YAP-Driven Malignant Reprogramming of Epithelial Stem Cells at Single Cell Resolution.

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ cell tracing approaches with spatiotemporally controlled oncogene activation and tumor suppressor inhibition to unveil the processes underlying oral epithelial progenitor cell reprogramming into cancer stem cells (CSCs) at single cell resolution. This revealed the rapid emergence of a distinct stem-like cell state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal (pEMT) invasive gene programs. Interestingly, CSCs harbor limited cell autonomous invasive capacity, but instead recruit myeloid cells to remodel the basement membrane and ultimately initiate tumor invasion. CSC transcriptional programs are conserved in human carcinomas and associated with poor patient survival. These findings illuminate the process of cancer initiation at single cell resolution, thus identifying candidate targets for early cancer detection and prevention.

Direct reprogramming of oral epithelial progenitor cells to cancer stem cells at single cell resolution in vivo.

Tumor initiation represents the initial step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Most studies investigating cancer-driving mechanisms in solid tumors rely on analyses of established malignant lesions, and thus cannot directly capture processes underlying the reprogramming of normal progenitor cells into cancer cells. Here, using spatiotemporally controlled oncogene expression in a genetically engineered system we demonstrate that concomitant YAP activation and HPV E6-E7 -mediated inhibition of tumor suppressive pathways is sufficient to rapidly reprogram oral epithelial progenitor cells (OEPCs) into cancer stem cells (CSCs). Single cell analyses of these nascent CSCs revealed hallmark transcriptional programs driving tumor initiation. Importantly, these CSC-enriched expression signatures distinguish normal tissue from malignant head and neck tumors and are associated with poor patient survival. Elucidating mechanisms underlying OEPC to CSC reprogramming may offer new insights to halt the conversion of premalignant cells into invasive carcinoma. HIGHLIGHTS: YAP and HPV E6-E7 reprogram oral epithelial progenitor cells into cancer stem cells. Single cell analyses reveal the transcriptional architecture of tumor initiation.CSC transcriptional programs distinguish normal tissue from carcinoma.CSC signatures are associated with poor head and neck cancer survival.

An outbreak of systemic chlamydiosis in farmed American alligators (Alligator mississippiensis).

Chlamydia spp are reported to causes systemic disease in a variety of hosts worldwide including few reports in crocodilians. Disease presentations vary from asymptomatic to fulminant disease, some of which are zoonotic. The aim of this study was to describe the pathological, immunohistochemical, and molecular findings associated with the occurrence of a previously unreported Chlamydia sp infection causing a major mortality event in farmed American alligators (Alligator mississippiensis). The outbreak presented with sudden death in juvenile alligators mainly associated with necrotizing hepatitis and myocarditis, followed by the occurrence of conjunctivitis after the initial high mortality event. The widespread inflammatory lesions in multiple organs correlated with intralesional chlamydial organisms identified via immunohistochemistry and confirmed by 23S rRNA-specific real-time quantitative polymerase chain reaction (qPCR) for Chlamydiaceae bacteria. By sequencing and phylogenetic analysis of the OmpA gene, this uncultured Chlamydia sp grouped closely with Chlamydia poikilothermis recently described in snakes. This study highlights the significance of such outbreaks in farmed populations. Enhanced epidemiological monitoring is needed to gain further insight into the biology of Chlamydia sp in alligators, disease dynamics, risk factors, and role of carrier animals.

Zebra-borne neurotropic equid herpesvirus 1 meningoencephalitis in a Thomson's gazelle ( Eudorcas thomsonii).

We describe the histopathologic, immunohistochemical, and molecular features of a case of meningoencephalitis in a Thomson's gazelle ( Eudorcas thomsonii) naturally infected with zebra-borne equid herpesvirus 1 (EHV-1) and the implications for the molecular detection of zebra-borne EHV-1. A 4-y-old female Thomson's gazelle was submitted for postmortem examination; no gross abnormalities were noted except for meningeal congestion. Microscopic evaluation demonstrated multifocal nonsuppurative meningoencephalitis with intranuclear eosinophilic and amphophilic inclusion bodies and EHV-9 antigen in neurons. PCR demonstrated the presence of a herpesvirus with a nucleotide sequence 99-100% identical to the corresponding sequences of zebra-borne EHV-1 and of EHV-9 strains. To determine whether EHV-1 or EHV-9 was involved, a PCR with a specific primer set for EHV-9 ORF59/60 was used. The sequence was identical to that of 3 recognized zebra-borne EHV-1 strains and 91% similar to that of EHV-9. This isolate was designated as strain LM2014. The partial glycoprotein G ( gG) gene sequence of LM2014 was also identical to the sequence of 2 zebra-borne EHV-1 strains (T-529 isolated from an onager, 94-137 from a Thomson's gazelle). The histologic lesions of encephalitis and antigen localization in this gazelle indicate prominent viral neurotropism, and lesions were very similar to those seen in EHV-1- and EHV-9-infected non-equid species. Histologic lesions caused by EHV-9 and zebra-borne EHV-1 are therefore indistinguishable.

Establishing a New Species Encephalitozoon pogonae for the Microsporidian Parasite of Inland Bearded Dragon Pogona vitticeps Ahl 1927 (Reptilia, Squamata, Agamidae).

The microsporidium parasitizing Inland Bearded Dragons Pogona vitticeps, and developing primarily in macrophages within foci of granulomatous inflammation of different organs, is described as a new species Encephalitozoon pogonae. Establishing the new species was based on sequencing the ITS-SSUrDNA region of the ribosomal gene and consequent SSUrDNA-inferred phylogenetic analyses, as well as on comparison of pathogenesis, host specificity, and ultrastructure among Encephalitozoon species and isolates. The new species is closely related to E. lacertae and E. cuniculi. Analysis of the literature suggests that this microsporidium has been reported previously as an unidentified microsporidian species or isolate of E. cuniculi and may represent a common infection in bearded dragons. All stages of E. pogonae develop in parasitophorous vacuoles. Uninucleate spores on methanol-fixed smears measured 2.1 × 1.1 µm, range 1.7-2.6 × 0.9-1.7 µm; on ultrathin sections spores measured 0.8-1.1 × 1.8-2.2 µm. Ultrastructural study revealed 3-6 polar filament coils, a mushroom-shaped polar disk, and a polar sac embracing half of the volume occupied by the lamellar polaroplast. In activated spores, polar filament everted eccentrically. The overall morphology and intracellular development of E. pogonae were similar to other Encepahalitozoon spp. We also review the existing data on microsporidia infecting reptiles.

Diethylstilbestrol decreased adrenal cholesterol and corticosterone in rats.

The synthetic oestrogen diethylstilbestrol (DES), which is known to bind oestrogen receptors (ERs), has been reported to have adverse effects on endocrine homeostasis; however, the molecular mechanisms underlying these effects are poorly understood. In this study, we treated rats with DES and found high levels of this compound in the liver, adrenal glands and pituitary gland, as compared with other tissues. We have also detected early adverse effects of DES in the adrenal glands. The adrenal glands of rats treated with DES (340 µg/kg body weight every 2 days) for 2 weeks showed increased weight and size and a decreased fat droplet size. Following 1 week of treatment with DES, the blood and adrenal corticosterone levels were substantially decreased without any histological alterations. The levels of the precursors for corticosteroid biosynthesis in the adrenal glands were also decreased, as determined using mass spectroscopy. Cholesterol, the principal material of corticosteroid biosynthesis, decreased substantially in the adrenal glands after only 1 week of treatment with DES. In conclusion, cholesterol insufficiency results in a reduction in adrenal corticosterone biosynthesis, which may lead to endocrine dysfunction, such as reproductive toxicity.

Testicular yolk sac tumor of myxomatous, reticular, and polyvesicular vitelline type in a newborn calf.

Yolk sac tumors (YSTs) are rare neoplasms of germ cell origin. In humans, the tumors primarily occur in the testes or ovaries, but occasionally develop at other sites. The neoplastic cells of YSTs form many histological patterns resembling embryonal structures, and the World Health Organization classification lists 11 such patterns: reticular, macrocystic, endodermal sinus, papillary, solid, glandular-alveolar, myxomatous, sarcomatoid, polyvesicular vitelline, hepatoid, and parietal. Among domestic animals, only 2 cases of YST, which were of testicular and abdominal cavity origin, have been reported in calves. In both cases, neoplastic cells had epithelial properties and disseminated metastases in the abdomen. In the present study, the enlarged testis of a newborn calf, which was subsequently diagnosed as YST and exhibited myxomatous, reticular, and polyvesicular vitelline histological patterns, is described. There was no metastasis in this case, and histological and immunohistochemical features varied from previous cases of YST.

Jejunal intussusception associated with lymphoma in a horse.

A 2-year-old Thoroughbred horse presented with acute onset of colitis, and the intussuscepted jejunum was surgically resected. A transmural mass protruding into the lumen was found at the leading edge of the intussusceptum. Based on histological and immunohistochemical examinations, the mass was diagnosed as diffuse large B-cell lymphoma with metastasis to the mesenteric lymph nodes. Anatomical localization of the mass in the intussusception and absence of other obvious underlying diseases indicated that the intussusception had occurred in association with the mass. To our knowledge, this case is the first report of equine intussusception associated with focal intestinal lymphoma.

Intestinal undifferentiated carcinoma in a red-crowned crane (Grus japonensis).

A 33-year-old red-crowned crane (Grus japonensis) had a diffuse intestinal thickening from the duodenum to colon. Microscopically, neoplastic cells were arranged in sheets and occasionally nests or cords without gland or squamous differentiation. Metastatic tumor cells were found in the lungs, heart, kidneys and adrenal glands. Immunohistochemically, the neoplastic cells were strongly positive for pan-cytokeratin and cytokeratin 8 and 18 and only partly positive for E-cadherin antibodies. Immunostaining for CD3 was positive in normal lymphocytes, and NSE was also positive in normal nerve fibers. But, the neoplastic cells were not immunoreactive to CD3 and NSE. Based on the histological features and the epithelial characteristics in the immunohistochemical stain, the present case was diagnosed as undifferentiated carcinoma originating from the intestine. Interestingly, the neoplastic cells showed a unique growth pattern; they never invaded the submucosa or muscularis throughout the intestine, whereas they spread lymphogenously or hematogenously to other organs.

Cutaneous lymphoplasmacytic lymphoma with systemic metastasis in a cat.

A lymphoplasmacytic lymphoma was diagnosed in a 12- year-old domestic cat that had a primary cutaneous mass involving the stomach, liver, kidneys, heart, abdominal wall, diaphragm, bone marrow and several lymph nodes. Histopathologically, the most characteristic feature of this tumor was the heterogeneity of cell components, such as small lymphocytes, well-differentiated plasma cells and plasmacytoid transformed lymphocytes. Amyloid was deposited in the skin, stomach, and several lymph nodes. Immunohistochemically, neoplastic small lymphocytes were positive for CD20, and well-differentiated plasma cells and plasmacytoid transformed lymphocytes were positive for λ-Ig light chains and MUM1/IRF-4. These results emphasize the importance of lymphoplasmacytic lymphoma as a differential diagnosis of extramedullary cutaneous plasmacytoma in cats.

Mycobacterium ulcerans infection in an Indian flap-shelled turtle (Lissemys punctata punctata).

We report an atypical mycobacterial infection in an Indian flap-shelled turtle, Lissemys punctata punctata, that died in an aquarium in Japan. At necropsy, the turtle showed multiple white nodules on the capsular surface and parenchyma of various organs such as the liver, spleen, intestine, and lung. Histologically, granulomatous inflammation surrounding a central zone of necrosis was observed. Sections stained by the Ziehl-Neelsen method revealed numerous acid-fast bacilli in the cytoplasm of macrophages and in the central area of necrosis. The organisms were identified as a mycobacterial species by PCR and nucleotide sequence analysis and revealed 98-100% homology to M. ulcerans. This is, to our knowledge, the first report of mycobacteriosis due to M. ulcerans in a turtle.

Two cases of bovine sarcoma in clinically long-standing lesions.

Histopathological examination of clinically long-standing lesions with durations of one year or more in the extremities of two cattle revealed the presence of sarcomas with distant metastases. In case 1, neoplastic cells were fusiform to pleomorphic, stained for no specific differentiation markers, and diagnosed as undifferentiated sarcoma. Neoplastic growth in case 2 was composed of spindle to histiocytoid cells and a significant number of multinucleated giant cells, both of which were immunoreactive to histiocyte markers, and diagnosed as giant cell malignant fibrous histiocytoma. Neoplastic cells of both cases were immunohistochemically positive for nitric oxide-related antigens, which were recognized as markers of inflammation-induced carcinogenesis in human and laboratory animals.

Systemic candidiasis and mesenteric mast cell tumor with multiple metastases in a dog.

A 5-year-old female miniature dachshund presenting with persistent vomiting and diarrhea had two concurrent rare pathological conditions: systemic candidiasis and mesenteric mast cell tumor with multiorgan metastases. Neoplastic mast cells formed mass in the mesentery of the cecal-colonic region and were also found in the liver, spleen, kidneys, lungs, adrenal grands, ovaries, bone marrow and other tissues. The cells had intracytoplasmic granules with metachromasia and were immunohistochemically positive for c-kit and histamine. Granulomatous lesions with fungal organisms were present in the heart, lungs, kidneys, pancreas, subserosal and surrounding adipose tissue of the duodenum, thyroid glands and mesenteric mass, and phagocytosed organisms were detected in the liver and bone marrow. Bacteriologically and immunohistochemically, the fungi were consistent with Candida albicans.

Suprascapular nerve paralysis due to streptococcal meningoradiculitis in a cow.

A cow, presenting with lameness with atrophy of the right supraspinatus and infraspinatus muscles, was clinically diagnosed with suprascapular nerve paralysis. Histological examination revealed necrosuppurative lesions with Gram-positive cocci arranged in chains in multiple organs, including the cardiac valves, lungs, muscles, joints, brain, cerebral and spinal meninges. Spinal meningitis progressed into the roots of the right 6th to 8th cervical nerves. The suprascapular nerve showed partial loss of nerve fibers in the periphery, and innervated muscle fibers were atrophied. Bacterial culture of the cerebrospinal fluid revealed the presence of Streptococcus pyogenes. From these findings, the suprascapular nerve paralysis in this case was considered to result from meningoradiculitis associated with systemic streptococcal infection. This is a rare bovine case of suprascapular nerve paralysis due to central nerve damage of infectious cause.