Andreas E. Kremer

Lysophosphatidic Acid Is a Potential Mediator of Cholestatic Pruritus

BACKGROUND & AIMS Pruritus is a common and disabling symptom in cholestatic disorders. However, its causes remain unknown. We hypothesized that potential pruritogens accumulate in the circulation of cholestatic patients and activate sensory neurons. METHODS Cytosolic free calcium ([Ca(2+)](i)) was measured in neuronal cell lines by ratiometric fluorometry upon exposure to serum samples from pruritic patients with intrahepatic cholestasis of pregnancy (ICP), primary biliary cirrhosis (PBC), other cholestatic disorders, and pregnant, healthy, and nonpruritic disease controls. Putative [Ca(2+)](i)-inducing factors in pruritic serum were explored by analytical techniques, including quantification by high-performance liquid chromatography/mass spectroscopy. In mice, scratch activity after intradermal pruritogen injection was quantified using a magnetic device. RESULTS Transient increases in neuronal [Ca(2+)](i) induced by pruritic PBC and ICP sera were higher than corresponding controls. Lysophosphatidic acid (LPA) could be identified as a major [Ca(2+)](i) agonist in pruritic sera, and LPA concentrations were increased in cholestatic patients with pruritus. LPA injected intradermally into mice induced scratch responses. Autotaxin, the serum enzyme converting lysophosphatidylcholine into LPA, was markedly increased in patients with ICP versus pregnant controls (P < .0001) and cholestatic patients with versus without pruritus (P < .0001). Autotaxin activity correlated with intensity of pruritus (P < .0001), which was not the case for serum bile salts, histamine, tryptase, substance P, or mu-opioids. In patients with PBC who underwent temporary nasobiliary drainage, both itch intensity and autotaxin activity markedly decreased during drainage and returned to preexistent levels after drain removal. CONCLUSIONS We suggest that LPA and autotaxin play a critical role in cholestatic pruritus and may serve as potential targets for future therapeutic interventions.

The biliary HCO3− umbrella: A unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies

This review focuses on the hypothesis that biliary HCO  3− secretion in humans serves to maintain an alkaline pH near the apical surface of hepatocytes and cholangiocytes to prevent the uncontrolled membrane permeation of protonated glycine‐conjugated bile acids. Functional impairment of this biliary HCO  3− umbrella or its regulation may lead to enhanced vulnerability of cholangiocytes and periportal hepatocytes toward the attack of apolar hydrophobic bile acids. An intact interplay of hepatocellular and cholangiocellular adenosine triphosphate (ATP) secretion, ATP/P2Y‐ and bile salt/TGR5‐mediated Cl−/ HCO  3− exchange and HCO  3− secretion, and alkaline phosphatase–mediated ATP breakdown may guarantee a stable biliary HCO  3− umbrella under physiological conditions. Genetic and acquired functional defects leading to destabilization of the biliary HCO  3− umbrella may contribute to development and progression of various forms of fibrosing/sclerosing cholangitis. (HEPATOLOGY 2010)

Serum autotaxin is increased in pruritus of cholestasis, but not of other origin, and responds to therapeutic interventions†‡

Pruritus is a seriously disabling symptom accompanying many cholestatic liver disorders. Recent experimental evidence implicated the lysophospholipase, autotaxin (ATX), and its product, lysophosphatidic acid (LPA), as potential mediators of cholestatic pruritus. In this study, we highlight that increased serum ATX levels are specific for pruritus of cholestasis, but not pruritus of uremia, Hodgkins disease, or atopic dermatitis. Treatment of patients with cholestasis with the bile salt sequestrant, colesevelam, but not placebo, effectively reduced total serum bile salts and fibroblast growth factor 19 levels, but only marginally altered pruritus intensity and ATX activity. Rifampicin (RMP) significantly reduced itch intensity and ATX activity in patients with pruritus not responding to bile salt sequestrants. In vitro, RMP inhibited ATX expression in human HepG2 hepatoma cells and hepatoma cells overexpressing the pregnane X receptor (PXR), but not in hepatoma cells in which PXR was knocked down. Treatment of severe, refractory pruritus by the molecular adsorbents recirculation system or nasobiliary drainage improved itch intensity, which, again, correlated with the reduction of ATX levels. Upon reoccurrence of pruritus, ATX activity returned to pretreatment values. Conclusion: Serum ATX activity is specifically increased in patients with cholestatic, but not other forms of, systemic pruritus and closely correlates with the effectiveness of therapeutic interventions. The beneficial antipruritic action of RMP may be explained, at least partly, by the PXR‐dependent transcriptional inhibition of ATX expression. Thus, ATX likely represents a novel therapeutic target for pruritus of cholestasis. (HEPATOLOGY 2012)

Fibroblast growth factor 21 is induced by endoplasmic reticulum stress.

Increased hepatic expression is held responsible for elevated serum levels of fibroblast growth factor 21 (FGF21) in non-alcoholic fatty liver disease (NAFLD) but the underlying molecular mechanism is unclear. In the present study we tested the postulate that the metabolic hormone FGF21 is regulated by endoplasmic reticulum (ER) stress, a condition that is observed in a number of diseases including NAFLD and results in activation of an adaptive response known as the unfolded protein response (UPR). ER stress stimuli were found to induce expression of Fgf21 mRNA in H4IIE hepatoma cells and in isolated rat hepatocytes. Moreover, intraperitoneal injection of the ER stressor tunicamycin induced hepatic Fgf21 expression in mice and resulted in marked elevation of serum FGF21 levels. The effect of ER stress on FGF21 expression could be mimicked by overexpression of ATF4, a transcriptional effector of the PERK-branch of the UPR. In silico analysis revealed the presence of two binding sites for ATF4 in the FGF21 promoter region. Combined disruption of these elements, abrogated FGF21 promoter activity induced by ER stress or ATF4 overexpression. These findings implicate the PERK/eIF2alpha/ATF4 cascade in ER stress regulation of FGF21. A consequence of this notion is that other intracellular stress signaling pathways that converge at eIF2alpha, can regulate FGF21 expression. Indeed, both nutrient (amino acid deprivation) and oxidative stress (arsenite) were found to induce Fgf21 expression in hepatoma cells and isolated rat hepatocytes. In conclusion, FGF21 expression is regulated by ER stress and additional intracellular stress signaling pathways. Our findings suggest that increased cellular stress in fatty livers may underlie the elevated FGF21 levels observed in patients with NAFLD.

Clinical Relevance of Circulating Nucleosomes in Cancer

Nucleosomes, complexes of DNA and histone proteins, are released during cell death into the blood circulation. Elevated serum and plasma levels have been found in various forms of cancer, but also in autoimmune diseases and acute situations such as stroke, trauma, and during sepsis. Here, the clinical relevance of circulating nucleosomes for diagnosis, staging, prognosis, and therapeutic monitoring of cancer is reviewed. Several studies have shown that levels of nucleosomes are significantly higher in serum and plasma of cancer patients in comparison to healthy controls. However, because of elevations of nucleosome levels in patients with benign diseases relevant for differential diagnosis, they are not suitable for cancer diagnosis. Concerning tumor staging, nucleosome levels correlate with tumor stage and presence of metastases in gastrointestinal cancer, but not in other tumor types. Prognostic value of circulating nucleosomes is found in lung cancer in univariate analyses, but not in multivariate analyses. Circulating nucleosomes are most informative for the monitoring of cytotoxic therapy. Strongly decreasing levels are mainly found in patients with remission of disease, whereas constantly high or increasing values are associated with progressive disease during chemo‐ and radiotherapy. In addition, therapy outcome is already indicated by the nucleosomal course during the first week of chemo‐ and radiotherapy in patients with lung, pancreatic, and colorectal cancer as well as in hematologic malignancies. Despite their non‐tumor‐specificity, kinetics of nucleosomes are valuable markers for the early estimation of therapeutic efficacy and may be helpful to adapting early cancer therapy in the future.

Pruritus in cholestasis: Facts and fiction

Pruritus is a common symptom in patients with cholestatic liver diseases such as primary biliary cirrhosis, primary sclerosing cholangitis, intrahepatic cholestasis of pregnancy, or hereditary pediatric cholestatic disorders and may accompany, although less frequently, many other liver diseases. Recent findings indicate that lysophosphatidic acid (LPA), a potent neuronal activator, and autotaxin (ATX; ectonucleotide pyrophosphatase/phosphodiesterase 2), the enzyme which forms LPA, may form a key element of the long‐sought pruritogenic signaling cascade in cholestatic patients suffering from itch. Serum ATX, but no other pruritogen candidate studied so far, correlates with pruritus intensity and responds to therapeutic interventions. In this comprehensive review, we provide a short update on actual insights in signal transmission related to pruritus and discuss pruritogen candidates in cholestasis. We also summarize evidence‐based and guideline‐approved as well as experimental therapeutic approaches for patients suffering from pruritus in cholestasis. (Hepatology 2014;60:399–407)

Pathogenesis and Treatment of Pruritus in Cholestasis

Pruritus is an enigmatic, seriously disabling symptom accompanying cholestatic liver diseases and a broad range of other disorders. Most recently, novel itchspecificneuronal pathways, itch mediators and their relevant receptors have been identified. In addition, new antipruritic therapeutic strategies have been developed and/or are under evaluation. This review highlights recent experimental and clinical findings focusing on the pathogenesis and actual treatment of pruritus in cholestatic liver disease. Evidence-based therapeutic recommendations, including the use of anion exchange resins cholestyramine, colestipol and colesevelam, the microsomal enzyme inducer rifampicin, the opioid receptor antagonists naltrexone and naloxone, and the serotonin reuptake inhibitor sertraline, are provided.

Increased frequencies of IL‐31‐producing T cells are found in chronic atopic dermatitis skin

Abstract:  Interleukin (IL)‐31 has been associated with pruritus, a characteristic feature of atopic dermatitis (AD). Local T cell responses may be responsible for the increased level of IL‐31 mRNA observed in AD. We investigated the frequency of IL‐31‐producing T cells in AD lesions, as well as their cytokine profile. T cells were isolated from chronic AD lesions, autologous blood and healthy donor skin. Intracellular expression of IL‐31, IFN‐γ, IL‐13, IL‐17 and IL‐22 was measured using flow cytometry. T cells from AD lesions contained significantly higher percentages of IL‐31‐producing T cells compared to autologous blood and donor skin. Many IL‐31‐producing T cells co‐produced IL‐13 and to lesser extent IL‐22, but rarely IFN‐γ or IL‐17. A substantial part of the IL‐31‐producing T cells did not co‐produce any of the other cytokines and could therefore not be linked to any of the known functionally different T cell subsets. The T cell infiltrates were also relatively enriched for Th2/Tc2 and Th22/Tc22 cells, while frequencies of Th1/Tc1 and Th17 cells were decreased. This is the first report describing the detection of IL‐31 at protein level in skin‐infiltrating T cells. We show here that T cells in chronic AD skin produce IL‐31 and that AD lesions contain increased levels of these IL‐31‐producing T cells. This suggests that a substantial part of previously reported increased IL‐31 mRNA levels in AD skin is T cell derived and that these cells may be involved in the pathogenesis of AD.

Receptors, cells and circuits involved in pruritus of systemic disorders

Pruritus is a sensory phenomenon accompanying a broad range of systemic disorders including hematologic and lymphoproliferative disorders, metabolic and endocrine diseases, solid tumours, and infectious diseases. The molecular mechanisms involved in itch sensation remain enigmatic in most of these diseases. However, from studies in patients and animal models a large number of mediators and receptors responsible for scratching behaviour have been identified in recent years. New insights into the interplay between neuronal and non-neuronal cells in the initiation, modulation and sensitization of itch sensation have been acquired. This review highlights the current knowledge of the molecular mechanism involved in pruritus of systemic disorders and summarizes the signalling pathways of biogenic amines, neuropeptides, proteases, eicosanoids, cytokines, opioids, endocannabinoids, neurotrophins, phospholipids and other signalling molecules participating in pruritus.

Nucleosomes in pancreatic cancer patients during radiochemotherapy.

Nucleosomes appear spontaneously in elevated concentrations in the serum of patients with malignant diseases as well as during chemo- and radiotherapy. We analyzed whether their kinetics show typical characteristics during radiochemotherapy and enable an early estimation of therapy efficacy. We used the Cell Death Detection Elisaplus (Roche Diagnostics) and investigated the course of nucleosomes in the serum of 32 patients with a local stage of pancreatic cancer who were treated with radiochemotherapy for several weeks. Ten of them received postsurgical therapy, 21 received primary therapy and 1 received therapy for local relapse. Blood was taken before the beginning of therapy, daily during the first week, once weekly during the following weeks and at the end of radiochemotherapy. The response to therapy was defined according to the kinetics of CA 19-9: a decrease of CA 19-9 ≧50% after radiochemotherapy was considered as ‘remission’; an increase of ≧100% (which was confirmed by two following values) was defined as ‘progression’. Patients with ‘stable disease’ ranged intermediately. Most of the examined patients showed a decrease of the concentration of nucleosomes within 6 h after the first dose of radiation. Afterwards, nucleosome levels increased rapidly, reaching their maximum during the following days. Patients receiving postsurgery, primary or relapse therapies did not show significant differences in nucleosome values during the time of treatment. Single nucleosome values, measured at 6, 24 and 48 h after the application of therapy, could not discriminate significantly between patients with no progression and those with progression of disease. However, the area under the curve of the first 3 days, which integrated all variables of the initial therapeutic phase, showed a significant correlation with the progression-free interval (p = 0.008). Our results indicate that the area under the curve of nucleosomes during the initial phase of radiochemotherapy could be valuable for the early prediction of the progression-free interval.