Percutaneous drainage and combined praziquantel-albendazole therapy: a novel approach for the treatment of simple echinococcal liver cysts.

Cystic echinococcosis (CE) is a worldwide helminthic zoonosis causing serious disease in humans. The WHO Informal Working Group on Echinococcosis recommends a stage-specific treatment approach of hepatic CE that facilitates the decision on what therapy option is most appropriate. Percutaneous aspiration, instillation of a scolicide, e.g., ethanol or hypertonic saline, and subsequent re-aspiration (PAIR) have been advocated for treating medium-size unilocular WHO-stage CE1 cysts. PAIR can pose a risk of toxic cholangitis because of spillage of ethanol in the case of a cysto-biliary fistula or of life-threatening hypernatriaemia when hypertonic saline is used. The purpose of our study is to develop an alternative, safe, minimally invasive method to treat CE1 cysts, avoiding the use of toxic topic scolicides.We opt for percutaneous drainage (PD) in four patients: the intrahepatic drainage catheter is placed under CT-fluoroscopy, intracystic fluid is aspirated, and the viability of intracystic echinococcal protoscolices is assessed microscopically. Oral praziquantel (PZQ) is added to albendazole (ABZ) instead of using topical scolicidals.Protoscolices degenerate within 5 to 10 days after PZQ co-medication at a cumulative dosage of 250 to 335 mg/kg, and the cysts collapse. The cysts degenerate, and no sign of spillage nor relapse is observed in the follow-up time of up to 24 months post-intervention.In conclusion, PD combined with oral PZQ under ABZ coverage is preferable to PAIR in patients with unilocular echinococcal cysts.

Evaluation of muscarinic acetylcholine receptor type 3 gene polymorphisms in patients with primary biliary cholangitis and primary sclerosing cholangitis.

AIM: Muscarinic acetylcholine receptor type 3-mediated signaling might be involved in the pathogenesis of chronic inflammatory biliary diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). The aim of the present study was to investigate the prevalence of five well-characterized specific single-nucleotide polymorphisms within the muscarinic acetylcholine receptor type 3 gene, CHRM3 (rs11578320, rs6690809, rs6429157, rs7548522, and rs4620530), in patients with PBC and PSC. Patients with chronic hepatitis C (CHC) and healthy individuals served as control cohorts. In the PBC cohort, baseline characteristics and response to ursodeoxycholic acid therapy applying established response criteria at 12 months after the initiation of treatment were evaluated according to the underlying CHRM3 genotype. METHODS: CHRM3 genotyping was carried out in 306 PBC patients, 205 PSC patients, 208 CHC patients, and 240 healthy controls from two independent German tertiary care university centers in Berlin and Leipzig, Germany. RESULTS: CHRM3 rs4620530 proportions in patients with PBC significantly differed from patients with PSC (P = 0.005), CHC (P = 0.009), and healthy controls (P = 0.008), primarily due to a substantial overrepresentation of the T allele in PBC (49.3% in PBC vs. 39.8% in PSC, 35.7% in CHC, and 40% in healthy controls), indicating a potential association of the rs4620530 T allele with PBC (OR 1.461, 95% CI 1.147-1.861, P = 0.002). Further analysis showed no association of CHRM3 single-nucleotide polymorphism rs4620530 with baseline characteristics and ursodeoxycholic acid treatment response in PBC. CONCLUSION: CHRM3 single-nucleotide polymorphism rs4620530 might confer an increased genetic risk for the development of PBC.

Interleukin 17, Produced by γδ T Cells, Contributes to Hepatic Inflammation in a Mouse Model of Biliary Atresia and Is Increased in Livers of Patients.

BACKGROUND & AIMS: Biliary atresia (BA) is a rare disease in infants, with unknown mechanisms of pathogenesis. It is characterized by hepatobiliary inflammatory, progressive destruction of the biliary system leading to liver fibrosis, and deterioration of liver function. Interleukin (IL) 17A promotes inflammatory and autoimmune processes. We studied the role of IL17A and cells that produce this cytokine in a mouse model of BA and in hepatic biopsy samples from infants with BA. METHODS: We obtained peripheral blood and liver tissue specimens from 20 patients with BA, collected at the time of Kasai portoenterostomy, along with liver biopsies from infants without BA (controls). The tissue samples were analyzed by reverse transcription quantitative polymerase chain reaction (PCR), in situ PCR, and flow cytometry analyses. BA was induced in balb/cAnNCrl mice by rhesus rotavirus infection; uninfected mice were used as controls. Liver tissues were collected from mice and analyzed histologically and by reverse transcriptase PCR; leukocytes were isolated, stimulated, and analyzed by flow cytometry and PCR analyses. Some mice were given 3 intraperitoneal injections of a monoclonal antibody against IL17 or an isotype antibody (control). RESULTS: Livers from rhesus rota virus-infected mice with BA had 7-fold more Il17a messenger RNA than control mice (P = .02). γδ T cells were the exclusive source of IL17; no T-helper 17 cells were detected in livers of mice with BA. The increased number of IL17a-positive γδ T cells liver tissues of mice with BA was associated with increased levels of IL17A, IL17F, retinoid-orphan-receptor C, C-C chemokine receptor 6, and the IL23 receptor. Mice that were developing BA and given antibodies against IL17 had lower levels of liver inflammation and mean serum levels of bilirubin than mice receiving control antibodies (191 µmol/L vs 78 µmol/L, P = .002). Liver tissues from patients with BA had 4.6-fold higher levels of IL17 messenger RNA than control liver tissues (P = .02). CONCLUSIONS: In livers of mice with BA, γδ T cells produce IL17, which is required for inflammation and destruction of the biliary system. IL17 is up-regulated in liver tissues from patients with BA, compared with controls, and might serve as a therapeutic target.

Shear wave elastography and shear wave dispersion imaging in primary biliary cholangitis-a pilot study.

BACKGROUND: Primary biliary cholangitis (PBC) is a chronic liver disease that can lead to liver fibrosis and cirrhosis. Two-dimensional shear wave elastography (2D-SWE) is a modern technique for fibrosis assessment. However, data regarding its performance in PBC is sparse. We aimed to characterize severity of liver disease in PBC patients using non-invasive 2D-SWE and the new methods of attenuation imaging (ATI) and shear wave dispersion imaging (SWD). METHODS: Twenty two PBC patients were examined with 2D-SWE, SWD and ATI, alongside established non-invasive fibrosis and steatosis assessment methods as well as liver function tests. RESULTS: Median 2D-SWE values were 1.48 m/s (range, 1.14-2.13 m/s) and 6.7 kPa (range, 3.8-14.7 kPa), respectively. Median SWD, ATI, transient elastography (TE) and controlled attenuation parameter (CAP) values were 13.9 m/s/kHz (range, 11.6-21 m/s/kHz), 0.57 dB/cm/MHz (range, 0.5-0.68 dB/cm/MHz), 7 kPa (range, 3.7-14.6 kPa), and 208 dB/m (range, 107-276 dB/m), respectively. 2D-SWE displayed a significant correlation with spleen length, platelet count, non-invasive fibrosis scores (FIB-4, APRI) and with TE. SWD correlated with alkaline phosphatase (ALP) levels, which is a prognostic marker in PBC. CONCLUSIONS: Our findings add further evidence that 2D-SWE is a reliable method for fibrosis assessment in PBC. Even though the cohort size was small, the correlation of SWD with the prognostic marker ALP suggests a potentially valuable role of this new non-invasive method in evaluating liver disease activity in PBC.

Evaluation of Inhibitory Antibodies against the Muscarinic Acetylcholine Receptor Type 3 in Patients with Primary Biliary Cholangitis and Primary Sclerosing Cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) constitute rare chronic inflammatory biliary diseases which likely comprise genetic, environmental and autoimmune factors. Specific inhibitory (auto-) antibodies against the muscarinic acetylcholine receptor type 3 (mAChR3 auto-ab) may contribute to the pathogenesis of chronic biliary inflammation by modulating mAChR3- mediated signaling. AIMS: The aim of this study was to analyze the prevalence and relevance of inhibitory mAChR3 auto-ab (mAChR3inh+ auto-ab) in a large cohort of PBC patients from two independent tertiary centers in Berlin and Leipzig in comparison to a large PSC cohort. Baseline parameters and response rates to standard treatment with ursodeoxycholic acid (UDCA) were characterized with respect to the individual mAChR3 auto-ab status. METHODS: In total, the study population comprised 437 PBC patients, 187 PSC patients and 80 healthy controls. Clinical and laboratory baseline characteristics were retrieved from medical records. The response to ursodeoxycholic acid (UDCA) therapy after 12 months of treatment was available in 176 PBC and 45 PSC patients. RESULTS: The prevalence of mAChR3inh+ auto-ab was significantly higher among PBC patients (11.2%, 49/437; p = 0.008 vs. healthy controls) and PSC patients (33.6%, 63/187; p < 0.0001 vs. healthy controls) compared to healthy controls (2.5%, 2/80), respectively. PBC patients with mAChR3inh+ auto-ab exhibited significantly higher levels of alkaline phosphatase (ALP) and bilirubin, which constitute established parameters for PBC risk stratification. Moreover, mAChR3inh+ PBC patients tended to show decreased response rates to UDCA therapy compared to PBC patients without mAChR3inh+ auto-ab (mAChR3- PBC). In contrast, PSC patients with mAChR3inh+ auto-ab showed no significant differences in laboratory findings compared to mAChR3 auto-ab negative (mAChR3-) PSC patients. CONCLUSION: MAChR3inh+ auto-ab might be involved in the pathogenesis and treatment response of chronic biliary inflammation in patients with PBC but not in patients with PSC.

Disease Severity in Moderate-to-Severe COVID-19 Is Associated With Platelet Hyperreactivity and Innate Immune Activation.

Background: Hemostasis and inflammation are both dysregulated in patients with moderate-to-severe coronavirus disease 2019 (COVID-19). Yet, both processes can also be disturbed in patients with other respiratory diseases, and the interactions between coagulation, inflammation, and disease severity specific to COVID-19 are still vague. Methods: Hospitalized patients with acute respiratory symptoms and with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-positive (COVpos) and SARS-CoV2-negative (COVneg) status were included. We assessed adenosine diphosphate (ADP)-, thrombin receptor activator peptide 6 (TRAP)-, and arachidonic acid (AA)-induced platelet reactivity by impedance aggregometry, as well as leukocyte subtype spectrum and platelet-leukocyte aggregates by flow cytometry and inflammatory cytokines by cytometric bead array. Results: ADP-, TRAP-, and AA-induced platelet reactivity was significantly higher in COVpos than in COVneg patients. Disease severity, assessed by sequential organ failure assessment (SOFA) score, was higher in COVpos than in COVneg patients and again higher in deceased COVpos patients than in surviving COVpos. The SOFA score correlated significantly with the mean platelet volume and TRAP-induced platelet aggregability. A larger percentage of classical and intermediate monocytes, and of CD4pos T cells (TH) aggregated with platelets in COVpos than in COVneg patients. Interleukin (IL)-1 receptor antagonist (RA) and IL-6 levels were higher in COVpos than in COVneg patients and again higher in deceased COVpos patients than in surviving COVpos. IL-1RA and IL-6 levels correlated with the SOFA score in COVpos but not in COVneg patients. In both respiratory disease groups, absolute levels of B-cell-platelet aggregates and NK-cell-platelet aggregates were correlated with ex vivo platelet aggegation upon stimulation with AA and ADP, respectively, indicating a universal, but not a COVID-19-specific mechanism. Conclusion: In moderate-to-severe COVID-19, but not in other respiratory diseases, disease severity was associated with platelet hyperreactivity and a typical inflammatory signature. In addition to a severe inflammatory response, platelet hyperreactivity associated to a worse clinical outcome in patients with COVID-19, pointing to the importance of antithrombotic therapy for reducing disease severity.

TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development.

TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in established tumor cell lines but not nontransformed cells. Herein, we demonstrate a role for the apoptosis-inducing TRAIL receptor (TRAIL-R) as a metastasis suppressor. Although mouse models employing tumor transplantation have shown that TRAIL can reduce tumor growth, autochthonous tumor models have generated conflicting results with respect to the physiological role of the TRAIL system during tumorigenesis. We used a multistage model of squamous cell carcinoma to examine the role of TRAIL-R throughout all steps of tumor development. DMBA/TPA-treated TRAIL-R-deficient mice showed neither an increase in number or growth rate of benign papillomas nor an increase in the rate of progression to squamous cell carcinoma. However, metastasis to lymph nodes was significantly enhanced, indicating a role for TRAIL-R specifically in the suppression of metastasis. We also found that adherent TRAIL-R-expressing skin carcinoma cells were TRAIL resistant in vitro but were sensitized to TRAIL upon detachment by inactivation of the ERK signaling pathway. As detachment from the primary tumor is an obligatory step in metastasis, this provides a possible mechanism by which TRAIL-R could inhibit metastasis. Hence, treatment of cancer patients with agonists of the apoptosis-inducing receptors for TRAIL may prove useful in reducing the incidence of metastasis.

Real-World Clinical Management of Patients with Primary Biliary Cholangitis-A Retrospective Multicenter Study from Germany.

BACKGROUND: Clinical practice guidelines for patients with primary biliary cholangitis (PBC) have been recently revised and implemented for well-established response criteria to standard first-line ursodeoxycholic acid (UDCA) therapy at 12 months after treatment initiation for the early identification of high-risk patients with inadequate treatment responses who may require treatment modification. However, there are only very limited data concerning the real-world clinical management of patients with PBC in Germany. OBJECTIVE: The aim of this retrospective multicenter study was to evaluate response rates to standard first-line UDCA therapy and subsequent Second-line treatment regimens in a large cohort of well-characterized patients with PBC from 10 independent hepatological referral centers in Germany prior to the introduction of obeticholic acid as a licensed second-line treatment option. METHODS: Diagnostic confirmation of PBC, standard first-line UDCA treatment regimens and response rates at 12 months according to Paris-I, Paris-II, and Barcelona criteria, the follow-up cut-off alkaline phosphatase (ALP) ≤ 1.67 × upper limit of normal (ULN) and the normalization of bilirubin (bilirubin ≤ 1 × ULN) were retrospectively examined between June 1986 and March 2017. The management and hitherto applied second-line treatment regimens in patients with an inadequate response to UDCA and subsequent response rates at 12 months were also evaluated. RESULTS: Overall, 480 PBC patients were included in this study. The median UDCA dosage was 13.2 mg UDCA/kg bodyweight (BW)/d. Adequate UDCA treatment response rates according to Paris-I, Paris-II, and Barcelona criteria were observed in 91, 71.3, and 61.3% of patients, respectively. In 83.8% of patients, ALP ≤ 1.67 × ULN were achieved. A total of 116 patients (24.2%) showed an inadequate response to UDCA according to at least one criterion. The diverse second-line treatment regimens applied led to significantly higher response rates according to Paris-II (35 vs. 60%, p = 0.005), Barcelona (13 vs. 34%, p = 0.0005), ALP ≤ 1.67 × ULN and bilirubin ≤ 1 × ULN (52.1 vs. 75%, p = 0.002). The addition of bezafibrates appeared to induce the strongest beneficial effect in this cohort (Paris II: 24 vs. 74%, p = 0.004; Barcelona: 50 vs. 84%, p = 0.046; ALP < 1.67 × ULN and bilirubin ≤ 1 × ULN: 33 vs. 86%, p = 0.001). CONCLUSION: Our large retrospective multicenter study confirms high response rates following UDCA first-line standard treatment in patients with PBC and highlights the need for close monitoring and early treatment modification in high-risk patients with an insufficient response to UDCA since early treatment modification significantly increases subsequent response rates of these patients.

Transforming growth factor-beta-mediated tumor necrosis factor-related apoptosis-inducing ligand expression and apoptosis in hepatoma cells requires functional cooperation between Smad proteins and activator protein-1.

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptotic cell death in normal and transformed hepatocytes. We recently identified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as an important mediator of TGF-beta-induced apoptosis in hepatoma cells. In this study, we have further explored the mechanism by which TGF-beta up-regulates TRAIL expression. The 5'-flanking region of the TRAIL gene was isolated and characterized. Deletion mutants of the 5'-untranslated region of the TRAIL gene revealed a region comprising nucleotides -1950 to -1100 responsible for TRAIL induction following treatment with TGF-beta. Within this region, we have identified an activator protein-1 (AP-1) site indispensable for TGF-beta-mediated induction of TRAIL. Activation of this AP-1 site is mediated by a JunD.FosB heterodimer. Expression of DNSmad4, DNJunD, or DNFosB significantly impairs TGF-beta-mediated activation of the TRAIL promoter. Furthermore, with tRNA interference targeting Smad4, junD, FosB, we could abolish TRAIL expression and, subsequently, TGF-beta-induced TRAIL-mediated apoptosis in hepatoma cells. Our results reveal a new AP-1 site within the TRAIL promoter functionally involved in TGF-beta-induced TRAIL expression and apoptosis in hepatomas and thus provide evidence for the underlying mechanism by which TGF-beta might regulate cell death in liver cancer.

Loss of inter-cellular cooperation by complete epithelial-mesenchymal transition supports favorable outcomes in basal breast cancer patients.

According to the sequential metastasis model, aggressive mesenchymal (M) metastasis-initiating cells (MICs) are generated by an epithelial-mesenchymal transition (EMT) which eventually is reversed by a mesenchymal-epithelial transition (MET) and outgrowth of life-threatening epithelial (E) macrometastases. Paradoxically, in breast cancer M signatures are linked with more favorable outcomes than E signatures, and M cells are often dispensable for metastasis in mouse models. Here we present evidence at the cellular and patient level for the cooperation metastasis model, according to which E cells are MICs, while M cells merely support E cell persistence through cooperation. We tracked the fates of co-cultured E and M clones and of fluorescent CDH1-promoter-driven cell lines reporting the E state derived from basal breast cancer HMLER cells. Cells were placed in suspension state and allowed to reattach and select an EMT cell fate. Flow cytometry, single cell and bulk gene expression analyses revealed that only pre-existing E cells generated E cells, mixed E/M populations, or stem-like hybrid E/M cells after suspension and that complete EMT manifest in M clones and CDH1-negative reporter cells resulted in loss of cell plasticity, suggesting full transdifferentiation. Mechanistically, E-M coculture experiments supported the persistence of pre-existing E cells where M cells inhibited EMT of E cells in a mutual cooperation via direct cell-cell contact. Consistently, M signatures were associated with more favorable patient outcomes compared to E signatures in breast cancer, specifically in basal breast cancer patients. These findings suggest a potential benefit of complete EMT for basal breast cancer patients.

Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival.

Breast cancer stem cells (CSCs) are thought to drive recurrence and metastasis. Their identity has been linked to the epithelial to mesenchymal transition (EMT) but remains highly controversial since--depending on the cell-line studied--either epithelial (E) or mesenchymal (M) markers, alone or together have been associated with stemness. Using distinct transcript expression signatures characterizing the three different E, M and hybrid E/M cell-types, our data support a novel model that links a mixed EM signature with stemness in 1) individual cells, 2) luminal and basal cell lines, 3) in vivo xenograft mouse models, and 4) in all breast cancer subtypes. In particular, we found that co-expression of E and M signatures was associated with poorest outcome in luminal and basal breast cancer patients as well as with enrichment for stem-like cells in both E and M breast cell-lines. This link between a mixed EM expression signature and stemness was explained by two findings: first, mixed cultures of E and M cells showed increased cooperation in mammosphere formation (indicative of stemness) compared to the more differentiated E and M cell-types. Second, single-cell qPCR analysis revealed that E and M genes could be co-expressed in the same cell. These hybrid E/M cells were generated by both E or M cells and had a combination of several stem-like traits since they displayed increased plasticity, self-renewal, mammosphere formation, and produced ALDH1+ progenies, while more differentiated M cells showed less plasticity and E cells showed less self-renewal. Thus, the hybrid E/M state reflecting stemness and its promotion by E-M cooperation offers a dual biological rationale for the robust association of the mixed EM signature with poor prognosis, independent of cellular origin. Together, our model explains previous paradoxical findings that breast CSCs appear to be M in luminal cell-lines but E in basal breast cancer cell-lines. Our results suggest that targeting E/M heterogeneity by eliminating hybrid E/M cells and cooperation between E and M cell-types could improve breast cancer patient survival independent of breast cancer-subtype.

Cancer cell-autonomous TRAIL-R signaling promotes KRAS-driven cancer progression, invasion, and metastasis.

Many cancers harbor oncogenic mutations of KRAS. Effectors mediating cancer progression, invasion, and metastasis in KRAS-mutated cancers are only incompletely understood. Here we identify cancer cell-expressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients.

Metastasis suppressor function of tumor necrosis factor-related apoptosis-inducing ligand-R in mice: implications for TRAIL-based therapy in humans?

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapy, as it can induce apoptosis specifically in tumor cells but not in normal cells. Although earlier mouse tumor studies revealed a strong tissue dependency of TRAIL and its death receptor in suppressing primary tumorigenesis or experimental metastases, we recently found that TRAIL-R inhibits lymph node metastases without affecting primary tumor formation in a mouse model of multistage skin tumorigenesis. This finding uncouples the role of TRAIL in primary tumorigenesis from metastasis formation, likely by sensitization of previously TRAIL-resistant tumor cells upon detachment, an early step required for metastasis formation. Therefore, TRAIL-R is a novel metastasis suppressor, suggesting that TRAIL-related tumor therapy might be most effective in primary tumors and early metastatic cancers, before selection for TRAIL resistance occurs.

Dietary isoflavones act on bone marrow osteoprogenitor cells and stimulate ovary development before influencing bone mass in pre-pubertal piglets.

Food containing soybeans provide isoflavone phytoestrogens that can preserve bone mass in postmenopausal women, and prevent bone loss in ovariectomized rats. But their effects on bone remain unclear, particularly on bone formation during growth. Two groups of eight pre-pubertal piglets were fed a basal or an isoflavone-enriched (S800) diet for 6 weeks. The S800 diet contained 800 mg SoyLifetrade mark/kg, providing 2.8 mg isoflavones/kg body weight/day. Several bones were collected and tested for bone strength and density. Bone marrow was collected from humeri together with blood samples and genital tracts. The plasma concentrations of isoflavones were increased in the pigs fed S800, but growth rate, body weight, plasma bone markers, bone mineral density, and strength were all unaffected. In contrast, cultured stromal cells from S800 pigs had more alkaline phosphatase-rich cells and mineralized nodules, secreted more osteocalcin, osteoprotegerin and RANK-L, synthesized more osteoprotegerin, and RANK-L. Cultured mononucleated nonadherent bone marrow cells from S800 pigs developed fewer tartrate-resistant acid phosphatase mononucleated cells (osteoclast progenitors) when cultured with 1,25(OH)(2)D(3), and resorbed a smaller area of dentine slices. Freshly isolated bone marrow osteoclast progenitors from S800 pigs had more caspase-3 cleavage activity, and synthesized less RANK. Both osteoclast and osteoblast progenitors had ERalpha and ERbeta, whose syntheses were stimulated by the S800 diet. The S800 piglets had heavier ovaries with more follicles, but their uterus weight was unaffected. We conclude that dietary isoflavones have no detectable effect on the bone mass of growing female piglets, but act on bone marrow osteoprogenitors via ERs--mainly ERbeta, and stimulate ovary development.

Signaling networks from Gbeta1 subunit to transcription factors and actin remodeling via a membrane-located ERbeta-related protein in the rapid action of daidzein in osteoblasts.

Although estrogen replacement has been the main therapy to prevent and treat osteoporosis, there are concerns about its safety. Phytoestrogens have attracted attention to their potential impacts in osteoporosis prevention and treatment. Among phytoestrogens, the isoflavone daidzein (Dz) acts on transcription via the intracellular estrogen receptors (ER), mainly ERbeta, in osteoblasts, but mimics only part of the estrogen effects. Since estradiol also exerts rapid effects in osteoblasts, we investigated the multistep processes involved in the rapid actions of low (1-100 pM) doses of daidzein. Dz bound to a membrane moiety, related to ERbeta since the calcium response to Dz was blocked by an anti-ERbeta antibody directed against the C-terminus, but not by a double-stranded siRNA specific for ERbeta. This protein was coupled to a pertussis toxin (PTX)-sensitive Gbeta1 subunit whose transducer was PLC-beta2, which triggered a rapid (5 sec) mobilization of calcium from the endoplasmic reticulum. Dz phosphorylated within 15 sec ERK1/2 whose phosphorylation involved two routes: Gbeta1/PLC-beta2/PKC/c-Raf-1/MEK1/2 and Gbeta1/PI3K/cSrc/c-Raf-1/MEK1/2 as shown using several inhibitors. Dz induced rapid (1 min) changes in the actin cytoskeleton via the two routes. The rapid (20 sec) phosphorylation of Elk-1 and CREB by Dz involved Gbeta1 and ERK1/2. All the processes were insensitive to the estradiol antagonist ICI 182,780. In conclusion, the rapid effects of Dz seem to be biologically relevant for the function of osteoblast in bone since the isoflavone activates transcription factors linked to early genes controlling cellular proliferation and differentiation, and modulates actin cytoskeleton which controls cell adhesion, division, or secretion.

Transforming growth factor beta can mediate apoptosis via the expression of TRAIL in human hepatoma cells.

Transforming growth factor beta (TGF-beta) has been shown to induce apoptotic cell death in normal and transformed hepatocytes. However, the exact mechanism through which TGF-beta induces cell death is still unknown. We examined a potential role of various death receptor/ligand systems in TGF-beta-induced apoptosis and identified the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a mediator of TGF-beta-induced apoptosis in hepatoma cells. TGF-beta-induced apoptosis is significantly impaired upon blockage of TRAIL. We show that TRAIL is upregulated in hepatoma cells upon treatment with TGF-beta, whereas TRAIL receptor levels remain unchanged. In conclusion, our results provide evidence that the TRAIL system is critically involved in TGF-beta-induced cell death in liver pathology.

A low dose of daidzein acts as an ERbeta-selective agonist in trabecular osteoblasts of young female piglets.

The role of estrogens and estrogen-like molecules, including isoflavones, in regulating bone cell activities is essential in understanding the etiology and treatment of post-menopausal osteoporosis. Although estrogen replacement (HRT) has been the main therapy used to prevent and treat osteoporosis, there are concerns about its safety. Isoflavones have attracted attention to their potential roles in osteoporosis prevention and treatment. We have compared the effects of the isoflavone daidzein (1 nM), which has no effect on tyrosine kinases, and 17beta-estradiol (1 nM) on the development and function of cultured osteoblasts isolated from long bones of young female piglets. Daidzein increased ALP activity, osteocalcin secretion, and mineralization, while E2 increased only ALP activity. The content of ERbeta and osteoprotegerin secretion by control cells gradually increased during osteoblast differentiation, whereas the ERalpha and RANK-L content decreased. Daidzein enhanced only the nuclear ERbeta whereas estradiol increased both ERalpha and ERbeta. Daidzein and estradiol increased osteoprotegerin and RANK-L secretion. Daidzein had a more pronounced effect than did estradiol. Daidzein and estradiol increased the membrane content of RANK-L and the nuclear content of runx2/Cbfa1. Daidzein enhanced the nuclear content of progesterone and vitamin D receptors but not as much as did estradiol. All the effects of daidzein were blocked by ICI 182,780. We conclude that a low concentration of daidzein may exert its anti-resorptive action by increasing the activity of porcine mature osteoblasts via ERbeta, by regulating runx2/Cbfa1 production, and by stimulating the secretion of key proteins involved in osteoclastogenesis, such as osteoprotegerin and RANK-ligand.

Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8.

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.

TNF-related apoptosis-inducing ligand mediates tumoricidal activity of human monocytes stimulated by Newcastle disease virus.

The Newcastle disease virus (NDV) has antineoplastic and immunostimulatory properties, and it is currently clinically tested in anticancer therapy. However, the tumoricidal mechanisms of NDV tumor therapy are not fully understood. The results presented here demonstrate that NDV-stimulated human monocytes (Mphi) kill various human tumor cell lines and that this tumoricidal activity is mediated by TRAIL. In contrast to soluble TRAIL-R2-Fc, soluble CD95-Fc and TNF-R2-Fc showed only minimal blocking of the antitumor effect. TRAIL expression is induced on human Mphi after stimulation with NDV and UV-inactivated NDV. These results show that TRAIL induction on human Mphi after NDV stimulation is independent from viral replication and that TRAIL mediates the tumoricidal activity of NDV-stimulated human Mphi.