Hypoxia directed migration of human naïve monocytes is associated with an attenuation of cytokine release: indications for a key role of CCL26.

BACKGROUND: Numerous tissue-derived factors have been postulated to be involved in tissue migration of circulating monocytes. The aim of this study was to evaluate whether a defined hypoxic gradient can induce directed migration of naïve human monocytes and to identify responsible autocrine/paracrine factors. METHODS: Monocytes were isolated from peripheral blood mononuclear cells, transferred into chemotaxis chambers and subjected to a defined oxygen gradient with or without the addition of CCL26. Cell migration was recorded and secretome analyses were performed. RESULTS: Cell migration recordings revealed directed migration of monocytes towards the source of hypoxia. Analysis of the monocyte secretome demonstrated a reduced secretion of 70% (19/27) of the analyzed cytokines under hypoxic conditions. The most down-regulated factors were CCL26 (- 99%), CCL1 (- 95%), CX3CL1 (- 95%), CCL17 (- 85%) and XCL1 (- 83%). Administration of recombinant CCL26 abolished the hypoxia-induced directed migration of human monocytes, while the addition of CCL26 under normoxic conditions resulted in a repulsion of monocytes from the source of CCL26. CONCLUSIONS: Hypoxia induces directed migration of human monocytes in-vitro. Autocrine/paracrine released CCL26 is involved in the hypoxia-mediated monocyte migration and may represent a target molecule for the modulation of monocyte migration in-vivo.

Human monocytes subjected to ischaemia/reperfusion inhibit angiogenesis and wound healing in vitro.

OBJECTIVES: The sequence of initial tissue ischaemia and consecutive blood flow restoration leads to ischaemia/reperfusion (I/R) injury, which is typically characterized by a specific inflammatory response. Migrating monocytes seem to mediate the immune response in ischaemic tissues and influence detrimental as well as regenerative effects during I/R injury. MATERIALS AND METHODS: To clarify the role of classical monocytes in I/R injury, isolated human monocytes were subjected to I/R in vitro (3 hours ischaemia followed by 24 hours of reperfusion). Cellular resilience, monocyte differentiation, cytokine secretion, as well as influence on endothelial tube formation, migration and cell recovery were investigated. RESULTS: We show that I/R supported an enhanced resilience of monocytes and induced intracellular phosphorylation of the prosurvival molecules Erk1/2 and Akt. FACS analysis showed no major alteration in monocyte subtype differentiation and surface marker expression under I/R. Further, our experiments revealed that I/R changes the cytokine secretion pattern, release of angiogenesis associated proteins and MMP-9 activity in supernatants of monocytes exposed to I/R. Supernatants from monocytes subjected to I/R attenuated endothelial tube formation as indicator for angiogenesis as well as endothelial cell migration and recovery. CONCLUSION: In summary, monocytes showed no significant change in cellular integrity and monocyte subtype after I/R. Functionally, monocytes might have a rather detrimental influence during the initial phase of I/R, suppressing endothelial cell migration and neoangiogenesis.

Allogeneic transplantation of programmable cells of monocytic origin (PCMO) improves angiogenesis and tissue recovery in critical limb ischemia (CLI): a translational approach.

BACKROUND: Employing growth factor-induced partial reprogramming in vitro, peripheral human blood monocytes can acquire a state of plasticity along with expression of various markers of pluripotency. These so-called programmable cells of monocytic origin (PCMO) hold great promise in regenerative therapies. The aim of this translational study was to explore and exploit the functional properties of PCMO for allogeneic cell transplantation therapy in critical limb ischemia (CLI). METHODS: Using our previously described differentiation protocol, murine and human monocytes were differentiated into PCMO. We examined paracrine secretion of pro-angiogenic and tissue recovery-associated proteins under hypoxia and induction of angiogenesis by PCMO in vitro. Allogeneic cell transplantation of PCMO was performed in a hind limb ischemia mouse model in comparison to cell transplantation of native monocytes and a placebo group. Moreover, we analyzed retrospectively four healing attempts with PCMO in patients with peripheral artery disease (PAD; Rutherford classification, stage 5 and 6). Statistical analysis was performed by using one-way ANOVA, Tukey's test or the Student's t test, p < 0.05. RESULTS: Cell culture experiments revealed good resilience of PCMO under hypoxia, enhanced paracrine release of pro-angiogenic and tissue recovery-associated proteins and induction of angiogenesis in vitro by PCMO. Animal experiments demonstrated significantly enhanced SO2 saturation, blood flow, neoangiogenesis and tissue recovery after treatment with PCMO compared to treatment with native monocytes and placebo. Finally, first therapeutic application of PCMO in humans demonstrated increased vascular collaterals and improved wound healing in patients with chronic CLI without exaggerated immune response, malignant processes or extended infection after 12 months. In all patients minor and/or major amputations of the lower extremity could be avoided. CONCLUSIONS: In summary, PCMO improve angiogenesis and tissue recovery in chronic ischemic muscle and first clinical results promise to provide an effective and safe treatment of CLI.

Generation of monocyte-derived insulin-producing cells from non-human primates according to an optimized protocol for the generation of PCMO-derived insulin-producing cells.

OBJECTIVE: The vision of potential autologous cell therapy for the cure of diabetes encourages ongoing research. According to a previously published protocol for the generation of insulin-producing cells from human monocytes, we analyzed whether the addition of growth factors could increase insulin production. This protocol was then transferred to a non-human primate model by using either blood- or spleen-derived monocytes. METHODS: Human monocytes were treated to dedifferentiate into programmable cells of monocytic origin (PCMO). In addition to the published protocol, PCMOs were then treated with either activin A, betacellulin, exendin 3 or 4. Cells were characterized by protein expression of insulin, Pdx-1, C-peptide and Glut-2. After identifying the optimal protocol, monocytes from baboon blood were isolated and the procedure was repeated. Spleen monocytes following splenectomy of a live baboon were differentiated and analyzed in the same manner and calculated in number and volume. RESULTS: Insulin content of human cells was highest when cells were treated with activin A and their insulin content was 13,000 µU/1 million cells. Insulin-producing cells form primate monocytes could successfully be generated despite using human growth factors and serum. Expression of insulin, Pdx-1, C-peptide and Glut-2 was comparable to that of human neo-islets. Total insulin content of activin A-treated baboon monocytes was 16,000 µU/1 million cells. CONCLUSION: We were able to show that insulin-producing cells can be generated from baboon monocytes with human growth factors. The amount generated from one spleen could be enough to cure a baboon from experimentally induced diabetes in an autologous cell transplant setting.

Imaging of acute heart-transplant rejection using 99m-Technetium labelled oligonucleotides against interleukin-2 mRNA in rats.

OBJECTIVES: Today, acute cardiac rejection is detected by endomyocardial biopsy, which harbours many risks. Thus, there is a necessity for less invasive methods. Since interleukin-2 (IL2) is over-expressed in acute graft rejection, we use radioactive DNA-fragments complementary to the mRNA of IL2 to detect graft rejection scintigraphically. METHODS: In a rat model of acute graft rejection, the oligonucleotide sequence complementary to the mRNA of IL2 is labelled with 99m-Technetium and injected intravenously. Scintigraphic and Geiger-counter activity of the transplants are evaluated and correlated with the current rejection classification of the International Society for Heart and Lung Transplantation (ISHLT). RESULTS: From the fourth postoperative day onwards, the scintigraphic images show a significant increase of radioactivity (p<0.05) in the rejected organs than in the accepted grafts. While scintigraphy is not significantly correlated with the standard rejections classification of the ISHLT, there is significant correlation between the ISHLT classification and radioactivity in the Geiger-counter analysis. CONCLUSIONS: Radioactively labelled anti-sense-oligonucleotides against mRNA of IL2 may be a promising approach for the detection of acute transplant rejection in vivo.

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.

Impact of the site of anastomosis after oncologic esophagectomy on quality of life--a prospective, longitudinal outcome study.

BACKGROUND: For patients undergoing oncologic surgery, the quality of life (QoL) is generally accepted as an important outcome parameter in addition to long-term survival, mortality, and complication rates. Our study focused on outcome in terms of QoL in patients with esophageal cancer, comparing the sites of anastomosis (cervical versus thoracic anastomosis). METHODS: In a prospective longitudinal single-center study from 1998 to 2005, 105 patients underwent surgery for esophageal cancer. To assess QoL the EORTC-QLQ-C-30 and a tumor-specific module were administered before surgery, at discharge, and three, six, 12, and 24 months after surgery. Clinical data were collected prospectively and follow-up was performed every six months. RESULTS: The histological type was squamous cell carcinoma in 51.4% of the cases, adenocarcinoma in 41.9%, and some other type in 6.7%. There was no significant difference between cervical and thoracic anastomosis with regard to morbidity, mortality, and survival rates (30% five-year survival rate), whereas tumor stage was a significant (p < 0.001) prognostic factor. Most QoL scores dropped significantly below baseline in the early postoperative period. Even though they recovered slowly during the follow-up period, they never reached preoperative levels again. There was no statistically significant difference in any of the QoL scales between patients with a cervical or a thoracic anastomosis. CONCLUSIONS: Esophageal resections are associated with significant deterioration of QoL, which persists during the follow-up period. The surgical technique and position of the esophagogastrostomy did not affect QoL deterioration.

Influence of sex and age on morphological organ damage after hemorrhagic shock.

Immune function after hemorrhagic shock (shock) and subsequent sepsis is proofed to be sex- and age-related, showing an enhanced immune function and better survival of young females and a deteriorating immune response in advanced age. However, it remains unclear if the observed sex- and age-related effects observed on the immune function mirror the histomorphological changes of the affected organs. To scrutinize a possible association, male and female CBA/J mice (young, 2-3 months; aged 18-19 months) were subjected to shock (35 + 5 mmHg for 90 min and fluid resuscitation) or sham operation. At 48 h after shock, histological specimen at definite sites were harvested (lung, small bowel, liver, and kidney) and immediately stored in 10% formalin. After paraffin embedding, hematoxylin-eosin stain and immunohistochemical stains (vascular cell adhesion molecule 1 [VCAM-1], cluster of differentiation 44 [CD44], signal transducers and activators of transcription 3 [STAT-3]) were performed. In both sexes, aged animals developed significantly increased (P < 0.05) tissue damage in all analyzed organs compared with young mice. Sex differences were noticed in the lungs of young mice, showing a significantly (P < 0.05) lower organ damage score in female animals. Sex-related differences were found for VCAM-1 and cluster of differentiation 44 expression, whereas age-related changes were observed for STAT-3. These results demonstrate that the severity of tissue damage caused by hemorrhagic shock is influenced by sex- and age-related effects. Variances in the VCAM-1 and STAT-3 expression suggest that improved immune function in female and young subjects may be responsible for less shock-induced tissue damage.

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 gamma (PPARG) are located in this interval. AIMS: To investigate APOBEC1 and PPARG as candidate genes for common symptomatic gallstone disease in humans. PATIENTS AND METHODS: Eight hundred and ten patients who underwent cholecystectomy for symptomatic gallstone disease (median age of onset 50) were compared with 718 sex-matched control individuals. An independent additional sample included 368 gallstone patients and 368 controls. Control individuals were sonographically free of gallstones. Haplotype tagging and all known coding single nucleotide polymorphisms were genotyped for PPARG (N=32) and APOBEC1 (N=11). RESULTS: The investigated high-risk patient sample provides a power of greater than 80% for the detection of odds ratios down to 1.45. No evidence of association of the two genes in the single-point tagging markers, coding variants and in the sliding window haplotype analysis was detected (all nominal single point P-values >0.04). A logistic regression analysis including age, sex and BMI as covariates was also negative (nominal P-values > or =0.08). CONCLUSIONS: In the investigated German samples, no evidence of association of APOBEC1 and PPARG with gallstone susceptibility was detected. Systematic fine mapping of the complete Lith6 region is required to identify the causative genetic variants for gallstone in mice and humans.

Small bowel cancer: single-centre results over a period of 12 years.

BACKGROUND/AIMS: Tumors of the small bowel are rare, accounting for about 3-6% of all gastrointestinal neoplasms. However, diagnosis and treatment are difficult and an ongoing challenge. METHODOLOGY: Follow-up and clinical data of 43 patients with small bowel cancer who underwent surgery at our hospital. RESULTS: Subgroups consisted of adenocarcinoma (n=16; 37.2%), neuroendocrine tumors (n=12; 27.9%), gastrointestinal stroma tumor (GIST) (n=10; 23.3%), lymphoma (n=3; 7%) and desmoid tumor (n=2; 4.6%). Tumor localizations were within duodenum (46.5%), jejunum (16.3%) and ileum (37.2%). Thirty patients were curatively operated, 13 for palliative treatment or diagnostic purpose. Adenocarcinoma patients showed preponderance of advanced tumor stages: stage I/II in 5 pts, III/IV in 11 patients. Stage distribution for patients with neuroendocrine tumors was 3 each for I and II and 6 for III. Localization was predominantly within the ileum (n=7). Overall survival after five/ten years was 48/37%. Patients with neuroendocrine tumors showed best survival results (75/57%), GIST patients 60/35% and adenocarcinoma (27% each). There was a strong trend towards better survival at early tumor stages in patients with adenocarcinoma and neuroendocrine tumors. CONCLUSIONS: Early diagnosis is essential for prognosis of small bowel malignancies. Cure is unlikely if lymph node or distant metastases have already developed.

Age- and gender-related differences of the immune function in a murine model of hemorrhagic shock: IL-10 restores immunodepression in aged females without reduction of mortality.

INTRODUCTION: Interleukin-10 (IL-10) treatment has been shown to have beneficial effects on the immune function after hemorrhagic shock and to improve survival after subsequent sepsis in young male mice, but not in young females. Although it was demonstrated that the immune function under these conditions is reversed with age, it remains unclear whether the observed gender-related effect of IL-10 treatment continues to exist in aged mice. MATERIALS AND METHODS: Aged male and female CBA/J mice (18-19 months) were subjected to hemorrhage (35 +/- 5 mmHg for 90 min) or sham operation. At resuscitation, each received either 10-microg recombinant murine (rm)IL-10 or placebo i.p. At 48 h after resuscitation, either the mice were killed and the plasma, splenic macrophages (sM phi), and splenocytes were harvested or polymicrobial sepsis was induced by cecal ligation and puncture (CLP). After CLP, either survival over 10 days was determined or, 4 h after CLP, tissues were again harvested and cytokine-released in vitro were assessed by enzyme-linked immunosorbent assay. RESULTS: Early IL-10 treatment restored depressed proinflammatory immune response (TNF-alpha, IL-1 beta) and Th1 response of splenocytes in aged females after hemorrhage, whereas having no effects or having suppressive effects in aged males. Subsequent sepsis combined with placebo treatment led to a significant suppression of proinflammatory cytokine release of sM phi and a significant increase of Th2 response in both males and females associated with high mortality (80-100%, respectively) after CLP. These effects were not influenced by early rmIL-10 treatment. CONCLUSION: After hemorrhage, early rmIL-10 treatment restored immune function in aged females, but not in males. However, in contrast to young mice, rmIL-10 treatment had no effect on survival and immune function after CLP in aged mice.

Small intestine transplantation today.

INTRODUCTION: Intestinal transplantation has become a life-saving therapy in patients with irreversible loss of intestinal function and complications of total parenteral nutrition. DISCUSSION: The 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. CONCLUSION: Thus, 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.

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 CD4CD25 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.

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.

WOFIE synergizes with calcineurin-inhibitor treatment and early steroid withdrawal in kidney transplantation.

BACKGROUND: According to the window of opportunity for immunologic engagement (WOFIE) concept, as designed by R. Calne, the authors prospectively analyzed the effect of a 72-hr immunosuppressive window on graft function in renal transplant recipients. METHODS: Immunosuppressive therapy comprised tacrolimus (trough levels, 5-8 ng/mL after day 8 posttransplantation), daclizumab (1 mg/kg body weight at day 0 and 2, 4, 6, and 8 weeks posttransplantation), mycophenolate mofetil (MMF) (1-2 g/day), and prednisolone. All patients received an induction therapy including daclizumab, prednisolone, and MMF. WOFIE patients were stopped from immunosuppression for 72 hr posttransplant. Steroids were withdrawn in both groups 12 to 16 weeks after transplantation and dual therapy with MMF and low-dose tacrolimus ensued. RESULTS: Thirty renal transplant recipients (16 in the WOFIE group and 14 in the control group) have been enrolled since May 2000. Patient and graft survival were 93.8% and 87.5%, respectively, in the WOFIE group and 100.0% and 92.9% in the control group, respectively. One patient in the WOFIE group died of cytomegalovirus infection with stable graft function. There were no grafts lost because of acute rejection or primary nonfunction in either group. Patients treated according to the WOFIE protocol revealed less acute rejection episodes during the time of observation (first biopsy-confirmed acute rejection rate 12.5% in the WOFIE group vs. 42.9% in the control cohort). Whereas 92.1% of the WOFIE patients were successfully discontinued from steroids, permanent steroid withdrawal was achieved in only 60% of the control cohort. CONCLUSIONS: Initial interruption of immunosuppression was associated with a decrease of acute graft rejections. Subsequently, the authors postulate a synergistic effect on the immunosuppressive efficiency of calcineurin inhibitors when combined with an initial drug-free window.

Regulation of biglycan gene expression by transforming growth factor-beta requires MKK6-p38 mitogen-activated protein Kinase signaling downstream of Smad signaling.

Several signaling pathways have been implicated in mediating TGF-beta1-induced extracellular matrix production and fibrosis. We have shown recently that induction of biglycan (BGN) expression by TGF-beta1 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-beta 1 to induce BGN mRNA, in addition to Smads, requires p38 MAPK signaling, because 1) pharmacological inhibitors of p38 dose-dependently inhibited the TGF-beta effect without significantly affecting the transcriptional activity of a constitutively active mutant of the TGF-beta type I receptor or Smad2 phosphorylation at concentrations up to 10 microm, 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-beta 1-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-beta 1-induced BGN mRNA expression, and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented the TGF-beta 1 effect on BGN mRNA. We further demonstrate that the (delayed) p38 activation by TGF-beta 1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-beta-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-beta1 induction of p38 activation and, as shown earlier, BGN mRNA expression; finally, re-expression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-beta-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation. These results clearly show that TGF-beta 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.

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.