Investigation of Serum Markers of Hepatic Fibrosis in Equids.

Liver disease is common in equine practice, and treatment and prognosis are dependent on histopathologic examination of biopsies. Liver biopsy is invasive and expensive which restricts its use. Serum markers are used to predict hepatic fibrosis in humans. This study aimed to investigate the enhanced liver fibrosis (ELF) test, based on serum Hyaluronic Acid (HA), procollagen III N-terminal peptide (PIIINP), and tissue inhibitor of metalloproteinase 1 (TIMP-1) to detect hepatic fibrosis in equids. Four groups were included; two with increased serum concentrations of liver-derived enzymes and a liver biopsy (group H; 10 horses and ponies and group D; 10 donkeys) and two without any evidence of liver disease (group HC; 10 horses and ponies and group DC; 10 donkeys). All samples were analyzed for concentrations of HA, PIINP, and TIMP-1. Given the failure to detect TIMP-1 in most subjects, a novel eELF (equid ELF) score was calculated, based on HA and PIIINP. HA and PIIINP concentrations and the eELF score, were compared with determined hepatic fibrosis. HA, PIIINP, and eELF were significantly greater in horses and ponies with a histopathologic fibrosis score ≥ 2 compared with those < 2. A similar observation was found with donkeys for HA and eELF. A significant correlation was found between fibrosis score and HA, PIIINP, and eELF for horses and ponies, and between fibrosis score and HA and eELF in donkeys. Serum HA and the eELF score might be useful serum markers to predict and monitor hepatic fibrosis in horses, ponies, and donkeys.

Shear wave velocity values measured by 2D-shear wave elastography are not different between awake and anesthetized cats without clinically significant hepatic fibrosis.

Two-dimensional shear wave elastography (2D-SWE) is widely used as a noninvasive method to quantify liver stiffness. In humans, liver stiffness approximates histologic hepatic fibrosis. While histology is the gold standard for diagnosing liver disease, 2D-SWE may be a minimally invasive alternative to biopsy in feline patients. The objectives of this prospective, observational, crossover study were trifold: (1) to assess the feasibility of performing 2D-SWE in awake cats, (2) to determine whether anesthesia altered shear wave velocity (SWV) measurements, and (3) to correlate hepatic stiffness with histologically quantified hepatic fibrosis. Eleven healthy, purpose-bred cats underwent 2D-SWE in awake and anesthetized states. SWV measurements were compared with histologic fibrosis measurements obtained from liver biopsies during the anesthetic period. The mean velocities were not significantly different between awake (1.47 ± 0.18 m/s) and anesthetized (1.47 ± 0.24 m/s) cats. Premedication and anesthetic drugs did not impact mean SWV. There was a higher variability in the SWV values in the awake group. The data points were reliably replicated, with an interquartile range of 0.24 and 0.32 in anesthetized and awake groups, respectively. There was moderate agreement between observers (intraclass correlation coefficient = 0.66). All cats had clinically insignificant fibrosis. There was no correlation between the SWV measurements and the histological fibrosis values. This study demonstrates that 2D-SWE is feasible in awake cats and that the anesthetic protocol employed did not significantly alter mean SWV. This work is the first to histologically validate normal SWV values in cats and show that 2D-SWE cannot differentiate minimal differences in feline hepatic fibrosis.

Liver fibrosis is a common pathological change in the liver of dairy cows with fatty liver.

Fatty liver (i.e., hepatic lipidosis) is a prevalent metabolic disorder in dairy cows during the transition period, characterized by excess hepatic accumulation of triglyceride (TG), tissue dysfunction, and cell death. Detailed pathological changes, particularly hepatic fibrosis, during fatty liver remain to be determined. Liver fibrosis occurs as a consequence of liver damage, resulting from the excessive accumulation of extracellular matrix, which distorts the architecture of the normal liver, compromising its normal synthetic and metabolic functions. Thus, we aimed to investigate liver fibrosis status and its potential causal factors including oxidative stress, hepatocyte apoptosis, and production of inflammatory cytokines in the liver of cows with fatty liver. Forty-five dairy cows (parity, 3-5) were selected, and liver biopsy and blood were collected on the second week postpartum (days in milk, 10-14 d). On the basis of the degree of lipid accumulation in liver, selected cows were categorized into normal (n = 25; TG <1% wet wt), mild fatty liver (n = 15; 1% ≤ TG <5% wet wt), and moderate fatty liver (n = 5; 5% ≤ TG <10% wet wt). Compared with normal cows, blood concentrations of nonesterified fatty acids and ß-hydroxybutyrate, along with alanine aminotransferase and aspartate aminotransferase activities, were greater in the cows with fatty liver (mild and moderate). Hepatic extracellular matrix deposition, as indicated by Picrosirius red staining, was greater in cows with fatty liver than those with normal ones. In addition, we observed an increased proportion of collagen type I fiber in extracellular matrix with increased lipid accumulation in the liver. Compared with normal cows, the area of α-smooth muscle actin (α-SMA)-positive staining along with the mRNA abundance of collagen type I α 1 (COL1A1), ACTA2 (gene encoding α-SMA), and transforming growth factor-ß (TGFB) were greater in cows with fatty liver. Compared with normal cows, hepatic contents of malondialdehyde, glutathione disulfide, and 8-isoprostane were greater, whereas total antioxidant capacity, the hepatic content of glutathione, and activities of antioxidant indicators, including superoxide dismutase, glutathione peroxidase, and catalase, were lower in cows with fatty liver. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and abundance of apoptosis-related molecules BAX, CASP3, CASP8, and CASP9 were greater in cows with fatty liver. However, mRNA abundance of the anti-apoptotic gene BCL2 did not differ. The mRNA abundance of pro-inflammatory cytokines including tumor necrosis factor-α (TNFA), interleukin-1ß (IL1B), and interleukin-6 (IL6) was greater in the liver of cows with fatty liver. Overall, the present study indicated that fibrosis is a common pathological response to liver damage and is associated with oxidative stress, hepatocyte death, and inflammation.

Research Note: Expression of IGF-1 and IGF-1 receptor proteins in skeletal muscle fiber types in chickens with hepatic fibrosis.

We investigated the expression of insulin-like growth factor 1 (IGF-1) and IGF-1 type 1 receptor (IGF-1R) in skeletal muscle fiber types in chickens with hepatic fibrosis induced by bile duct ligation (BDL). Eleven hens, approximately 104 weeks old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM; n = 7) groups. In BDL hens, histopathology revealed marked bile duct proliferation and liver fibrosis. The cross-sectional area (CSA) of myofibers from both the pectoralis (PCT) muscles significantly decreased in the BDL group compared with the SHAM group (P < 0.01). In contrast, the CSA of myofibers from the femorotibialis lateralis (FTL) muscle did not decrease in the BDL group. Type I fibers were large, round, and hypertrophic. Elongated type IIA and IIB fibers were also present. For IGF-1 immunostaining, the immunoreaction intensity was higher in the PCT in the BDL group than the SHAM group. Within the BDL group, type I fibers from FTL had a stronger immunoreaction intensity than the type II fibers. For IGF-1R immunostaining, the intensity of the immunoreactions was similar within the PCT in the BDL group compared with the SHAM group. For FTL, type I fibers had stronger reactions to IGF-1R than type II fibers in the BDL group. These results suggest that type I fibers express both IGF-1 and IGF-1R and become hypertrophic in chickens with hepatic fibrosis.

Gastroduodenal ulceration in dogs with liver disease.

BACKGROUND: Liver disease is frequently cited as a cause of gastroduodenal ulceration (GDU) in dogs but studies regarding GDU and liver disease are limited. OBJECTIVES: To document the presence of GDU in dogs with liver disease. ANIMALS: Forty dogs that underwent liver biopsy, computed tomographic (CT) angiography or both at the University of Florida Small Animal Hospital to diagnose congenital or acquired liver disease. METHODS: Cross-sectional study. Dogs had gastroduodenoscopy performed with photographic and video documentation in a standardized fashion. Lesions (hemorrhage, erosions, ulcers) in the esophagus, stomach, and duodenum were scored based on a grading scale. Presence of esophageal varices was recorded. Dogs were categorized into 4 groups according to cause of liver disease (inflammatory disease, cirrhosis, congenital, other). Presence or absence of ulcers, erosions or both as well as total endoscopic scores were compared among groups. RESULTS: Forty dogs were enrolled with the following distribution: 13 congenital, 13 inflammatory, 3 cirrhosis, and 11 other. Four dogs had GDU (10%; 95% confidence interval [CI], 3%-24%) and 6 dogs had erosions (15%; 95% CI, 6%-30%). No difference was found in total endoscopic score (P = .21) or in the proportion of dogs with ulcers, erosions or both versus those without (P = .25) among the groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Gastroduodenal ulceration was found in 10% of dogs with liver disease in this population. Additional studies are warranted to confirm these findings in larger numbers of dogs with specific disease etiologies.

Relationships between congenital peritoneopericardial diaphragmatic hernia or congenital central diaphragmatic hernia and ductal plate malformations in dogs and cats.

OBJECTIVE: To characterize the association between peritoneopericardial diaphragmatic hernia (PPDH) or congenital central diaphragmatic hernia (CCDH) and ductal plate malformations (DPMs) in dogs and cats. ANIMALS: 18 dogs and 18 cats with PPDH or CCDH and 19 dogs and 18 cats without PPDH or CCDH. PROCEDURES: Evaluation of clinical details verified PPDH or CCDH and survival times. Histologic features of nonherniated liver samples were used to categorize DPM. Immunohistochemical staining for cytokeratin-19 distinguished bile duct profiles per portal tract and for Ki-67-assessed cholangiocyte proliferation. Histologic features of herniated liver samples from PPDH or CCDH were compared with those of pathological controls (traumatic diaphragmatic hernia, n = 6; liver lobe torsion, 6; ischemic hepatopathy, 2). RESULTS: DPM occurred in 13 of 18 dogs with the proliferative-like phenotype predominating and in 15 of 18 cats with evenly distributed proliferative-like and Caroli phenotypes. Congenital hepatic fibrosis DPM was noted in 3 dogs and 2 cats and renal DPM in 3 dogs and 3 cats. No signalment, clinical signs, or clinicopathologic features discriminated DPM. Kaplan Meier survival curves were similar in dogs and cats. Bile duct profiles per portal tract in dogs (median, 5.0; range, 1.4 to 100.8) and cats (6.6; 1.9 to 11.0) with congenital diaphragmatic hernias significantly exceeded those in healthy dogs (1.4; 1.2 to 1.6) and cats (2.3; 1.7 to 2.6). Animals with DPM lacked active cholangiocyte proliferation. Histologic features characterizing malformative bile duct profiles yet without biliary proliferation were preserved in herniated liver lobes in animals with DPM. CONCLUSIONS AND CLINICAL RELEVANCE: DPM was strongly associated with PPDH and CCDH. Because DPM can impact health, awareness of its coexistence with PPDH or CCDH should prompt biopsy of nonherniated liver tissue during surgical correction of PPDH and CCDH.

Resting and postprandial serum bile acid concentrations in dogs with liver disease.

BACKGROUND: Serum bile acids (SBAs) are frequently measured in dogs. However, there is limited data comparing SBAs in different liver diseases diagnosed according to standardized histological criteria. OBJECTIVES: To compare resting and postprandial SBAs, and determine their sensitivity and specificity, for various liver diseases in dogs. ANIMALS: Three hundred and forty-one client-owned dogs with suspected liver disease that had a liver biopsy and SBAs measured. METHODS: Multicenter retrospective study. Cases were classified according to standardized histological criteria. The sensitivity and specificity of resting and postprandial SBAs for the diagnosis of each liver disease, and all liver diseases combined, were calculated. RESULTS: The median resting SBAs were highest in dogs with cirrhosis (98.8 µmol/L; range, 6-135) and congenital circulatory anomalies (CCa; 79.45 µmol/L; 0.3-705). The highest median postprandial concentrations were found in CCa (126 µmol/L; 0-726) and chronic hepatitis (CH; 54.3 µmol/L; 0-260). Using the cut-off value of 10 µmol/L, the highest sensitivities of resting SBAs were recorded in dogs with CCa (87.5%; 95% confidence interval, 76.8-94.4) and CH (81.1%; 71.5-88.6). The sensitivities of postprandial SBAs were the highest in cholangitis (100%; 47.8-100.0) and CCa (91.1%; 78.8-97.5). The specificities of resting and postprandial SBAs for all diseases were 49.3% (37.6-61.1) and 29.7% (15.9-47.0), respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Postprandial SBAs are more sensitive but less specific than resting SBAs for the diagnosis of liver disease. There were dogs in all categories of liver disease with resting SBAs <10 and >90 µmol/L. Therefore, careful interpretation of both normal and elevated values is required.

Extrahepatic biliary obstruction can interfere with hepatic fibrosis prediction using two-dimensional shear wave elastography in dogs.

Two-dimensional shear wave elastography (2D-SWE) can be used to quantitatively evaluate the elastic modulus of the liver as shear wave velocity (SWV), which can noninvasively predict clinically relevant hepatic fibrosis in both dogs and humans. However, extrahepatic biliary obstruction (EHBO), regardless of the presence of clinically relevant hepatic fibrosis, can influence SWVs in humans and thus may interfere with hepatic fibrosis prediction using 2D-SWE in dogs. The aim of this prospective, observational, and one-group pretest-posttest study is to investigate whether SWV measured by 2D-SWE displays a difference between dogs with and without EHBO. A total of 20 dogs were included (7 with EHBO and 13 with gallbladder pathology but no EHBO) that underwent preoperative SWV measurement using 2D-SWE. In all dogs, stages of hepatic fibrosis were evaluated histopathologically using a scoring scheme. In addition, postoperative SWVs in dogs with EHBO relieved via laparotomy were also evaluated. The median (range) SWVs in the dogs with and without EHBO were 1.91 (1.81-2.54) m/s and 1.57 (1.37-1.64) m/s, respectively. Although there was no significant difference in the histopathological hepatic fibrosis stages between the dogs with and without EHBO, the preoperative SWVs in the dogs with EHBO were significantly higher than in dogs without EHBO (P = .0004), and SWVs were found to decrease significantly after surgery (P = .0097). This study demonstrates that EHBO can increase the SWV of dogs without clinically relevant hepatic fibrosis and can interfere with the prediction of noninvasive hepatic fibrosis using 2D-SWE.

Skeletal Muscle Fibre Type Changes in an Avian Model of Hepatic Fibrosis.

We investigated the susceptibility of type I and type II skeletal myofibres to atrophy in hens with hepatic fibrosis induced by bile duct ligation (BDL). Seven hens, approximately 2 years old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM) (n = 3) groups. Mean body weight and mean liver weight as a percentage of mean body weight were significantly lower in the BDL group than in the SHAM group at 4 weeks post surgery (P = 0.002, P = 0.005, respectively). Mean plasma aspartate aminotransferase activity was slightly higher, while total cholesterol (P <0.001), total bilirubin (P = 0.022) and NH3 (P = 0.048) concentrations were significantly higher in the BDL group than in the SHAM group. Liver lesions were induced in all hens in the BDL group. The weights of the pectoralis (PCT) (P = 0.049) and flexor perforans et perforatus digiti III (FPPD III) muscles (P = 0.006) as a percentage of body weight were significantly decreased in the BDL group. A significantly reduced mean myofibre cross-sectional area in the PCT of BDL hens (P = 0.005) was indicative of atrophy. No significant differences were observed in the fibre type composition of the PCT, supracoracoideus or FPPD III muscles between the SHAM and BDL groups. However, there was an approximate 43% increase in the number of type I fibres in the femorotibialis lateralis of the BDL group and small angular type II fibres and large round type I fibres in this muscle were characteristic of peripheral neuropathy. The results suggest that type II fibres are more susceptible to atrophy than type I fibres in this model of hepatic fibrosis.

A study on causes of cattle liver condemnation at an abattoir in Omdurman area, Khartoum State, Sudan.

BACKGROUND: Information obtained from abattoirs on the causes of liver condemnation is important in preventing the spread of diseases and for promoting food security. The current study reviews three years (2009 to 2011) postmortem inspection records of cattle slaughtered at an abattoir in Omdurman, Khartoum State, Sudan. The aim was to determine the prevalence of diseases and conditions that lead to liver condemnation. RESULTS: From a total of 234,175 cattle slaughtered, 8,910 (3.8%) livers were condemned due to several diseases/conditions mainly fasciolosis, cysticercosis, necrosis, abscess, calcification, hemorrhages, liver cirrhosis, hydatidosis, and other miscellaneous causes. Collectively, fasciolosis was the leading cause of liver condemnation and was responsible for 51.6 % of total liver condemnations followed by necrosis (18.6%), and cysticercosis (13.5%). CONCLUSIONS: Because of their zoonotic nature, the observed high frequency of some detected diseases/conditions is thought to pose a public health risk among consumers. This survey could be used as a regional baseline for future monitoring of control programmers against these liver diseases.

Usefulness of noninvasive shear wave elastography for the assessment of hepatic fibrosis in dogs with hepatic disease.

BACKGROUND: Two-dimensional shear wave elastography (2D-SWE) can noninvasively evaluate hepatic elastic modulus as shear wave velocity (SWV). Additionally, it may predict the presence of clinical relevant hepatic fibrosis (≥F2) in dogs with hepatic disease. OBJECTIVES: To investigate whether SWV measured by 2D-SWE can differentiate between dogs with (≥F2) and without (F0-1) clinically relevant hepatic fibrosis. ANIMALS: Twenty-eight client-owned dogs with hepatic disease and 8 normal healthy Beagle dogs were enrolled. METHODS: In this cross-sectional prospective study, SWVs were measured using 2D-SWE in all dogs. Hepatic fibrosis stages and necroinflammatory activity grades were histopathologically evaluated using a histological scoring scheme that was adapted from the Ishak schema used in human medicine. RESULTS: Median SWVs were significantly higher in dogs with clinically relevant hepatic fibrosis (2.04 m/s; range, 1.81-2.26 m/s) than in healthy dogs (1.51 m/s; range, 1.44-1.66 m/s; P = .007), and dogs without clinically relevant hepatic fibrosis (1.56 m/s; range, 1.37-1.67 m/s; P < .001). However, no significant difference was found in the SWVs between dogs without clinically relevant hepatic fibrosis and healthy dogs (P = .99). Furthermore, median SWVs were not significantly different among dogs with necroinflammatory activity, those without necroinflammatory activity, and healthy dogs (Kruskal-Wallis test, P = .12). CONCLUSIONS AND CLINICAL IMPORTANCE: The 2D-SWE may be useful for predicting the presence of hepatic fibrosis in dogs with hepatic disease.

Use of serum biomarkers in staging of canine hepatic fibrosis.

Accurate staging of hepatic fibrosis (HF) is important for treatment and prognosis of canine chronic hepatitis. HF scores are used in human medicine to indirectly stage and monitor HF, decreasing the need for liver biopsy. We developed a canine HF score to screen for moderate or greater HF. We included 96 dogs in our study, including 5 healthy dogs. A liver biopsy for histologic examination and a biochemistry profile were performed on all dogs. The dogs were randomly split into a training set of 58 dogs and a validation set of 38 dogs. A HF score that included alanine aminotransferase, alkaline phosphatase, total bilirubin, potassium, and gamma-glutamyl transferase was developed in the training set. Model performance was confirmed using the internal validation set, and was similar to the performance in the training set. The overall sensitivity and specificity for the study group were 80% and 70% respectively, with an area under the curve of 0.80 (0.71-0.90). This HF score could be used for indirect diagnosis of canine HF when biochemistry panels are performed on the Konelab 30i (Thermo Scientific), using reagents as in our study. External validation is required to determine if the score is sufficiently robust to utilize biochemical results measured in other laboratories with different instruments and methodologies.

Predicting copper toxicosis: relationship between the ATP7A and ATP7B gene mutations and hepatic copper quantification in dogs.

This preliminary study summarizes the genotypes of 42 Labrador Retrievers and Labrador Retriever-Golden Retriever crosses and phenotypes a subset of ten of these dogs that are homozygous mutant, heterozygous, or homozygous normal for mutations in the ATP7A and ATP7B genes that have been associated with the development of copper toxicosis in Labrador Retrievers. The purpose of this study is to evaluate whether there is a correlation between ATP7A and ATP7B genotypes and clinical evidence of hepatic pathology in young, asymptomatic Labrador Retrievers. We evaluated serum ALT levels, hepatic copper concentrations, and hepatic histopathology from ten offspring where both parents had a least one copy of the ATP7B mutation. Five were homozygous mutant, four were heterozygous, and one was homozygous normal for comparison. None had increased serum ALT activity. All dogs homozygous for the ATP7B mutation had elevated hepatic copper concentrations compared to dogs heterozygous for the ATP7B mutation regardless of sex or presence of an ATP7A mutation with the mean hepatic copper concentration being 1464 ppm (reference range 100-330 ppm). Mean hepatic copper concentration in homozygous normal and heterozygous dogs was 328 ppm. In this preliminary analysis, we found that dogs that carry two copies of the ATP7B mutation have abnormally elevated hepatic copper levels despite having normal serum ALT activity. Our findings support the hypothesis that the ATP7B DNA test can predict defects in hepatic copper metabolism. Veterinarians can test for the ATP7B gene mutation to identify Labrador Retrievers at risk for copper toxicosis so that they can take steps to prevent development of copper-associated chronic hepatitis in their patients.

Evaluation of potential serum biomarkers of hepatic fibrosis and necroinflammatory activity in dogs with liver disease.

BACKGROUND: Serum interleukin 6 (IL-6), chemokine ligand 2 (CCL2), C-reactive protein (CRP), and the ratio of aspartate transaminase to alanine transaminase (AST:ALT) have been correlated with fibrosis and necroinflammatory activity in humans with various hepatopathies. HYPOTHESIS/OBJECTIVES: To determine whether increases in serum IL-6, CCL2, CRP, or AST:ALT were associated with moderate to severe fibrosis or necroinflammatory activity in dogs with various hepatopathies. ANIMALS: Forty-four client-owned dogs with clinical evidence of liver disease and 10 healthy purpose-bred dogs, all undergoing liver biopsies by laparoscopy or laparotomy. METHODS: Measurement of serum IL-6, CCL2, CRP, AST, and ALT before scheduled liver biopsy and evaluation of liver histopathology using the METAVIR scoring system used in human medicine, blinded to clinical presentation. RESULTS: Median serum IL-6 was approximately twice as high in dogs with high fibrosis scores (15.5 pg/mL; range, 1.4 to 235 pg/mL) compared to dogs with low fibrosis scores (7.6 pg/mL; range, 1.4 to 148.1 pg/mL), with marginal significance (P = .05). Median serum CCL2 was significantly higher in dogs with active necroinflammation (444 pg/mL; range, 144 to 896 pg/mL) compared to dogs without detectable necroinflammation (326 pg/mL; range, 59 to 1692 pg/mL; P = .008), but with considerable overlap between groups. Neither serum CRP nor AST:ALT ratios were significantly different based on fibrosis or necroinflammatory scores. CONCLUSIONS AND CLINICAL IMPORTANCE: Because of substantial variability among dogs, single measurements of IL-6 and CCL2 have limited diagnostic utility for identifying fibrosis or necroinflammation, respectively, in dogs with various chronic liver diseases. The value of these biomarkers should be explored further in monitoring response to treatment in individual dogs with chronic hepatopathies.

Hepatic fibropoiesis in dogs naturally infected with Leishmania (Leishmania) infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-ß in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-ß in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Hepatic Fibrosis in Dogs.

Hepatic fibrosis is commonly diagnosed in dogs, often as a sequela to chronic hepatitis (CH). The development of fibrosis is a crucial event in the progression of hepatic disease that is of prognostic value. The pathophysiology of hepatic fibrosis in human patients and rodent models has been studied extensively. Although less is known about this process in dogs, evidence suggests that fibrogenic mechanisms are similar between species and that activation of hepatic stellate cells is a key step. Diagnosis and staging of hepatic fibrosis in dogs requires histopathological examination of a liver biopsy specimen. However, performing a liver biopsy is invasive and assessment of fibrotic stage is complicated by the absence of a universally accepted staging scheme in veterinary medicine. Serum biomarkers that can discriminate among different fibrosis stages are used in human patients, but such markers must be more completely evaluated in dogs before clinical use. When successful treatment of its underlying cause is feasible, reversal of hepatic fibrosis has been shown to be possible in rodent models and human patients. Reversal of fibrosis has not been well documented in dogs, but successful treatment of CH is possible. In human medicine, better understanding of the pathomechanisms of hepatic fibrosis is leading to the development of novel treatment strategies. In time, these may be applied to dogs. This article comparatively reviews the pathogenesis of hepatic fibrosis, its diagnosis, and its treatment in dogs.

Caracterização do imunofenótipo das células envolvidas no processo fibrótico de fígados bovinos cronicamente infectados por Fasciola hepatica / Immunophenotype characterization of the cells involved in the fibrotic process of bovine livers chronically infected by Fasciola hepatica

A fasciolose é uma doença parasitária causada por trematódeo do gênero Fasciola sp., que pode ocasionar fibrose hepática. Objetivou-se caracterizar o imunofenótipo das células que participam da fibrogênese de fígados bovinos frente à infecção por F. hepatica. Foram utilizados fragmentos dos lobos direito e esquerdo de 74 fígados bovinos com fasciolose. Os fragmentos foram submetidos a processamento histológico, coloração com tricrômico de Masson e imuno-histoquímica. Utilizaram-se análise estatística descritiva e teste de correlação de Spearmann com 5% de probabilidade. Na classificação do grau de fibrose, observou-se prevalência do grau 1, com associação positiva e significativa entre o grau de fibrose e o lobo hepático esquerdo (ρ=0,41; P<0,0001). Os imunofenótipos observados foram células estreladas hepáticas (CEHs) no parênquima e miofibroblastos (MFs) no espaço porta (EP). Não foram encontrados fibroblastos. Não houve correlação significativa entre o grau de fibrose e a quantidade de CEH nos lobos hepáticos, direito e esquerdo. Verificou-se aumento do número de estruturas portais, bem como do número de camadas circundando cada estrutura no EP, contudo não houve influência de qualquer estrutura sobre o grau de fibrose hepática (P>0,05). Concluiu-se que as células CEH e os MFs participam da fibrogênese de fígados bovinos com fasciolose crônica.(AU)

Fascioliasis is a parasitic disease caused by a fluke of the genus Fasciola sp., which can lead to end liver fibrosis. This study aimed to characterize the immunophenotype of cells that participate in the fibrogenesis of livers of cattle that face infection by F. hepatica. Fragments of the right and left lobes of 74 cattle livers with fascioliasis were used. The fragments were subjected to histological analysis, Masson's trichrome special stain, and immunohistochemistry. A descriptive statistical analysis was used, with a 5% probability in Spearman correlation test. The classification of degree of fibrosis revealed prevalence of grade 1, with a positive and significant association between the degree of fibrosis and the left hepatic lobe (ρ = 0.41; p <0.0001). The observed immunophenotypes corresponded to hepatic stellate cells (HSCs) in the parenchyma and myofibroblasts (MFs) in the portal tract (PT). Fibroblasts were not found. There was no significant correlation between the degree of fibrosis and the amount of HSC in right and left hepatic lobes. There was an increase in the number of portal structures, as well as in the number of layers surrounding each structure of the PT, but there was no influence of any structure of the PT on the degree of liver fibrosis (P>0.05). HSCs and MFs were concluded to play a role in the fibrogenesis of cattle livers with chronic fascioliasis.(AU)

Evaluation of hyaluronic acid, procollagen type III N-terminal peptide, and tissue inhibitor of matrix metalloproteinase-1 as serum markers of canine hepatic fibrosis.

The only way to diagnose hepatic fibrosis in dogs is by histological assessment of a liver biopsy specimen. As this technique is invasive and susceptible to sampling variation, serum biomarkers are used to detect hepatic fibrosis in humans. The objective of this study was to assess the utility of hyaluronic acid (HA), procollagen type III N-terminal peptide (PIIINP), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as serum markers of canine hepatic fibrosis. Serum samples were collected from 47 dogs with histologically confirmed hepatobiliary disease and 24 healthy dogs in order to measure concentrations of HA, PIIINP, and TIMP-1. Hepatic fibrosis was staged using a 5-point scoring scheme. There was no correlation between serum concentrations of HA or PIIINP and the severity of hepatic fibrosis. There was a negative correlation between serum concentration of TIMP-1 and the severity of hepatic fibrosis (rs = -0.33; P = 0.036). It was not possible to use serum concentrations of HA, PIIINP, or TIMP-1 to discriminate between dogs with absent-to-moderate hepatic fibrosis and those with marked-to-very-marked fibrosis. The results of this study do not support the utility of measuring serum concentrations of HA, PIIINP, or TIMP-1 for diagnosing canine hepatic fibrosis. Further studies are needed to support this finding.

Le seul moyen de diagnostiquer la fibrose hépatique chez les chiens est par évaluation histologique d'un échantillon de biopsie hépatique. Étant donné que cette technique est invasive et sujette à variation dans l'échantillonnage, chez l'humain des marqueurs sériques sont utilisés pour détecter la fibrose hépatique. L'objectif de la présente étude était d'évaluer l'utilité de l'acide hyaluronique (AH), du peptide N-terminal du pro-collagène de type III (PNPIII), et de l'inhibiteur tissulaire de la métalloprotéinase-1 matricielle (ITMP-1) comme marqueurs sériques de la fibrose hépatique canine. Des échantillons de sérums ont été récoltés de 47 chiens avec une pathologie hépatobiliaire confirmée histologiquement et 24 chiens en santé afin de mesurer les concentrations d'AH, PNPIII, et ITMP-1. La fibrose hépatique a été catégorisée en utilisant un schéma de pointage en cinq points. Il n'y avait pas de corrélation entre les concentrations sériques d'AH ou de PNPIII et la sévérité de fibrose hépatique. Il y avait une corrélation négative entre la concentration sérique d'ITMP-1 et la sévérité de la fibrose hépatique (rs = −0,33; P = 0,036). Il n'était pas possible d'utiliser les concentrations sériques d'AH, de PNPIII, ou d'ITMP-1 afin de différencier les chiens avec une fibrose hépatique absente à modérée de ceux avec une fibrose marquée à très marquée. Les résultats de la présente étude ne permettent pas de justifier l'utilité de mesurer les concentrations sériques d'AH, de PNPIII, ou d'ITMP-1 pour le diagnostic de la fibrose hépatique canine. Des études supplémentaires sont requises pour soutenir cette trouvaille.(Traduit par Docteur Serge Messier).

Sinusoidal endothelial cell and hepatic stellate cell phenotype correlates with stage of fibrosis in chronic liver disease in dogs.

We evaluated the extent of hepatic fibrosis in chronic liver disease of dogs using a modification of Ishak's staging criteria for human chronic liver disease, and examined the association of stage of fibrosis with immunophenotypic markers of transdifferentiation of hepatic sinusoidal endothelial cells and hepatic stellate cells. Formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained liver biopsy specimens from 45 case dogs with chronic liver disease and 55 healthy control dogs were scored for the presence and extent of fibrosis. This stage score for fibrosis strongly correlated with upregulated von Willebrand factor (vWF) expression in lobular sinusoidal endothelial cells (Spearman correlation coefficient [SCC] = 0.57, p < 0.05). Immunoreactivity for vWF factor was identified in 68.9% of case biopsies, varying in distribution from periportal to diffuse, whereas vWF immunoreactivity was identified in only 14.5% of control specimens, and was restricted to the immediate periportal sinusoids. The majority of both case and control biopsies exhibited similar prominent lobular perisinusoidal expression of alpha-smooth muscle actin (α-SMA). A minority of specimens (17.8% of case biopsies, 1.8% of control biopsies) exhibited low perisinoidal α-SMA expression, and there was a weak negative correlation between α-SMA expression and stage of fibrosis (SCC = -0.29, p = 0.0037). These results document a method for staging the severity of fibrosis in canine liver biopsies, and show a strong association between fibrosis and increased expression of vWF in hepatic sinusoidal endothelial cells.

Immunohistochemical study of hepatic fibropoiesis associated with canine visceral leishmaniasis.

Hepatic fibropoiesis has been confirmed in canine visceral leishmaniasis. In fibrotic disease, hepatic stellate cells (HSC) play an important role in fibropoiesis, undergoing activation by TGF-ß to acquire characteristics of myofibroblasts. These cells show extensive capacity for proliferation, motility, contractility, collagen synthesis and extracellular matrix component synthesis. The aim of this work was to identify markers of HSC activation in 10 symptomatic and 10 asymptomatic dogs naturally infected with Leishmania (Leishmania) infantum. Eight uninfected dogs were used as controls. Alpha-actin (α-SMA), vimentin and cytokeratin were investigated by immunohistochemistry as HSC markers. The cytokine TGF-ß in tissue was also evaluated by immunohistochemistry. All infected dogs showed higher numbers of reticular fibres than controls. Fibropoiesis found in infected dogs was always associated with the presence of parasites and chronic granulomatous hepatitis. Positive correlation was found among fibropoiesis, parasite tissue load and expression of α-SMA. There was no correlation between fibropoiesis, vimentin and cytokeratin markers. The expression of cytokine TGF-ß was higher in infected dogs than in controls, but not significantly different between symptomatic and asymptomatic dogs. These results confirm previous work describing the intense hepatic fibropoiesis in dogs naturally infected with Leishmania infantum, but now associated them with overexpression of TGF-ß, where α-SMA may be a superior marker for activated HSC cells in CVL.