Intestinal Microbiome in Dogs with Chronic Hepatobiliary Disease: Can We Talk about the Gut-Liver Axis?

The gut-liver axis represents a current topic in human medicine. Extensive research investigates the gut microbiome (GM) modifications in relation to various kinds of chronic hepatobiliary diseases (CHD), with many mechanisms and therapeutical implications recognized. Those aspects in veterinary medicine are still quite unexplored. The aim of the present study was to evaluate GM in dogs diagnosed with CD. Comparison among CHD dogs were made considering some clinical and biochemical variables (lipemia and alanine-aminotransferase activities), presence of cholestasis or endocrine disorders, diet). Sixty-five dogs were prospectively enrolled with clinical and hematobiochemical evaluation and 16S-RNA GM sequencing assessed. Dogs that received antibiotics and/or pre/pro/symbiotics administration were excluded. Deeper GM alteration was observed between dogs with or without ultrasonographic and biochemical cholestatic CHD. Cholestasis was associated with a decrease in several bacterial taxa, including Clostridium hiranonis, Fusobacterium, Megamonas, Ruminococcus faecis, Turicibacter, and higher levels of Escherichia/Shigella and Serratia. Thus, the alteration in bile flow and composition, typical of cholestasis, may directly affect the local intestinal microbial environment. For the management of dogs with CHD and especially cholestatic CHD, clinicians should be aware that gut-liver interaction may lead to dysbiosis.

Analysis of Intestinal Microbiota and Metabolic Pathways before and after a 2-Month-Long Hydrolyzed Fish and Rice Starch Hypoallergenic Diet Trial in Pruritic Dogs.

Intestinal microbiota alterations were described in allergic individuals and may improve with diets. Farmina Ultra Hypo (FUH), a hydrolyzed fish/rice starch hypoallergenic diet, is able to improve clinical signs in allergic dogs. Study objectives were to determine microbiota differences in allergic dogs before and after feeding with FUH for eight weeks. Forty skin allergic dogs were evaluated clinically before and after the diet. Unresponsive dogs were classified as canine atopic dermatitis (CAD); responsive dogs relapsing after challenge with previous foods were classified as being food reactive (AFR), and those not relapsing as doubtful (D). Sequencing of feces collected pre- and post-diet was performed, with comparisons between and within groups, pre- and post-diet, and correlations to possible altered metabolic pathways were sought. Microbiota in all dogs was dominated by Bacteroidota, Fusobacteriota, Firmicutes and Proteobacteria, albeit with large interindividual variations and with some prevalence changes after the diet. In general, bacteria producing short-chain fatty acids were increased in all samples. CAD dogs showed pre-and post-diet microbiota patterns different from the other two groups. Bacteria taxa were enriched post-diet only in the AFR group. Changes in metabolic pathways were observed mainly in the CAD group. FUH may be able to improve intestinal microbiota and thus clinical signs of skin allergy.

Polycomb group ring finger protein 6 suppresses Myc-induced lymphomagenesis.

Max is an obligate dimerization partner for the Myc transcription factors and for several repressors, such as Mnt, Mxd1-4, and Mga, collectively thought to antagonize Myc function in transcription and oncogenesis. Mga, in particular, is part of the variant Polycomb group repressive complex PRC1.6. Here, we show that ablation of the distinct PRC1.6 subunit Pcgf6-but not Mga-accelerates Myc-induced lymphomagenesis in Eµ-myc transgenic mice. Unexpectedly, however, Pcgf6 loss shows no significant impact on transcriptional profiles, in neither pre-tumoral B-cells, nor lymphomas. Altogether, these data unravel an unforeseen, Mga- and PRC1.6-independent tumor suppressor activity of Pcgf6.

Cooperation Between MYC and ß-Catenin in Liver Tumorigenesis Requires Yap/Taz.

BACKGROUND AND AIMS: Activation of MYC and catenin beta-1 (CTNNB1, encoding ß-catenin) can co-occur in liver cancer, but how these oncogenes cooperate in tumorigenesis remains unclear. APPROACH AND RESULTS: We generated a mouse model allowing conditional activation of MYC and WNT/ß-catenin signaling (through either ß-catenin activation or loss of APC - adenomatous polyposis coli) upon expression of CRE recombinase in the liver and monitored their effects on hepatocyte proliferation, apoptosis, gene expression profiles, and tumorigenesis. Activation of WNT/ß-catenin signaling strongly accelerated MYC-driven carcinogenesis in the liver. Both pathways also cooperated in promoting cellular transformation in vitro, demonstrating their cell-autonomous action. Short-term induction of MYC and ß-catenin in hepatocytes, followed by RNA-sequencing profiling, allowed the identification of a "Myc/ß-catenin signature," composed of a discrete set of Myc-activated genes whose expression increased in the presence of active ß-catenin. Notably, this signature enriched for targets of Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz), two transcriptional coactivators known to be activated by WNT/ß-catenin signaling and to cooperate with MYC in mitogenic activation and liver transformation. Consistent with these regulatory connections, Yap/Taz accumulated upon Myc/ß-catenin activation and were required not only for the ensuing proliferative response, but also for tumor cell growth and survival. Finally, the Myc/ß-catenin signature was enriched in a subset of human hepatocellular carcinomas characterized by comparatively poor prognosis. CONCLUSIONS: Myc and ß-catenin show a strong cooperative action in liver carcinogenesis, with Yap and Taz serving as mediators of this effect. These findings warrant efforts toward therapeutic targeting of Yap/Taz in aggressive liver tumors marked by elevated Myc/ß-catenin activity.

Therapeutic synergy between tigecycline and venetoclax in a preclinical model of MYC/BCL2 double-hit B cell lymphoma.

High-grade B cell lymphomas with concurrent activation of the MYC and BCL2 oncogenes, also known as double-hit lymphomas (DHL), show dismal prognosis with current therapies. MYC activation sensitizes cells to inhibition of mitochondrial translation by the antibiotic tigecycline, and treatment with this compound provides a therapeutic window in a mouse model of MYC-driven lymphoma. We now addressed the utility of this antibiotic for treatment of DHL. BCL2 activation in mouse Eµ-myc lymphomas antagonized tigecycline-induced cell death, which was specifically restored by combined treatment with the BCL2 inhibitor venetoclax. In line with these findings, tigecycline and two related antibiotics, tetracycline and doxycycline, synergized with venetoclax in killing human MYC/BCL2 DHL cells. Treatment of mice engrafted with either DHL cell lines or a patient-derived xenograft revealed strong antitumoral effects of the tigecycline/venetoclax combination, including long-term tumor eradication with one of the cell lines. This drug combination also had the potential to cooperate with rituximab, a component of current front-line regimens. Venetoclax and tigecycline are currently in the clinic with distinct indications: Our preclinical results warrant the repurposing of these drugs for combinatorial treatment of DHL.

Transcriptional integration of mitogenic and mechanical signals by Myc and YAP.

Mammalian cells must integrate environmental cues to determine coherent physiological responses. The transcription factors Myc and YAP-TEAD act downstream from mitogenic signals, with the latter responding also to mechanical cues. Here, we show that these factors coordinately regulate genes required for cell proliferation. Activation of Myc led to extensive association with its genomic targets, most of which were prebound by TEAD. At these loci, recruitment of YAP was Myc-dependent and led to full transcriptional activation. This cooperation was critical for cell cycle entry, organ growth, and tumorigenesis. Thus, Myc and YAP-TEAD integrate mitogenic and mechanical cues at the transcriptional level to provide multifactorial control of cell proliferation.

Primary fibrosarcoma of the urinary bladder in a cat: follow-up after incomplete surgical excision.

CASE SUMMARY: An 11-year-old female spayed domestic shorthair cat was presented with haematuria of 2 months' duration followed by pollakiuria and stranguria. A firm, non-painful mass in the urinary bladder was palpated. Abdominal radiographs and ultrasound were suggestive of a urinary neoplasia. During explorative laparotomy, a partial cystectomy and surgical debulking were performed. Histopathology and immunostaining were consistent with a fibrosarcoma. The cat was discharged 10 days after surgery with a residual mass of about 1.8 cm on ultrasound re-examination. The cat was not given adjuvant therapy. The cat was euthanased 8 months after surgery because of tumour invasion of the urinary trigone and subsequent ureter dilation, hydronephrosis and severe azotaemia. RELEVANCE AND NOVEL INFORMATION: Malignant urinary fibrosarcoma in this cat appeared to be only locally invasive. Palliative surgery without adjuvant postoperative chemotherapy in this cat resulted in an 8 month period of good quality of life.

Fibrosarcoma of the urinary bladder in a cat.

CASE SUMMARY: A 5-year-old female spayed domestic shorthair cat was presented with haematuria, pollakiuria and stranguria of 2 months' duration, and a firm non-painful mass in the urinary bladder was palpated. Abdominal radiographs showed thickening and irregular cranial margins of the urinary bladder wall. Abdominal ultrasound showed a vascularised mass of mixed echogenicity almost entirely occupying the urinary bladder lumen. During explorative laparotomy, the mass appeared pedunculated and was totally excised. Histopathology was characterised by infiltration of the mucosal, submucosal and muscular layers by proliferated atypical mesenchymal cells; immunochemistry confirmed the diagnosis of fibrosarcoma. The cat was discharged with normal urination 5 days after surgery. The owner declined any imaging follow-up but reported the cat to be free of any clinical signs at 16 months after surgery. RELEVANCE AND NOVEL INFORMATION: To the best of our knowledge, this is the first case of primary fibrosarcoma of the urinary bladder in the cat. Fibrosarcoma should be included in the differential diagnosis of urinary bladder neoplasia.

Sodium glucose cotransporter 1 ligand BLF501 as a novel tool for management of gastrointestinal mucositis.

BACKGROUND: Recent studies demonstrated that engagement of sodium glucose transporter 1 (SGLT-1) by orally administered D-glucose protects the intestinal mucosa from lipopolysaccharide (LPS)-induced injury. We tested whether SGLT-1 engagement might protect the intestinal mucosa from doxorubicin (DXR)- and 5-fluorouracil (5-FU)-induced injury in animal models mimicking acute or chronic mucositis. METHODS: Mice were treated intraperitoneally with DXR, alone or in combination with 5-FU, and orally with BLF501, a glucose-derived synthetic compound with high affinity for SGLT-1. Intestinal mucosal epithelium integrity was assessed by histological analysis, cellular proliferation assays, real-time PCR gene expression assays and Western blot assays. Student's t-test (paired two-tailed) and χ2 analyses were used for comparisons between groups. Differences were considered significant at p < 0.05. RESULTS: BLF501 administration in mice treated with DXR and/or 5-FU decreased the injuries to the mucosa in terms of epithelial integrity and cellular proliferative ability. Co-treatment with BLF501 led to a normal expression and distribution of both zonula occludens-1 (ZO-1) and beta-catenin, which were underexpressed after treatment with either chemotherapeutic agent alone. BLF501 administration also restored normal expression of caspase-3 and ezrin/radixin/moesin (ERM), which were overexpressed after treatment with DXR and 5-FU. In SGLT1-/- mice, BLF501 had no detectable effects. BLF501 administration in wild-type mice with growing A431 tumors did not modify antitumor activity of DXR. CONCLUSIONS: BLF501-induced protection of the intestinal mucosa is a promising novel therapeutic approach to reducing the severity of chemotherapy-induced mucositis.

Reduced diversity of immunoglobulin and T-cell receptor gene rearrangements in chronic inflammatory gastrointestinal diseases in dogs.

Inflammatory bowel disease has a multifactorial etiology in dogs as it does in humans. Evidence has been accumulated showing an abnormal response of the immune system, mostly represented by lymphocyte infiltration in the lamina propria of the gastrointestinal tract and in the epithelium, likely driven by chronic antigenic stimulation against luminal microorganisms. A relevant role is also ascribed to the genetic predisposition typical of some canine breeds. The role of chronic antigenic stimulation is still under debate. It may be responsible for selective pressure on the lymphoid population, favouring the emergence of some lymphocyte clones. This cross-sectional study is aimed at investigating the immunoglobulin and T-cell receptor gene rearrangements in a group of dogs affected by inflammatory bowel disease. The database of a referral Veterinary Laboratory was investigated. Based upon the histological evaluation of the bioptic samples collected during endoscopy, 54 canine cases met the WSAVA criteria for diagnosing IBD and were included in the study. The histological slides were retrieved and the gDNA was purified using protocols for formalin-fixed tissue. The gDNA was PCR amplified using fluorescent-labelled primers specific for canine immunoglobulin and T-cell receptor gene rearrangements; the PCR products were analysed with fragment analysis by means of capillary electrophoresis on an automatic sequencer (GeneScanning). In 47/54 (87.3%) cases, it was possible to amplify the gDNA. Twenty-one patients out of 47 (44.7%) showed polyclonal patterns in both the immunoglobulin and the T-cell receptors, 18/47 (38.3%) showed at least one oligoclonal pattern without monoclonal ones while 8/47 (17.0%) cases showed an Ig (7/47; 14.9%) or TCR (1/47: 2.1%) monoclonal pattern. These findings indicate that reduced diversity of the immunoglobulin and T-cell receptor repertoire occurs in canine inflammatory bowel disease. The reduced diversity correlated significantly with the severity of the histological lesions and carried a significantly increased risk of death. Beside its possible role as a reliable ancillary assay, immunoglobulin and T-cell receptor GeneScanning analysis points to the possible role of aberrant chronic antigenic stimulation, leading to clonal expansion of certain lymphocyte subsets in the pathogenesis of canine IBD.

Inflammatory polyps of the nasal turbinates of cats: an argument for designation as feline mesenchymal nasal hamartoma.

Inflammatory polyps of the nasal turbinates (IPNT) in cats are benign growths that are histologically distinct from feline nasopharyngeal polyps. Most cats with IPNT are presented at less than 1 year of age with sneezing, noisy breathing and epistaxis, but without mucoid or mucopurulent nasal discharge. Histologically, IPNT are characterised by the presence of woven bone as part of the proliferating stroma and erythrocyte-filled spaces. These unique histological features are analogous to nasal hamartomas (NH) of children, specifically chondromesenchymal hamartoma (NCMH) and sinonasal fibro-osseous hamartoma (SFOH), which also result in signs of nasal obstruction, sneezing and epistaxis. In our study, clinical and histopathological features in five cats with IPNT were compared with published descriptions of NH in children. We conclude that the terminology 'feline mesenchymal nasal hamartoma' provides a more accurate description of the disease currently termed IPNT, and has the added advantage of being consistent with its human counterpart.

Analysis of prognostic factors associated with injection-site sarcomas in cats: 57 cases (2001-2007).

OBJECTIVE: To identify prognostic factors in cats with injection-site sarcomas (ISSs). DESIGN: Retrospective case series. ANIMALS: 57 cats with ISSs. PROCEDURES: Medical records of cats were reviewed with regard to sex, age, anatomic site of tumor, tumor size, histologic grade, excision of a primary tumor versus excision of a recurrent ISS, use of excision alone versus excision plus adjuvant therapy, local tumor recurrence, and development of distant metastasis to predict overall survival time (ie, time from tumor excision to death). RESULTS: In univariate analyses, local recurrence and development of distant metastasis were significantly associated with survival time in cats. On multivariate analysis, development of distant metastasis remained a significant prognostic factor. Histologic grade was associated with distant metastasis, with cats having grade 3 tumors being significantly more likely to develop metastasis than cats with grade 1 and 2 tumors. Factors associated with local recurrence of ISSs were not identified. CONCLUSIONS AND CLINICAL RELEVANCE: The development of distant metastasis, which may occur later during the course of the disease, was identified as a prognostic factor for overall survival time in cats with ISSs. In addition, cats with histologic grade 3 ISSs should be considered for further interventional studies with chemotherapy to prevent the high rate of distant metastasis.

Reflux esophagitis in three cats associated with metaplastic columnar esophageal epithelium.

Gastroesophageal reflux is a relatively common condition in dogs and cats and may lead to secondary reflux esophagitis. A consequence of chronic gastroesophageal reflux that is well described in humans is Barrett's esophagus, which is the replacement of the normal squamous epithelium of the distal esophagus with metaplastic columnar epithelium. Three cats with clinical and endoscopic signs of chronic esophagitis had metaplastic columnar epithelium on biopsy of the distal esophageal mucosa. Suspected underlying causes were cardial incompetence and sliding hiatal hernia. Two cats had complete resolution of the clinical signs after treatment. One cat was euthanized.