Identification of chronic stress biomarkers in dairy cows.

Stress in dairy herds can occur from multiple sources. When stress becomes chronic because of a long duration and inability of animals to adapt, it is likely to deeply affect the emotional state, health, immunity, fertility and milk production of cows. While assessing chronic stress in herds would be beneficial, no real consensus has emerged from the literature regarding the indicators of interest. The goal of this study was to compare and evaluate potential biomarkers for chronic stress after inducing stress over a 4-week period through severe overstocking, restricted access to feed and isolated unusual events. A total of 30 cows were involved in the experiment and two similar groups were constituted. Over a 4-week period, the 15 cows of the stress group were housed in overstocked conditions, with 4.6 m2 per cow, including resting and feeding areas. In this area, only seven individual places at the feeding area were available for the 15 cows to generate competition for feed access. Twice during the trial and over a period of 2 h, an additional stress was induced by moving cows to an unfamiliar barn and diffusion of stressing noises (dog barking). Meanwhile, the 15 cows of the control group stayed in the original barn, with more than 10 m2 per cow and more individual places at the feeding area than cow number. On a weekly basis, several variables considered as potential biomarkers for chronic stress were recorded. Collected data were analysed using single trait linear repeated mixed models. No differences were observed regarding milk yield, BW of cows or body condition score but the milk loss was more pronounced in the stress group. The activity was more heterogeneous and the rumination of cows was lower in the stress group. The heart rate was lower in the stress group and showed more heterogeneity at the end of the stress period. No differences were observed regarding salivary cortisol, blood glucose, ß-endorphin, thyroxine and leucocyte profile. A higher level of hair cortisol and blood fructosamine were observed in the stress group at the end of the stress period. Regarding the practical use of the highlighted biomarkers, milk loss may be an effective and easy way to detect general problems, including stress. The blood fructosamine and the hair cortisol concentrations are promising indicators to assess chronic stress in commercial farms.

Point Shear Wave Elastography by Acoustic Radiation Force Impulse Quantification in Comparison to Transient Elastography for the Noninvasive Assessment of Liver Fibrosis in Chronic Hepatitis C: A Prospective International Multicenter Study.

PURPOSE: The aim of the present prospective European multicenter study was to demonstrate the non-inferiority of point shear wave elastography (pSWE) compared to transient elastography (TE) for the assessment of liver fibrosis in patients with chronic hepatitis C. MATERIALS AND METHODS: 241 patients with chronic hepatitis C were prospectively enrolled at 7 European study sites and received pSWE, TE and blood tests. Liver biopsy was performed with histological staging by a central pathologist. In addition, for inclusion of cirrhotic patients, a maximum of 10 % of patients with overt liver cirrhosis confirmed by imaging methods were allowed by protocol (n = 24). RESULTS: Owing to slower than expected recruitment due to a reduction of liver biopsies, the study was closed after 4 years before the target enrollment of 433 patients with 235 patients in the 'intention to diagnose' analysis and 182 patients in the 'per protocol' analysis. Therefore, the non-inferiority margin was enhanced to 0.075 but non-inferiority of pSWE could not be proven. However, Paired comparison of the diagnostic accuracy of pSWE and TE revealed no significant difference between the two methods in the 'intention to diagnose' and 'per protocol' analysis (0.81 vs. 0.85 for F ≥ 2, p = 0.15; 0.88 vs. 0.92 for F ≥ 3, p = 0.11; 0.89 vs. 0.94 for F = 4, p = 0.19). Measurement failure was significantly higher for TE than for pSWE (p = 0.030). CONCLUSION: Non-inferiority of pSWE compared to TE could not be shown. However, the diagnostic accuracy of pSWE and TE was comparable for the noninvasive staging of liver fibrosis in patients with chronic hepatitis C.

Acoustic radiation force impulse imaging for non-invasive assessment of liver fibrosis in chronic hepatitis B.

Acoustic radiation force impulse (ARFI) imaging is a novel ultrasound-based elastography method that is integrated in a conventional ultrasound machine. It might provide an alternative method to transient elastography for the noninvasive assessment of liver fibrosis. While previous studies have shown comparable diagnostic accuracy of ARFI to transient elastography in chronic hepatitis C, the aim of the present prospective multicenter study was to evaluate ARFI for the assessment of liver fibrosis in chronic hepatitis B. ARFI imaging involves the mechanical excitation of tissue using short-duration acoustic pulses to generate localized displacements in tissue. The displacements result in shear-wave propagation which is tracked using ultrasonic, correlation-based methods and recorded in m/s. In the present international prospective study, patients infected with chronic hepatitis B received ARFI imaging, blood tests and if available transient elastography. The results were compared to liver biopsy as reference method analysed by a central pathologist. In 92 of 114 patients, a comparison of ARFI with transient elastography was possible. ARFI imaging and transient elastography correlated significantly with histological fibrosis stage. The diagnostic accuracy expressed as areas under ROC curves for ARFI imaging and transient elastography was 0.75 and 0.83 for the diagnosis of significant fibrosis (F ≥ 2), 0.93 and 0.94 for the diagnosis of severe fibrosis (F ≥ 3), and 0.97 and 0.93 for the diagnosis of liver cirrhosis, respectively. No significant difference was found between ARFI and transient elastography. ARFI imaging is a reliable ultrasound-based method for the assessment of advanced stages of liver fibrosis in chronic hepatitis B.

Real-time tissue elastography versus FibroScan for noninvasive assessment of liver fibrosis in chronic liver disease.

PURPOSE: Transient elastography (FibroScan, [TE]) and serum fibrosis markers such as the FibroTest (FT) are established methods for the noninvasive staging of liver fibrosis. A study using real-time elastography (HI-RTE), which is integrated in a conventional ultrasound system, was recently published with comparable results to transient elastography. The aim of the present study was to validate real-time elastography using the formulas calculated in previous studies and to compare the results to transient elastography and FibroTest for the noninvasive assessment of liver fibrosis. MATERIALS AND METHODS: One hundred and thirty-four patients with chronic liver disease and either histological assessment of liver fibrosis (n = 112) or proven liver cirrhosis (n = 22) were included in the study. All patients received TE, HI-RTE, and biochemical evaluation on the same day as presentation. The calculation of the elasticity score of real-time elastography was performed in accordance with the two previously published studies. RESULTS: The Spearman correlation coefficient between transient elastography, real-time elastography and FibroTest with the histological Chevallier score was statistically significant with 0.78, 0.34, and 0.67, respectively (p < 0.01). The diagnostic accuracy expressed as areas under ROC curves was 0.84, 0.69 and 0.85 for the diagnosis of significant fibrosis (F > or = 2), and 0.97, 0.65, and 0.83 for the diagnosis of cirrhosis, respectively. CONCLUSION: Real-time elastography in its present form cannot replace transient elastography for noninvasive assessment of liver fibrosis.

[Liver metastases: incidence and histogenesis]. / Lebermetastasen: Inzidenz und histogenetische Einordnung.

Metastases are the most common malignant tumors of the liver. In the files of the Institute of Pathology of the University of Cologne 12,161 liver tissue cases are registered. Of them, 1,357 cases (11.2%) showed tumors or tumor like masses. Liver metastases of solid tumors were the largest group of the neoplasias with 611 cases (5.0%) followed by hepatocellular carcinoma (380 cases; 3.1%). Other entities were rare and include cholangiolar carcinoma (0.5%), vascular tumors (0.4%), lymphomas (0.4%), focal nodular hyperplasias (0.36%) and liver cell adenomas (0.23%). Adenocarcinomaa are the largest group of metastases with 400 cases (65.5%). 48.2% of this group were metastases of colorectal cancer, 13.5% of pancreatic cancer, 13% of breast cancer, 6.2% of gastric cancer, 4.5% of lung cancer and 3.7% of esophageal cancer. Neuroendocrine carcinomas are the second largest group with 16% of liver filiae. Other entities were rarely found. Metastases in cirrhotic livers are seldom. The gross findings, the histology, the differential diagnosis including immunohistochemistry and the value of the liver biopsy is discussed.

Fulminant liver failure induced by hepatosplenic alphabeta T-cell lymphoma.

Since survival rates of fulminant liver failure are low, early consideration of liver transplantation in patients developing hepatic encephalopathy due to progressive liver failure is mandatory. Rapid diagnostic work-up is necessary to identify the underlying disease and to rule out contraindications to liver transplantation. We report the case of a 35-year-old patient presenting with fulminant hepatitis and a four-week history of biopsy-proven autoimmune hepatitis. Despite high-dose steroid-treatment liver function progressively worsened and hepatic encephalopathy rapidly developed. Histopathologic evaluation of a liver biopsy specimen revealed necrotizing hepatitis and rare atypical lymphocytes. Surgical biopsy specimens confirmed the suspicion of an aggressive hepatosplenic alphabeta T-cell lymphoma which represents a contraindication to liver transplantation.

Immunoreactivity of cytokeratins (CK7, CK20) and mucin peptide core antigens (MUC1, MUC2, MUC5AC) in adenocarcinomas, normal and metaplastic tissues of the distal oesophagus, oesophago-gastric junction and proximal stomach.

AIMS: Adenocarcinomas of the distal oesophagus and especially the oesophago-gastric junction have shown an increasing incidence during the last decade. Definition of subgroups according to different sites of development, histogenesis or aetiology may prove to be valuable for clinical diagnosis and treatment. Previous studies have shown differences in cytokeratin patterns between Barrett's metaplasia of the oesophagus and intestinal metaplasia in the stomach. The aim of our study was to investigate whether the expression of certain cytokeratins (CK7, CK20) and mucins (MUC1, MUC2, MUC5AC) exhibit clear-cut patterns, thus allowing a subclassification of adenocarcinomas of the oesophago-gastric junction. The possibility of a relationship between antigen expression and the presence or absence of Barrett's metaplastic epithelium was also studied. METHODS AND RESULTS: CK7, CK20, MUC1, MUC2 and MUC5AC were visualized in six adenocarcinomas of the distal oesophagus, 29 adenocarcinomas of the oesophago-gastric junction and eight adenocarcinomas of the proximal stomach. CK7, CK20 and MUC1 were strongly expressed in the great majority of all neoplasms under study, whereas MUC2 and MUC5AC were absent or only faintly detectable. CK20 exhibited a significantly stronger expression in poorly differentiated tumours (G3) and MUC1 immunoreactivity correlated with tubular and papillary versus signet-ring cell histopathology. Other statistically significant correlations between antigens and histopathological features (pTNM stage, grading, histopathological subtype, presence/absence of Barrett's epithelium) were not observed. CONCLUSIONS: According to our results, most adenocarcinomas of the oesophago-gastric junction show a CK7+, CK20+, MUC1+ phenotype irrespective of the presence or absence of Barrett's epithelium. The immunohistochemical data suggest a similar histogenesis of these tumours.

[Extramedullary initial manifestations of acute myeloid leukemia (AML)]. / Extramedulläre Erstmanifestationen von akuten myeloischen Leukämien (AML).

Extramedullary myeloblastic tumors, so-called myelosarcomas (granulocytic sarcomas, chloromas) have been reported only sporadically in the pertinent literature which reflects their rather infrequent occurrence. These lesions may accompany the initial manifestation or signal relapse of acute myeloid leukemia (AML) or coincide with blastic transformation of a chronic myeloproliferative disorder. However, even more rarely, primary myelosarcomas may precede AML by months or years or may be associated with myelodysplastic syndromes (MDS) that never progress to manifest leukemia. In a retrospective evaluation a clinicopathological study on these latter two variants of isolated extramedullary manifestations of AML was performed to elucidate certain aspects of site involvement and histopathology by application of enzyme and immunohistochemistry. For this reason, we selected 6 patients presenting with a myelosarcoma in combination with MDS and 12 patients revealing only uncharacteristic reactive changes of the bone marrow. Of these patients 8 developed AML following an observation time of up to 2 years. Focal leukemic infiltrates were most often localized in the skin ( n=4), oral mucosa ( n=4), lymph nodes ( n=3), gastrointestinal tract ( n=3) or pleura and retroperitoneum ( n=3 each). Myelosarcomas were usually regarded by the clinicians as putative malignant lymphomas unless further evaluation, especially involving chloroacetate esterase reactions as well as immunostaining with a panel of antibodies reactive with lysozyme, myeloperoxidase, CD68, CD43, CD56, CD117 and CD34 proved their true nature. Although at that time bone marrow findings were inconclusive, a straightforward diagnosis was reached by considering the possibility of a (primary) myelosarcoma in these patients.

[Deposition of keinoid fibers in gastrointestinal stromal tumors of the small intestine. Report of two cases and review of the literature]. / Ablagerung von Skeinoidfasern in gastrointestinalen Stromatumoren des Dünndarms. Zwei Fallberichte und Literaturübersicht.

Skeinoid fibers are interstitial collections of a pathological collagen, most often seen in gastrointestinal stromal tumors. They were first described in 1991. We report two cases of intestinal stromal tumors, one in an exceptionally young patient with excessive skeinoid fiber deposition. The microscopic as well as the ultrastructural findings of skeinoid fibers are demonstrated and their role is discussed considering the newest literature.

Prevalence, distribution, and viral load of human papillomavirus 16 DNA in tonsillar carcinomas.

BACKGROUND: Oncogenic human papillomaviruses (HPV) DNA have repeatedly been observed in many head and neck carcinomas (HNSCCs), and HPV infections are currently considered a possible factor in the etiology of these tumors. However, the reported prevalences of HPV-DNA in HNSCC are variable. In the current study the authors used highly sensitive polymerase chain reactions (PCRs) to analyze the occurrence of viral sequences in 98 carefully stratified HNSCCs. The authors determined the load and localization of HPV DNA in a subset of tonsillar carcinomas and their metastases. METHODS: Nested PCR and an HPV16 specific single step PCR were used to screen 98 HNSCCs for HPV DNA for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. In two patients HPV16 subtypes in tonsillar carcinomas and their metastases were compared by amplification and sequencing of the long control region of the virus. In a subset of HPV16 positive tonsillar carcinomas and their metastases, localization and viral load were determined using laser assisted microdissection and real time fluorescent PCR, respectively. RESULTS: Altogether 25 HNSCCs (26%) were found to be HPV positive. Stratified according to the tumor localization, the frequency of HPV positive lesions was 18% in the oral cavity, 45% for oropharynx, 25% for hypopharynx, 8% for nasopharynx, and 7% for larynx. The highest HPV DNA prevalence (58%) was found in tonsillar carcinomas. The high risk HPV type 16 was found in 84% of positive HNSCCs, in 14% of which EV-associated HPVs were detected. Human papillomavirus sequences were detected in 64% of biopsies with normal mucosa from 11 patients with positive carcinomas. As a control group, 14 tumor free tonsils were analyzed. In none of these specimens were HPV sequences detected. Viral long transcriptional control region sequences in homologous metastases were identical with those in primary tumors and the load values in both locations were roughly comparable. Viral loads differed substantially in different areas of one tumor. Statistical evaluation of data related to clinicopathologic parameters showed a significant linkage of HPV with tonsillar carcinomas compared to other locations. Furthermore, a significant correlation of HPV status of tonsillar carcinomas with tumor grading and alcohol consumption was found. CONCLUSIONS: Our study shows a preferential association of HPV-DNA with tonsillar carcinomas. The data support the view of HPV negative and positive tonsillar carcinomas being different tumor entities and conventional cancer risk factors being of less importance in HPV-infected individuals. The HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Data on quantification of HPV16 in tonsillar tumors and their metastases showed mean viral loads comparable to other HPV associated malignancies.

Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levels of transforming growth factor-beta.

Although many patients with chronic viral hepatitis C infection suffer from progressive liver disease, the rate of fibrosis progression is highly variable and some patients do not show any measurable progression. However, our ability to predict which patients progress is very limited. Since transforming growth factor-beta (TGF-beta) is a key mediator of liver fibrogenesis, we assessed the predictive role of TGF-beta for fibrogenesis in chronic hepatitis C. We studied 39 patients with chronic hepatitis C in whom two liver biopsies were taken at least 12 months apart, and who did not receive therapy during this period. TGF-beta was measured by bioassay and by ELISA in serum samples taken at the time of the first biopsies, and TGF-beta was determined semiquantitatively by immunostaining of liver biopsy sections. Fibrosis was scored blinded in the biopsy samples by two pathologists independently. There was a close correlation between TGF-beta serum levels and the rate of fibrosis progression. Patients with no progression of fibrosis had significantly lower (59 ng/mL +/- 22) TGF-beta serum levels than patients with progressive disease (115 ng/mL +/- 20), and a TGF-beta level below 75 ng/mL was predictive for stable disease. Immunohistology for TGF-beta in biopsy samples was also predictive for progressive liver disease with fibrosis progression found in those patients displaying staining of hepatocytes and sinusoidal cells. No such correlation was found with other markers such as procollagen III peptide, viral load or transaminase levels. These results further support the role of TGF-beta in liver fibrogenesis, and offer an opportunity to predict clinical disease progression, which may help in selecting patients who are in need of therapeutic interventions.

[Liver biopsy in chronic hepatitis C. Histopathology, molecular diagnostic analysis, and implications for therapeutic management]. / Bioptische Diagnostik der Hepatitis C. Morphologie, Molekularpathologie und klinisch-therapeutische Implikationen.

Histological evaluation of liver biopsies is the gold standard for assessing the severity of liver disease in chronic hepatitis C. In conjunction with anamnestic and serological data, it provides critical information regarding prognosis and therapeutic consequences. Grading of the necroinflammatory activity and staging of the manifested fibrosis according to well-established scoring systems are the essential parameters of the histopathological report. The frequency of chronic hepatitis C necessitates awareness of coinciding liver damage by nutritional or drug toxicity, bile duct diseases, and hereditary hemochromatosis. Detection of hepatitis C virus (HCV) RNA directly from the formalin fixed, paraffin-embedded biopsy specimen is highly sensitive and offers help in cases of equivocal results of serological testing for HCV.

[Diagnosis of seronegative chronic hepatitis in tissue]. / Diagnostik der "seronegativen" chronischen Hepatitis am Gewebe.

The evaluation of a liver biopsy in chronic hepatitis should make a statement on the etiology and report the degree of activity and stage of the disease. The category of so called seronegative chronic hepatitis may include cases of chronic hepatitis C or infection with other viruses such as the Epstein-Barr virus (EBV) and cytomegalovirus (CMV), cases of marker-negative autoimmune hepatitis as well as drug-induced injury and Wilson's disease in younger patients. In order to establish the diagnosis, sensitive techniques of molecular biology should be applied as well as copper staining by histochemistry. Exact and detailed histopathologic analysis can reveal certain features of autoimmune hepatitis or drug injury.

[Highly differentiated hepatocellular tumors. Concepts, criteria, and differential diagnosis]. / Hochdifferenzierte hepatozelluläre Tumoren. Konzepte, Kriterien und Differenzialdiagnose.

Well-differentiated hepatocellular tumors represent a difficult diagnostic problem in hepatopathology with rising clinical impact. The differential diagnosis mainly includes well-differentiated hepatocellular carcinoma (HCC) and its premalignant precursor lesion, the dysplastic nodule (DN), in addition to multiacinar regenerative nodule as well as hepatocellular adenoma (LCA) and focal nodular hyperplasia (FNH). Optimized diagnosis of these lesions is based on exact histomorphological analysis, a close interdisciplinary cooperation as well as good clinical and anamnestic information. Histopathological differential diagnosis requires the search for specific characteristics and detailed analyses of the subtle cytological differences, the histoarchitecture, and also the surrounding nontumorous liver tissue. Special techniques may be helpful in selected cases but currently are of limited importance.

Nodular regenerative hyperplasia of the liver: case report of a 13-year-old girl and review of the literature.

BACKGROUND: Nodular regenerative hyperplasia (NRH) of the liver is a multi-acinar regenerative nodular lesion in a non-cirrhotic liver. It is a rare entity, especially in children, and remains of unknown aetiology. OBJECTIVE: NRH is often seen in association with other diseases or drug intake. In half of patients it is complicated by portal hypertension. Radiologically, its nodular appearance may look like neoplasia. RESULTS: We report a case of NRH with enormous hepatomegaly and multiple huge nodules. CONCLUSION: We wish to emphasise the importance of open wedge biopsy to establish diagnosis, since the prognosis of NRH in the absence of portal hypertension is good. Complications such as rupture of a nodule are rare.

Therapy of prednisone-refractory collagenous colitis with budesonide.

Collagenous colitis is a rare cause of chronic watery diarrhea. No effective standard treatment has yet been established. Based upon anecdotal reports some anti-inflammatory and symptomatic drugs seem to have some therapeutic efficacy. Prednisone is widely believed to be the most effective treatment. Here we describe three female patients with histologically confirmed collagenous colitis refractory to therapy with prednisone. Each had received prednisone with a high starting bolus and lower dose maintenance therapy for their disease. However, definite clinical remission could not be achieved. After the administration of 3 x 3 mg/day controlled ileal release (CIR) capsules of budesonide the symptoms resolved immediately. The mean follow-up after beginning budesonide was 11 months (range 7-18). Two patients are still on budesonide. One had had a quick relapse of diarrhea after stopping her treatment. Budesonide therapy was therefore resumed. She has remained symptom-free on a lower daily dose of 2 x 3 mg/day budesonide. One patient has been in remission for more than 1 year after a 3-month course of budesonide. Budesonide is a topically acting steroid with rapid absorption, high receptor affinity, and low systemic bioavailability, thus causing almost no side effects. As yet only few case reports have been published on the use of budesonide for collagenous colitis. We present here the first three cases of prednisone refractory collagenous colitis successfully treated with budesonide.

Detection of hepatitis C virus in paraffin-embedded liver biopsies of patients negative for viral RNA in serum.

The diagnosis of hepatitis C is based on serological testing for antibodies against various epitopes of the hepatitis C virus (HCV) and detection of HCV RNA in serum, because anti-HCV antibodies alone cannot discriminate patients who are infectious from those who have resolved the infection. If HCV RNA is not detected, which is the case in at least 20% of enzyme immunoassay (EIA)-positive patients, diagnosis remains unclear in a state of disease possibly well suited for therapeutic intervention. Therefore, we investigated if detection of HCV antigens or HCV RNA in routinely processed, formalin-fixed and paraffin-embedded (ffpe) liver biopsy specimens of patients positive for anti-HCV, but negative for HCV RNA in serum, could confirm diagnosis in this serological constellation. We detected HCV RNA by reverse-transcription polymerase chain reaction (RT-PCR) in 27 (61%) of 44 ffpe liver biopsies from EIA-positive, but HCV-RNA-seronegative, patients. Testing of 18 of these biopsies by a panel of polyclonal antibodies against structural and nonstructural HCV proteins revealed positive immunostaining in 6 cases (33%), which were also positive by RT-PCR. Most biopsies showed necroinflammation compatible with chronic hepatitis C, and the detection of tissue HCV RNA correlated significantly with a higher grade of inflammatory activity. Detectability of HCV RNA did not correlate with HCV subtype. In conclusion, the search for HCV RNA by RT-PCR within the liver biopsy specimen can establish rapid and unequivocal diagnosis of hepatitis C in at least 60% of anti-HCV antibody-positive patients who are seronegative for HCV RNA, and thus may help to avoid repeated testing and delayed therapy. Tissue RT-PCR may also be more efficient than serological testing for surveillance of interferon therapy response, because ongoing chronic active hepatitis C is clearly demonstrated in the absence of detectable serum HCV RNA.