A case of olfactory ganglioneuroblastoma in a dog: immunohistochemical comparison with olfactory neuroepithelia and olfactory neuroblastomas.

In the present study, histopathological and immunohistochemical findings of olfactory ganglioneuroblastoma in a dog were compared to those of canine olfactory neuroepithelia and neuroblastomas. Olfactory ganglioneuroblastoma consists of ganglion cell-like tumor cells with Schwannian stroma and neuroblast-like tumor cells. Immunohistochemically, ganglion cell-like tumor cells were immunopositive for synaptophysin, ß3-tubulin, and tyrosine hydroxylase, Schwannian stroma was immunopositive for GFAP and SOX2, and neuroblast-like tumor cells were immunopositive for OLIG2, ß3-tubulin, SOX2, cytokeratin AE1/AE3, and p63. The immunohistochemical results of olfactory neuroepithelia and olfactory neuroblastomas were similar to those of neuroblast-like tumor cells. These results suggest that the ganglion cell-like tumor cells in the present case have a sympathetic neuron immunophenotype, whereas neuroblast-like tumor cells have an olfactory neuroepithelial immunophenotype.

Inhibition of prefrontal cortex parvalbumin interneurons mitigates behavioral and physiological sequelae of chronic stress in male mice.

Chronic stress leads to hypofunction of the medial prefrontal cortex (mPFC), mechanisms of which remain to be determined. Enhanced activation of GABAergic of parvalbumin (PV) expressing interneurons (INs) is thought to play a role in stress-induced prefrontal inhibition. In this study, we tested whether chemogenetic inhibition of mPFC PV INs after chronic stress can rescue chronic stress-related behavioral and physiological phenotypes. Mice underwent 2 weeks of chronic variable stress (CVS) followed by a battery of behavioral tests known to be affected by chronic stress exposure, e.g. an open field (OF), novel object recognition (NOR), tail suspension test (TST), sucrose preference test (SPT), and light dark (LD) box. Inhibitory DREADDs were actuated by 3 mg/kg CNO administered 30 min prior to each behavioral test. CVS caused hyperactivity in the OF, reduced sucrose preference in the SPT (indicative of enhanced anhedonia), and increased anxiety-like behavior in the LD box. Inhibition of PV IN after stress mitigated these effects. In addition, CVS also resulted in reduced thymus weight and body weight loss, which were also mitigated by PV IN inhibition. Our results indicate that chronic stress leads to plastic changes in PV INs that may be mitigated by chemogenetic inhibition. Our findings implicate cortical GABAergic INs as a therapeutic target in stress-related diseases.

Development of Aptamers for RNase Inactivation in Xtract-Free™ Sample Collection and Transport Medium.

There is a significant need to develop new environmentally friendly, extraction-free sample collection mediums that can effectively preserve and protect genetic material for point-of-care and/or self-collection, home-collection, and mail-back testing. Systematic evolution of ligands by exponential enrichment (SELEX) was used to create anti-ribonuclease (RNase) deoxyribonucleic acid (DNA) aptamers against purified RNase A conjugated to paramagnetic carboxylated beads. Following eight rounds of SELEX carried out under various stringency conditions, e.g., selection using Xtract-Free™ (XF) specimen collection medium and elevated ambient temperature of 28 °C, a panel of five aptamers was chosen following bioinformatic analysis using next-generation sequencing. The efficacy of aptamer inactivation of RNase was assessed by monitoring ribonucleic acid (RNA) integrity via fluorometric and real-time RT-PCR analysis. Inclusion of aptamers in reaction incubations resulted in an 8800- to 11,200-fold reduction in RNase activity, i.e., digestion of viral RNA compared to control. Thus, anti-RNase aptamers integrated into XF collection medium as well as other commercial reagents and kits have great potential for ensuring quality intact RNA for subsequent genomic analyses.

Cytoplasmic aggregation of TDP43 and topographic correlation with tau and α-synuclein accumulation in the rTg4510 mouse model of tauopathy.

In patients with TDP43 proteinopathy, phosphorylated TDP43 (p-TDP43) accumulates in the cytoplasm of neurons. The accumulation of p-TDP43 has also been reported in patients with tauopathy and α-synucleinopathy. We investigated spatiotemporal changes in p-TDP43 accumulation in the brains of rTg4510 mice that overexpressed human mutant tau (P301L) and exhibited hyperphosphorylated tau (hp-tau) and phosphorylated αSyn (p-αSyn) accumulation. Immunohistochemically, p-TDP43 aggregates were observed in the cytoplasm of neurons, which increased with age. A significant positive correlation was observed between the number of cells with p-TDP43 aggregates and hp-tau and p-αSyn aggregates. Suppression of the human mutant tau (P301L) expression by doxycycline treatment reduces the accumulation of p-TDP43, hp-tau, and p-αSyn. Proteinase K-resistant p-TDP43 aggregates were found in regions with high hp-tau, and p-αSyn accumulation. Western blotting of the sarkosyl-insoluble fraction revealed bands of monomeric TDP43 and p-TDP43. These results indicate that the accumulation of mouse p-TDP43 is associated with the accumulation of human mutant tau (P301L) in rTg4510 mouse brains. The accumulation of hp-tau and p-αSyn may promote sarkosyl-insoluble p-TDP43 aggregates that are resistant to proteinase K. The synergistic effects of tau, TDP43, and αSyn may be involved in the pathology of proteinopathies, leading to the accumulation of multiple abnormal proteins.

Efficacy and adverse events of L-Asparaginase administration as a first-line treatment for feline large-cell gastrointestinal lymphoma.

L-Asparaginase (L-Asp) is often used to induce remission in feline large-cell gastrointestinal lymphoma (LCGIL). However, no study has evaluated the efficacy and adverse events following the initial use of this drug as a first-line treatment in feline LCGIL. We retrospectively reviewed medical records of cats with LCGIL treated with L-Asp to induce remission. This study included 43 cats. The response rate (RR) after the first administration of L-Asp was 37.2% (Complete remission: 7.0%, partial remission: 30.2%). RR was significantly higher in cases with primary gastric lesions (64.3%) than in those with primary intestinal lesions (24.1%) (P=0.018), and it was also higher in cases without anemia (57.1%) than those with anemia (15.0%) (P=0.009). The most common adverse event was hyperammonemia, which occurred in 10 of 12 cases where we could compare plasma ammonia concentrations before and after the first dose of L-Asp. Plasma phosphate concentrations were also significantly increased (P<0.001) within 24 hr after the first dose. Decreased appetite, vomiting, and diarrhea were also observed in five, three, and seven cases, respectively, and Grade 3 or higher gastrointestinal signs were observed as adverse events in three cases. The median overall survival of all cats was 150 days (range, 5-1,065 days), and the median progression-free survival was 104 days (range, 2-978 days). In conclusion, L-Asp was effective to induce remission, and severe adverse events were uncommon in feline LCGIL.

US commercial health plan coverage dynamics in pulmonary arterial hypertension therapies.

BACKGROUND: Health plan coverage is central to patient access to care, especially for rare, chronic diseases. For specialty drugs, coverage varies, resulting in barriers to access. Pulmonary arterial hypertension (PAH) is a rare, progressive, and fatal disease. Guidelines suggest starting or rapidly escalating to combination therapy with drugs of differing classes (phosphodiesterase 5 inhibitors [PDE5is], soluble guanylate cyclase stimulators [sGC stimulators], endothelin receptor antagonists [ERAs], and prostacyclin pathway agents [PPAs]). OBJECTIVE: To assess the variation in commercial health plan coverage for PAH treatments and how coverage has evolved. To examine the frequency of coverage updates and evidence cited in plan policies. METHODS: We used the Tufts Medical Center Specialty Drug Evidence and Coverage database, which includes publicly available specialty drug coverage policies. Overall, and at the drug and treatment class level, we identified plan-imposed coverage restrictions beyond the drug's US Food and Drug Administration label, including step therapy protocols, clinical restrictions (eg, disease severity), and prescriber specialty requirements. We analyzed variation in coverage restrictiveness and how coverage has changed over time. We determined how often plans update their policies. Finally, we categorized the cited evidence into 6 different types. RESULTS: Results reflected plan coverage policies for 13 PAH drugs active between August 2017 and August 2022 and issued by 17 large US commercial health plans, representing 70% of covered lives. Coverage restrictions varied mainly by step therapy protocols and prescriber restrictions. Seven plans had step therapy protocols for most drugs, 9 for at least one drug, and 1 had none. Ten plans required specialist (cardiologist or pulmonologist) prescribing for at least one drug, and 7 did not. Coverage restrictions increased over time: the proportion of policies with at least 1 restriction increased from 38% to 73%, and the proportion with step therapy protocols increased from 29% to 46%, with generics as the most common step. The proportion of policies with step therapy protocols increased for every therapy class with generic availability: 18% to 59% for ERAs, 33% to 77% for PDE5is, and 33% to 43% for PPAs. The proportion of policies with prescriber requirements increased from 24% to 48%. Plans updated their policies 58% of the time annually and most often cited the 2019 CHEST clinical guidelines, followed by randomized controlled trials. CONCLUSIONS: Plan use of coverage restrictions for PAH therapies increased over time and varied across both drugs and plans. Inconsistency among health plans may complicate patient access and reduce the proportion who can persist on PAH treatments.

Activation of the Wnt/ß-catenin signaling pathway and CTNNB1 mutations in canine intestinal epithelial proliferative lesions.

Nuclear expression of ß-catenin has been reported in canine intestinal epithelial tumors (IETs) and colorectal inflammatory polyps (CIPs) with dysplastic epithelia. However, the role of the Wnt/ß-catenin signaling pathway in these lesions remains unclear. To investigate the association between the nuclear ß-catenin expression and the activation of the Wnt/ß-catenin signaling pathway, immunohistochemistry and mutation analyses were conducted on 64 IETs and 20 CIPs. IETs and CIPs with ß-catenin nuclear and/or cytoplasm immunolabeling were classified as ß-catenin (+). The immunostaining scores of c-Myc and Cyclin D1 and Ki-67 index were significantly higher in ß-catenin (+) cases than in ß-catenin (-) cases. Identical APC mutations (p.E154D and p.K155X) were detected in 6/41 ß-catenin (+) IETs; all 6 of IETs with APC mutations were Jack Russell Terriers. CTNNB1 mutations were detected in 29/42 ß-catenin (+) IETs, 3/11 ß-catenin (+) CIPs, and 2/22 ß-catenin (-) IETs, most of which were hotspots associated with human colorectal carcinoma. In one Miniature Dachshund diagnosed with a CIP that subsequently developed into an IET, the same CTNNB1 mutation was detected in both lesions. The immunohistochemical results suggest that cell proliferative activity in ß-catenin (+) cases may be associated with activation of the Wnt/ß-catenin signaling pathway. The mutation analysis results suggest that CTNNB1 mutations may be associated with cytoplasmic ß-catenin accumulation in IET and CIP. Furthermore, the dysplastic epithelium in CIP may progress to IET through the activation of the Wnt/ß-catenin signaling pathway by the CTNNB1 mutation.

Mechanically induced localisation of SECONDARY WALL INTERACTING bZIP is associated with thigmomorphogenic and secondary cell wall gene expression.

Plant growth requires the integration of internal and external cues, perceived and transduced into a developmental programme of cell division, elongation and wall thickening. Mechanical forces contribute to this regulation, and thigmomorphogenesis typically includes reducing stem height, increasing stem diameter, and a canonical transcriptomic response. We present data on a bZIP transcription factor involved in this process in grasses. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus following mechanostimulation. Classical touch-responsive genes were upregulated in B. distachyon roots following touch, including significant induction of the glycoside hydrolase 17 family, which may be unique to grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. SWIZ overexpression resulted in plants with reduced stem and root elongation. These data further define plant touch-responsive transcriptomics and physiology, offering insights into grass mechanotranduction dynamics.

Japanese Encephalitis Virus and Schizophyllum commune Co-Infection in a Harbor Seal in Japan.

The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has a wide host range, extending from pigs and ardeid birds to opportunistic dead-end hosts, such as humans and horses. However, JEV encephalitis infections in aquatic mammals are rare, with only two cases in seals reported to date. Here, we report a lethal case of JEV and Schizophyllum commune co-infection in an aquarium-housed harbor seal in Japan. We isolated JEV from the brain of the dead seal and characterized its phylogeny and pathogenicity in mice. The virus isolate from the seal was classified as genotype GIb, which aligns with recent Japanese human and mosquito isolates as well as other seal viruses detected in China and Korea, and does not exhibit a unique sequence trait distinct from that of human and mosquito strains. We demonstrated that the seal isolate is pathogenic to mice and causes neuronal symptoms. These data suggest that seals should be considered a susceptible dead-end host for circulating JEV in natural settings.

Development and Evaluation of an Immunoinformatics-Based Multi-Peptide Vaccine against Acinetobacter baumannii Infection.

Multi-drug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen associated with hospital-acquired infections. Due to its environmental persistence, virulence, and limited treatment options, this organism causes both increased patient mortality and incurred healthcare costs. Thus, prophylactic vaccination could be ideal for intervention against MDR Acinetobacter infection in susceptible populations. In this study, we employed immunoinformatics to identify peptides containing both putative B- and T-cell epitopes from proteins associated with A. baumannii pathogenesis. A novel Acinetobacter Multi-Epitope Vaccine (AMEV2) was constructed using an A. baumannii thioredoxin A (TrxA) leading protein sequence followed by five identified peptide antigens. Antisera from A. baumannii infected mice demonstrated reactivity to rAMEV2, and subcutaneous immunization of mice with rAMEV2 produced high antibody titer against the construct as well as peptide components. Immunization results in increased frequency of IL-4-secreting splenocytes indicative of a Th2 response. AMEV2-immunized mice were protected against intranasal challenge with a hypervirulent strain of A. baumannii and demonstrated reduced bacterial burden at 48 h. In contrast, all mock vaccinated mice succumbed to infection within 3 days. Results presented here provide insight into the effectiveness of immunoinformatic-based vaccine design and its potential as an effective strategy to combat the rise of MDR pathogens.

Diffuse large B-cell lymphoma with DNA copy number changes in a Japanese black calf.

A 2-month-old Japanese Black calf exhibited mandibular and superficial cervical lymph node swelling. Fine needle aspiration cytology of the superficial cervical lymph node revealed large lymphoblast-like cells with mitoses. Hematological examination revealed remarkable lymphocytosis with atypical lymphocytes. Increased activities of serum total lactate dehydrogenase and thymidine kinase were detected. At necropsy, generalized swelling of lymph nodes was observed. Histopathological analysis revealed diffuse proliferation of medium-sized round centroblastic neoplastic cells that were positive for CD20, CD79α, PAX5, and BLA-36, and negative for CD3, CD5, CD10, and CD34. The calf was diagnosed with centroblastic diffuse large B-cell lymphoma (DLBCL) based on these findings. Analysis of DNA copy number variation revealed an increased copy number for the GIMAP family relative to that in healthy cattle. Moreover, decreases in copy numbers of GBP-1, MIR3141, OR5P1E, and PTPRG relative to those in healthy cattle were also observed. Because DNA copy number variation represent a major contribution to the somatic mutation landscapes in human tumors, these findings suggest that DNA copy number changes might have contributed to the onset of DLBCL in the present case.

The relationships of platelet-derived growth factor, microvascular proliferation, and tumor cell proliferation in canine high-grade oligodendrogliomas: Immunohistochemistry of 45 tumors and an AFOB-01 xenograft mouse model.

High-grade oligodendroglioma (HGOG) is the most common type of glioma in dogs and expresses platelet-derived growth factor receptor-α (PDGFR-α). Microvascular proliferation is often observed in HGOG. Therefore, the present study investigated the functional relationships between PDGFR-α, microvascular proliferation, and tumor cell proliferation in canine HGOG. The expression of PDGFR-α and PDGF-subunit A (PDGF-A) in tumor cells, as well as endothelial cells and pericytes of tumor-associated microvascular proliferations, in 45 canine HGOGs were examined immunohistochemically. Microvascular proliferation was observed in 24/45 cases (53%). PDGFR-α expression in tumor cells and microvascular proliferations was observed in 45/45 (100%) and 2/24 cases (8%), respectively. Furthermore, PDGF-A expression in tumor cells and microvascular proliferations was detected in 13/45 (29%) and 24/24 cases (100%), respectively. In vitro, stimulation of the canine HGOG cell line AOFB-01 with PDGF-A showed that the doubling time of AOFB-01 cells was significantly shorter with PDGF-A than without PDGF-A. Crenolanib (a PDGFR inhibitor) inhibited AOFB-01 cell proliferation. In vivo, the AOFB-01 xenograft mouse model was treated with crenolanib. Tumor xenografts were smaller in crenolanib-treated mice than in untreated control mice. PDGFR-α expression in tumor cells and PDGF-A expression in microvascular proliferations and tumor cells suggest autocrine and paracrine effects of PDGF-A in canine HGOG. The results of in vitro assays indicate that canine HGOG expresses functional PDGFR-α, which responds to PDGF-A. Therefore, PDGF-A produced by microvascular proliferations and tumor cells may promote the proliferation of PDGFR-α-expressing tumor cells in canine HGOG. PDGFR-α signaling has potential as a therapeutic target.

A case of Exophiala dermatitidis-induced phaeohyphomycosis in a cat with multiple intra-abdominal masses.

Pus discharge containing black granular materials (1-2 mm in diameter) was found in the abdominal skin of a 13-year-old sterilized female cat. Abdominal ultrasonography revealed a large intra-abdominal mass with abundant blood flow beneath the skin lesion. Laparotomy revealed a large mass that adhered to the spleen and left kidney. Similar small lesions were found in the abdominal wall and mesentery. The masses were surgically removed along with the spleen and kidney. Histopathologically, the mass lesions consisted of granulomas with lesional pigmented fungi, and the cat was diagnosed with phaeohyphomycosis. Uisng genetic analysis, the Exophiala dermatitidis was identified as the causative pathogen.

Association of a novel dystrophin (DMD) genetic nonsense variant in a cat with X-linked muscular dystrophy with a mild clinical course.

X-linked muscular dystrophy in cats (FXMD) is an uncommon disease, with few reports describing its pathogenic genetic variants. A 9-year-old castrated male domestic shorthair cat was presented with persistent muscle swelling and breathing difficulty from 3 years of age. Serum activity of alanine aminotransferase, aspartate transaminase, and creatine kinase were abnormally high. Physical and neurological examinations showed muscle swelling in the neck and proximal limb, slow gait, and occasional breathing difficulties. Electromyography showed pseudomyotonic discharges and complex repetitive discharges with a "dive-bomber" sound. Histopathology revealed muscle necrosis and regeneration. Whole-genome sequencing identified a novel and unique hemizygous nonsense genetic variant, c.8333G > A in dystrophin (DMD), potentially causing a premature termination codon (p.Trp2778Ter). Based on a combination of clinical and histological findings and the presence of the DMD nonsense genetic variant, this case was considered FXMD, which showed mild clinical signs and long-term survival, even though immunohistochemical characterization was lacking.

Path integration deficits are associated with phosphorylated tau accumulation in the entorhinal cortex.

Alzheimer's disease is a devastating disease that is accompanied by dementia, and its incidence increases with age. However, no interventions have exhibited clear therapeutic effects. We aimed to develop and characterize behavioural tasks that allow the earlier identification of signs preceding dementia that would facilitate the development of preventative and therapeutic interventions for Alzheimer's disease. To this end, we developed a 3D virtual reality task sensitive to the activity of grid cells in the entorhinal cortex, which is the region that first exhibits neurofibrillary tangles in Alzheimer's disease. We investigated path integration (assessed by error distance) in a spatial navigation task sensitive to grid cells in the entorhinal cortex in 177 volunteers, aged 20-89 years, who did not have self-reported dementia. While place memory was intact even in old age, path integration deteriorated with increasing age. To investigate the relationship between neurofibrillary tangles in the entorhinal cortex and path integration deficit, we examined a mouse model of tauopathy (P301S mutant tau-overexpressing mice; PS19 mice). At 6 months of age, PS19 mice showed a significant accumulation of phosphorylated tau only in the entorhinal cortex, associated with impaired path integration without impairments in spatial cognition. These data are consistent with the idea that path integration deficit is caused by the accumulation of phosphorylated tau in the entorhinal cortex. This method may allow the early identification of individuals likely to develop Alzheimer's disease.

Harmonizing the Generation and Pre-publication Stewardship of FAIR Image data.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

Pan-tumour analysis of COX-2 expression in dogs.

Cyclooxgenase-2 (COX-2) is associated with inflammatory microenvironment and tumour progression. COX-2 expression was reported in canine tumours, and anti-COX treatment showed therapeutic effects in selected tumour types. Currently, direct comparisons between different tumour types or reports were impossible due to varying evaluation protocols. Additionally, COX-2 expression in relatively uncommon tumours were yet to be evaluated. Here, we analysed COX-2 expression across various tumour types in dogs in a consistent protocol, aiming to revisit accumulated evidence in the field and report novel candidate tumours for anti-COX therapy. COX-2 expression in 32 histological types of tumours, which consisted of 347 samples in total, was investigated using immunohistochemistry followed by the Belshaw's method scoring (range: 0-12). More than the half of the samples expressed COX-2 in mast cell tumours, transitional cell carcinoma in the urinary tract, squamous cell carcinoma, liposarcoma, and melanoma, with COX-2 median scores ranging from 1-8. On the other hand, <20% tissues expressed COX-2 in the half of tumour types investigated. Overall COX-2 positive rate was 27%. In conclusion, the results confirmed COX-2 expression in the well-known COX-2-expresing tumour types and suggested novel candidate tumours for anti-COX-2 therapy. At the same time, overall COX-2 expression was low, and inter- and intra-histology heterogeneity was apparent. This study will provide a foundation reference for future research in canine tumours.

Identification and characterization of dystrophin-locus-derived testis-specific protein: A testis-specific gene within the intronic region of the rat dystrophin gene.

The mammalian X chromosome exhibits enrichment in genes associated with germ cell development. Previously, we generated a rat model of Becker muscular dystrophy (BMD) characterized by an in-frame mutation in the dystrophin gene, situated on the X chromosome and responsible for encoding a protein crucial for muscle integrity. Male BMD rats are infertile owing to the absence of normal spermatids in the epididymis. Within the seminiferous tubules of BMD rats, elongated spermatids displayed abnormal morphology. To elucidate the cause of infertility, we identified a putative gene containing an open reading frame situated in the intronic region between exons 6 and 7 of the dystrophin gene, specifically deleted in male BMD rats. This identified gene, along with its encoded protein, exhibited specific detection within the testes, exclusively localized in round to elongated spermatids during spermiogenesis. Consequently, we designated the encoded protein as dystrophin-locus-derived testis-specific protein (DTSP). Given the absence of DTSP in the testes of BMD rats, we hypothesized that the loss of DTSP contributes to the infertility observed in male BMD rats.

Establishment and characterization of a novel cell line and xenotransplant mouse model derived from feline colorectal adenocarcinoma.

Colorectal adenocarcinoma is an aggressive malignant tumor in cats that frequently metastasizes to the lymph nodes and/or distant organs. However, research on feline colorectal adenocarcinoma is limited, and experimental models have not been established. A novel cell line, FeLeco-G7, was established from the lymph node of a 12-year-old spayed female Maine Coon cat with metastatic colorectal adenocarcinoma. FeLeco-G7 cells were polygonal with abundant cytoplasm and adherent growth. The population-doubling time was approximately 28.3 hours, and the mean number of chromosomes was 37.6±0.1 per cell (ranging between 32 and 41). Consistent with the original tumor, FeLeco-G7 cells were immunopositive for cytokeratin (CK) 20 and CDX2, and immunonegative for CD10 and CK7. Nuclear accumulation of ß-catenin was rarely observed. Mutation analysis suggested TP53 gene alterations. A subcutaneous injection of FeLeco-G7 cells into immunodeficient mice resulted in the formation of a mass at the injection site without the development of metastatic lesions. An orthotopic (intrarectal) transplantation of FeLeco-G7 cells caused cachexia and diffuse involvement of the rectal mucosa in one of the 3 mice and the formation of masses around the rectum in the other 2 mice. Metastases to the regional lymph nodes and lungs were detected in three of the 3 and one of the 3 mice, respectively. The histological findings and immunohistochemical features of these masses were similar to those of the original tumor. These results suggest that FeLeco-G7 cells and the orthotopically transplanted mouse model are valuable tools for further molecular and therapeutic research on feline colorectal adenocarcinoma.

Intraepithelial lymphocytes are associated with epithelial injury in feline intestinal T-cell lymphoma.

Our previous study indicated that cytotoxicity of intraepithelial lymphocytes is a poor prognostic factor in feline intestinal T-cell lymphoma (FITL), but the effect of cytotoxic lymphocytes on mucosal epithelium is still unknown. Thus, we investigated the association between cytotoxic lymphocytes and mucosal epithelium in 71 cases of feline intestinal T-cell lymphoma (FITL): epithelial injury, basement membrane injury, cleaved-caspase-3 positivity of epithelial cells, and the number and Ki67 positivity of intraepithelial lymphocytes in granzyme B (GRB)+ and GRB- FITLs were evaluated. Epithelial injury score and the number of intraepithelial lymphocytes in granzyme B (GRB)+ FITL were significantly higher than those of GRB- FITL (P<0.05, P<0.05), but no significant differences were found in the basement membrane injury score, the percentage of cleaved-caspase-3+ epithelial cells, and the percentage of Ki67+ intraepithelial lymphocytes. There was a significant correlation between the epithelial injury score and the number of intraepithelial lymphocytes (P<0.05), but no significant correlation was observed between the epithelial injury score and Ki67+ percentage of intraepithelial lymphocytes. Because epithelial cell cleaved-caspase-3 positivity was observed in FITL, regardless of GRB expression in lymphocytes, GRB-mediated apoptosis may not contribute to epithelial injury in FITL. The association between increased number of intraepithelial lymphocytes and epithelial injury suggests that intraepithelial lymphocytes infiltration may contribute to epithelial injury in FITL.