Fred Faendrich

Regulation of Biglycan Gene Expression by Transforming Growth Factor-β Requires MKK6-p38 Mitogen-activated Protein Kinase Signaling Downstream of Smad Signaling

Several signaling pathways have been implicated in mediating TGF-β1-induced extracellular matrix production and fibrosis. We have shown recently that induction of biglycan (BGN) expression by TGF-β1 depended on a functional Smad pathway (Chen, W.-B., Lenschow, W., Tiede, K., Fischer, J. W., Kalthoff, H., and Ungefroren, H. (2002) J. Biol. Chem.277, 36118–36128). Here, we present evidence that the ability of TGF-β1 to induce BGN mRNA, in addition to Smads, requires p38 MAPK signaling, because 1) pharmacological inhibitors of p38 dose-dependently inhibited the TGF-β effect without significantly affecting the transcriptional activity of a constitutively active mutant of the TGF-β type I receptor or Smad2 phosphorylation at concentrations up to 10 μm, 2) the up-regulation of BGN mRNA was preceded by a delayed increase in the phosphorylation of p38 and its upstream activator MKK6 in TGF-β1-treated PANC-1 cells, 3) inhibition of the p38 pathway by stable retroviral transduction with a dominant negative mutant of either p38 or MKK6 reduced TGF-β1-induced BGN mRNA expression, and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented the TGF-β1 effect on BGN mRNA. We further demonstrate that the (delayed) p38 activation by TGF-β1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-β-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-β1 induction of p38 activation and, as shown earlier, BGN mRNA expression; finally, re-expression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-β-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation. These results clearly show that TGF-β induction of BGN expression in pancreatic cells requires activation of MKK6-p38 MAPK signaling downstream of Smad signaling and provide a mechanistic clue to the up-regulation of BGN seen in inflammatory response-related fibrosis and desmoplasia.

Wortmannin inhibits growth of human non-small-cell lung cancer in vitro and in vivo

Abstract Background and aims. Recently we demonstrated that phosphatidylinositol 3-kinase (PI3K) is overexpressed in human lung cancer. This study evaluated whether the PI3K inhibiting agent wortmannin affects proliferation of human lung cancer cells in vitro and in vivo. Methods. Effects of exposure of human non-small-cell lung cancer (NSCLC) cells (KNS-62, Colo-699) to wortmannin were investigated in vitro by proliferation, cytotoxicity, and DNA fragmentation assays. In vivo we examined the effects of blocking PI3K by wortmannin prior to xenotransplantation of human NSCLC cells into SCID-bg mice and the effect of systemic wortmannin administration following intrapulmonary xenotransplantation of human NSCLC. Results. Exposure of KNS-62 and Colo-699 lung cancer cells to wortmannin inhibited proliferation in correlation to concentration in vitro. In vivo the blocking of PI3K by wortmannin prior to xenotransplantation caused a significant delay in the growth of subcutaneously induced tumors. Systemic wortmannin administration increased mean survival after intrapulmonary xenotransplantation of human NSCLC significantly by 38% and 47%. Conclusions. These data suggest inhibition of PI3K activity as a potential target for treatment of human NSCLC. Systemic toxicity of wortmannin requires development of improved PI3K inhibitors with favorable pharmacological properties.

WOFIE stimulates regulatory T cells: a 2-year follow-up of renal transplant recipients.

Background. Initial interruption of immunosuppression for 72 hr was analyzed in renal transplant recipients according to Calne et al.s “window of opportunity for immunologic engagement” (WOFIE) concept. Methods. This pilot study was designed as a randomized, open-label, prospective trial of 40 recipients (20 in the WOFIE group, 20 in the control group) of cadaveric kidney transplants who were followed up for 2 years. Immunosuppression comprised tacrolimus (trough levels 5–8 ng/mL), daclizumab (1 mg per kilogram of body weight on day 0 and after 2, 4, 6, and 8 weeks), mycophenolate mofetil (1–2 g/day), and prednisolone (maintenance dose of 10 mg/day). After induction with daclizumab, prednisolone, and mycophenolate mofetil, immunosuppression was interrupted for 72 hr in the WOFIE group. Steroid withdrawal followed in both groups within 12 to 16 weeks posttransplant. Results. Patient and graft survival did not differ significantly between the two cohorts. However, the WOFIE group experienced less acute rejection episodes and developed better graft function. Although all but one of the patients in the WOFIE group successfully discontinued steroid treatment, permanent steroid withdrawal was achieved in only 76.4% of the control group. After daclizumab discontinuation, the WOFIE group demonstrated an increase of CD4+CD25+ T cells in peripheral blood (P<0.05 vs. control group), which was stable over time and strongly correlated with a significantly higher expression level of Foxp3-mRNA. Conclusions. Initial interruption of immunosuppression for 72 hr correlates with the induction of regulatory immunologic mechanisms and allows early and reliable minimization of immunosuppressive treatment.

Inhibition of Interleukin-6-Transsignaling via gp130-Fc in Hemorrhagic Shock and Sepsis

BACKGROUND Immune function after hemorrhagic shock and subsequent sepsis is characterized by an early proinflammatory burst of IL-6, and high IL-6 levels have been linked to high mortality after trauma and in sepsis. Trans-signaling is defined as the activation of cells that do not express the membrane bound IL-6 receptor by the complex of IL-6 and the soluble IL-6 receptor (sIL-6R). Gp130-Fc is able to bind the IL-6/sIL-6R complex, and beneficial effects of IL-6 blockade in chronic inflammatory diseases have been shown. The first aim of this study was to investigate the potential effect of a gp130 blockade via the gp130-Fc antibody causing impairment of IL-6 signaling. The second aim was to find out what role the IL-6/sIL-6R complex can play in the context of hemorrhagic shock and subsequent sepsis as an acute inflammatory disease. MATERIAL AND METHODS Male CBA/J mice were subjected to hemorrhagic shock (35+/-5 mmHg for 90min and fluid resuscitation) or sham operation. At resuscitation each animal received either 0.5mg gp130-Fc or placebo (PL) i.p. At 48 h after resuscitation, both splenocytes and peritoneal macrophages (pMphi) were harvested or polymicrobial sepsis was induced by cecal ligation and puncture. Survival over 10 d was determined. Release of IL-6, TNF-alpha, and IL-10 of pMphi and release of IL-2, IL-10, and IFN-gamma of splenocytes was assessed by ELISA. Proliferation of splenocytes and their morphologic damage were determined. RESULTS Binding of the IL-6/sIL-6R complex by gp130-Fc led to significant lower IL-6 levels compared with placebo treated animals. Placebo treated males showed depressed proinflammatory immune response (IL-2, IL-6) after hemorrhagic shock. While splenocyte proliferation was significantly reduced directly after hemorrhagic shock and restored after 48 h by gp130-Fc, pMphi cytokine release was not influenced. Finally, survival appeared to be unaffected. CONCLUSION Transsignaling does not seem to play a pivotal role in the development of the immune dysfunction and mortality in our model of hemorrhage and subsequent sepsis.

Small intestine transplantation today

IntroductionIntestinal transplantation has become a life-saving therapy in patients with irreversible loss of intestinal function and complications of total parenteral nutrition.DiscussionThe patient and graft survival rates have improved over the last years, especially after the introduction of tacrolimus and rapamycin. However, intestinal transplantation is more challenging than other types of solid organ transplantation due to its large amount of immune competent cells and its colonization with microorganisms. Moreover, intestinal transplantation is still a low volume procedure with a small number of transplanted patients especially in Germany. A current matter of concern is the late referral of intestinal transplant candidates.ConclusionThus, patients often present after onset of life-threatening complications or advanced cholestatic liver disease. Earlier timing of referral for candidacy might result in further improvement of this technique in the near future.

Investigation of the Lith6 candidate genes APOBEC1 and PPARG in human gallstone disease

Background: Genetic susceptibility contributes to the aetiology of gallbladder diseases as shown by multiple epidemiological studies. A major gallstone susceptibility locus (Lith6) was identified in 2003 by quantitative trait locus mapping in mice. Two attractive positional and functional candidate genes in apolipoprotein B mRNA‐editing protein (APOBEC1) and peroxisome proliferator‐activated receptor γ (PPARG) are located in this interval.

Feasibility and Early Results of Interstitial Intensity-Modulated HDR/PDR Brachytherapy (IMBT) with/without Complementary External-Beam Radiotherapy and Extended Surgery in Recurrent Pelvic Colorectal Cancer

Background:A new multimodality treatment concept consisting of extended resection and postoperative fractionated intensity-modulated interstitial brachytherapy (IMBT) was introduced for pelvic recurrence of colorectal carcinoma.Patients and Methods:46 patients received extended resection and single plastic tubes were sutured directly onto the tumor bed. IMBT was started within 2 weeks postoperatively with a median dose of 24.5 Gy (5–35 Gy). Patients were treated either with high-dose-rate brachytherapy (HDR; n = 23) or with pulsed-dose-rate brachytherapy (PDR; n = 23). 25 patients received complementary 45-Gy external-beam irradiation (EBRT) to the pelvic region after explanting the plastic tubes.Results:Median follow-up was 20.6 months (7–107 months) and mean patient survival 25.7 ± 25.8 months (median 17, range 1–107 months). After 5 years overall survival, disease-free survival and local control rate were 23%, 20% and 33%, significantly influenced by the resectional state. There was a trend in favor of PDR compared to HDR, which reached statistical significance in patients who had not received additional EBRT.Conclusion:The combination of extended surgery and postoperative interstitial IMBT is feasible and offers effective interdisciplinary treatment of recurrent colorectal cancer. In this small and inhomogeneous cohort of patients PDR seems to be more effective than HDR, particularly when application of complementary EBRT is not possible. None of the patients who required resection of distant metastasis survived > 2 years in this study.Hintergrund:Zur Behandlung von Beckenrezidiven bei kolorektalen Karzinomen wurde die erweiterte chirurgische Resektion mit anschließender postoperativer fraktionierter intensitätsmodulierter interstitieller Brachytherapie (IMBT) als neuartige interdisziplinäre Behandlungsform entwickelt und in einer nicht randomisierten Beobachtungsstudie untersucht.Patienten und Methodik:46 Patienten erhielten eine erweiterte chirurgische Resektion im kleinen Becken mit direkter Aufnähung von Brachytherapieapplikatoren auf das clipmarkierte Tumorbett. Die Brachytherapie wurde postoperativ innerhalb von 2 Wochen mit einer mittleren Dosis von 24,5 Gy (5–35 Gy) begonnen. Die Patienten wurden entweder mit High-Dose-Rate-(HDR-)Brachytherapie (n = 23) oder mit Pulsed-Dose-Rate-(PDR-)Brachytherapie (n = 23) behandelt. Bei 25 Patienten wurde eine zusätzliche perkutane Bestrahlung des Beckens bis 45 Gy durchgeführt.Ergebnisse:Der mediane Nachbeobachtungszeitraum betrug 20,6 Monate (7–107 Monate) und das mittlere Überleben 25,7 ± 25,8 Monate (median 17, Bereich 1–107 Monate). Nach 5 Jahren lagen das Gesamtüberleben bei 23%, das krankheitsspezifische Überleben bei 20% und die lokale Tumorkontrolle bei 33%; sie wurden signifikant vom Resektionsstatus beeinflusst. Es zeigte sich ein Trend zugunsten der PDR-Brachytherapie im Vergleich zur HDR-Brachytherapie; dieser Trend war statistisch signifikant für Patienten, die keine zusätzliche perkutane Strahlentherapie erhielten.Schlussfolgerung:Die Kombination aus erweiterter chirurgischer Resektion und postoperativer interstitieller IMBT bietet eine wirksame Behandlungsmöglichkeit zur Therapie von Beckenrezidiven kolorektaler Karzinome. Hierbei scheint in diesem kleinen und sehr inhomogenen Patientengut die PDR-Therapie effektiver als die HDR-Therapie zu sein, insbesondere wenn keine zusätzliche perkutane Radiatio möglich ist. In dieser Studie überlebte keiner der Patienten, bei denen eine Resektion von Fernmetastasen erforderlich war, > 2 Jahre.

M2033 Blockade of TNF Alpha Reduces Pancreatic Tumor Growth and Metastasis in a Xenotransplantation Model

Inflammation and inflammatory cytokines accompany most solid cancers including pancreatic ductal adenocarcinoma (PDAC). In the present work we investigated the role of the major proinflammatory cytokine TNFalpha for the malignancy of PDAC cells In Vitro and In Vivo. In Vitro, TNFalpha strongly increased invasiveness of Colo357-, BxPc3and PancTuIcells while the anti-proliferative effect showed to be moderate. In Vivo, TNF alpha provoked a severe enhanced tumor growth and metastasis in mice bearing orthotopically growing PDAC. Inhibition of TNFalpha with infliximab or etanercept affected proliferation and invasiveness of PDAC cells In Vitro and revealed a strong anti-tumoral effects in SCID mice with Colo357-, BxPc3or PancTuI-tumors. Furthermore, after surgical resection of the PDAC tumors, we determined an even stronger therapeutic effect for both anti-TNF compounds. Infliximab-treatment reduced the volume of recurrent tumor by 70% and etanercept by almost 60%. Furthermore, both drugs strongly reduced the number of liver metastases. Thus, tumor cell-derived TNFalpha plays a profound role in malignancy of PDAC and inhibition of TNFalpha represents a promising therapeutic option particularly in adjuvant therapy after subtotal pancreatectomy.