ScoMorphoFISH: A deep learning enabled toolbox for single-cell single-mRNA quantification and correlative (ultra-)morphometry.

Increasing the information depth of single kidney biopsies can improve diagnostic precision, personalized medicine and accelerate basic kidney research. Until now, information on mRNA abundance and morphologic analysis has been obtained from different samples, missing out on the spatial context and single-cell correlation of findings. Herein, we present scoMorphoFISH, a modular toolbox to obtain spatial single-cell single-mRNA expression data from routinely generated kidney biopsies. Deep learning was used to virtually dissect tissue sections in tissue compartments and cell types to which single-cell expression data were assigned. Furthermore, we show correlative and spatial single-cell expression quantification with super-resolved podocyte foot process morphometry. In contrast to bulk analysis methods, this approach will help to identify local transcription changes even in less frequent kidney cell types on a spatial single-cell level with single-mRNA resolution. Using this method, we demonstrate that ACE2 can be locally upregulated in podocytes upon injury. In a patient suffering from COVID-19-associated collapsing FSGS, ACE2 expression levels were correlated with intracellular SARS-CoV-2 abundance. As this method performs well with standard formalin-fixed paraffin-embedded samples and we provide pretrained deep learning networks embedded in a comprehensive image analysis workflow, this method can be applied immediately in a variety of settings.

Dipeptidyl peptidase 3 modulates the renin-angiotensin system in mice.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase involved in degrading oligopeptides with 4-12 amino acid residues. It has been associated with several pathophysiological processes, including blood pressure regulation, pain signaling, and cancer cell defense against oxidative stress. However, the physiological substrates and the cellular pathways that are potentially targeted by DPP3 to mediate these effects remain unknown. Here, we show that global DPP3 deficiency in mice (DPP3-/-) affects the renin-angiotensin system (RAS). LC-MS-based profiling of circulating angiotensin peptides revealed elevated levels of angiotensin II, III, IV, and 1-5 in DPP3-/- mice, whereas blood pressure, renin activity, and aldosterone levels remained unchanged. Activity assays using the purified enzyme confirmed that angiotensin peptides are substrates for DPP3. Aberrant angiotensin signaling was associated with substantially higher water intake and increased renal reactive oxygen species formation in the kidneys of DPP3-/- mice. The metabolic changes and altered angiotensin levels observed in male DPP3-/- mice were either absent or attenuated in female DPP3-/- mice, indicating sex-specific differences. Taken together, our observations suggest that DPP3 regulates the RAS pathway and water homeostasis by degrading circulating angiotensin peptides.

Glucagon-Like Peptide-1 Receptor Agonism Improves Nephrotoxic Serum Nephritis by Inhibiting T-Cell Proliferation.

Glucagon-like peptide (GLP)-1 analogs such as liraglutide improved albuminuria in patients with type 2 diabetes in large randomized controlled trials. One of the suspected mechanisms is the anti-inflammatory potential of GLP-1 receptor (Glp1r) agonism. Thus, the anti-inflammatory action of Glp1r agonism was tested in a nondiabetic, T-cell-mediated murine model of nephrotoxic serum nephritis (NTS). The role of Glp1r in NTS was evaluated by using Glp1r-/- mice or C57BL/6 mice treated with liraglutide. In vitro, murine T cells were stimulated in the presence of liraglutide or vehicle. Glp1r-/- mice displayed increased renal infiltration of neutrophils and T cells after induction of NTS. Splenocyte proliferation and TH1 cytokine transcription were increased in spleen and lymph nodes of Glp1r-/- mice. Liraglutide treatment significantly improved the renal outcome of NTS in C57BL/6 mice by decreasing renal infiltration and proliferation of T cells, which resulted in decreased macrophage infiltration. In vitro, T cells stimulated in the presence of liraglutide showed decreased proliferation of TH1 and TH17 cells. Liraglutide blocked glycolysis in T cells and decreased their Glut1 mRNA expression. Together, Glp1r agonism protects mice from a T-cell-dependent glomerulonephritis model by inhibition of T-cell proliferation, possibly by interacting with their metabolic program. This mechanism may explain in part the renoprotective effects of Glp1r agonism in diabetic nephropathy.

FXR-dependent Rubicon induction impairs autophagy in models of human cholestasis.

BACKGROUND & AIMS: Cholestasis comprises a spectrum of liver diseases characterized by the accumulation of bile acids. Bile acids and activation of the farnesoid X receptor (FXR) can inhibit autophagy, a cellular self-digestion process necessary for cellular homeostasis and regeneration. In mice, autophagy appears to be impaired in cholestasis and induction of autophagy may reduce liver injury. METHODS: Herein, we explored autophagy in human cholestasis in vivo and investigated the underlying molecular mechanisms in vitro. FXR chromatin immunoprecipitation-sequencing and qPCR were performed in combination with luciferase promoter studies to identify functional FXR binding targets in a human cholestatic liver sample. RESULTS: Autophagic processing appeared to be impaired in patients with cholestasis and in individuals treated with the FXR ligand obeticholic acid (OCA). In vitro, chenodeoxycholic acid and OCA inhibited autophagy at the level of autophagosome to lysosome fusion in an FXR-dependent manner. Rubicon, which inhibits autophago-lysosomal maturation, was identified as a direct FXR target that is induced in cholestasis and by FXR-agonistic bile acids. Genetic inhibition of Rubicon reversed the bile acid-induced impairment of autophagic flux. In contrast to OCA, ursodeoxycholic acid (UDCA), which is a non-FXR-agonistic bile acid, induced autophagolysosome formation independently of FXR, enhanced autophagic flux and was associated with reduced Rubicon levels. CONCLUSION: In models of human cholestasis, autophagic processing is impaired in an FXR-dependent manner, partly resulting from the induction of Rubicon. UDCA is a potent inducer of hepatic autophagy. Manipulating autophagy and Rubicon may represent a novel treatment concept for cholestatic liver diseases. LAY SUMMARY: Autophagy, a cellular self-cleansing process, is impaired in various forms of human cholestasis. Bile acids, which accumulate in cholestatic liver disease, induce Rubicon, a protein that inhibits proper execution of autophagy. Ursodeoxycholic acid, which is the first-line treatment option for many cholestatic liver diseases, induces hepatic autophagy along with reducing Rubicon.

The conundrum of postpartum thrombotic Microangiopathy: case report and considerations for management.

BACKGROUND: Microangiopathic hemolytic anemias and thrombocytopenias in pregnant or postpartum women constitute an interdisciplinary diagnostic and therapeutic challenge in the evaluation of thrombotic microangiopathies (TMA), where urgent care must be considered. CASE PRESENTATION: We here report the case of a 21-year-old Somali woman, who was delivered by emergency caesarean section at 35 weeks of gestational age with acute dyspnea, placental abruption and gross edema due to severe preeclampsia/HELLP syndrome. After delivery, she developed acute kidney failure and thrombotic microangiopathy as revealed by kidney biopsy. The lack of early response to plasma exchange prompted extensive laboratory workup. Ultimately, the patient completely recovered with negative fluid balance and control of severe hypertension. CONCLUSIONS: This case report emphasizes the importance to differentiate between primary TMA syndromes and microangiopathic hemolytic anemias due to systemic disorders. Delayed recovery from preeclampsia/HELLP syndrome and malignant hypertension can clinically mimic primary TMA syndromes in the postpartum period.

A rare case of cancer-to-cancer metastasis: breast cancer to renal cell cancer : Case report and review of literature.

BACKGROUND: Cancer-to-cancer metastasis is very rare with less than 50 cases described in literature. This article reports a case of breast cancer with synchronous metastasis to clear cell renal cell cancer. CASE DESCRIPTION: A 79-year-old woman was diagnosed with a bilateral breast carcinoma. Sonographic staging investigation of the abdomen revealed a 6 cm wide expansion of the right kidney. Bilateral mastectomy and nephrectomy of the right kidney was performed. The histology revealed a clear cell renal cell carcinoma and in the center of the tumor a 0.5 cm metastasis of the breast cancer. The patient's comorbidities and performance status precluded chemotherapy und she received palliative radiotherapy, targeted monoclonal antibody therapy and antihormonal treatment. CONCLUSIONS: Even if cancer-to-cancer metastasis is a very rare phenomenon, the simultaneous or consecutive finding of a renal tumor in women with breast cancer should be carefully evaluated.

Blockade of prostaglandin E2 receptor 4 ameliorates nephrotoxic serum nephritis.

Prostaglandin E2 (PGE2) signaling is known to modulate inflammation and vascular resistance. Receptors of PGE2 [E-type prostanoid receptors (EP)] might be an attractive pharmacological target in immune-mediated diseases such as glomerulonephritis. We hypothesized that selective EP4 antagonism improves nephrotoxic serum nephritis (NTS) by its anti-inflammatory properties. Mice were subjected to NTS and treated with the EP4 antagonist ONO AE3-208 (10 mg·kg body wt-1·day-1] or vehicle starting from disease initiation. In one set of experiments, treatment was started 4 days after NTS induction. Tubular epithelial cells were evaluated in vitro under starving conditions. EP4 antagonist treatment significantly improved the NTS phenotype without affecting blood pressure levels. Remarkably, the improved NTS phenotype was also observed when treatment was started 4 days after NTS induction. EP4 antagonism decreased tubular chemokine (C-X-C motif) ligand ( Cxcl) 1 and Cxcl-5 expression and thereby subsequently reduced interstitial neutrophil infiltration into the kidney. In vitro, tubular epithelial cells increasingly expressed Cxcl-5 mRNA and Cxcl-5 protein when treated with PGE2 or an EP4 agonist under starving conditions, which was blunted by EP4 antagonist treatment. Together, EP4 antagonism improves the NTS phenotype, probably by decreasing mainly Cxcl-5 production in tubular cells, thereby reducing renal neutrophil infiltration.

Lysyl oxidase-like protein 2 (LOXL2) modulates barrier function in cholangiocytes in cholestasis.

BACKGROUND & AIMS: The lysyl oxidase-like protein 2 (LOXL2) promotes stabilization of the extracellular matrix, chemotaxis, cell growth and cell mobility. We aimed to (i) identify stimuli of LOXL2 in cholangiopathies, (ii) characterize the effects of LOXL2 on biliary epithelial cells' (BECs) barrier function, (iii) compare LOXL2 expression in primary sclerosing cholangitis (PSC), primary biliary cholangitis, and disease controls, and (iv) to determine LOXL2 expression and its cellular sources in four mouse models of cholangiopathies. METHODS: Cultured murine BECs were challenged with well-known triggers of cellular senescence, hypoxia, phospholipid-deficient Abcb4-/- mouse bile and chenodeoxycholic acid and investigated for LOXL2, SNAIL1 and E-cadherin expression and transepithelial electrical resistance with and without LOX-inhibition. In vivo, LOXL2 expression was studied in PSC livers, and controls and mouse models. We compared LOXL2 serum levels in patients with PSC, secondary SC, primary biliary cholangitis, and controls. RESULTS: Cellular senescence, hypoxia, Abcb4-/- bile and chenodeoxycholic acid induced LOXL2 and SNAIL1 expression, repressed E-cadherin expression, and significantly reduced transepithelial electrical resistance in BECs. Notably, all of the pathological changes could be recovered via pharmacological LOX-inhibition. Mouse models showed induced LOXL2 expression in the portal region and in association with ductular reaction. LOXL2 serum levels were significantly elevated in patients with cholangiopathies. In PSC, LOXL2 expression was located to characteristic periductal onion skin-type fibrosis, ductular reaction, Kupffer cells, and fibrotic septa. Importantly, in PSC, LOXL2 overexpression was paralleled by E-cadherin loss in BECs from medium-sized bile ducts. CONCLUSIONS: Reactive BECs produce LOXL2, resulting in increased tight junction permeability, which can be ameliorated by pharmacological LOX-inhibition in vitro. Reactive BECs, portal myofibroblasts, and Kupffer cells are the main sources of LOXL2 in cholangiopathies. LAY SUMMARY: In this study, we investigate the role of lysyl oxidase-like protein 2 (LOXL2), an enzyme pivotal in the development of organ fibrosis, in the pathogenesis of cholangiopathies (diseases of bile ducts), such as primary sclerosing cholangitis. We found LOXL2 to be expressed in association with bile duct epithelial injury and uncovered mechanisms for its upregulation and the subsequent effects in vitro and in vivo. Our findings support testing of anti-LOXL2 treatment strategies for patients with primary sclerosing cholangitis.

Cholemic nephropathy - Historical notes and novel perspectives.

Acute kidney injury is common in patients with liver disease and associated with significant morbidity and mortality. Besides bacterial infections, fluid loss, and use of nephrotoxic drugs AKI in liver disease may be triggered by tubular toxicity of cholephiles. Cholemic nephropathy, also known as bile cast nephropathy, supposedly represents a widely underestimated but important cause of renal dysfunction in cholestasic or advanced liver diseases with jaundice. Cholemic nephropathy describes impaired renal function along with characteristic histomorphological changes consisting of intratubular cast formation and tubular epithelial cell injury directed towards distal nephron segments. The underlying pathophysiologic mechanisms are not entirely understood and clear defined diagnostic criteria are still missing. This review aims to summarize (i) the present knowledge on clinical and morphological characteristics of cholemic nephropathy, (ii) available preclinical models, (iii) potential pathomechanisms especially the potential role of bile acids, and (iv) future diagnostic and therapeutic strategies for cholemic nephropathy. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Genetic loss of the muscarinic M3 receptor markedly alters bile formation and cholestatic liver injury in mice.

AIM: Hepatic innervation represents a potentially underestimated regulator of liver function and regeneration. The muscarinic 3 receptor (M3 -R) is the primary cholangiocyte receptor for the afferent parasympathetic innervation of bile ducts. We aimed to determine the specific role of the M3 -R in bile formation and models for cholestatic liver disease in mice. METHODS: We compared bile flow and composition in M3 -R knock-out mice (M3 -R-/- ) and wild type littermates (WT). Furthermore, we compared liver inury of M3 -R-/- and WT mice after 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding, a well-characterized preclinical model of cholestatic liver disease. To analyze the possible role of the M3 -R as a therapeutic target, we treated 4-week-old Mdr2-/- mice, a preclinical model for sclerosing cholangitis, with the M3 -R agonist bethanechol for 4 weeks. RESULTS: M3 -R-/- mice showed significantly reduced bile flow compared to WT mice, most likely due to decreased biliary HCO3- secretion. However, even aged M3 -R-/- mice did not spontaneously develop liver injury or cholestasis. Challenging M3 -R-/- and WT littermates with DDC feeding showed substantially aggravated liver injury in M3 -R-/- mice. After 4 weeks bethanechol treatment, Mdr2-/- mice showed less liver injury compared to controls. CONCLUSION: Our experimental findings suggest that M3 -R-signalling significantly influences bile formation. Loss of the M3 -R increases susceptibility to cholestatic injury in DDC-fed mice. Since treatment of Mdr2-/- mice with a M3 -R agonist decreases liver injury, M3-R signaling may represent a therapeutic target in specific cholangiopathies.

NorUrsodeoxycholic acid ameliorates cholemic nephropathy in bile duct ligated mice.

BACKGROUND & AIMS: Severe cholestasis may cause cholemic nephropathy that can be modeled in common bile duct ligated (CBDL) mice. We aimed to explore the therapeutic efficacy and mechanisms of norursodeoxycholic acid (norUDCA) in cholemic nephropathy. METHODS: In 8-week CBDL mice fed with norUDCA (prior or post CBDL) or chow we evaluated serum urea levels, urine cytology and urinary neutrophil gelatinase associated lipocalin (uNGAL), kidney and liver tissue quantification of fibrosis by hydroxyproline content and gene chip expression looking at key genes of inflammation and fibrosis. Moreover, we comprehensively analysed bile acid profiles in liver, kidney, serum and urine samples. RESULTS: NorUDCA-fed CBDL mice had significantly lower serum urea and uNGAL levels and less severe cholemic nephropathy as demonstrated by normal urine cytology, significantly reduced tubulointerstitial nephritis, and renal fibrosis as compared to controls. NorUDCA underwent extensive metabolism to produce even more hydrophilic compounds that were significantly enriched in kidneys. CONCLUSION: NorUDCA ameliorates cholemic nephropathy due to the formation of highly hydrophilic metabolites enriched in kidney. Consequently, norUDCA may represent a medical treatment for cholemic nephropathy. LAY SUMMARY: The term cholemic nephropathy describes renal dysfunction together with characteristic morphological alterations of the kidney in obstructive cholestasis that can be mimicked by ligation of the common bile duct in mice. Feeding the hydrophilic bile acid norUDCA to bile duct ligated mice leads to a significant amelioration of the renal phenotype due to the formation of highly hydrophilic metabolites enriched in the kidney and may therefore represent a medical treatment for cholemic nephropathy.

Anal verrucous carcinoma is not related to infection with human papillomaviruses and should be distinguished from giant condyloma (Buschke-Löwenstein tumour).

AIMS: Verrucous carcinoma (VC) is a variant of well-differentiated squamous cell carcinoma and in the anal region is regarded as synonymous with giant condyloma (Buschke-Löwenstein tumour) (BLT). Aetiology, diagnostic criteria and clinical behaviour of both lesions are controversial. Recent studies suggest that VC at other sites is not associated with human papillomaviruses (HPV). We hypothesized that anal VC is also not related to HPV, while BLT is a HPV-induced lesion. METHODS AND RESULTS: Ten cases of VC and four cases of BLT were included. Several techniques were used for HPV detection: in-situ hybridization for HPV6, 11, 16 and 18, six different polymerase chain reaction (PCR) protocols for detection of at least 89 HPV types from alpha-, beta-, gamma- and mu-PV genera and in-situ hybridization for high-risk HPV E6/E7 mRNA; p16 immunohistochemistry and morphometric analysis were also performed. Alpha-, gamma- and mu-PVs were not found in any case of VC, while HPV6 was detected in all cases of BLT. p16 overexpression was not present in any of the lesions. Among microscopic features, only the absence of koilocytosis and enlarged spinous cells seem to be useful to distinguish VC from BLT. CONCLUSIONS: Our results suggest that anal VC, similarly to VC at other sites, is not associated with HPV infection, and must be distinguished from BLT, which is associated with low-risk HPV. Only with well-set diagnostic criteria will it be possible to ascertain clinical behaviour and optimal treatment for both lesions.

Lymph node retrieval in colorectal cancer: determining factors and prognostic significance.

PURPOSE: The study aimed to analyze clinicopathological factors that determine the extent of lymph node retrieval and to evaluate its prognostic impact in patients with colorectal cancer (CRC). METHODS: The number of retrieved lymph nodes was analyzed in 381 CRC specimens. Lymph node count was related to different clinicopathological variables by binary logistic regression. Progression-free survival (PFS) and cancer-specific survival (CSS) were determined using the Kaplan-Meier method and Cox regression models. RESULTS: The median number of retrieved lymph nodes was 20 (mean 21 ± 10, range 1-65) in right-sided, 13 (16 ± 10, 1-66) in left-sided, and 15 (18 ± 11, 3-64) in rectal tumors. The number of retrieved lymph nodes was independently associated with T-classification (p < 0.001), N-classification (p = 0.014), and tumor size (p = 0.005) as well as right-sided tumor location (p = 0.012). There was no association with age, sex, tumor grade, mismatch-repair status, and lymph or blood vessel invasion. The longer the surgical specimen, the higher were the numbers of retrieved and positive lymph nodes (p < 0.001, respectively). In patients with locally advanced (T3/T4) tumors (n = 283), analysis of more than 12 lymph nodes was independently associated with PFS (HR = 0.63, p = 0.025) and CSS (HR = 0.54, p = 0.004). In the subset of T3/T4 N0 patients (n = 130), analysis of more than 12 lymph nodes similarly proved to be an independent predictor of outcome (PFS, HR = 0.48, p = 0.046; OS, HR = 0.41, p = 0.026). CONCLUSION: The number of retrieved lymph nodes is associated with higher tumor stage, tumor size, and right-sided location. Low lymph node count indicates adverse outcome in patients with locally advanced (T3/T4) disease.

Tumour budding with and without admixed inflammation: two different sides of the same coin?

BACKGROUND: Tumour budding is an adverse prognostic indicator in colorectal cancer (CRC). Marked overall peritumoural inflammation has been associated with favourable outcome and may lead to the presence of isolated cancer cells due to destruction of invading cancer cell islets. METHODS: We assessed the prognostic significance of tumour budding and peritumoural inflammation in a cohort of 381 patients with CRC applying univariate and multivariate analyses. RESULTS: Patients with high-grade budding and marked inflammation had a significantly better outcome compared with patients with high-grade budding and only mild inflammation. Outcome in these cases, however, was still worse compared with cases with low-grade budding, in which the extent of peritumoural inflammation had no further prognostic effect. CONCLUSIONS: Tumour budding proved to be a powerful prognostic variable in patients with CRC. Scattering of invading cancer cell islets by marked overall peritumoural inflammation seems to have a minor role.

Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Inclusion of cytological features in tumor grading improves prognostic stratification of patients with colorectal cancer.

PURPOSE: Tumor grade is a traditional prognostic parameter in colorectal cancer. Remarkably, however, there is still no generally accepted consensus how to perform tumor grading. In this study, we systematically compared the prognostic value of traditional grading based upon histological features, that is, gland formation alone with grading based upon both histological and cytological features, such as nuclear pleomorphism and anaplasia ("alternative grade"). METHODS: Three hundred eighty-one tumors of randomly selected patients were retrospectively reviewed. Traditional and alternative tumor grades were related to various clinicopathological features and to progression-free and cancer-specific survival applying both univariate and multivariate testing. RESULTS: Traditional and alternative tumor grades were significantly associated with T and N classification, tumor size, lymphovascular invasion, as well as both progression-free and cancer-specific survival. In Cox's proportional hazards regression models, the alternative grade was superior to the traditional tumor grade and was significantly associated with progression-free survival (hazard ratio 1.57, 95% confidence interval 1.04-2.35; p = 0.031), independent of patients' age and gender, T and N classification, and lymphovascular invasion. Likewise, patients with tumors with high alternative grade were more likely to die of disease (hazard ratio 1.30, 95% confidence interval 0.85-2.00), but this difference was not statistically significant (p = 0.22). CONCLUSIONS: Tumor grade based upon both histological and cytological features was superior to grade based upon histological features alone and proved to be an independent prognostic parameter. Thus, tumor grade based upon both histological and cytological features may help to improve prognostic stratification and may thereby affect clinical decision-making and patient management.

Peritumoral eosinophils predict recurrence in colorectal cancer.

In colorectal cancer, the presence and extent of eosinophil granulocyte infiltration may render important prognostic information. However, it remains unclear whether an increasing number of eosinophils might simply be linked to the overall inflammatory cell reaction or represent a self-contained, antitumoral mechanism that needs to be documented and promoted therapeutically. Peri- and intratumoral eosinophil counts were retrospectively assessed in 381 primary colorectal cancers from randomly selected patients. Tumors were diagnosed in American Joint Committee on Cancer (AJCC)/Union Internationale Contre le Cancer (UICC) stage I in 21%, stage II in 32%, stage III in 33%, and stage IV in 14%. Presence and extent of eosinophils was related to various histopathological parameters as well as patients' outcome. Overall, peri- and intratumoral eosinophils were observed in 86 and 75% cancer specimens. The peritumoral eosinophil count correlated strongly with the intratumoral eosinophil count (R=0.69; P<0.001) and with the intensity of the overall inflammatory cell reaction (R=0.318; P<0.001). Both increasing peri- and intratumoral eosinophil counts were significantly associated with lower T and N classification, better tumor differentiation, absence of vascular invasion, as well as improved progression-free and cancer-specific survival. However, only peritumoral eosinophils, but not intratumoral, were an independent prognosticator of favorable progression-free (hazard ratio 0.75; 95% confidence interval 0.58-0.98; P=0.04) and cancer-specific survival (hazard ratio 0.7; 95% confidence interval 0.52-0.93; P=0.01)-independent of the intensity of overall inflammatory cell reaction. This was also found for patients with AJCC/UICC stage II disease, wherein the presence of peritumoral eosinophils was significantly associated with favorable outcome. In conclusion, the number of peritumoral eosinophils had a significant favorable impact on prognosis of colorectal cancer patients independent of the overall tumor-associated inflammatory response. Evaluation of peritumoral eosinophils represents a promising readily assessable tool and should therefore routinely be commented on in the pathology report.

Potential of nor-Ursodeoxycholic Acid in Cholestatic and Metabolic Disorders.

24-nor-ursodeoxycholic acid (norUDCA) is a side-chain shortened derivate of ursodeoxycholic acid (UDCA). Since norUDCA is only ineffectively conjugated with glycine or taurine, it has specific physicochemical and therapeutic properties distinct from UDCA. Nonamidated norUDCA undergoes cholehepatic shunting enabling 'ductular targeting' and inducing a bicarbonate-rich hypercholeresis, with cholangioprotective effects. At the same time it has direct anti-inflammatory, antilipotoxic, anti fibrotic, and antiproliferative properties targeting various liver cell populations. norUDCA appears to be one of the most promising novel treatment approaches targeting the liver and the bile duct system at multifactorial and multicellular levels. This review article is a summary of a lecture given at the XXIII International Bile Acid Meeting (Falk Symposium 194) on 'Bile Acids as Signal Integrators and Metabolic Modulators' held in Freiburg, October 8-9, 2014, and summarizes the recent progress with norUDCA as a novel therapeutic approach in cholestatic and metabolic (liver) disorders.