RLL5, an F-box-containing protein, involved in preventing transgene silencing and in maintaining global DNA methylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) and ROS1-dependent active DNA demethylation pathways are antagonistic processes that dynamically regulate site-specific methylation. In this study, we obtained a mutant with reduced luciferase (LUC) luminescence by genetic screening, which was named rll5-1 (for reduced LUC luminescence 5-1). The rll5-1 mutant showed narrower, frizzled and curly leaves, and the low-LUC-luminescence phenotype in the rll5-1 mutant can be largely restored by DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Map-based cloning coupled with genome resequencing data revealed that a nucleotide substitution of G to A was found at the 124th bp of ORF of At4G10190, leading to an aspartate-to-asparagine change at position 42 in such a protein. Bisulfite sequencing data indicated that DNA methylation of 3' region of the double 35S promoter that drives the LUC expression was appreciably increased. Further analysis revealed that there were 4747 hypo-DMRs and 936 hyper-DMRs found in the rll5-1 genome, and the hypo-DMRs was predominantly distributed on TEs, which appeared to stem from the downregulation of a few RdDM pathway genes and DNA methyltransferase genes. Closer inspection demonstrated that there were 1229 hypo-DMRs commonly shared among rll5-1, nrpd1-3 and nrpe1-11, and a total of 1349 hypo-DMRs were common to rll5-1 and cmt2 mutants. Thus, these studies demonstrate the roles of RLL5 in preventing transgene silencing and in maintaining genome-wide DNA methylation in a direct/indirect or locus-specific manner.

c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1.

The c-Jun N-terminal protein kinases (JNKs) JNK1 and JNK2 can act as either tumor suppressors or pro-oncogenic kinases in human cancers. The isoform-specific roles for JNK1 and JNK2 in human pancreatic cancer are still unclear, the question which should be addressed in this project. Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 clones were established either expressing either JNK1 or -2 shRNA in a stable manner. Basal anchorage-dependent and -independent cell growth, single-cell movement, and invasion using the Boyden chamber assay were analyzed. Xenograft growth was assessed using an orthotopic mouse model. All seven tested pancreatic cancer cell lines expressed JNKs as did human pancreatic cancer samples determined by immunohistochemistry. Pharmacological, unspecific JNK inhibition (SP600125) reduced cell growth of all cell lines but PANC-1. Especially inhibition of JNK2 resulted in overall increased oncogenic potential with increased proliferation and invasion, associated with alterations in cytoskeleton structure. Specific inhibition of JNK1 revealed opposing functions. Overall, JNK1 and JNK2 can exert different functions in human pancreatic cancer and act as counter players for tumor invasion. Specifically modulating the activity of JNKs may be of potential therapeutic interest in the future.

Opposing Oncogenic Functions of p38 Mitogen-activated Protein Kinase Alpha and Beta in Human Pancreatic Cancer Cells.

BACKGROUND/AIM: The p38 family of mitogen-activated protein kinases (MAPK) includes four isoforms: p38α, -ß, -γ and -δ. The aim of this study was to elucidate possible functions of p38α and p38ß in human pancreatic cancer. MATERIALS AND METHODS: Isoform expression was determined in seven human pancreatic cancer cell lines. After shRNA based selective knockdown of p38α and p38ß, in vitro growth and migration as well as in vivo tumorigenicity were assessed. RESULTS: All pancreatic cancer cells expressed p38 isoforms. Knockdown of p38α and p38ß inhibited in vitro growth. Migration was markedly reduced in p38α shRNA expressing clones, but not altered by p38ß knockdown. While in vivo inhibition of p38ß decreased tumor formation and growth, the knockdown of p38α significantly enhanced tumorigenicity. CONCLUSION: p38 MAPKs may exert isoform specific functions in pancreatic cancer. Selective targeting may contribute to individualized treatment of pancreatic cancer in the future.

Inhibition of PI3K signalling increases the efficiency of radiotherapy in glioblastoma cells.

Glioblastoma, the most common primary brain tumour, is also considered one of the most lethal cancers per se. It is highly refractory to therapeutic intervention, as highlighted by the mean patient survival of only 15 months, despite an aggressive treatment approach, consisting of maximal safe surgical resection, followed by radio- and chemotherapy. Radiotherapy, in particular, can have effects on the surviving fractions of tumour cells, which are considered adverse to the desired clinical outcome: It can induce increased cellular proliferation, as well as enhanced invasion. In this study, we established that differentiated glioblastoma cells alter their DNA repair response following repeated exposure to radiation and, therefore, high single-dose irradiation (SD-IR) is not a good surrogate marker for fractionated dose irradiation (FD-IR), as used in clinical practice. Integrating irradiation into a combination therapy approach, we then investigated whether the pharmacological inhibition of PI3K signalling, the most abundantly activated survival cascade in glioblastoma, enhances the efficacy of radiotherapy. Of note, treatment with GDC-0941, which blocks PI3K-mediated signalling, did not enhance cell death upon irradiation, but both treatment modalities functioned synergistically to reduce the total cell number. Furthermore, GDC-0941 not only prevented the radiation-induced increase in the motility of the differentiated cells, but further reduced their speed below that of untreated cells. Therefore, combining radiotherapy with the pharmacological inhibition of PI3K signalling is a potentially promising approach for the treatment of glioblastoma, as it can reduce the unwanted effects on the surviving fraction of tumour cells.

Radiation and Brain Tumors: An Overview.

The use of radiation is an essential part of both modern cancer diagnostic assessment and treatment. Next-generation imaging devices create 3D visualizations, allowing for better diagnoses and improved planning of precision treatment. This is particularly important for primary brain cancers such as diffuse intrinsic pontine glioma or the most common primary brain tumor, glioblastoma, because radiotherapy is often the only treatment modality that offers a significant improvement in survival and quality of life. In this review, we give an overview of the different imaging techniques and the historic role of radiotherapy and its place in modern cancer therapy. Finally, we discuss three key areas of risks associated with the use of ionizing radiation: (1) brain tumor induction mainly as a consequence of the diagnostic use of radiation; (2) cognitive decline as a consequence of treating childhood brain tumors as an example of long term consequences often neglected in favor of highlighting secondary primary cancers; and (3) pro-proliferative and pro-invasive alterations that occur in tumor cells that survive radiotherapy. Throughout the discussion, we highlight areas of potential future research.

Blocking distinct interactions between Glioblastoma cells and their tissue microenvironment: A novel multi-targeted therapeutic approach.

Due to the highly invasive nature of Glioblastoma (GB), complete surgical resection is not feasible, while motile tumour cells are often associated with several specific brain structures that enhance treatment-resistance. Here, we investigate the therapeutic potential of Disulfiram and Carbenoxolone, that inhibit two distinct interactions between GB and the brain tissue microenvironment: stress-induced cell-matrix adhesion and gap junction mediated cell-cell communication, respectively. Increase in cell numbers of tumour-initiating cells, which are cultured in suspension as cell clusters, and adherent differentiated cells can be blocked to a similar extent by Carbenoxolone, as both cell populations form gap junctions, but the adherent differentiated cells are much more sensitive to Disulfiram treatment, which - via modulation of NF-κB signalling - interferes with cell-substrate adhesion. Interestingly, inducing adhesion in tumour-initiating cells without differentiating them does not sensitize for Disulfiram. Importantly, combining Disulfiram, Carbenoxolone and the standard chemotherapeutic drug Temozolomide reduces tumour size in an orthotopic mouse model. Isolating GB cells from their direct environment within the brain represents an important addition to current therapeutic approaches. The blockage of cellular interactions via the clinically relevant substances Disulfiram and Carbenoxolone, has distinct effects on different cell populations within a tumour, potentially reducing motility and/or resistance to apoptosis.

The effects of PI3K-mediated signalling on glioblastoma cell behaviour.

The PI3K/Akt/mTOR signalling network is activated in almost 90% of all glioblastoma, the most common primary brain tumour, which is almost invariably lethal within 15 months of diagnosis. Despite intensive research, modulation of this signalling cascade has so far yielded little therapeutic benefit, suggesting that the role of the PI3K network as a pro-survival factor in glioblastoma and therefore a potential target in combination therapy should be re-evaluated. Therefore, we used two distinct pharmacological inhibitors that block signalling at different points of the cascade, namely, GDC-0941 (Pictilisib), a direct inhibitor of the near apical PI3K, and Rapamycin which blocks the side arm of the network that is regulated by mTOR complex 1. While both substances, at concentrations where they inhibit their primary target, have similar effects on proliferation and sensitisation for temozolomide-induced apoptosis, GDC-0941 appears to have a stronger effect on cellular motility than Rapamycin. In vivo GDC-0941 effectively retards growth of orthotopic transplanted human tumours in murine brains and significantly prolongs mouse survival. However, when looking at genetically identical cell populations that are in alternative states of differentiation, i.e. stem cell-like cells and their differentiated progeny, a more complex picture regarding the PI3K/Akt/mTOR pathway emerges. The pathway is differently regulated in the alternative cell populations and, while it contributes to the increased chemo-resistance of stem cell-like cells compared to differentiated cells, it only contributes to the motility of the latter. Our findings are the first to suggest that within a glioblastoma tumour the PI3K network can have distinct, cell-specific functions. These have to be carefully considered when incorporating inhibition of PI3K-mediated signals into complex combination therapies.

Circulating adipokine levels and prognostic value in septic patients.

BACKGROUND: Adipokines have a wide range of effects and are linked to sepsis and septic shock. The aim of the present study was to describe the changes in adipokine levels in septic patients in relation to patients' preseptic adipokine levels. Furthermore, we examined adipokines as prognostic markers. METHODS: Fourteen consecutive critically ill patients meeting the clinical criteria for severe sepsis or septic shock 3 days up to 1 month after major visceral surgery were enrolled prospectively. Plasma adipokines were measured preoperatively, 1 and 4 days after diagnosis of severe sepsis or septic shock following elective surgery. RESULTS: Median plasma adiponectin levels were lowered and resistin and leptin levels elevated in sepsis compared with preseptic plasma levels. MCP-1, C-reactive protein and white blood cell count were higher in septic compared with preseptic patients. Survivors had significantly higher preseptic adipokine levels than non-survivors. Adiponectin levels of survivors decreased significant (on average by 33 %) at day one after onset of sepsis compared with preseptic levels. In contrast, median adiponectin levels of patients dying during sepsis showed a slight increase (11 %). Median BMI of survivors was 30 kg/m(2), median BMI of non-survivors was 25, respectively. CONCLUSIONS: Adipokine levels change during the course of sepsis. Higher preseptic adiponectin levels and decreasing adiponectin levels after onset of sepsis are associated with survival of sepsis. Survival of overweight and obese patients was higher than in normal weight patients. Changes in adiponektin levels could be a prognostic marker for outcome of severe sepsis/septic shock following surgery.

Sanger Sequencing for BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples.

Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.

Interaction of titanium, zirconia and lithium disilicate with peri-implant soft tissue: study protocol for a randomized controlled trial.

BACKGROUND: Against the background of increasing use of dental implants, and thus an increasing prevalence of implant-associated complications, a deeper understanding of the biomolecular mechanisms in the peri-implant tissue is needed. Peri-implant soft tissue is in direct contact with transmucosal dental implant abutments. The aim of this trial is to distinguish the biomolecular and histological interactions of various dental abutment materials with peri-implant soft tissue. METHODS/DESIGN: The study is designed as a prospective, randomized, investigator-initiated clinical pilot trial with blinded assessment. We will ultimately include 24 eligible patients who opt for implant treatment to replace a single missing posterior tooth. Three months after implantation (submerged procedure), the study begins with the second-stage surgery. Each of the 24 patients will be given three different transmucosal abutments (zirconia, lithium disilicate, titanium) consecutively. The sequence in which the three materials are used is randomized. Peri-implant crevicular fluid is sampled weekly around the respective abutment for biomolecular analyses. After one month of wearing time, the stamping press from the second-stage surgery is used to gain a narrow gingival ring biopsy around the abutment for immunohistochemical analyses. The next abutment is then inserted. The same procedure is used for all three abutments. After sampling is completed, the patients will receive a definitive crown. The primary outcome measure of the trial is biomolecular detection of specific markers in the peri-implant crevicular fluid: matrix metalloproteinase 8, interleukin- 1ß, polymorphonuclear elastase, and myeloid-related protein MRP8/14 (calprotectin). Secondary outcome measures include immunohistochemical analyses and clinical parameters. DISCUSSION: The study design will allow us to perform correlation analyses between the clinical indices with biomarkers' expression in the interface of the transmucosal abutments and the peri-implant soft tissue. A deeper understanding of the three abutment materials' interactions with peri-implant soft tissue will help us understand the formation mechanisms of implant-associated complications and then develop prevention strategies. TRIAL REGISTRATION: The trial is registered at the German Clinical Trial Register and the International Clinical Trials Registry Platform by the WHO under DRKS00006555 (Registered on 27 October 2014).

A Potential Role for the Inhibition of PI3K Signaling in Glioblastoma Therapy.

Glioblastoma multiforme (GBM) is the most common primary brain tumor and among the most difficult to treat malignancies per se. In almost 90% of all GBM alterations in the PI3K/Akt/mTOR have been found, making this survival cascade a promising therapeutic target, particular for combination therapy that combines an apoptosis sensitizer, such as a pharmacological inhibitor of PI3K, with an apoptosis inducer, such as radio- or chemotherapy. However, while in vitro data focusing mainly on established cell lines has appeared rather promising, this has not translated well to a clinical setting. In this study, we analyze the effects of the dual kinase inhibitor PI-103, which blocks PI3K and mTOR activity, on three matched pairs of GBM stem cells/differentiated cells. While blocking PI3K-mediated signaling has a profound effect on cellular proliferation, in contrast to data presented on two GBM cell lines (A172 and U87) PI-103 actually counteracts the effect of chemotherapy. While we found no indications for a potential role of the PI3K signaling cascade in differentiation, we saw a clear and strong contribution to cellular motility and, by extension, invasion. While blocking PI3K-mediated signaling concurrently with application of chemotherapy does not appear to be a valid treatment option, pharmacological inhibitors, such as PI-103, nevertheless have an important place in future therapeutic approaches.

Role of alveolar macrophages in the regulation of local and systemic inflammation after lung contusion.

BACKGROUND: Blunt chest trauma is an injury that enhances the morbidity and mortality rate, particularly in the context of polytrauma. Our previous studies showed local and systemic inflammatory alterations after blunt chest trauma in mice. This study was designed to determine whether alveolar macrophages (AMΦ) have an alleviative role in this posttraumatic inflammation. METHODS: AMΦ of male C3H/HeN mice were depleted by instillation of clodronate liposomes into the lung before blunt chest trauma induced by a single blast wave. In bronchoalveolar lavage, lung homogenates, plasma, and cell culture supernatants of Kupffer cells, peripheral blood mononuclear cells, splenic macrophages, and splenocytes isolated 2 hours or 24 hours after chest trauma mediator concentrations were determined by multiplex assay or enzyme-linked immunosorbent assay. RESULTS: In bronchoalveolar lavage, AMΦ depletion led to increased monocyte chemoattractant protein 1 and regulated and normal T cell expressed and secreted (RANTES) concentrations as well as an attenuated increase of interleukin 6 concentrations after chest trauma. Bronchoalveolar lavage keratinocyte-derived chemokine concentrations increased in nontraumatized but AMΦ-depleted animals with no further change after chest trauma. Cytokine concentrations in lung homogenates were altered in the same way as in bronchoalveolar lavage early after trauma. In the plasma of AMΦ-depleted animals, interleukin 6 concentrations were slightly decreased after chest trauma. Depletion of AMΦ abrogated the trauma-induced decrease of Kupffer cell chemokine release. Cytokine concentrations of blood monocytes, splenic macrophages, and splenocyte supernatants were not influenced by AMΦ depletion. CONCLUSION: These depletion experiments show that AMΦ ameliorate the inflammatory response after blunt chest trauma. Taken together, this study gives relevant insights into the regulative role of AMΦ during the local and systemic inflammation after lung contusion.

Inhibition of NF-κB signaling ablates the invasive phenotype of glioblastoma.

UNLABELLED: Glioblastoma multiforme, the most common primary brain tumor, is highly refractory to therapy, mainly due to its ability to form micrometastases, which are small clusters or individual cells that rapidly transverse the brain and make full surgical resection impossible. Here, it is demonstrated that the invasive phenotype of glioblastoma multiforme is orchestrated by the transcription factor NF-κB which, via metalloproteinases (MMP), regulates fibronectin processing. Both, cell lines and tumor stem cells from primary glioblastoma multiforme, secrete high levels of fibronectin which when cleaved by MMPs forms an extracellular substrate. Subsequently, forming and interacting with their own microenvironment, glioblastoma multiforme cells are licensed to invade their surroundings. Mechanistic study revealed that NF-κB inhibition, either genetically or pharmacologically, by treatment with Disulfiram, significantly abolished the invasive phenotype in the chick chorioallantoic membrane assay. Furthermore, having delineated the underlying molecular mechanism of glioblastoma multiforme invasion, the potential of a disulfiram-based therapy was revealed in a highly invasive orthotrophic glioblastoma multiforme mouse model. IMPLICATIONS: This study defines a novel therapeutic approach that inhibits micrometastases invasion and reverts lethal glioblastoma into a less aggressive disease.

Combined inhibition of HER1/EGFR and RAC1 results in a synergistic antiproliferative effect on established and primary cultured human glioblastoma cells.

Glioblastoma is the most frequent brain tumor of glial origin in adults. With the best available standard-of-care, patients with this disease have a life expectancy of only approximately 15 months after diagnosis. Because the EGF receptor (HER1/EGFR) is one of the most commonly dysregulated oncogenes in glioblastoma, HER1/EGFR-targeted agents, such as erlotinib, were expected to provide a therapeutic benefit. However, their application in the clinical setting failed. Seeking an explanation for this finding, we previously identified several candidate genes for resistance of human glioblastoma cell lines toward erlotinib. On the basis of this panel of genes, we aimed at identifying drugs that synergistically enhance the antiproliferative effect of erlotinib on established and primary glioblastoma cell lines. We found that NSC23766, an inhibitor of RAC1, enhanced the antineoplastic effects of erlotinib in U87MG, T98MG, and A172MG glioblastoma cell lines for the most part in a synergistic or at least in an additive manner. In addition, the synergistic antiproliferative effect of erlotinib and NSC23766 was confirmed in primary cultured cells, indicating a common underlying cellular and molecular mechanism in glioblastoma. Therefore, agents that suppress RAC1 activation may be useful therapeutic partners for erlotinib in a combined targeted treatment of glioblastoma.

A novel low temperature PCR assured high-fidelity DNA amplification.

As previously reported, a novel low temperature (LoTemp) polymerase chain reaction (PCR) catalyzed by a moderately heat-resistant (MHR) DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94-96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV) type 52 (HPV-52) as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'-5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.

Human cytomegalovirus infection of M1 and M2 macrophages triggers inflammation and autologous T-cell proliferation.

Macrophages (MΦ) are first targets during human cytomegalovirus (HCMV) infection and are thought to be crucial for viral persistence and dissemination. However, since MΦ are also a first line of defense and key modulators of the immune response, these cells are at the crossroad between protection and viral pathogenesis. To date, the MΦ-specific contribution to the immune response against HCMV is still poorly understood. In view of the opposite roles of M1 and M2 MΦ during initiation and resolution of the immune response, we characterized the effects of HCMV infection on classically activated M1 MΦ and alternatively activated M2 MΦ. Although HCMV susceptibility was higher in M2 MΦ, HCMV established a productive and persistent infection in both types of MΦ. Upon HCMV encounter, both types of MΦ acquired similar features of classical activation and secreted high levels of proinflammatory cytokines and chemokines. As a functional consequence, conditioned media obtained from HCMV-infected M1 and M2 MΦ potently activated freshly isolated monocytes. Finally, compared to HCMV-infected monocyte-derived dendritic cells, infected M1 and M2 MΦ were more efficient in stimulating proliferation of autologous T cells from HCMV-seropositive donors at early times (24 h) postinfection, while the MΦ immunostimulatory properties were reduced, but not abrogated, at later times (72 h postinfection). In summary, our findings indicate that MΦ preserve proper antigen presentation capacity upon HCMV infection while enhancing inflammation, thus suggesting that MΦ play a role in the maintenance of the large HCMV-specific T-cell repertoire in seropositive individuals.

Interactions of pancreatic cancer and stellate cells are mediated by FGFR1-III isoform expression.

BACKGROUND/AIMS: Human pancreatic cancer is characterised by an extensive desmoplastic reaction. Activation of pancreatic stellate cells (PSCs) and their interactions with pancreatic cancer cells seem to be of essential importance in this process. Expression of fibroblast growth factor (FGF) receptor (FGFR) 1 splice variants may be of special interest in this communication as they are known to modulate the malignant phenotype of pancreatic cancer. The aim of the present study was to characterize interactions between PSCs and pancreatic cancer cells focusing on the Ig-domain III variants of fibroblast growth factor (FGF) receptor (FGFR) 1. METHODOLOGY: Expression of FGF ligands and FGFR1-III isoforms was determined by immunoblotting and specific RT-PCR analysis, respectively. RESULTS: PSCs and COLO-357, MIA PaCa-2, and PANC-1 pancreatic cancer cells expressed and secreted FGF2 and FGF5. Both FGFR1-III isoforms were coexpressed in PSCs and cancer cells. Conditioned medium of COLO-357 cells induced expression of both FGFR1- III isoforms in PSCs. In contrast, conditioned medium of PSCs induced FGFR1-IIIc, but reduced FGFR1- IIIb expression in the cancer cells. Neutralizing the effects of FGFs by heparin-sepharose precipitation abolished these effects completely. FGF2 and other growth factors secreted by PSCs resulted in upregulation of FGFR1-IIIc and downregulation of FGFR1-IIIb in pancreatic cancer cells. CONCLUSIONS: We identified in this study a mechanism based on tumor-stroma interactions involving PSCs that can contribute to enhance the malignant phenotype of human pancreatic cancer.

Inhaled hydrogen sulfide induces suspended animation, but does not alter the inflammatory response after blunt chest trauma.

The treatment of acute lung injury and septic complications after blunt chest trauma remains a challenge. Inhaled hydrogen sulfide (H2S) may cause a hibernation-like metabolic state, which refers to an attenuated systemic inflammatory response. Therefore, we tested the hypothesis that inhaled H2S-induced suspended animation may attenuate the inflammation after pulmonary contusion. Male Sprague-Dawley rats were subjected to blunt chest trauma (blast wave) or sham procedure and subsequently exposed to a continuous flow of H2S (100 ppm) or control gas for 6 h. Body temperature and activity were measured by an implanted transmitter. At 6, 24, or 48 h after trauma, animals were killed, and the cellular contents of bronchoalveolar lavage (BAL) as well as cytokine concentrations in BAL, plasma, and culture supernatants of blood mononuclear cells, Kupffer cells, splenic macrophages, and splenocytes were determined. Hydrogen sulfide inhalation caused a significant reduction in body temperature and activity. The trauma-induced increase in alveolar macrophage counts was abrogated 48 h after trauma when animals received H2S, whereas the trauma-induced increase in neutrophil counts was unaltered. Furthermore, H2S inhalation partially attenuated the mediator release in BAL and culture supernatants of Kupffer cells as well as splenic cells; it altered plasma cytokine concentrations but did not affect the trauma-induced changes in mononuclear cell culture supernatants. These findings indicate that inhaled H2S induced a reduced metabolic expenditure and partially attenuated inflammation after trauma. Nevertheless, in contrast to hypoxic- or pathogen-induced lung injury, H2S treatment appears to have no protective effect after blunt chest trauma.

Alveolar macrophage phagocytosis is enhanced after blunt chest trauma and alters the posttraumatic mediator release.

Blunt chest trauma is known to induce a pulmonary invasion of short-lived polymorphonuclear neutrophils and apoptosis of alveolar epithelial type 2 (AT2) cells. Apoptotic cells are removed by alveolar macrophages (AMΦ). We hypothesized that chest trauma alters the phagocytic response of AMΦ as well as the mediator release of AMΦ during phagocytosis. To study this, male Sprague-Dawley rats were subjected to blunt chest trauma. Phagocytosis assays were performed in AMΦ isolated 2 or 24 h after trauma with apoptotic cells or opsonized beads. Phagocytosis of apoptotic AT2 cells by unstimulated AMΦ was significantly increased 2 h after trauma. At 24 h, AMΦ from traumatized animals, stimulated with phorbol-12-myristate-13-acetate, ingested significantly more apoptotic polymorphonuclear neutrophils than AMΦ from sham animals. Alveolar macrophages after trauma released significantly higher levels of tumor necrosis factor α, macrophage inflammatory protein 1α, and cytokine-induced neutrophil chemoattractant 1 when they incorporated latex beads, but significantly lower levels of interleukin 1ß and macrophage inflammatory protein 1α when they ingested apoptotic cells. In vivo, phagocytosis of intratracheally instilled latex beads was decreased in traumatized rats. The bronchoalveolar lavage concentrations of the phagocytosis-supporting surfactant proteins A and D after blunt chest trauma were slightly decreased, whereas surfactant protein D mRNA expression in AT2 cells was significantly increased after 2 h. These findings indicate that chest trauma augments the phagocytosis of apoptotic cells by AMΦ. Phagocytosis of opsonized beads enhances and ingestion of apoptotic cells downregulates the immunologic response following lung contusion. Our data emphasize the important role of phagocytosis during posttraumatic inflammation after lung contusion.

Identification of c-FLIP(L) and c-FLIP(S) as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells.

BACKGROUND: Evasion of apoptosis is a hallmark of pancreatic cancer. However, the underlying mechanisms are still only partly understood and may involve antiapoptotic proteins such as c-FLIP. Here, the role of c-FLIP in the regulation of death receptor-mediated apoptosis in pancreatic cancer was investigated. METHODS: Expression of c-FLIP(L) and c-FLIP(S) was analysed in primary pancreatic carcinoma samples, pancreatic carcinoma cell lines and primary tumour cells together with its function as a regulator of death receptor-induced apoptosis by knockdown and overexpression studies and through modulation by chemotherapeutics. RESULTS: c-FLIP is expressed in pancreatic intraepithelial neoplasm (PanIN) lesions and in pancreatic ductal adenocarcinomas, whereas normal pancreatic ducts were consistently negative for c-FLIP. Simultaneous downregulation of c-FLIP(L) and c-FLIP(S) as well as individual knockdown of either isoform by RNA interference significantly enhances TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)- and CD95-induced caspase activation and caspase-dependent apoptosis. Also, pretreatment with chemotherapeutic drugs--that is, 5-fluorouracil (5-FU), cisplatin or gemcitabine--downregulates c-FLIP and renders cells sensitive to death receptor-triggered apoptosis. Similarly, primary cultured pancreatic cancer cells are primed for TRAIL-induced apoptosis by pre-exposure to 5-FU or cisplatin. Mechanistic studies revealed that 5-FU-mediated suppression of c-FLIP results in increased TRAIL-induced recruitment and activation of caspase-8 at the death-inducing signalling complex (DISC), leading to caspase-3 activation and caspase-dependent cell death. Overexpression of c-FLIP(L) rescues cells from 5-FU- or cisplatin-mediated sensitisation for TRAIL-induced apoptosis, indicating that c-FLIP suppression is a key event in this chemotherapy-mediated sensitisation to TRAIL. Further, concomitant neutralisation of c-FLIP and XIAP acts in concert to potentiate TRAIL-induced apoptosis. CONCLUSIONS: Both the long and the short isoform of the antiapoptotic protein c-FLIP are critical regulators of death receptor-induced apoptosis in pancreatic carcinoma cells and are suppressed by chemotherapeutics. Targeting either c-FLIP(L) or c-FLIP(S) is sufficient to promote death receptor-induced apoptosis in pancreatic carcinoma cells. These findings have important implications for the design of TRAIL-based combination protocols in pancreatic cancer.