Generation of three CRISPR/Cas9 edited human induced pluripotent stem cell lines (DHMi005-A-5, DHMi005-A-6 and DHMi005-A-7) carrying a Holt-Oram Syndrome patient-specific TBX5 mutation with known cardiac phenotype and a FLAG-tag after exon 9 of the TBX5 gene.

TBX5 is a transcription factor (TF) playing essential role during cardiogenesis. It is well known that TF mutations possibly result in non- or additional binding of the DNA due to conformational changes of the protein. We introduced a Holt-Oram Syndrome (HOS) patient-specific TBX5 mutation c.920_C > A heterozygously in a healthy induced pluripotent stell cell (iPSC) line. This TBX5 mutation results in conformational changes of the protein and displayed ventricular septal defects in the patient itself. Additionally we introduced a FLAG-tag on the TBX5 mutation-carrying allele. The resulting heterozygous TBX5-FLAG iPSC lines are a powerful tool to investigate altered TF activity bonding.

Generation of two CRISPR/Cas edited human induced pluripotent stem cell lines (DHMi005-A-3 and DHMi005-A-4) carrying a FLAG-tag after exon 9 of the TBX5 gene.

Although TBX5 plays a major role during human cardiogenesis and initiates and controls limb development, many of its interactions with genomic DNA and the resulting biological consequences are not well known. Existing anti-TBX5-antibodies work very inefficiently in certain applications such as ChIP-Seq analysis. To circumvent this drawback, we introduced a FLAG-tag sequence into the TBX5 locus at the end of exon 9 prior to the stop codon by CRISPR/Cas9. The expressed TBX5-FLAG fusion protein can effectively be precipitated by anti-FLAG antibodies. Therefore, these gene-edited iPSC lines represent powerful cellular in vitro tools to unravel TBX5:DNA interactions in detail.

Establishment of an induced pluripotent stem cell line DHMi005-A from a healthy male proband.

We generated an induced pluripotent stem cell (iPSC) line from a healthy male 29-year-old proband. Adipose fibroblasts (AFs) were reprogrammed using Sendai virus. Generated iPSCs showed typical stem cell morphology. From passage 9 on, iPSCs were free of virus. Pluripotency in the iPSCs was verified and spontaneous differentiation showed expression of all three germ layers. Karyotyping indicated no anomalies for the generated iPSCs. Many patient-specific iPSCs are generated from subcutaneous fat fibroblasts obtained during surgical procedure. The described control iPSC line was generated equally and therefore serves as an ideal control for adipose-fibroblast-based patient-specific iPSC lines in disease modeling.

Establishment of a patient-specific induced pluripotent stem cell line DHMi004-A from a male Holt-Oram syndrome patient with verified TBX5 mutation.

The Holt-Oram syndrome (HOS) is a rare autosomal dominant disorder, mostly based on mutations in the TBX5 gene. Patients show malformation of at least one upper limb along with congenital heart defects. The established induced pluripotent stem cell (iPSC) line was generated from a patient displaying pronounced and typical features of HOS and carrying a single-nucleotide change c.920_C>A leading to an amino acid change from proline to threonine at amino acid position 85, which appeared de novo. Adipose fibroblasts from the patient were reprogrammed using Sendai virus. Pluripotency of the iPSCs was fully demonstrated.

Corpuls CPR Generates Higher Mean Arterial Pressure Than LUCAS II in a Pig Model of Cardiac Arrest.

According to the European Resuscitation Council guidelines, the use of mechanical chest compression devices is a reasonable alternative in situations where manual chest compression is impractical or compromises provider safety. The aim of this study is to compare the performance of a recently developed chest compression device (Corpuls CPR) with an established system (LUCAS II) in a pig model. Methods. Pigs (n = 5/group) in provoked ventricular fibrillation were left untreated for 5 minutes, after which 15 min of cardiopulmonary resuscitation was performed with chest compressions. After 15 min, defibrillation was performed every 2 min if necessary, and up to 3 doses of adrenaline were given. If there was no return of spontaneous circulation after 25 min, the experiment was terminated. Coronary perfusion pressure, carotid blood flow, end-expiratory CO2, regional oxygen saturation by near infrared spectroscopy, blood gas, and local organ perfusion with fluorescent labelled microspheres were measured at baseline and during resuscitation. Results. Animals treated with Corpuls CPR had significantly higher mean arterial pressures during resuscitation, along with a detectable trend of greater carotid blood flow and organ perfusion. Conclusion. Chest compressions with the Corpuls CPR device generated significantly higher mean arterial pressures than compressions performed with the LUCAS II device.

Pressure shift freezing as potential alternative for generation of decellularized scaffolds.

Background. Protocols using chemical reagents for scaffold decellularization can cause changes in the properties of the matrix, depending on the type of tissue and the chemical reagent. Technologies using physical techniques may be possible alternatives for the production grafts with potential superior matrix characteristics. Material and Methods. We tested four different technologies for scaffold decellularization. Group 1: high hydrostatic pressure (HHP), 1 GPa; Group 2: pressure shift freezing (PSF); Group 3: pulsed electric fields (PEF); Group 4: control group: detergent (SDS). The degree of decellularization was assessed by histological analysis and the measurement of residual DNA. Results. Tissue treated with PSF showed a decellularization with a penetration depth (PD) of 1.5 mm and residual DNA content of 24% ± 3%. HHD treatment caused a PD of 0.2 mm with a residual DNA content of 28% ± .4%. PD in PEF was 0.5 mm, and the residual DNA content was 49% ± 7%. In the SDS group, PD was found to be 5 mm, and the DNA content was determined at 5% ± 2%. Conclusion. PSF showed promising results as a possible technique for scaffold decellularization. The penetration depth of PSF has to be optimized, and the mechanical as well as the biological characteristics of decellularized grafts have to be evaluated.

[Successful individualized and targeted therapy of an NSCLC patient with Gefitinib based on a predictive assessment of the EGF-receptor mutation status]. / Erfolgreiche individualisierte zielgerichtete Gefitinib-Therapie beim NSCLC nach prädiktiver Mutationsanalyse des EGF Rezeptors.

BACKGROUND: Molecular alterations in the tyrosine kinase (TK) domain of the human epidermal growth factor receptor (EGFR) have been correlated with tumour remission upon treatment with the TK inhibitor Gefitinib in non-small cell lung cancer (NSCLC). We have retrospectively investigated the correlation of point mutations with clinical response and, based on our retrospective results, used predictive molecular assessment as the basis for treatment in one patient. METHODS: Mutational analysis was performed in 11 NSCLC-patients (10 responders, 1 non-responder) among 62 patients treated with Gefitinib within an expanded access program. RESULTS: Activating molecular alterations were found in 8/11 investigated samples (point mutations in exons 18 and 21, deletions in exon 19). All molecular changes were found in adenocarcinoma or bronchioloalveolar carcinoma. The tumours of two male responders with squamous cell carcinoma showed either a wild-type sequence or carried a nonsense mutation. In one patient treatment with Gefitinib after prospective assessment of mutations resulted in tumour remission and thus proved to be predictive. CONCLUSIONS: Mutations in the EGFR TK domain correlate with the clinical response to Gefitinib. The predictive assessment of molecular alterations may thus be helpful for treatment decisions in selected cases. A clinical response to Gefitinib is nevertheless also found in patients with wild-type EGFR.

[Non-small-cell lung cancer third-line therapy with gefitinib]. / Drittlinientherapie mit Gefitinib beim nicht-kleinzelligen Bronchialkarzinom.

The EGFR-inhibition via tyrosine-kinase-inhibitor gefitinib (Iressa) constitutes a new way to treat non-small-cell lung cancer. Recent research results enable us to better understand the basics of molecular targeted therapy (MTT). These results are helpful to re-interpret the clinical results obtained so far for gefitinib and to consider for the first time in a predictive manner factors for the selection of patients suitable for therapy. Three case reports are presented in this paper which illustrate that -- with view to the results from translational research -- the use of gefitinib offers an efficient new therapeutic modality for the treatment of chemotherapy-resistant non-small-cell lung cancer.

Peri/nuclear localization of intact insulin-like growth factor binding protein-2 and a distinct carboxyl-terminal IGFBP-2 fragment in vivo.

Insulin-like growth factor binding protein-2 (IGFBP-2) as one of the most important IGFBPs has never been assessed in the intracellular compartment in vivo. Since there is evidence for novel intracellular functions of distinct IGFBPs, we investigated the presence of IGFBP-2 inside the cell. In peri/nuclear fractions of various tissues isolated from IGFBP-2 transgenic and non-transgenic mice we were able to show the presence of intact IGFBP-2. In addition, we demonstrate the presence of a highly conserved carboxyl-terminal IGFBP-2 fragment in the peri/nuclear fraction by using different peptide-induced antibodies. In pancreatic sections, confocal microscopy revealed the presence of IGFBP-2 on the nuclear surface but not within the nucleus. Our findings suggest novel functions of intact IGFBP-2 and IGFBP-2 fragments within the cell.

Insulin-like growth factor-binding protein-4 inhibits growth of the thymus in transgenic mice.

Numerous in vitro studies have demonstrated that IGF-binding protein (IGFBP)-4 is a consistent inhibitor of IGF actions. In order to investigate the functions of IGFBP-4 in vivo, transgenic mice were generated by microinjection of a transgene, in which the murine Igfbp4 cDNA is driven by the H-2K(b) promoter, and followed by a splicing cassette and polyadenylation signal of the human beta-globin gene. Transgene mRNA was expressed ubiquitously, and elevated IGFBP-4 protein was detected in the spleen, thymus, kidney and lung of transgenic mice. The activities of serum IGFBPs were not changed in transgenic mice. Immunohistochemical studies revealed transgene expression predominantly in the thymic medulla and red pulp of the spleen. Body weight and the weights of the spleen, kidney and lung of transgenic mice were not different from controls. In contrast, the thymus of transgenic mice showed a significantly reduced weight and cortex volume. In transgenic thymus and spleen, cell proliferation was inhibited and apoptosis was stimulated. Transgenic mice showed normal T- and B-cell development and normal basal plasma immunoglobulin levels. In conclusion, overexpression of IGFBP-4 inhibits growth of the thymus. IGFBP-4 excess inhibits cell proliferation and stimulates apoptosis in lymphoid tissues, but does not affect lymphocyte development. These findings suggest that IGFBP-4 is a potential growth inhibitor of lymphoid tissues.

IGF-binding protein-4: biochemical characteristics and functional consequences.

IGFs have multiple functions regarding cellular growth, survival and differentiation under different physiological and pathological conditions. IGF effects are modulated systemically and locally by six high-affinity IGF-binding proteins (IGFBP-1 to -6). Despite their structural similarity, each IGFBP has unique properties and exhibits specific functions. IGFBP-4, the smallest IGFBP, exists in both non-glycosylated and N-glycosylated forms in all biological fluids. It is expressed by a wide range of cell types and tIssues, and its expression is regulated by different mechanisms in a cell type-specific manner. IGFBP-4 binds IGF-I and IGF-II with similar affinities and inhibits their actions under almost all in vitro and in vivo conditions. In this review, we summarize the available data regarding the following aspects of IGFBP-4: genomic organization, protein structure-function relationship, expression and its regulation, as well as IGF-dependent and -independent actions. The biological significance of IGFBP-4 for reproductive physiology, bone formation, renal pathophysiology and cancer is discussed.

Increased activity of catalase in tumor cells overexpressing IGFBP-2.

Elevated levels of IGFBP-2 are found in serum and tissues under various stressful conditions and in many malignancies. In previous studies, we have shown that overexpression of IGFBP-2 results in increased tumorigenic potential in Y-1 mouse adrenocortical tumor cells, and that these effects are presumably mediated through IGF-independent mechanisms. Here, we show that highly proliferative IGFBP-2-overexpressing Y-1 cells, but not control Y-1 cells, grow to very high cell densities. In order to evaluate whether the increased cell densities in IGFBP-2-transfected Y-1 cells were accompanied by alterations in the oxidative stress system, we analyzed the effect of IGFBP-2 overexpression on the activity of various antioxidative enzymes in two malignant cell lines. Among the tested antioxidative enzymes (catalase, superoxide-dismutase, glutathione peroxidase, glutathione S-transferase), only catalase enzyme activity was significantly higher in IGFBP-2-transfected Y-1 mouse adrenocortical tumor cells and in IGFBP-2-transfected human colon tumor cells (Caco-2) compared to control-transfected Y-1 and Caco-2 cells and non-tumor 293 human epithelial cells. However, overexpression of catalase in malignant cells did not result in increased resistance to oxidative stress as measured by cell viability and protein oxidation after treatment of the cells with hydrogen peroxide. This might be due to an upregulation of the GST enzyme activity after treatment with H (2)O (2) that we observed selectively in the control-transfected Y-1 cells and which might compensate for the higher catalase activity in the IGFBP-2 overexpressing cells. In summary, we found a strong and selective upregulation of the catalase activity in IGFBP-2 overexpressing malignant Y-1 and Caco-2 cell lines that might contribute to the highly malignant phenotype of IGFBP-2 overexpressing tumors through as yet unknown mechanisms.

Prognostic significance of cytokine modulation in non-small cell lung cancer.

Increased production of immunosuppressive interleukin-10 (IL-10) by non-small cell lung cancer (NSCLC) and increased serum IL-10 concentrations in NSCLC-patients have recently been correlated to reduced survival. We earlier demonstrated suppression of IL-2 secretion in whole blood cell cultures of NSCLC-patients. We now analyzed the influence of IL-2 secretion on survival in NSCLC-patients and the influence of IL-10 on IL-2 secretion. The correlation of the IL-2 producing ability of whole blood cells in response to PHA in 90 NSCLC-patients at the time of diagnosis to survival was calculated by Crit-level, the Kaplan-Meier method and the log-rank test. With a cut-off value of IL-2 production of 1,100 pg/ml by whole blood cells the difference in survival was significant with a p-value of 0.014. In the group with high and low IL-2, median survival was 14.1 and 9.7 months, respectively. In the subgroup of 33 surgically-treated patients the difference in survival was significant with a p-value of 0.011. In 14 patients with surgical resection of the tumor and high IL-2 at diagnosis and 19 patients with surgical resection, but low IL-2 at diagnosis, median survival was 86.2 and 11.3 months, respectively. Secretion of IL-2 in whole blood cell cultures from healthy individuals was inhibited in a dose-dependent manner upon addition of IL-10. Taken together, suppression of IL-2 secretion has prognostic significance for survival of NSCLC-patients and may be mediated by tumor-derived IL-10.

Mutation of the RGD sequence does not affect plasma membrane association and growth inhibitory effects of elevated IGFBP-2 in vivo.

Using insulin-like growth factor-binding protein-2 (IGFBP-2) transgenic mice (D mice) as a model of elevated IGFBP-2 expression, which is often found in unphysiological conditions, we found association of IGFBP-2 to purified plasma membranes of many organs. To determine whether the RGD (Arg-Gly-Asp) motif of IGFBP-2 mediates cell surface binding in vivo, we mutated the RGD motif of IGFBP-2 into an RGE (Arg-Gly-Glu) sequence and produced transgenic mice (E mice) which express elevated amounts of mutated IGFBP-2. Our data demonstrate that in vivo IGFBP-2 cell surface association is not dependent on the RGD motif and that mutation of this sequence does not alter growth inhibitory effects of IGFBP-2.

IGF-binding protein-5: flexible player in the IGF system and effector on its own.

The multiple activities of IGF-I and -II are modulated by a family of IGF-binding proteins (IGFBP-1 to -6). Although structurally related, each IGFBP has unique properties and exerts specific functions. IGFBP-5 is the most conserved IGFBP across species and was identified as an essential regulator of physiological processes in bone, kidney and mammary gland. In addition, IGFBP-5 appears to play a decisive role in the control of proliferation of specific tumour cell types. In many situations IGFBP5 exerts biological activities in the absence of IGFs, indicating the existence of IGF-independent actions. This concept was supported by the unexpected localisation of IGFBP-5 in the nucleus and the description of IGFBP-5-specific membrane-bound IGFBP-5 receptor(s). The scope of this review is to summarise the available information about the structure of IGFBP-5 and the regulation of its expression. Furthermore, the potential significance of IGFBP-5 in the regulation of physiological processes will be critically analysed in the light of recent experimental data.

Galectin-8 expression decreases in cancer compared with normal and dysplastic human colon tissue and acts significantly on human colon cancer cell migration as a suppressor.

BACKGROUND AND AIMS: Galectins are beta-galactoside binding proteins. This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells. In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features. This evident gap has prompted us to extend galectin analysis beyond the two prototypes. The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts. The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines. METHODS: Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with alpha- and beta-D-galactose, alpha-D-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy. The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction. In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy. In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy. RESULTS: A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue. Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3-4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1-2/N0/M0). The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo. Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate. In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo. CONCLUSIONS: Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts. In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin. Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus on galectins-1 and -3.

Cytokine secretion: clinical relevance of immunosuppression in non-small cell lung cancer.

Increased production of immunosuppressive IL-10 by non-small cell lung cancer (NSCLC) and increased plasma IL-10 concentrations in NSCLC-patients have recently been correlated to reduced survival. We earlier demonstrated suppression of IL-2 secretion in NSCLC-patients. We now analyzed the influence of IL-2 suppression on survival in NSCLC-patients and influence of IL-10 on IL-2 secretion. The correlation of the IL-2-concentration in whole blood cell cultures from 90 NSCLC-patients at the time of diagnosis to survival was analyzed by using crit-level, the Kaplan-Meier method and the log-rank test. IL-2 secretion capacity at the time of diagnosis significantly influenced survival in NSCLC-patients. With a cut-off value for IL-2 of 1100 pg/ml, the difference in survival was significant with a P-value of 0.014 in the whole patient group. In the subgroup of surgically treated patients (n=33), survival was different with a P-value of 0.011. Moreover, secretion of IL-2 was inhibited in a dose-dependent manner upon addition of IL-10 in whole blood cell cultures from normal individuals. Thus, suppression of IL-2 secretion is predictive for survival of NSCLC-patients and may be mediated by tumor-derived IL-10.

Insulin-like growth factor-binding protein-5 inhibits growth and induces differentiation of mouse osteosarcoma cells.

The precise role of insulin-like growth factor-binding protein-5 (IGFBP-5) in regulating the growth of tumor cells, especially of bone-derived malignant cells, is not well understood. We have investigated the biological activity of IGFBP-5 by transfecting OS/50-K8 mouse osteosarcoma cells with an expression vector containing the osteocalcin promoter and the complete mouse IGFBP-5 cDNA (OC-IGFBP-5). Overexpression of IGFBP-5 mRNA and secretion of increased amounts of bioactive protein in conditioned media were demonstrated in different clones. For the analysis of cell proliferation, three clones exhibiting high levels of IGFBP-5 expression were selected and compared to a mock clone and to nontransfected parental cells. IGFBP-5-secreting clones displayed reduced proliferation under both anchorage-dependent and -independent conditions (P < 0.05). The increase in proliferation observed in IGFBP-5-secreting clones after addition of exogenous IGF was significantly lower than that observed in mock-transfected or parental cells. A similar result was obtained with long[R3]IGF-I which has a low affinity for all IGFBPs, suggesting that the inhibitory effect of IGFBP-5 is only partially IGF-dependent. OC-IGFBP-5-transfected clones expressed significantly higher amounts of osteocalcin mRNA (P < 0.05) and secreted more osteocalcin protein than a mock clone or parental OS-50/K8 cells. Thus, part of the growth-inhibiting effect of IGFBP-5 may be due to an induction of differentiation in these cells.