Inhibition of Basal and Stimulated Release of Endothelin-1 from Guinea Pig Tracheal Epithelial Cells in Culture by Beta 2-adrenoceptor Agonists and Cyclic AMP Enhancers

The effects of cyclic AMP-related compounds and beta adrenoceptor agonists on the basal and lipopolysaccharide (LPS)-stimulated release of endothelin-1 (ET-1) from guinea-pig tracheal epithelial cells (GPTEpCs) in culture were studied. Forskolin (a potent activator of adenylyl cyclase), 8-bromo-cyclic AMP (a cyclic AMP analogue), salbutamol and salmeterol (two beta 2-adrenoceptor agonists), were used to increase cyclic AMP levels. Cultured GPTEpCs released ET-1 continuously over a 24 h incubation period. The values reached 1,938 ± 122 pg/mg of total cell proteins after 24 h. LPS (10 μg/ml) significantly stimulated the release of ET-1 by 1.6- to 1.8-fold, up to 1,262 ± 56 pg/mg total cell proteins after an 8 h incubation period. Compound 8-bromo-cyclic AMP (10⁻⁵, 10⁻⁴ and 10⁻³ M) reduced the basal release of ET-1 from GPTEpCs by up to 31% (P < 0.01) and the LPS stimulated release by up to 42% (P < 0.05), after an 8 h incubation period. Forskolin (10⁻⁶, 10⁻⁵ and 10⁻⁴ M) also inhibited the basal release of ET-1 by up to 28% (P < 0.05) and LPS-stimulated release of ET-1 by up to 50% (P < 0.05), after an 8 h incubation period. At the concentration of 10⁻⁵ M, forskolin increased cyclic AMP levels in GPTEpCs by 17-fold (P < 0.001) in the medium, 15 min after the beginning of the incubation. Salbutamol (10⁻⁸ to 10⁻⁶ M) had no effect on the basal production and release of ET-1 after 8 h. Conversely, this short acting beta 2-adrenoceptor agonist significantly reduced LPS-mediated increase of ET-1 production by up to 55% (P < 0.05) after an 8 h incubation period. Salmeterol (10⁻⁹ M to 10⁻⁵ M) inhibited basal and LPS-stimulated production and release of ET-1 after an 8 h incubation period (between 44 and 51%, P < 0.01). Both salbutamol and salmeterol (10⁻⁶ M) increase cyclic AMP levels by five- and twofold, respectively (P < 0.05). In summary, these observations indicate that beta 2-adrenoceptor agonists or cyclic AMP enhancers can modulate both basal and more markedly, the enhanced production of ET-1 from LPS-activated guinea pig airway EpCs. In addition, these compounds increase cyclic AMP levels in the cells. It is suggested that there is a correlation between cyclic AMP increase and inhibition of ET-1 release by guinea pig airway EpCs. Since ET-1 production was shown to be elevated in asthmatic subjects and in patients suffering from other inflammatory lung disorders, the inhibition of its production by beta adrenoceptor agonists, such as salbutamol and salmeterol, could be added to their therapeutical benefits.     

Citicoline is not protective in experimental models of Huntington's disease

We have evaluated the neuroprotective effects of citicoline in relevant phenotypic models of Huntington's disease induced by either the mitochondrial inhibitor 3-nitropropionic acid or the N-methyl-d-aspartate agonist quinolinic acid, which, respectively, reproduce the metabolic defect or the excitotoxicity seen in the disease. We found that citicoline failed to reverse behavioural and histological alterations induced by both neurotoxins. In addition, citicoline did not reduce PC12 cell death induced by the expression of an N-terminal fragment of mutated Huntingtin. Altogether, our results suggest that citicoline is not a potential therapeutic agent for the treatment of Huntington's disease.     

Specific isolation of disseminated cancer cells: a new method permitting sensitive detection of target molecules of diagnostic and therapeutic value

Molecular studies of rare cells, such as circulating cancer cells, require efficient pre-enrichment steps to obtain a pure population of target cells for further characterization. We have developed a two-step approach, starting with immunomagnetic enrichment, followed by specific isolation of individual, easily identifiable bead-rosetted target cells using a new semi-automated CellPick system. With this procedure, 1–50 live target cells can now be isolated. As a model system, we spiked a small number of tumor cells into millions of normal mononuclear cells (MNCs). Efficient isolation of pure target cells was obtained by use of the CellPick system, and the nature of isolated, bead-rosetted cells was verified by use of FISH. Single breast cancer cells were picked directly into an RNA preserving lysis buffer, reverse transcribed, and PCR amplified with two cDNA specific primer sets. With the isolated cells we consistently obtained both ubiquitously expressed and tumor cell specific PCR products. We also performed a successful mutation analysis of single cells using PCR and cycling temperature capillary electrophoresis (CTCE). This may have significant clinical implications in cancer and in other diseases, e.g. in characterizing micrometastatic cancer cells in blood and lymph nodes to help identifying patients who most likely will respond to therapies like tyrosine kinase inhibitors and compounds targeting specific mutations. By use of the CellPick system it is possible to specifically isolate bead-rosetted or otherwise labelled target cells from a heterogeneous cell population for further molecular characterization.     

Attitudes of German infertile couples towards preimplantation genetic diagnosis for different uses: a comparison to international studies

BACKGROUND: In Germany, preimplantation genetic diagnosis (PGD) is currently not legal, but there is still a controversial debate about legalization. Studies about the attitudes of infertile couples towards PGD are rare. METHODS: A survey was conducted with 265 German infertile couples about knowledge, attitudes and prospective use of PGD. The influence of independent variables associated with approval of PGD is analysed by binary logistic regression. RESULTS: Sixty percent of respondents have heard about PGD. Eighty-seven percent support a general legalization of PGD in Germany for severe, early-onset genetic diseases. Seventy-four percent consider PGD morally acceptable. Sixty percent supported legalizing PGD for HLA-matching. But only a minority approved PGD to test for non-health-related traits. Respondents with a higher education level were the least supportive to all uses of PGD. CONCLUSIONS: Our results suggest that German infertile couples are as liberal towards PGD for health-related uses as in other western countries. They would legalize and use PGD to raise the rates to get pregnant and to avoid severe diseases of the offspring. Taking the opinions of German infertile couples into consideration could help redefine and reframe the public debate towards legalization of PGD and the moral status of the embryo in Germany.     

Phylogenetic comparison of the S10 genes of recent isolates of bluetongue virus from the United States and French Martinique Island

The sequences of the S10 genes of 28 recent isolates (1994-2004) of bluetongue virus (BTV) from the United States (US) and French Martinique Island (2006) in the Caribbean Basin were compared in phylogenetic analyses to those of viruses previously isolated in the same regions. Although the analyses segregated the recent virus isolates from the two regions into distinct topotype clusters, the analyses also confirm that viruses from the US and the Caribbean Basin/Central America can share similar S10 genes despite the fact that distinct constellations of BTV serotypes occur in the two regions.     

Rigid spine syndrome (vacuolar variant). A quantitative electromyograhic study

BACKGROUND: Quantitative electromyography (EMG) using different needle techniques was performed as part of a complete systems review on 9 patients diagnosed with the vacuolar variant of rigid spine syndrome (RSS). Purpose: To establish statistically: (1) correlations between clinical features (patient age, disease duration, degree of weakness) and quantitative EMG; (2) correlation between different EMG parameters; (3) differences in quantitative EMG comparing patients with a healthy control group; and (4) correlate EMG with muscle pathology findings. METHODS: Nerve conduction studies and needle EMG (motorunit analysis, MacroEMG, SFEMG) were performed on Mm. triceps brachii and tibialis anterior according to standard techniques on 9 RSS patients. RESULTS: Nerve conduction studies were normal. Overall, clinical parameters did not correlate well with motorunit analysis and MacroEMG results. Motorunit analysis and MacroEMG results were significantly different comparing patients with controls. Motorunit analysis and MacroEMG correlated well with muscle biopsy findings. SFEMG results were within normal limits. CONCLUSION: Comprehensive EMG study results were compatible with a benign myopathic process. Results were consistent amongst patients within the study group, but differed significantly from the control group. Stable neuromuscular junction physiology did not accurately reflect evidence of muscle fiber degeneration and regeneration observed on muscle biopsies.     

Facioscapulohumeral muscular dystrophy. Multimodal evoked potentials and electroretinogram

BACKGROUND: Clinical or subclinical abnormalities of the central nervous system (CNS) have been reported in a range of primary muscle diseases, including the muscular dystrophies. PURPOSE: To ascertain by neurophysiologic techniques evidence of CNS dysfunction in a relatively large, homogeneous group of patients with facioscapulohumeral muscular dystrophy (FSHD). METHODS: Standard evoked potential (EP) and electroretinogram techniques were used to study the visual, auditory and somatosensory pathways in 20 patients with FSHD. RESULTS: Abnormal values were recorded in 70% (14/20) of patients, specifically of visual pathways (4/20), brainstem auditory pathways (4/20), median (5/20) and tibial nerve (2/20) somatosensory pathways, and of the retina (2/18). Abnormal results did not correlate with clinical parameters of patient age, disease duration and degree of weakness. CONCLUSIONS: Any process that caused CNS conduction delays was tentatively associated with FSHD. Sensorineural hearing deficit and vascular retinopathy were rare causes of abnormal EPs. Peripheral conduction delay of the arms was ascribed to mechanical factors secondary to shoulder girdle weakness. Progress in genetics and molecular pathogenesis of FSHD may shed further insight into the association between the myodystrophic process and nervous system abnormalities.     

Magnetic silica extraction for low-viremia human immunodeficiency virus type 1 genotyping

Nucleic acid extraction and human immunodeficiency virus type 1 (HIV-1) genotyping using the NucliSens miniMAG platform and the TruGene HIV-1 genotyping kit gave HIV-1 sequence data from HIV-1-negative plasma spiked with 100 copies/ml reference HIV-1 RNA and from low-viremia clinical samples (<500 copies/ml) without the need for ultracentrifugation or nested second-round PCR.     

Cloning and expression in E. coli of a functional Fab fragment obtained from single human lymphocyte against anthrax toxin

Human lymphocytes derived from the blood of a donor immunized with anthrax vaccine were isolated and enriched for B-cells by Nycoprep density centrifugation. Individual anti-anthrax protective antigen (PA) B-cells were isolated by fluorescence activated cell sorting with fluorescence-labeled recombinant PA (rPA). The RNA from sorted single B-cells was extracted using plant total RNA as the carrier prior to purification by Nanoprep RNA isolation columns and then cDNA was prepared. Donor specific human Fab primer sets were developed based on rapid amplification of 5'-complementary DNA end results. Heavy chain and light chain of human Fab were amplified from the donor single B-cells by PCR. The amplified heavy and light chain were then cloned into the expression vector pASK-IBA2 and expressed in Escherichia coli (E. coli). The chains combined in vivo to form a functional Fab which was then purified as one protein. The human Fab antibodies produced by this technique were functional when tested in Western blots where the rPA was the target as well as in ELISA. This approach allowed us to obtain human Fab that retained the natural heavy and light chain pairing, which is supposed to have a high antigen-binding affinity.     

Generation of llama single-domain antibodies against methotrexate, a prototypical hapten

Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L. A single band around 16,000Da corresponding to VHH fragments was found after analysis by SDS-PAGE and Western blotting, while competition ELISA demonstrated selective binding to soluble MTX. Surface plasmon resonance (SPR) analysis showed that anti-MTX VHH domains had affinities in the nanomolar range (29-515nM) to MTX-serum albumin conjugates. The genes encoding anti-MTX VHH were found by IMGT/V-QUEST to be similar to the previously reported llama and human IGHV germline genes. The V-D and D-J junction rearrangements in the seven anti-MTX CDR3 sequences indicate that they were originated from three distinct progenitor B cells. Our results demonstrate that camelid single-domain antibodies are capable of high affinity binding to low molecular weight hydrosoluble haptens. Furthermore, these anti-MTX VHH give new insights on how the antigen binding repertoire of llama single-domain antibody can provide combining sites to haptens in the absence of a VL. This type of single-domain antibodies offers advantages compared to murine recombinant antibodies in terms of production rate and sequence similarity to the human IGHV3 subgroup genes.