canine species. We establish that, in principle, diagnostic tests used for identifying infected individuals of other species can effectively be used in the dog. Further work is urgently needed to establish the sensitivity and specificity of the testing approach used in this study for future clinical application.

Comparison of Fluid-Fluid Interfacial Areas Measured with X-ray Microtomography and Interfacial Partitioning Tracer Tests for the same Samples.

Two different methods are currently used for measuring interfacial areas between immiscible fluids within 3-D porous media, high-resolution microtomographic imaging and interfacial partitioning tracer tests (IPTT). Both methods were used in this study to measure non-wetting/wetting interfacial areas for a natural sand. The microtomographic imaging was conducted on the same packed columns that were used for the IPTTs. This is in contrast to prior studies comparing the two methods, for which in all cases different samples were used for the two methods. In addition, the columns were imaged before and after the IPTTs to evaluate the potential impacts of the tracer solution on fluid configuration and attendant interfacial area. The interfacial areas measured using IPTT are ~5 times larger than the microtomographic-measured values, which is consistent with previous work. Analysis of the image data revealed no significant impact of the tracer solution on NAPL configuration or interfacial area. Other potential sources of error were evaluated, and all were demonstrated to be insignificant. The disparity in measured interfacial areas between the two methods is attributed to the limitation of the microtomography method to characterize interfacial area associated with microscopic surface roughness due to resolution constraints.

The impact of transitions between two-fluid and three-fluidphases on fluid configuration and fluid-fluid interfacial areain porous media.

Multiphase-fluid distribution and flow is inherent in numerous areas of hydrology. Yet, pore-scale characterization of transitions between two and three immiscible-fluids is limited. The objective of this study was to examine the impact of such transitions on the pore-scale configuration of organic liquid in a multi-fluid system comprising natural porous media. Three-dimensional images of an organic liquid (trichloroethene) in two-phase (organic-liquid/water) and three-phase (air/organic-liquid/water) systems were obtained using X-ray microtomography before and after drainage and imbibition. Upon transition from a two-phase to a three-phase system, a significant portion of the organic liquid (intermediate wetting fluid) was observed to exist as lenses and films in contact with air (nonwetting fluid). In these cases, the air was either encased by or contiguous to the organic liquid. The presence of air resulted in an increase in the surface-area-to-volume ratios for the organic-liquid blobs. Upon imbibition, the air was displaced downgradient, and concomitantly, the morphology of the organic-liquid blobs no longer in contact with air reverted to that characteristic of a two-phase distribution (i.e., more spherical blobs and ganglia). This change in morphology resulted in a reduction in the surface-area-to-volume ratio. These results illustrate the impact of transitions between two-phase and three-phase conditions on fluid configuration, and they demonstrate the malleable nature of fluid configuration under dynamic, multiphase-flow conditions. The results have implications for characterizing and modeling pore-scale flow and mass-transfer processes.

Expression of putative markers of pluripotency in equine embryonic and adult tissues.

Expression of several putative markers of pluripotency (OCT4, SOX2, NANOG, LIN28A, REX1, DNMT3B and TERT) was examined in a range of equine tissues, including early embryos, induced pluripotent stem cells (iPSCs), testis, adipose- and bone marrow-derived mesenchymal stromal cells (MSCs), and keratinocytes. Transcript levels of all markers were highest in embryos and iPSCs and, except for SOX2, were very low or undetectable in keratinocytes. Mean expression levels of all markers were lower in testis than in embryos or iPSCs and, except for DNMT3B, were higher in testis than in MSCs. Expression of OCT4, NANOG and DNMT3B, but not the other markers, was detected in MSCs. Of all markers analysed, only LIN28A, REX1 and TERT were associated exclusively with pluripotent cells in the horse.