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.

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.

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.

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.

Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.

The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy.

Use of prior vaccinations for the development of new vaccines.

There is currently a need for vaccine development to improve the immunogenicity of protective epitopes, which themselves are often poorly immunogenic. Although the immunogenicity of these epitopes can be enhanced by linking them to highly immunogenic carriers, such carriers derived from current vaccines have not proven to be generally effective. One reason may be related to epitope-specific suppression, in which prior vaccination with a protein can inhibit the antibody response to new epitopes linked to the protein. To circumvent such inhibition, a peptide from tetanus toxoid was identified that, when linked to a B cell epitope and injected into tetanus toxoid-primed recipients, retained sequences for carrier but not suppressor function. The antibody response to the B cell epitope was enhanced. This may be a general method for taking advantage of previous vaccinations in the development of new vaccines.

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.

Secretion of bioactive granulocyte-macrophage colony-stimulating factor by human colorectal carcinoma cells.

Secretion of several cytokines by colorectal carcinoma cells has been substantiated. These do not include granulocyte-macrophage colony-stimulating factor (GM-CSF) thus far. We show that the supernatant of two human colorectal carcinoma cell lines, LS1034 and SW480, stimulates proliferation of GM-CSF-dependent M07e cells. The activity was constitutively secreted by LS1034 cells and could be induced by serum-free culture conditions in SW480 cells. Addition of a neutralizing anti-GM-CSF antibody completely inhibited this activity. Preabsorption with anti-GM-CSF antibody removed all M07e growth-stimulating activity from LS1034 and SW480 supernatant. Western blot analysis revealed the presence of GM-CSF in LS1034 supernatant. Our results indicate that human colorectal carcinoma cells secrete indeed biologically active GM-CSF.

Overexpression of insulin-like growth factor-binding protein-2 results in increased tumorigenic potential in Y-1 adrenocortical tumor cells.

Increased concentrations of insulin-like growth factor-binding protein-2 (IGFBP-2) have been observed in human malignancies including adrenocortical carcinomas. To elucidate the functional consequences of IGFBP-2 overexpression, we have stably transfected the cDNA of murine IGFBP-2 in mouse adrenocortical tumor cells (Y-1). Long-term overexpression of IGFBP-2 was associated with significant morphological alterations, enhanced cell proliferation, and increased cloning efficiency as compared with mock transfected control cells. The enhanced proliferation of IGFBP-2 secreting clones was independent of exogenous insulin-like growth factors (IGFs). These data suggest that elevated levels of IGFBP-2 may contribute to the highly malignant phenotype of adrenocortical cancer by a thus far unknown, presumably IGF-independent, mechanism.

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver.

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.

Mapping of the p56lck-mediated phosphorylation of GAP and analysis of its influence on p21ras-GTPase activity in vitro.

The protein tyrosine kinase p56lck and other members of the src family can transduce signals from activated cell-surface receptors. As we showed earlier the GTPase-activating protein (GAP), a regulator of p21ras, is a substrate of p56lck. Here, tryptic peptides of p56lck-phosphorylated GAP were generated and analyzed by two-dimensional thin layer chromatography and mass spectroscopy. Results revealed that p56lck phosphorylates GAP specifically on Tyr-460 in vitro and in vivo. The effect of tyrosine phosphorylation of GAP on its GTPase-activating activity versus p21ras was then tested using a p21ras-dependent GTPase assay system. Our results demonstrate that p56lck-mediated tyrosine phosphorylation of GAP is not sufficient to change directly its effect on the intrinsic GTPase activity of p21ras.

Lack of interleukin-2 (IL-2) dependent growth of TAC positive T-ALL/NHL cells is due to the expression of only low affinity receptors for IL-2.

Binding of interleukin-2 (IL-2) to high affinity receptors on activated normal T cells was shown to be the essential step in induction of proliferation of such cells. The finding of abundant IL-2 receptors on malignant T cells in adult T cell leukemia suggested a deregulation of the IL-2/IL-2 receptor system and was assumed to account for aberrant growth in malignant disorders of T cells. In this study we use malignant T cells from nine patients with the clinical diagnosis of T-ALL or T-NHL and did not detect IL-2 dependent growth under conditions in which normal T cells responded to IL-2. IL-2 receptors comparable in numbers to activated T cells were found on T-ALL/T-NHL cells stimulated with PHA and PMA. However, binding studies using radiolabeled IL-2 indicated that the receptors present on malignant T cells were not able to bind to IL-2 with high affinity. Therefore, if IL-2 is involved in the proliferation of malignant T cells, its mechanism of growth regulation may be different from the one for normal T cells. Alternatively, IL-2 may not play a role in the regulation of growth of malignant T cells in vitro.

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text]

Growth inhibition in giant growth hormone transgenic mice by overexpression of insulin-like growth factor-binding protein-2.

To clarify the role of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) in postnatal growth regulation, we crossed hemizygous CMV-IGFBP-2 transgenic mice with hemizygous PEPCK-bGH transgenic mice, which are characterized by serum GH levels in the range of 2 microgram/ml. Four genetic groups were obtained: animals carrying both transgenes (GB), the GH (G) or the IGFBP-2 transgene (B), and nontransgenic controls (C). Male offspring were analyzed for serum levels of IGF-I, for serum and tissue levels of IGFBP-2, and for body and organ growth. Serum IGF-I levels were 2- to 3-fold increased (P < 0.001) in the GH-overexpressing groups, with no difference between G and GB mice. Serum IGFBP-2 levels were 4- to 9-fold (P < 0.001) increased both in B and GB vs. C and G mice. Western immunoblot analysis did not reveal differences in tissue IGFBP-2 levels between B and GB mice. IGFBP-2 levels were highest in pancreas, followed by skeletal muscle, heart, kidney, brain, skin, and spleen. No elevation of IGFBP-2 was found in the liver. Body weight gain of G and GB mice was significantly increased vs. C and B mice, resulting in almost 2-fold increased body weights at the age of 15 weeks. However, there was a significant reduction in body weight of GB vs. G mice (17%; P < 0.001) and of B vs. C mice (13%; P < 0.05). This was primarily caused by a marked reduction of carcass weight (GB vs. G, 27%; B vs. C, 21%; P < 0.001). Measurements of nose-rump-length, organ (brain, heart, spleen, liver, pancreas, kidney), and tissue weights (skin, carcass, abdominal fat) in 5- and 15-week-old mice revealed several indications that the growth-inhibiting effect of IGFBP-2 overexpression was more marked in high-GH/IGF-I mice: 1) At 5 weeks of age, GB mice displayed a significant reduction of all growth parameters except for the weight of abdominal fat, when compared with G mice, whereas only brain weight was significantly reduced in B vs. C mice. 2) In 15-week-old animals, a significant reduction in all growth parameters, except for spleen and abdominal fat weights, was seen in GB vs. G mice, whereas only nose-rump-length and the weights of carcass and brain were significantly reduced in B vs. C mice. Our study demonstrates, for the first time, the potential of IGFBP-2 to inhibit GH-stimulated growth in giant transgenic mice, providing further evidence for an inhibitory effect of this IGFBP in vivo.

Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain.

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) has been shown to inhibit IGF-dependent cell proliferation in a number of in vitro studies. However, no in vivo model of IGFBP-2 overexpression has been established so far. Therefore, we have generated transgenic mice, in which expression of a mouse IGFBP-2 complementary DNA is controlled by the cytomegalovirus (CMV) promoter. In two independent transgenic strains, transgene expression was highest in pancreas and stomach, followed by skeletal muscle, heart, colon, spleen, adipose tissue, brain, and kidney. Within the pancreas, IGFBP-2 expression was found in the islets but not in the exocrine part. Serum IGFBP-2 levels of CMV-IGFBP-2 transgenic mice were about 3-fold (P < 0.05) increased, compared with controls, whereas serum levels of IGF-I and IGF-II were unaffected by IGFBP-2 overexpression. Fasted serum glucose and fasted insulin levels were slightly reduced in transgenic mice, compared with controls. Postprandial serum glucose insulin levels were not affected by the genotype. At days later than 23, body weights of transgenic mice were significantly (P < 0.05) reduced in both sexes, compared with nontransgenic littermates. This reduction in body weight was mainly attributable to significantly (P < 0.05) lower carcass weights of CMV-IGFBP-2 transgenic vs. control mice. In contrast, absolute organ weights were not (or only as a tendency) reduced, except for the weight of the spleen, which was significantly (P < 0.05) lower in male transgenic than in control mice. Our data suggest that IGFBP-2 represents a negative regulator of postnatal growth in mice, potentially by reducing the bioavailability of IGF-I.

Postnatal overexpression of insulin-like growth factor II in transgenic mice is associated with adrenocortical hyperplasia and enhanced steroidogenesis.

The influence of postnatal insulin-like growth factor II (IGF-II) overexpression on adrenal growth and function was investigated in 3-month-old male phosphoenolpyruvate carboxykinase (PEPCK) promoter human IGF-II transgenic mice, which are characterized by 4-to 6-fold elevated postnatal IGF-II serum levels. Plasma corticosterone levels of PEPCK-IGF-II transgenic mice were 2-fold higher than in age- and sex-matched controls, both in the morning (7.4 +/- 1.5 vs. 17.8 +/- 3.9 ng/ml, P < 0.01) and in the evening (33.3 +/- 6.5 vs. 65.3 +/- 12 ng/ml, P < 0.01). When PEPCK-IGF-II transgenic mice were subjected to an ACTH challenge, corticosterone levels were stimulated 6-fold, to 396 +/- 17 ng/ml after 60 min, compared with 230 +/- 24 ng/ml in the control group. In contrast to corticosterone, plasma ACTH levels were similar in transgenic and control mice, excluding an indirect effect of IGF-II at the hypothalamic or pituitary level. In vitro, the basal and ACTH-induced corticosterone production of adrenal glands from transgenic mice was higher (2-fold and 1.8-fold, respectively) than that of control organs. However, when normalized for adrenal weight, the in vitro corticosterone secretion was similar in both groups. At autopsy, adrenal weights of transgenic mice were significantly greater than those of control adrenal glands (3.3 +/- 0.2 vs. 2.0 +/- 0.2 mg, P < 0.01, n = 10). Furthermore, a local expression of human IGF-II could be demonstrated in transgenic adrenal glands by RT-PCR, whereas in normal adult mice, no adrenal expression of IGF-II was detected. Stereological investigation of adrenal glands from another set of PEPCK-IGF-II transgenic mice and controls (6-month-old males) demonstrated that the increase in adrenal weight in transgenic mice is mainly caused by a 50% increase in the number of zona fasciculata cells, whereas cell volume and zonation of transgenic adrenal glands remained unchanged. In conclusion, our data indicate that postnatal overexpression of IGF-II induces an increased adrenal weight and elevated corticosterone serum levels, presumably by a direct mitogenic effect of IGF-II on adrenocortical fasciculata cells.

Galectins are differentially expressed in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas, and significantly modulate tumor astrocyte migration.

Galectins, a family of mammalian lectins with specificity to beta-galactosides, are involved in growth-regulatory mechanisms and cell adhesion. A relationship is assumed to exist between the levels of expression of galectins and the level of malignancy in human gliomas. A comparative study of this aspect in the same series of clinical samples is required to prove this hypothesis. Using computer-assisted microscopy, we quantitatively characterized by immunohistochemistry the levels of expression of galectins-1, -3 and -8 in 116 human astrocytic tumors of grades I to IV. Extent of transcription of galectins-1, -3, and -8 genes was investigated in 8 human glioblastoma cell lines by means of RT-PCR techniques. Three of these cell lines were grafted into the brains of nude mice in order to characterize in vivo the galectins-1, -3 and -8 expression in relation to the patterns of the tumor invasion of the brain. The role of galectin-1, -3 and -8 in tumor astrocyte migration was quantitatively determined in vitro by means of computer-assisted phase-contrast videomicroscopy. The data indicate that the levels of galectin-1 and galectin-3 expression significantly change during the progression of malignancy in human astrocytic tumors, while that of galectin-8 remains unchanged. These three galectins are involved in tumor astrocyte invasion of the brain parenchyma since their levels of expression are higher in the invasive parts of xenografted glioblastomas than in their less invasive parts. Galectin-3, galectin-1, and to a lesser extent galectin-8, markedly stimulate glioblastoma cell migration in vitro. Since bands for the transcripts of human galectins-2, -4 and -9 were apparently less frequent and intense in the 8 human glioblastoma cell lines, this system provides an excellent model to assign defined roles to individual galectins and delineate overlapping and distinct functional aspects.

Insulin-like growth factor-binding protein-2 inhibits proliferation of human embryonic kidney fibroblasts and of IGF-responsive colon carcinoma cell lines.

So far, the physiological role of insulin-like growth factor binding protein-2 (IGFBP-2) has not been demonstrated directly. Therefore, we transfected 293 cells with an expression vector containing the CMV promoter and the complete cDNA of mouse IGFBP-2. Secretion of bioactive IGFBP-2 into conditioned medium was demonstrated by Western ligand and Western immunoblotting and quantified by specific RIA. For the analysis of cell proliferation three clones exhibiting either high or low/no IGFBP-2 expression were selected and compared to non-transfected parental 293 cells. IGFBP-2 secreting clones displayed reduced conversion of thiazolyl blue when compared to negative clones or non-transfected parental 293 cells (P < 0.01). The lower growth activity measured in the IGFBP-2 secreting clones was compensated in great part by the administration of exogenous IGF-I or -II. Conditioned media of IGFBP-2 secreting clones inhibited growth of IGF-responsive colon tumor cell lines (LS513, HT-29) while those of negative clones did not. In addition, conditioned medium from a clone expressing high levels of IGFBP-2 inhibited anchorage-independent growth of LS513 and HT-29 cells. In contrast, growth of an IGF-unresponsive tumor cell line (Co-115) was not affected by the conditioned media. We hypothesize that IGFBP-2 might sequester the IGFs and thus prevent them from transferring their mitogenic signals.

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.

Tumour-derived, endocrine, exogenous and therapeutic factors differentially modulate cytokine secretion in whole blood cell culture.

Following our previous results which showed that TGF-beta 1 suppressed the secretion of certain cytokines, we investigated the effects of different endogenous and exogenous factors on cytokine secretion in whole blood cell culture by using an enzyme-linked immunosorbent assay (ELISA) for measurement of cytokine concentrations. Several molecules including dexamethasone, noradrenaline (NA) and ethanol differentially inhibited mitogen-induced cytokine secretion. Dexamethasone and noradrenaline suppressed secretion of IL-2, IFN alpha, IFN gamma, TNF alpha, IL-1 alpha and IL-1 beta. beta-Endorphin and Leu-Enkephalin had no significant influence on cytokine secretion. Suppression of cytokine secretion by TGF-beta 1 was further intensified significantly and dose dependently by addition of noradrenaline. GM-CSF stimulated the secretion of IL-1 alpha, IL-1 beta and TNF gamma, but had no influence on the secretion of IL-2, IFN alpha and IFN gamma. G-CSF, IL-3 and SCF did not significantly influence secretion of all cytokines tested. Thus, endogenous and exogenous factors differentially influence cytokine secretion by immunocompetent cells.