PubMed Health⌕ Search

Biomedical subjects

W Poller

Publications and source records attributed to W Poller.

At least 37 records · Page 2Linked to original sources
PubMed1999

Analysis of expression kinetics and activity of a new B-domain truncated and full-length FVIII protein in three different cell lines.

Transient expression of full-length wild-type (wt) and a new B-domain truncated (deltaB) FVIII has been investigated in three eukaryotic cell lines (HEK-293, COS, CHO). When expressed in CHO cells, both FVIII proteins reached the same peak antigen levels, whereas in HEK-293 and COS cells those of FVIII/deltaB were up to sixfold those of FVIII/wt. Investigation of specific activity of the recombinant FVIII proteins using a chromogenic and a one-stage assay in addition to the FVIII-antigen ELISA revealed large variations: In HEK-293 cells specific activity of FVIII/deltaB measured with both assays was higher than that of FVIII/wt. In COS cells specific activity of both FVIII proteins was higher measured in the one-stage assay than in the chromogenic assay. In CHO cells both FVIII proteins had similar specific activity in each assay. In summary, expression kinetics and specific activity of conditioned medium vary depending on cell type used.

Animals↗
PubMed1999

Molecular characterisation of the defective alpha 1-antitrypsin alleles PI Mwurzburg (Pro369Ser), Mheerlen (Pro369Leu), and Q0lisbon (Thr68Ile).

Deficiency of the serine proteinase inhibitor (serpin) alpha 1-antitrypsin (alpha 1AT) is the most common autosomal recessive genetic disorder in Northern Europe. alpha 1AT is the physiological regulator of the proteolytic enzyme neutrophil elastase and severe deficiency states are associated with an increased risk of developing chronic obstructive pulmonary disease (COPD) as a consequence of chronic proteolytic damage to the lungs. Among the known mutations of the alpha 1AT gene causing severe alpha 1AT deficiency and COPD a few alleles are also associated with liver disease. When expressed in cell cultures, all these particular alleles cause intracellular alpha 1AT accumulation which appears to be a prerequisite for the development of hepatic injury. Liver disease is seen in only a small fraction of all patients carrying such alleles, however. The reason for this is not completely clear, but there is evidence that PI ZZ individuals 'susceptible' to liver disease carry an additional defect affecting protein degradation in the endoplasmic reticulum (ER). We characterise a newly identified defective alpha 1AT allele PI Mwürzburg (Pro369 [CCC] to Ser [TCC]) associated with a complete intracellular transport block in cell cultures in vitro. The allele PI Mheerlen, a previously described different amino acid substitution in the same position as PI Mwürzburg (Pro369 [CCC] to Leu [CTC]) is shown to cause complete retention of the mutant alpha 1AT in the ER, too, whereas in the recently described mutant allele PI Q0lisbon (Thr68 [ACC] to Ile [ATC]) a significantly reduced alpha 1AT secretion from the cells was observed. Adenovirus-mediated recombinant expression of mutant Mwürzburg and Mheerlen, and of wild-type alpha 1AT in mouse liver in vivo showed that the mutant human proteins were not secreted into the mouse plasma, in contrast with human wild-type alpha 1AT which circulated at high concentrations over several weeks. In summary, all transportation deficient alpha 1ATs analysed have the potential to cause lung disease in the homozygous state or in heterozygous carriers of another deficiency allele, and they may also cause liver disease in certain patients. The mutant PI Mwürzburg and Mheerlen alpha 1ATs are completely retained within synthesising cells, and the molecular defect of transportation in these two alleles may be similar to that in the common PI Z allele. The molecular defect in the PI Q0lisbon allele (Thr68Ile) shows similarity with the immediately neighbouring Mmineral springs mutation (Gly67Glu).

Adult↗
PubMed1999

'Autoreplication' of the vector genome in recombinant adenoviral vectors with different E1 region deletions and transgenes.

High transgene stabilities of 1 year and more have been reported in immunodeficient hosts after adenovirus-mediated gene transfer. Transgene persistence of this duration could be due to inherently high stability of the episomal viral vector DNA. An alternative explanation would be limited 'autoreplication' of transgenic vector DNA, just sufficient to counteract slow but continuous degradation within the host cells. Autoreplication could occur in the absence of any production of infectious virus particles, based on residual activity of the adenoviral DNA replication system only. To test this hypothesis, a series of DNA metabolic labeling studies in non-permissive cells cultures transfected with different vectors was conducted. Due to extensive E1 region deletions none of the vectors was able to produce viral progeny in non-permissive cells. Vectors fell into two categories, however, with respect to their autoreplication potential. Neosynthesis of vector DNA in non-permissive vector-transfected cells was readily detectable in 'type A', but not in 'type B' vectors. In addition to their different transgene expression cassettes, vector DNA sequencing showed a less extensive E1 deletion in type A (nucleotides 453-3333 of wild-type virus) as compared to type B vectors (nucleotides 325-3523). Autoreplication was also associated with high transcriptional activity of several viral genes (E1B-14k, adenoviral DNA polymerase, single-strand DNA-binding protein, E4-25k), in contrast to type B vectors. In addition to these 'wild-type' transcripts, 'irregular' recombinant transcripts were detected in autoreplication vectors which contained the transgenic cDNA in conjunction with adenoviral vector sequences. Exogenous or cryptic promotors may (under certain conditions) enhance the transcriptional activity of a vector in such a way that autoreplication occurs. Conditions determining the level of transcriptional enhancement (extent of E1 deletion, type of promoter and transgene, etc) need to be further defined before rational design of adenovectors with high autoreplication capacity becomes possible. In summary, we have shown autoreplication to be a novel feature of certain E1-deleted adenovectors with likely relevance for their stability in vivo, but also with possibly adverse consequences for target cell function or vector immunogenicity. Full characterization of adenoviral vector systems should therefore include a description of their autoreplication capacity.

Adenoviridae↗
PubMed1999

Expression of coxsackie adenovirus receptor and alphav-integrin does not correlate with adenovector targeting in vivo indicating anatomical vector barriers.

Recombinant adenoviral vectors are broadly applied in gene therapy protocols. However, adenovector-mediated gene transfer has limitations in vivo. One of these is the low gene transfer rate into organs other than the liver after systemic intravenous vector injection. Local direct injection into the target organ has been used as one possible solution, but increases necessary equipment and methodology and is traumatic to the target. Wild-type adenovirus infection as well as adenovector-mediated gene transfer depends on virus interaction with the Coxsackie adenovirus receptor (CAR) mediating virus attachment to the cell surface, and on interaction with alphavbeta3 and alphavbeta5 integrins mediating virus entry into the cell. In order to assess the receptor-associated potential of different tissues to act as adenovector targets, we have therefore determined CAR and alphav-integrin expression in multiple organs from different species. In addition, we have newly determined several human, rat, pig and dog CAR-mRNA sequences. Sequence comparison and structural analyses of known and of newly determined sequences suggests a potential adenovirus binding site between amino acids 29 and 128 of the CAR. With respect to the virus receptor expression patterns we found that CAR-mRNA expression was extremely variable between different tissues, with the highest levels in the liver, whereas alphav-integrin expression was far more homogenous among different organs. Both CAR and alphav-integrin showed similar expression patterns among different species. There was no correlation, however, between the adenovector expression patterns after intravenous, intracardiac and aortic root injection, respectively, and the virus receptor patterns. In summary, many organs carry both receptors required to make them potential adenovector targets. In sharp contrast, their actual targeting clearly indicates that adenovirus receptor expression is necessary but not sufficient for vector transfer after systemic injection. The apparently very important role of anatomical barriers, in particular the endothelium, requires close attention when developing non-traumatic, organ-specific gene therapy protocols.

Adenoviridae↗
PubMed1999

Highly sensitive and species-specific assay for quantification of human transgene expression levels.

During the past few years great efforts have been made to construct and to test human factor VIII (hFVIII) and IX (hFIX) vectors suitable for haemophilia gene therapy in vivo. However, little is known about the molecular mechanisms of persistence and shut-off of transgene expression in the target organs after gene transfer using recombinant adenoviral vectors. To evaluate low transgene mRNA levels in different tissues, especially at long times after the gene transfer, the common northern blot method is often not sensitive enough. For this reason we developed a new, highly sensitive and species-specific method for hFIX mRNA quantification and employed it in mice treated with an adenoviral vector (Ad5CMVFIX) expressing human FIX. In addition to its very high sensitivity (lowest detection level=1 fg RNA), the method was shown to be strictly species-specific, since hFIX mRNA signals were never detected in untreated mice. In a long-term study of 18 vector-treated mice we compared the human FIX:Ag levels in the mouse plasma, the human FIX mRNA levels and human FIX vector DNA concentrations in the mouse liver. We found that a slow but continuous decrease of hFIX:Ag levels in mouse plasma was associated with a corresponding decrease of hFIX mRNA levels in the liver. However, the Ad5CMVFIX vector DNA levels did not decrease to a comparable degree, suggesting that the decrease of human FIX:Ag levels in mouse plasma is, to a significant extent, also caused by CMV promotor shut-off and only to a minor degree by loss of vector DNA.

Adenoviridae↗
PubMed1998

Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP-dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells.

An overactive renin-angiotensin-aldosterone system (RAAS) has a central role in the pathogenesis of hypertension and cardiac hypertrophy, precursors of cardiac failure. Natriuretic peptides and NO acting through their second messenger, cGMP, increase natriuresis and diuresis, and inhibit renin release; however the mechanism by which this inhibition of the RAAS system functions is obscure. We recently reported cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), elucidated its first known function of inhibiting the cystic fibrosis transmembrane conductance regulator in rat intestine, and initially described its location in rat kidney juxtaglomerular (JG) cells, the ascending thin limb, and the brush border of proximal tubules. Here, we demonstrate inhibition of isoproterenol- or forskolin-stimulated renin release by 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP), a selective activator of cGK, and prevention of this inhibition by a selective inhibitor of cGK, Rp-8-pCPT-cGMPS. In systems of differing complexity, inhibition by 8-pCPT-cGMP was nearly complete in isolated perfused kidney and microdissected afferent arterioles but only approximately 25% in isolated JG cells. Expression of either cGK II or cGK I in JG cells by using adenoviral vectors enhanced the inhibition of forskolin-stimulated renin release by 8-pCPT-cGMP to 50%. Our results indicate that cGK II, and possibly cGK I, can mediate cGMP inhibitory effects on renin release and are physiological components of the cGMP signal transduction system which opposes the RAAS.

Animals↗
PubMed1998

[Precapillary pulmonary hypertension of uncertain etiology].

HISTORY AND CLINICAL FINDINGS: A 39-year-old man was hospitalized for investigation of increasing dyspnoea for 3 month. On admission he was found to have bilateral ankle oedema, an enlarged liver and loud systolic murmur over the lower sternum. INVESTIGATIONS: There were signs of right heart strain/hypertrophy on the chest radiogram and echocardiogram. After treatment of right heart failure cardiac catheterization indicated moderate precapillary pulmonary hypertension (PH) with a mean pulmonary artery pressure (PAPm) of 24 mm Hg and pulmonary vascular resistance (PVR) of 470 dyn.s.cm-5 at rest. All known causes having been excluded, the PH was classified as idiopathic. TREATMENT AND COURSE: Evidence of acute pulmonary vascular reactivity was obtained with nitric oxide (NO) inhalation and oral diltiazem, a calcium-channel blocker. The latter, at a dosage of 3 x 120 mg daily, had after 13 days achieved a persisting reduction of PVR at rest and a reduction in PAP rise during exercise. CONCLUSION: After exclusion of other causes, the acute right heart failure was found to be due to primary pulmonary hypertension. The therapeutic efficacity of diltiazem as a vasodilator can be predicted from the response to inhaled NO.

Adult↗
PubMed1998

Lumpectomy and radiation therapy for the treatment of intraductal breast cancer: findings from National Surgical Adjuvant Breast and Bowel Project B-17.

PURPOSE: In 1993, findings from a National Surgical Adjuvant Breast and Bowel Project (NSABP) trial to evaluate the worth of radiation therapy after lumpectomy concluded that the combination was more beneficial than lumpectomy alone for localized intraductal carcinoma-in-situ (DCIS). This report extends those findings. PATIENTS AND METHODS: Women (N = 818) with localized DCIS were randomly assigned to lumpectomy or lumpectomy plus radiation (50 Gy). Tissue was removed so that resected specimen margins were histologically tumor-free. Mean follow-up time was 90 months (range, 67 to 130). Size and method of tumor detection were determined by central clinical, mammographic, and pathologic assessment. Life-table estimates of event-free survival and survival, average annual rates of occurrence for specific events, relative risks for event-specific end points, and cumulative probability of specific events comprising event-free survival are presented. RESULTS: The benefit of lumpectomy plus radiation was virtually unchanged between 5 and 8 years of follow-up and was due to a reduction in invasive and noninvasive ipsilateral breast tumors (IBTs). Incidence of locoregional and distant events remained similar in both treatment groups; deaths were only infrequently related to breast cancer. Incidence of noninvasive IBT was reduced from 13.4% to 8.2% (P = .007), and of invasive IBT, from 13.4% to 3.9% (P < .0001). All cohorts benefited from radiation regardless of clinical or mammographic tumor characteristics. CONCLUSION: Through 8 years of follow-up, our findings continue to indicate that lumpectomy plus radiation is more beneficial than lumpectomy alone for women with localized, mammographically detected DCIS. When evaluated according to the mammographic characteristics of their DCIS, all groups benefited from radiation.

Breast Neoplasms↗
PubMed1996

Stabilization of transgene expression by incorporation of E3 region genes into an adenoviral factor IX vector and by transient anti-CD4 treatment of the host.

Complex interactions between replication deficient adenoviral vectors (Ad5) and the immune system of the host influence the stability of transgenes in vivo. Vector-infected cells are attacked by diverse cellular immune mechanisms which limit transgene persistence. On the other hand, the products of several E3 region genes of wild-type adenovirus can suppress host immune reactions by interference with the expression of MHC class I molecules and by other mechanisms. We have developed an adenoviral vector for human factor IX (Ad5E3+FIX) which carries the E3 region of wild-type adenovirus, and an E3-deleted vector of otherwise similar structure (ad5 delta E3FIX). Intravenous injection of Ad5E3+FIX in C57BI/6 mice resulted in expression levels up to 6000 ng/ml of recombinant human factor IX in the mouse plasma and in enhanced transgene stability as compared with the vector Ad5 delta E3FIX. Whereas expression from E3-deleted vectors was essentially turned off 8 weeks after the gene transfer, the vector Ad5E3+FIX3+FIX supported transgene expression with therapeutic levels of human factor IX in the mouse plasma for > 4 months. The enhanced stability of the vector Ad5E3+FIX appears to be a consequence of efficient E3 region-mediated suppression of the host's antivector immune response. As an additional approach to improving transgene stability the influence of transient CD4+ T cell depletion of the host was investigated. CD4+ cytotoxic T lymphocytes contribute to the clearance of adenovirus-infected cells and play a pivotal role in the activation of CD8+ cytotoxic T cells and as helper T cells in the formation of human adenovirus neutralizing antibodies (HANA). Transient anti-CD4 treatment of the host limited to the time of vector injection resulted in a significant prolongation of transgene expression from the factor IX vector Ad5E3+FIX and a luciferase vector Ad5Luc. The combination of transient anti-CD4 treatment of the host and integration of a complete E3 region in an adenoviral vector resulted in markedly improved transgene stability after gene transfer to the liver (therapeutic factor IX levels for > 6 months).

Adenoviridae↗
PubMed1995

Differential recognition of alpha 1-antitrypsin-elastase and alpha 1-antichymotrypsin-cathepsin G complexes by the low density lipoprotein receptor-related protein.

Two multifunctional receptors, low density lipoprotein receptor-related protein (LRP) and gp330, have been implicated in the cellular uptake and degradation of a wide spectrum of functionally diverse ligands including plasma lipoproteins, proteases, and proteinase-inhibitor complexes. The two receptors show distinct tissue-specific expression patterns, suggesting different physiological functions. We have examined the cellular degradation of two serine proteinase inhibitor (serpin)-protease complexes, alpha 1-antitrypsin-neutrophil elastase (alpha 1AT.NEL) and alpha 1-antichymotrypsin-cathepsin G (alpha 1ACT.CathG) by normal murine fibroblasts (MEF) expressing LRP, and by a mutant fibroblast cell line (PEA13) which is genetically deficient for LRP. alpha 1AT.NEL complexes bound to LRP on ligand blots and were degraded efficiently by the MEF cells, but not by PEA13 cells. Degradation of the complexes was also significantly reduced by antibodies directed against LRP, further suggesting that fibroblasts require LRP for the cellular uptake and degradation of alpha 1AT.NEL complexes. In contrast to alpha 1AT.NEL, MEF cells did not degrade alpha 1ACT.CathG complexes. However, these complexes were rapidly degraded by the rat embryonal carcinoma cell line L2p58 which abundantly expresses gp330, raising the possibility that the alpha 1ACT.CathG complex might be recognized by gp330. Both complexes were efficiently metabolized by the hepatoma cell line HepG2, presumably involving the serpin-enzyme complex receptor. The differential recognition of serpin-protease complexes by fibroblasts and hepatoma cells, however, indicates that LRP, gp330, and the serpin-enzyme complex receptor are distinct proteins.

Animals↗
PubMed1995

A missense mutation in the glucagon receptor gene is associated with non-insulin-dependent diabetes mellitus.

Non-insulin-dependent diabetes mellitus (NIDDM) affects about 5% of the world population. The disease presents a polygenic mode of inheritance, but mechanisms and genes involved in late-onset NIDDM are largely unknown. We report the association of a single heterozygous Gly to Ser missense mutation in the glucagon receptor gene with late-onset NIDDM. This mutation was highly associated with NIDDM in a pooled set of French and Sardinian patients (chi 2 = 14.4, P = 0.0001) and showed some evidence for linkage to diabetes in 18 sibships from 9 French pedigrees (chi 2 = 6.63, P < 0.01). Receptor binding studies using cultured cells expressing the Gly40Ser mutation demonstrate that this mutation results in a receptor which binds glucagon with a three-fold lower affinity compared to the wild type receptor.

DNA Primers↗
PubMed1994

Six mutations in the glucokinase gene identified in MODY by using a nonradioactive sensitive screening technique.

We have reported that 56% of French families with maturity-onset diabetes of the young (MODY) carry a mutation in the glucokinase gene (GCK). Therefore, we have established a quick and sensitive nonradioactive technique (with the PhastSystem based on single-strand conformation polymorphism [SSCP] analysis) to routinely screen the 12 exons of GCK for mutations. We have studied GCK in 12 young hyperglycemic patients with a strong family history of type II diabetes. SSCP variants were observed in 6 of those 12 patients (50%), which cosegregated with diabetes in five families where DNA from additional members was available. Direct sequencing identified a 10-bp (base pair) deletion in exon 3; a 33-bp deletion at the exon 5/intron 5 junction, including the two consensus bases (GT) of the donor splice site; a nonsense mutation in exon 5 (Arg186-->Stop) in a Black-African family, which has been identified previously in a Caucasian family; and three missense mutations: Thr209-->Met209 in exon 6, Gly261-->Glu261 in exon 7, and Arg36-->Trp36 in exon 2. The missense mutation in exon 2 was found only in the second and third generation of the tested family but not in the first. To our knowledge, this is the first time that a de novo mutation of GCK is reported within a family. All six families carrying a mutation in GCK were typical MODY and most of their affected members had a mild form of diabetes. This nonradioactive SSCP technique may be useful to routinely diagnose glucokinase deficiency, which is an important cause of hyperglycemia among young type II diabetic patients.

Adult↗
PubMed1994

Identification and DNA sequence analysis of 15 new alpha 1-antitrypsin variants, including two PI*Q0 alleles and one deficient PI*M allele.

We have investigated the molecular basis of 15 new alpha 1-antitrypsin (alpha 1AT) variants. Phenotyping by isoelectric focusing (IEF) was used as a screening method to detect alpha 1AT variants at the protein level. Genotyping was then performed by sequence analysis of all coding exons, exon-intron junctions, and the hepatocyte-specific promoter region including exon Ic. Three of these rare variants are alleles of clinical relevance, associated with undetectable or very low serum levels of alpha 1AT:the PI*Q0saarbruecken allele generated by a 1-bp C-nucleotide insertion within a stretch of seven cytosines spanning residues 360-362, resulting in a 3' frameshift and the acquisition of a stop codon at residue 376; a point mutation in the PI*Q0lisbon allele, resulting in a single amino acid substitution Thr68(ACC)-->Ile(ATC); and an in-frame trinucleotide deletion delta Phe51 (TTC) in the highly deficient PI*Mpalermo allele. The remaining 12 alleles are associated with normal alpha 1AT serum levels and are characterized by point mutations causing single amino acid substitutions in all but one case. This exception is a silent mutation, which does not affect the amino acid sequence. The limitation of IEF compared with DNA sequence analysis, for identification of new variants, their generation by mutagenesis, and the clinical relevance of the three deficiency alleles are discussed.

Alleles↗
PubMed1993

A leucine-to-proline substitution causes a defective alpha 1-antichymotrypsin allele associated with familial obstructive lung disease.

Using denaturing gradient gel electrophoresis and direct sequencing of amplified genomic DNA, we have identified two defective mutants of the human alpha 1-antichymotrypsin (ACT) gene associated with chronic obstructive pulmonary disease (COPD). A leucine 55-to-proline substitution causing a defective ACT allele (Bochum-1) was observed in a family with COPD in three subsequent generations. Another mutation, proline 229-to-alanine (Bonn-1), was associated with ACT serum deficiency in four patients with a positive family history. These mutations were not detected among 100 healthy control subjects, suggesting a possible pathogenetic role of ACT gene defects in a subset of patients with COPD.

Amino Acid Sequence↗
PubMed1993

The molecular basis of alpha 1-antichymotrypsin deficiency in a heterozygote with liver and lung disease.

Alpha 1-antichymotrypsin (alpha 1-ACT) is a serine proteinase inhibitor (serpin) with cathepsin G, mast cell chymase and chymotrypsin as target enzymes. We present the case of a middle-aged man with low plasma levels of alpha 1-ACT, asthma with progression to emphysema, and chronic HCV positive liver disease with selective accumulation of alpha 1-ACT in hepatocytes. This secretory defect is analogous to that seen in Pi Z alpha 1-antitrypsin deficiency. The molecular basis of alpha 1-ACT deficiency in this patient has been characterized by direct sequencing of the alpha 1-ACT genes from the patient and his father. A C-->G transversion in exon III causing a 229Pro-->Ala substitution is proposed to cause a conformational change resulting in abnormal transport through the RER. This mutation was found in one of 20 additional tested patients with chronic obstructive lung disease, but in no control. Two additional polymorphisms of the gene have been identified in unrelated healthy individuals with normal plasma alpha 1-ACT levels. The alpha 1-ACT deficiency state may predispose to obstructive lung disease and influence the course of liver disease. Identification of a specific mutation allows identification of heterozygotes for this deficiency allowing future evaluation of its clinical significance.

Amino Acid Sequence↗