SIV-induced Translocation of Bacterial Products in the Liver Mobilizes Myeloid Dendritic and Natural Killer Cells Associated With Liver Damage.

Disruption of the mucosal epithelium during lentivirus infections permits translocation of microbial products into circulation, causing immune activation and driving disease. Although the liver directly filters blood from the intestine and is the first line of defense against gut-derived antigens, the effects of microbial products on the liver are unclear. In livers of normal macaques, minute levels of bacterial products were detectable, but increased 20-fold in simian immunodeficiency virus (SIV)-infected animals. Increased microbial products in the liver induced production of the chemoattractant CXCL16 by myeloid dendritic cells (mDCs), causing subsequent recruitment of hypercytotoxic natural killer (NK) cells expressing the CXCL16 receptor, CXCR6. Microbial accumulation, mDC activation, and cytotoxic NK cell frequencies were significantly correlated with markers of liver damage, and SIV-infected animals consistently had evidence of hepatitis and fibrosis. Collectively, these data indicate that SIV-associated accumulation of microbial products in the liver initiates a cascade of innate immune activation, resulting in liver damage.

Squamous cell carcinoma of the anal sac in a spotted hyena (Crocuta crocuta).

A 25-yr-old spayed female spotted hyena (Crocuta crocuta) developed intermittent right pelvic limb lameness that persisted following conservative medical therapy. No obvious musculoskeletal lesions were noted on initial physical exam; however, spinal radiography was suspicious for possible intervertebral degenerative joint disease or discospondylitis. Despite prolonged medical therapy, the lameness progressed to minimal weight bearing and marked muscle atrophy of the right pelvic limb. Electromyography showed spontaneous activity in the muscles of right sciatic nerve distribution. Sensory and motor nerve conduction velocities in the right tibial and peroneal nerves were undetectable and markedly reduced, respectively. A magnetic resonance imaging (MRI) scan revealed a large, space-occupying mass on the right side of the sacrum and pelvis. Antemortem fine-needle aspiration of the mass and postmortem histopathology resulted in diagnosis of a high-grade squamous cell carcinoma of the anal sac. Squamous cell carcinoma of the anal sac is very rare in domestic dogs and previously unreported in spotted hyenas.

Orthotopic ureterocele with concurrent ureteral urothelial carcinoma in a dog.

A ureterocele is a rare congenital anomaly with cystic dilation of the terminal segment of the ureter entirely within the bladder (orthotopic) or associated with ectopic ureter (ectopic). Its aetiology has not been fully clarified; however, it may involve genetic or acquired factors. Urothelial carcinoma (UC) is the most common type of canine urinary tract neoplasm, among which over 90% of cases are invasive. The non-papillary (flat) non-infiltrating form accounts for a very small percentage of canine UCs and is considered carcinoma in situ (CIS). The neoplastic cells of CIS remain within the ureteral mucosa and do not breach the basement membrane. UCs originating from the canine ureter are extremely rare, and no report of a ureteral UC concurrently occurring with a ureterocele has been reported. A 7-year-old castrated male Maltese dog weighing 3.5 kg was referred with a 2-week history of lethargy, anorexia, pollakiuria and intermittent panting. The dog underwent open surgery for removal of bladder calculi 2 years prior, and at the time of the surgery, no other urinary system abnormalities were identified. Ultrasonographic and computed tomographic scans revealed a severely enlarged right kidney and ureter with a ureterocele on the ipsilateral side. A diagnosis of an orthotopic ureterocele causing hydronephrosis and hydroureter was established. Complete nephroureterectomy and ureterocelectomy using the marsupialisation technique were performed. The postoperative histological examination of the excised tissues showed a multifocal carcinoma in situ (non-papillary non-infiltrating UC) in the proximal ureter and a fluid-filled kidney with a thin rim of fibrotic renal tissue. No neoplastic changes were observed in the ureterocele tissue. Postoperatively, the dog recovered rapidly without complications except temporary urinary incontinence, and no evidence of tumour recurrence was detected by ultrasonography performed 6 months after surgery. This case report describes the first case of a dog with an orthotopic ureterocele and ureteral UC, which occurred concurrently at the ipsilateral side of the ureter. The condition was successfully managed with a nephroureterectomy and partial ureterocelectomy.

Agreement in Histological Assessment of Mitotic Activity Between Microscopy and Digital Whole Slide Images Informs Conversion for Clinical Diagnosis.

Validating digital pathology as substitute for conventional microscopy in diagnosis remains a priority to assure effectiveness. Intermodality concordance studies typically focus on achieving the same diagnosis by digital display of whole slide images and conventional microscopy. Assessment of discrete histological features in whole slide images, such as mitotic figures, has not been thoroughly evaluated in diagnostic practice. To further gauge the interchangeability of conventional microscopy with digital display for primary diagnosis, 12 pathologists examined 113 canine naturally occurring mucosal melanomas exhibiting a wide range of mitotic activity. Design reflected diverse diagnostic settings and investigated independent location, interpretation, and enumeration of mitotic figures. Intermodality agreement was assessed employing conventional microscopy (CM40×), and whole slide image specimens scanned at 20× (WSI20×) and at 40× (WSI40×) objective magnifications. An aggregate 1647 mitotic figure count observations were available from conventional microscopy and whole slide images for comparison. The intraobserver concordance rate of paired observations was 0.785 to 0.801; interobserver rate was 0.784 to 0.794. Correlation coefficients between the 2 digital modes, and as compared to conventional microscopy, were similar and suggest noninferiority among modalities, including whole slide image acquired at lower 20× resolution. As mitotic figure counts serve for prognostic grading of several tumor types, including melanoma, 6 of 8 pathologists retrospectively predicted survival prognosis using whole slide images, compared to 9 of 10 by conventional microscopy, a first evaluation of whole slide image for mitotic figure prognostic grading. This study demonstrated agreement of replicate reads obtained across conventional microscopy and whole slide images. Hence, quantifying mitotic figures served as surrogate histological feature with which to further credential the interchangeability of whole slide images for primary diagnosis.

Automated Computational Detection, Quantitation, and Mapping of Mitosis in Whole-Slide Images for Clinically Actionable Surgical Pathology Decision Support.

BACKGROUND: Determining mitotic index by counting mitotic figures (MFs) microscopically from tumor areas with most abundant MF (hotspots [HS]) produces a prognostically useful tumor grading biomarker. However, interobserver concordance identifying MF and HS can be poorly reproducible. Immunolabeling MF, coupled with computer-automated counting by image analysis, can improve reproducibility. A computational system for obtaining MF values across digitized whole-slide images (WSIs) was sought that would minimize impact of artifacts, generate values clinically relatable to counting ten high-power microscopic fields of view typical in conventional microscopy, and that would reproducibly map HS topography. MATERIALS AND METHODS: Relatively low-resolution WSI scans (0.50 µm/pixel) were imported in grid-tile format for feature-based MF segmentation, from naturally occurring canine melanomas providing a wide range of proliferative activity. MF feature extraction conformed to anti-phospho-histone H3-immunolabeled mitotic (M) phase cells. Computer vision image processing was established to subtract key artifacts, obtain MF counts, and employ rotationally invariant feature extraction to map MF topography. RESULTS: The automated topometric HS (TMHS) algorithm identified mitotic HS and mapped select tissue tiles with greatest MF counts back onto WSI thumbnail images to plot HS topographically. Influence of dye, pigment, and extraneous structure artifacts was minimized. TMHS diagnostic decision support included image overlay graphics of HS topography, as well as a spreadsheet and plot of tile-based MF count values. TMHS performance was validated examining both mitotic HS counting and mapping functions. Significantly correlated TMHS MF mapping and metrics were demonstrated using repeat analysis with WSI in different orientation (R 2 = 0.9916) and by agreement with a pathologist (R 2 = 0.8605) as well as through assessment of counting function using an independently tuned object counting algorithm (OCA) (R 2 = 0.9482). Limits of agreement analysis support method interchangeability. MF counts obtained led to accurate patient survival prediction in all (n = 30) except one case. By contrast, more variable performance was documented when several pathologists examined similar cases using microscopy (pair-wise correlations, rho range = 0.7597-0.9286). CONCLUSIONS: Automated TMHS MF segmentation and feature engineering performance were interchangeable with both observer and OCA in digital mode. Moreover, enhanced HS location accuracy and superior method reproducibility were achieved using the automated TMHS algorithm compared to the current practice employing clinical microscopy.

The effects of aspirated thickened water on survival and pulmonary injury in a rabbit model.

OBJECTIVES/HYPOTHESIS: Liquid thickeners are one of the most frequently utilized treatment strategies for persons with oropharyngeal swallowing dysfunction. The effect of commercially available thickeners on lung injury is uncertain. The purpose of this study was to compare the effects of aspiration of water alone, xanthan gum (XG)-thickened water, and cornstarch (CS)-thickened water on survival and lung morphology in a rabbit model. STUDY DESIGN: Animal model. Prospective small animal clinical trial. METHODS: Adult New Zealand White rabbits (n = 24) were divided into three groups of eight rabbits. The groups underwent 3 consecutive days of 1.5 mL/kg intratracheal instillation of water (n = 8), XG-thickened water (n = 8), and CS-thickened water (n = 8). The animals were euthanized on day 4, and survival and pulmonary histopathology were compared between groups. RESULTS: In all, 12.5% of rabbits (n = 8) instilled with CS-thickened water survived until the endpoint of the study (day 4). All animals instilled with water (n = 8) or XG-thickened water (n = 8) survived. A mild increase in intra-alveolar hemorrhage was observed for the animals instilled with CS-thickened water compared to the other groups (P < .05). In the groups that survived to the endpoint of the study, instillation of water thickened with XG resulted in greater pulmonary inflammation, pulmonary interstitial congestion, and alveolar edema than water alone (P < .05). CONCLUSIONS: These data suggest that 3 consecutive days of 1.5 mg/kg of aspirated CS-thickened water are fatal, and that XG-thickened water is more injurious than aspirated water alone. Additional research is necessary to further delineate the dangers of aspirated thickened liquids. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:327-331, 2018.

Euthanasia by CO2 inhalation affects potassium levels in mice.

We and others frequently have noted serum potassium levels of 8.0 +/- 0.85 mEq/L or greater in laboratory mice; this concentration has even been published as the upper limit of a 'normal' reference range. However, if bone fide, this potassium concentration would be incompatible with life in all species. We investigated conditions frequently encountered in the research setting to distinguish artifactual from true hyperkalemia. Variables evaluated included site of collection, time allowed for clot formation before serum separation, time elapsed between collection and analysis of samples collected in a serum separator tube, precollection method of anesthesia, and euthanasia technique. Serum potassium was measured from 75 C57BL/6NTac 10-wk-old female mice and divided into at least 5 mice per variable. Animals were euthanized by exsanguination immediately after terminal CO2 or ketamine-xylazine (KX) administration. Mice euthanized with CO2 had higher mean serum potassium (7.0 +/- 0.5 mEq/L) and range serum potassium (6.0 to 8.1 mEq/L) than did KX-treated mice. CO2 inhalation resulted in significantly lower blood pH (6.9 +/- 0.1), higher pCO2 (153.3 +/- 38.8 mm Hg), and higher lactate levels (3.9 +/- 0.9 mmol/L) than did KX anesthesia followed by exsanguination. These results suggest that antemortem respiratory acidosis from CO2 administration causes artifactual hyperkalemia in mice. Therefore, blood collection under KX anesthesia is preferable over CO2 inhalation to obtain accurate potassium values from mice.