Renal alterations in cats (Felis catus) housed in shelters and affected by systemic AA-amyloidosis: Clinicopathological data, histopathology, and ultrastructural features.

AA-amyloidosis is frequent in shelter cats, and chronic kidney disease is the foremost cause of death. The aims were to describe kidney laboratory and microscopic findings in shelter cats with AA-amyloidosis. Cats were included if kidney specimens were collected post-mortem and laboratory data were available within 6 months before death. Renal lesions were evaluated with optical and electron microscopy. Mass spectrometry was used to characterize amyloid. Nine domestic short-hair cats were included; 4 females and 5 males with a median age of 8 years (range = 2-13). All cats had blood analyses and urinalyses available. Serum creatinine concentrations were increased in 6 cats and symmetric dimethylarginine was increased in all of the cats. All of the cats had proteinuria. Eight of 9 cats had amyloid in the medulla, and 9 had amyloid in the cortex (glomeruli). All cats had amyloid in the interstitium. Six cats had concurrent interstitial nephritis and 1 had membranoproliferative glomerulonephritis. All cats had extrarenal amyloid deposits. Amyloid was AA in each case. In conclusion, renal deposition of amyloid occurs in both cortex and medulla in shelter cats and is associated with azotemia and proteinuria. Renal involvement of systemic AA-amyloidosis should be considered in shelter cats with chronic kidney disease. The cat represents a natural model of renal AA-amyloidosis.

Tumor Immune Microenvironment and Its Clinicopathological and Prognostic Associations in Canine Splenic Hemangiosarcoma.

Tumor cells can induce important cellular and molecular modifications in the tissue or host where they grow. The idea that the host and tumor interact with each other has led to the concept of a tumor microenvironment, composed of immune cells, stromal cells, blood vessels, and extracellular matrix, representing a unique environment participating and, in some cases, promoting cancer progression. The study of the tumor immune microenvironment, particularly focusing on the role of tumor-infiltrating lymphocytes (TILs), is highly relevant in oncology due to the prognostic and therapeutic significance of TILs in various tumors and their identification as targets for therapeutic intervention. Canine splenic hemangiosarcoma (HSA) is a common tumor; however, its immune microenvironment remains poorly understood. This retrospective study aimed to characterize the histological and immunohistochemical features of 56 cases of canine splenic HSA, focusing particularly on tumor-infiltrating lymphocytes (TILs). We assessed the correlations between the lymphocytic response, the macroscopic and histological characteristics of the tumor, and the survival data. Our study demonstrated that FoxP3 distribution was associated with tumor-related death and survival, while the CD20 count was associated with metastasis. This study provides an in-depth characterization of the tumor immune microenvironment in canine splenic HSA and describes potential prognostic factors.

Deformable Vesicles with Edge Activators for the Transdermal Delivery of Non-Psychoactive Cannabinoids.

BACKGROUND: Transdermal delivery of highly lipophilic molecules is challenging due to the strong barrier function of the skin. Vesicles with penetration enhancers are safe and efficient systems that could improve the transdermal delivery of non-psychoactive cannabinoids such as cannabidiol and desoxy-cannabidiol. In the last decades, research interest in desoxy-cannabidiol as a potent drug with anti-nociceptive properties has risen. Still, its scarce market availability poses a limit for both research and clinical applications. Therefore, it is necessary to improve the synthesis to produce sufficient amounts of desoxy-cannabidiol. Moreover, also the formulation aspects for this drug are challenging and require to be addressed to meet an efficient delivery to the patients. OBJECTIVE: This work aimed to develop innovative phospholipid-based vesicles with propylene glycol (PG), oleic acid (OA), or limonene as edge activators, for the transdermal delivery of highly lipophilic drugs such as non-psychoactive cannabinoids. In particular, desoxy-cannabidiol was selected thanks to its anti-nociceptive activity, and its synthesis was improved enhancing the stereoselectivity of its synthon's production. METHODS: Desoxy-cannabidiol was synthesized by Lewis acid-mediated condensation of p-mentha-2,8-dien- 1-ol and m-pentylphenol, improving the stereoselectivity of the first synthon's production. Transethosomes containing 20-50% w/w PG, 0.4-0.8% w/w OA, or 0.1-1% w/w limonene were optimized and loaded with cannabidiol or desoxy-cannabidiol (0.07-0.8% w/w, 0.6-7.0 mg/mL). Ex-vivo studies were performed to assess both the skin permeation and accumulation of the cannabinoids, as well as the penetration depth of fluorescein- loaded systems used as models. RESULTS: An enantioselective bromination was added to the pathway, thus raising the production yield of pmentha- 2,8-dien-1-ol to 81% against 35%, and the overall yield of desoxy-cannabidiol synthesis from 12% to 48%. Optimized transethosomes containing 0.6 mg/mL cannabinoids were prepared with 1:10 PG:lipid weight ratio, 0.54 OA:lipid molar ratio, and 0.3 limonene:lipid molar ratio, showing good nanometric size (208 ± 20.8 nm - 321 ± 26.3 nm) and entrapment efficiency (> 80%). Ex-vivo tests showed both improved skin permeation rates of cannabinoids (up to 21.32 ± 4.27 µg/cm2 cannabidiol), and skin penetration (depth of fluorescein up to 240 µm, with PG). CONCLUSION: Desoxy-cannabidiol was successfully produced at high yields, and formulated into transethosomes optimized for transdermal delivery. Loaded vesicles showed improved skin penetration of desoxy-cannabidiol, cannabidiol and a lipophilic probe. These results suggest the potential of these carriers for the transdermal delivery of highly lipophilic drugs.

Renal amyloid-A amyloidosis in cats: Characterization of proteinuria and biomarker discovery, and associations with kidney histology.

BACKGROUND: Amyloid A (AA) amyloidosis is a protein misfolding disease arising from serum amyloid A (SAA). Systemic AA amyloidosis recently was shown to have a high prevalence in shelter cats in Italy and was associated with azotemia and proteinuria. OBJECTIVES: Investigate urine protein profiles and diagnostic biomarkers in cats with renal AA amyloidosis. ANIMALS: Twenty-nine shelter cats. METHODS: Case-control study. Cats with renal proteinuria that died or were euthanized between 2018 and 2021 with available necropsy kidney, liver and spleen samples, and with surplus urine collected within 30 days before death, were included. Histology was used to characterize renal damage and amyloid amount and distribution; immunohistochemistry was used to confirm AA amyloidosis. Urine protein-to-creatinine (UPC) and urine amyloid A-to-creatinine (UAAC) ratios were calculated, and sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) and liquid chromatography-mass spectrometry (LC-MS) of proteins were performed. RESULTS: Twenty-nine cats were included. Nineteen had AA amyloidosis with renal involvement. Cats with AA amyloidosis had a higher UPC (median, 3.9; range, 0.6-12.7 vs 1.5; 0.6-3.1; P = .03) and UAAC ratios (median, 7.18 × 10-3 ; range, 23 × 10-3 -21.29 × 10-3 vs 1.26 × 10-3 ; 0.21 × 10-3 -6.33 × 10-3 ; P = .04) than unaffected cats. The SDS-AGE identified mixed-type proteinuria in 89.4% of cats with AA amyloidosis and in 55.6% without AA amyloidosis (P = .57). The LC-MS identified 63 potential biomarkers associated with AA amyloidosis (P < .05). Among these, urine apolipoprotein C-III was higher in cats with AA amyloidosis (median, 1.38 × 107 ; range, 1.85 × 105 -5.29 × 107 vs 1.76 × 106 ; 0.0 × 100 -1.38 × 107 ; P = .01). In the kidney, AA-amyloidosis was associated with glomerulosclerosis (P = .02) and interstitial fibrosis (P = .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Renal AA amyloidosis is associated with kidney lesions, increased proteinuria and increased urine excretion of SAA in shelter cats. Additional studies are needed to characterize the role of lipid transport proteins in the urine of affected cats.

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/ß-Catenin and Hippo Pathways.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high lethality, and new therapeutics as well as new spontaneous animal models are needed to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors that share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, its active metabolite berberrubine, and eight analogs, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogs on cell viability via the induction of apoptosis, also identifying their role in inhibiting the Wnt/ß-catenin pathway and activating the Hippo signals with a downstream reduction in CTGF expression. In particular, the berberine analogs NAX035 and NAX057 show the highest therapeutic efficacy, deserving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spontaneous neoplastic diseases are needed, aiming at improving veterinary treatments of cancer as well as translational cancer research.

Histological evaluation of the distribution of systemic AA-amyloidosis in nine domestic shorthair cats.

Amyloidosis is a group of protein-misfolding disorders characterized by the accumulation of amyloid in organs, both in humans and animals. AA-amyloidosis is considered a reactive type of amyloidosis and in humans is characterized by the deposition of AA-amyloid fibrils in one or more organs. In domestic shorthair cats, AA-amyloidosis was recently reported to be frequent in shelters. To better characterize this pathology, we report the distribution of amyloid deposits and associated histological lesions in the organs of shelter cats with systemic AA-amyloidosis. AA-amyloid deposits were identified with Congo Red staining and immunofluorescence. AA-amyloid deposits were then described and scored, and associated histological lesions were reported. Based on Congo Red staining and immunofluorescence nine shelter cats presented systemic AA-amyloidosis. The kidney (9/9), the spleen (8/8), the adrenal glands (8/8), the small intestine (7/7) and the liver (8/9) were the organs most involved by amyloid deposits, with multifocal to diffuse and from moderate to severe deposits, both in the organ parenchyma and/or in the vascular compartment. The lung (2/9) and the skin (1/8) were the least frequently involved organs and deposits were mainly focal to multifocal, mild, vascular and perivascular. Interestingly, among the organs with fibril deposition, the stomach (7/9), the gallbladder (6/6), the urinary bladder (3/9), and the heart (6/7) were reported for the first time in cats. All eye, brain and skeletal muscle samples had no amyloid deposits. An inflammatory condition was identified in 8/9 cats, with chronic enteritis and chronic nephritis being the most common. Except for secondary cell compression, other lesions were not associated to amyloid deposits. To conclude, this study gives new insights into the distribution of AA-amyloid deposits in cats. A concurrent chronic inflammation was present in almost all cases, possibly suggesting a relationship with AA-amyloidosis.

Toward Improving Triplet Energy Transfer from Tetracene to Silicon Using a Covalently Bound Tetracene Seed Layer.

Silicon solar cells are operating close to the theoretical maximum efficiency limit. To increase their efficiency beyond this limit, it is necessary to decrease energy losses occurring for high-energy photons. A sensitizing layer of singlet-fission material can in principle double the current generated by high-energy photons, and significantly reduce energy losses from high-energy photons within the solar cell. Here, we construct a model of such a solar cell, using Si(111) surfaces and tetracene. To increase the energy transfer between the two layers, a series of tetracene derivatives was synthesized, and the molecules were covalently attached onto the silicon surface as a seed layer. Using X-ray diffraction, a shift in crystal structure and ordering of the tetracene close to the seed layer can be observed. Unfortunately, the effect on the energy transfer was limited, showing a need for further investigations into the effect of the seed layer.

AA-amyloidosis in cats (Felis catus) housed in shelters.

Systemic AA-amyloidosis is a protein-misfolding disease characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. A high prevalence of AA-amyloidosis has been reported in captive cheetahs and a horizontal transmission has been proposed. In domestic cats, AA-amyloidosis has been mainly described in predisposed breeds but only rarely reported in domestic short-hair cats. Aims of the study were to determine AA-amyloidosis prevalence in dead shelter cats. Liver, kidney, spleen and bile were collected at death in cats from 3 shelters. AA-amyloidosis was scored. Shedding of amyloid fibrils was investigated with western blot in bile and scored. Descriptive statistics were calculated. In the three shelters investigated, prevalence of AA-amyloidosis was 57.1% (16/28 cats), 73.0% (19/26) and 52.0% (13/25), respectively. In 72.9% of cats (35 in total) three organs were affected concurrently. Histopathology and immunofluorescence of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B = 0.026, CI95% = 0.007-0.046; p = 0.010). AA-amyloidosis was very frequent in shelter cats. Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease. In conclusion, AA-amyloidosis was very frequent in shelter cats and those staying longer had more deposits. The cat may represent a natural model of AA-amyloidosis.

Epithelial-to-Mesenchymal Transition and Phenotypic Marker Evaluation in Human, Canine, and Feline Mammary Gland Tumors.

Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties. EMT has been closely associated with cancer cell aggressiveness. The aim of this study was to evaluate the mRNA and protein expression of EMT-associated markers in mammary tumors of humans (HBC), dogs (CMT), and cats (FMT). Real-time qPCR for SNAIL, TWIST, and ZEB, and immunohistochemistry for E-cadherin, vimentin, CD44, estrogen receptor (ER), progesterone receptor (PR), ERBB2, Ki-67, cytokeratin (CK) 8/18, CK5/6, and CK14 were performed. Overall, SNAIL, TWIST, and ZEB mRNA was lower in tumors than in healthy tissues. Vimentin was higher in triple-negative HBC (TNBC) and FMTs than in ER+ HBC and CMTs (p < 0.001). Membranous E-cadherin was higher in ER+ than in TNBCs (p < 0.001), whereas cytoplasmic E-cadherin was higher in TNBCs when compared with ER+ HBC (p < 0.001). A negative correlation between membranous and cytoplasmic E-cadherin was found in all three species. Ki-67 was higher in FMTs than in CMTs (p < 0.001), whereas CD44 was higher in CMTs than in FMTs (p < 0.001). These results confirmed a potential role of some markers as indicators of EMT, and suggested similarities between ER+ HBC and CMTs, and between TNBC and FMTs.

Characterization and function of extracellular vesicles in a canine mammary tumour cell line: Ultracentrifugation versus size exclusion chromatography.

Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles involved in many biological processes such as tumour progression. For years, ultracentrifugation (UC) has been considered the gold standard for EV isolation but limited purity and integrity allowed the diffusion of alternative techniques. In this study, EVs were isolated from a canine mammary tumour cell line using UC and size exclusion chromatography (SEC) and analysed for size and concentration by nanoparticle tracking analysis (NTA) and for protein expression by western blot (WB). EV autocrine effect on cell proliferation, migration and invasiveness was then evaluated in vitro. In all samples, particles were in the EV size range (50-1000 nm), with a higher concentration in UC than in SEC samples (1011 and 1010 particles/ml respectively), and expressed EV markers (Alix, CD9). Functional assays did not show statistically significant difference among conditions, but EV treatment slightly increased cell proliferation and invasiveness and treatment with SEC-isolated EVs slightly enhanced cell migration compared to UC-isolated EVs. In conclusion, the main differences between the two isolation techniques are the quantity of the final EV-product and slight differences on EV functionality, which should be further explored to better highlight the real autocrine effect of tumoral EVs.

Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter.

AA amyloidosis is a systemic disease characterized by deposition of misfolded serum amyloid A protein (SAA) into cross-ß amyloid in multiple organs in humans and animals. AA amyloidosis occurs at high SAA serum levels during chronic inflammation. Prion-like transmission was reported as possible cause of extreme AA amyloidosis prevalence in captive animals, e.g. 70% in cheetah and 57-73% in domestic short hair (DSH) cats kept in zoos and shelters, respectively. Herein, we present the 3.3 Å cryo-EM structure of AA amyloid extracted post-mortem from the kidney of a DSH cat with renal failure, deceased in a shelter with extreme disease prevalence. The structure reveals a cross-ß architecture assembled from two 76-residue long proto-filaments. Despite >70% sequence homology to mouse and human SAA, the cat SAA variant adopts a distinct amyloid fold. Inclusion of an eight-residue insert unique to feline SAA contributes to increased amyloid stability. The presented feline AA amyloid structure is fully compatible with the 99% identical amino acid sequence of amyloid fragments of captive cheetah.

Resizing of the Gastric Pouch for Weight Regain after Laparoscopic Roux-en-Y Gastric Bypass and One-Anastomosis Gastric Bypass: Is It a Valid Option?

Introduction: The laparoscopic resizing of the gastric pouch (LPR) has recently been proposed as a revisional technique in the case of weight regain (WR) after gastric bypass procedures. The aim of this study was to report our experience with LPR for WR. Materials and Methods: All patients with WR ≥ 25% after gastric bypass and with a dilated gastric pouch and/or gastrojejunal anastomosis who underwent LPR between January 2017 and January 2022 were retrospectively reviewed. From a radiological point of view, a gastric pouch was considered dilated when its volume was calculated at >80 cm3 for LRYGB and >200 cm3 for OAGB upon a 3D-CT scan. The endoscopic criterion considered both the diameter of the gastrojejunal anastomosis and the gastric pouch volume. All anastomoses > 20 mm for LRYGB and >40 mm for OAGB were considered dilated, while a gastric pouch was considered endoscopically dilated when the retrovision maneuver with the gastroscope was easily performed. These selection criteria were arbitrarily established on the basis of both our personal experience and literature data. Results: Twenty-three patients had LPR after a Roux-en-Y gastric bypass or one-anastomosis gastric bypass. The mean BMI at LPR was 36.3 ± 4.7 kg/m2. All patients underwent LPR, while the resizing of the GJA was also performed in 3/23 (13%) cases, and hiatoplasty was associated with the resizing of the pouch in 6/23 cases (26.1%). The mean BMI at the last follow-up was 29.3 ± 5.8 kg/m2. The difference between the BMI before resizing and the BMI at the last follow-up visit was statistically significant (p = 0.00005). The mean %TWL at 24.2 ± 16.1 months was 19.6 ± 9%. Comorbidities had an overall resolution and/or improvement rate of 47%. The mean operative time was 71.7 ± 21.9 min. The conversion rate was nil. Postoperative complications occurred in two cases (8.7%). Conclusions: In our series, LPR for WR showed good results in weight loss and in improvement/resolution of comorbidities, with an acceptable complication rate and operative time. Only further studies with a greater cohort of patients and a longer postoperative follow-up will be able to highlight the long-term benefits of this technique.

Multidrug resistant Enterococcus faecium isolate from cholangitis/cholecystitis in a dog.

Mucocele and cholecystitis were diagnosed in a 10 year-old Shetland-sheepdog presenting aspecific clinical signs. Blood examinations and ultrasonography investigation were performed before to surgical approach, which allowed to collect biopsies and samples for bacteriological analyses. In addition, the patient was subjected to cholecystectomy. A multidrug resistant Enterococcus faecium was isolated from the gallbladder specimens. On the basis of antimicrobial susceptibility test, nitrofurantoin was used. The correct bacteriological diagnosis is necessary to set up effective therapy, influencing the patient's prognosis and improving the recovery time.

Trauma workload during COVID19 lockdown: an analysis of incidence in 4 million people.

BACKGROUND: Aim of this study is to report the trauma workload during COVID19 lockdown in a region of four million people and to compare it with the same period in 2019. METHODS: The regional register for A&E admissions and hospitalizations has been reviewed in order to compare the number of A&D admission, the triage colour codes rates, aetiology of trauma, number of patients hospitalized for trauma, number of fractures that required surgery, type of fractures and injuries and mean patients' age. RESULTS: During lockdown 7314 patients were admitted in A&E, while 22,508 patients were admitted in 2019. In 2020 and 2019 triage codes were respectively distributed as follows: red code 0.1% vs 0.2%, yellow code 8.9% vs 6.3%, green code 84% vs 84.7% and white code 6% vs 8.8%. (p = 0.042). The number of hospitalized patients for trauma was 670 in 2020, while in 2019 was 1774 (p = 0.02). The most common fracture that required surgery was femur fracture (409 in 2020 vs 635 in 2019); fracture subtype distribution and mean age of the patients were significantly different in the two groups (respectively p < 0.01 and p = 0.02). CONCLUSIONS: One month of lockdown showed a 68% decrease in the number of A&E visits and a 74% decrease of fractures that required surgery. Femur fracture showed the lowest decrease moving from 635 to 409 units but increasing their incidence rate (42 to 61%).

A Recurrent Inflammatory Myofibroblastic Tumor-like Lesion of the Splenic Capsule in a Kitten: Clinical, Microscopic and Ultrastructural Description.

OBJECTIVE: To describe the findings of an unusual splenic tumor in a kitten. METHODS: A grossly multinodular mass arising from the splenic capsule of a 7-month-old male Havana kitten was echographically detected and surgically removed by splenectomy, then analyzed microscopically and ultrastructurally. RESULTS: The mass showed features of an inflammatory myofibroblastic tumor. TREATMENT AND OUTCOME: Two months after surgical excision, the mass recurred in the same intra-abdominal area but disappeared after 2 months of anti-inflammatory therapy. Follow-up at 18 months after surgery revealed resolution of the disease. CLINICAL RELEVANCE: Inflammatory myofibroblastic tumor in cats have been rarely reported and are usually in the orbital region. In the present report, an unusual multinodular gross presentation, a recurrence over time, and a favorable clinical course, are described.