Computer-assisted mitotic count using a deep learning-based algorithm improves interobserver reproducibility and accuracy.

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intraobserver discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying or classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, computer-assisted review by pathologists may ensure reliability. In the present study, we compared partial (MC-ROI preselection) and full (additional visualization of MF candidates and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole-slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MFs, and improving classification against imposters. The interobserver consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected hotspot MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study demonstrate that computer assistance may lead to more reproducible and accurate MCs in ccMCTs.

Correction: Macrophage-expressed IFN-ß Contributes to Apoptotic Alveolar Epithelial Cell Injury in Severe Influenza Virus Pneumonia.

[This corrects the article DOI: 10.1371/journal.ppat.1003188.].

Macrophage-expressed IFN-ß contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia.

Influenza viruses (IV) cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs) has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM)-expressed IFN-ß significantly contributes to IV-induced alveolar epithelial cell (AEC) injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis-inducing ligand (TRAIL). Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-ß release in AM in a protein kinase R- (PKR-) and NF-κB-dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-ß and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-ß-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury.

Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

BACKGROUND: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. RESULTS: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. CONCLUSION: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Synthesis of porcine pCLCA2 protein during late differentiation of keratinocytes of epidermis and hair follicle inner root sheath.

Despite the discovery of the widely expressed CLCA (chloride channel regulators, calcium-activated) proteins more than 15 years ago, their seemingly diverse functions are still poorly understood. With the recent generation of porcine animal models for cystic fibrosis (CF), members of the porcine CLCA family are becoming of interest as possible modulators of the disease in the pig. Here, we characterize pCLCA2, the porcine ortholog of the human hCLCA2 and the murine mCLCA5, which are the only CLCA members expressed in the skin. Immunohistochemical studies with a specific antibody against pCLCA2 have revealed a highly restricted pCLCA2 protein expression in the skin. The protein is strictly co-localized with filaggrin and trichohyalin in the granular layer of the epidermis and the inner root sheath of the hair follicles, respectively. No differences have been observed between the expression patterns of wild-type pigs and CF transmembrane conductance regulator(-/-) pigs. We speculate that pCLCA2 plays an as yet undefined role in the structural integrity of the skin or, possibly, in specialized functions of the epidermis, including barrier or defense mechanisms.

Histological Characterization of Feline Bladder Urothelial Carcinoma.

Urothelial (transitional cell) carcinoma (UC) is the most common type of bladder cancer in humans, dogs and cats, although the incidence in cats is comparatively low. This retrospective study details the histopathological features of UC of the urinary bladder in 38 samples from 35 cats. Of the 38 samples, eight had a papillary architecture and in nine the tumour cells formed tubular or acinar structures. Tumour cell invasion of the bladder wall varied from confinement within the lamina propria or submucosa to transmural or extending to the serosa. The tumour stroma varied from sparse to abundant, with a scirrhous, myxomatous or mucinous appearance in eleven cases, three cases and one case, respectively. The degrees of tumour cell necrosis and inflammation were highly variable. We confirm that the histopathological features of bladder UC in cats have many similarities to the corresponding tumours in dogs and humans.

Peptidoglycan Recognition Protein 3 Does Not Alter the Outcome of Pneumococcal Pneumonia in Mice.

Pneumococci frequently cause community-acquired pneumonia, a disease with high mortality rates, particularly in young children and in the elderly. Endogenous antimicrobial peptides and proteins such as PGLYRP3 may contribute to the progression and outcome of this disease. Since increasing antibiotic resistant strains occur all over the world, these endogenous antimicrobial molecules are interesting new targets for future therapies. In this study, the expression pattern of PGLYRP3 was analyzed in alveolar epithelial cells, alveolar macrophages and neutrophils. Additionally, the function of PGLYRP3 during Streptococcus pneumoniae-induced pneumonia was investigated in a murine pneumococcal pneumonia model using PGLYRP3KO mice. PGLYRP3 is expressed in all selected cell types but pneumococcus-dependent induction of PGLYRP3 was observed only in neutrophils and alveolar macrophages. Interestingly, there were no significant differences in the bacterial loads within the lungs, the blood or the spleens, in the cytokine response, the composition of immune cells and the histopathology between wild type and PGLYRP3KO mice. Finally, we could neither observe significant differences in the clinical symptoms nor in the overall survival. Collectively, PGLYRP3 seems to be dispensable for the antibacterial defense during pneumococcal pneumonia.

No evidence of Sarcocystis calchasi involvement in mammalian meningoencephalitis of unknown origin.

Sarcocystis calchasi has recently been identified as the cause of pigeon protozoal encephalitis, PPE, a lethal brain disease in pigeons and parrots. While only avian species have been identified so far to be susceptible to this pathogen as definitive or intermediate hosts, we speculated whether mammals may be susceptible as well, as in Sarcocystis neurona and other related apicomplexan parasites. Specifically, we hypothesized its involvement in mammalian meningoencephalitis of unknown origin, MUO. A total of 143 archived formalin fixed, paraffin embedded brain samples with MUO from dogs, cats, pigs, cattle, sheep, guinea pigs, horses, goats, mice, raccoon, ferret, hamster, mink and maned wolf were examined pathohistologically and by PCR for parasitic stages or DNA, respectively, of Sarcocystis calchasi or other apicomplexan parasites. All samples had non-suppurative, lymphoplasmacytic and/or granulomatous encephalitis or meningoencephalitis typical of MUO with many similarities to PPE in pigeons. However, neither parasitic structures nor DNA of Sarcocystis calchasi or other apicomplexan parasites were detected. It thus appears that, despite histological similarities between mammalian MUO and pigeon PPE and despite seemingly high prevalence of PPE and a persistent threat by Sarcocystis calchasi in pigeons, based on histopathology and PCR there is no evidence for a role of this parasite in MUO in mammals as intermediate or aberrant hosts.

Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

The porcine chloride channel calcium-activated family member pCLCA4a mirrors lung expression of the human hCLCA4.

Pig models of cystic fibrosis (CF) have recently been established that are expected to mimic the human disease closer than mouse models do. The human CLCA (originally named chloride channels, calcium-activated) member hCLCA4 is considered a potential modifier of disease severity in CF, but its murine ortholog, mCLCA6, is not expressed in the mouse lung. Here, we have characterized the genomic structure, protein processing, and tissue expression patterns of the porcine ortholog to hCLCA4, pCLCA4a. The genomic structure and cellular protein processing of pCLCA4a were found to closely mirror those of hCLCA4 and mCLCA6. Similar to human lung, pCLCA4a mRNA was strongly expressed in porcine lungs, and the pCLCA4a protein was immunohistochemically detected on the apical membranes of tracheal and bronchial epithelial cells. This stands in sharp contrast to mouse mCLCA6, which has been detected exclusively in intestinal epithelia but not the murine lung. The results may add to the understanding of species-specific differences in the CF phenotype and support the notion that the CF pig model may be more suitable than murine models to study the role of hCLCA4.

Tissue and cellular expression patterns of porcine CFTR: similarities to and differences from human CFTR.

Emerging porcine models of cystic fibrosis (CF) are expected to mimic the human disease more closely than current mouse models do. However, little is known of the tissue and cellular expression patterns of the porcine CF transmembrane conductance regulator (pCFTR) and possible differences from human CFTR (hCFTR). Here, the expression pattern of pCFTR was systematically established on the mRNA and protein levels. Using specific anti-pCFTR antibodies, the majority of the protein was immunohistochemically detected on paraffin-embedded sections and on cryostate sections in the apical cytosol of intestinal crypt epithelial cells, nasal, tracheal, and bronchial epithelial cells, and other select, mostly glandular epithelial cells. Confocal laser scanning microscopy with co-localization of the Golgi marker 58K localized the protein in the cytosol between the Golgi apparatus and the apical cell membrane with occasional punctate or diffuse staining of the apical membrane. The tissue and cellular distribution patterns were confirmed by RT-PCR from whole tissue lysates or select cells after laser capture microdissection. Thus, expression of pCFTR was found to largely resemble that of hCFTR except for the kidney, brain, and cutaneous glands, which lack expression in pigs. Species-specific differences between pCFTR and hCFTR may become relevant for future interpretations of the CF phenotype in pig models.

Genomic, tissue expression, and protein characterization of pCLCA1, a putative modulator of cystic fibrosis in the pig.

Recent studies have identified members of the CLCA (chloride channels, calcium-activated) gene family as potential modulators of the cystic fibrosis (CF) phenotype, but differences between the human and murine CLCA genes and proteins may limit the use of murine CF models. Recently established pig models of CF are expected to mimic the human disease more closely than the available mouse models do. Here, we characterized the porcine CLCA gene locus, analyzed the expression pattern and protein processing of pCLCA1, and compared it to its human ortholog, hCLCA1. The porcine CLCA gene family is located on chromosome 4q25, with a broad synteny with the human and murine clca gene loci, except for a pig-specific gene duplication of pCLCA4. Using pCLCA1-specific antibodies, the protein was immunohistochemically localized in mucin-producing cells, including goblet cells and mucinous glands in the respiratory and alimentary tracts. Similar to hCLCA1, biochemical characterization of pCLCA1 identified a secreted soluble protein that could serve as an extracellular signaling molecule or functional constituent of the protective mucous layers. The results suggest that pCLCA1 shares essential characteristics of hCLCA1, supporting the pig model as a promising tool for studying the modulating role of pCLCA1 in the complex pathology of CF.