Cutaneous gene therapy – an update

In this paper we review various strategies for gene transfer into the skin, their applications and show some of our own examples. The skin is the most accessible somatic tissue. Inherited skin diseases, such as epidermolysis bullosa and ichthyosis, and various systemic metabolic disorders are under investigation as potential candidate diseases for cutaneous gene transfer. Research is directed against genetic therapy of wounds and malignancies as well. So far, cutaneous gene transfer only has been used experimentally, but several clinical trials are under preparation.     

Hitting bacteria at the heart of the central dogma: sequence-specific inhibition

An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of different human cancers and other human diseases. Applying antisense gene silencing and exploiting RNA interference (RNAi) are highly developed approaches in many eukaryotic systems. But in bacteria RNAi is absent, and gene silencing by antisense compounds is not nearly as well developed, despite its great potential and the intriguing possibility of applying antisense molecules in the fight against multiresistant bacteria. Recent breakthrough and current status on the development of antisense gene silencing in bacteria including especially phosphorothioate oligonucleotides (PS-ODNs), peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) will be presented in this review.     

Microfibrillar-Associated Protein 4: A Potential Biomarker for Screening for Liver Fibrosis in a Mixed Patient Cohort

MethodspMFAP4 was measured in samples from 351 drug users attending treatment centres and from 248 acutely hospitalized medical patients with mixed diagnoses. Linear and logistic multivariate regression analyses were performed and nonparametric receiver operating characteristic-curves for cirrhosis were used to estimate cut-off points for pMFAP4. Univariate and subgroup analyses were performed using non-parametric methods.ResultspMFAP4 increased significantly with liver fibrosis score. pMFAP4 was significantly associated with chronic viral infection in the drug users and with transient elastography in both cohorts. In the mixed patient cohort, pMFAP4 was significantly increased among patients with a previous diagnosis of liver disease or congestive heart failure compared to patients with other diagnoses.ConclusionspMFAP4 has the potential to be used as an outreach-screening tool for liver fibrosis in drug users and in mixed medical patients. pMFAP4 level is positively associated with transient elastography, but additional studies are warranted to validate the possible use of pMFAP4 in larger cohorts and in combination with transient elastography.     

Standardized activities of daily living in presence of sub-acute low-back pain: A pilot study

The aim of this pilot study was to investigate how sub-acute low-back pain (LBP) patients differed with respect to control in movements and muscle activation during standardized tasks representing daily living activities, and explore relationships between cognition and measured motor performance. Linear and nonlinear parameters were computed from kinetics, kinematics and muscle activity recorded for 12 sub-acute patients and 12 healthy matched controls during trunk flexion, sit-to-stand from a chair and lifting a box. Cognitive variables were collected to explore relationships with biomechanical parameters. For trunk flexion, left external abdominal oblique muscle activity level was lower for patients compared with controls (p < 0.05), whereas sample entropy (complexity) was higher (p < 0.05). Normalized mutual information was lower for patients compared with controls for left and right erector spinae (p < 0.05). Level of activity of left external abdominal oblique correlated negatively with cognitive ignoring and positively with catastrophizing (p ? 0.05), and catastrophizing also correlated positively with functional connectivity of abdominal muscles (p < 0.05). Signs of reorganization in muscle activation pointed towards different synergistic actions in trunk muscles in sub-acute LBP patients compared with controls. The interplay with maladaptive cognition suggested that in the subacute stage of LBP, both biomechanical and cognitive factors should be taken into account.     

Pressure load: the main factor for altered gene expression in right ventricular hypertrophy in chronic hypoxic rats

Background

The present study investigated whether changes in gene expression in the right ventricle following pulmonary hypertension can be attributed to hypoxia or pressure loading.

Methodology/Principal Findings

To distinguish hypoxia from pressure-induced alterations, a group of rats underwent banding of the pulmonary trunk (PTB), sham operation, or the rats were exposed to normoxia or chronic, hypobaric hypoxia. Pressure measurements were performed and the right ventricle was analyzed by Affymetrix GeneChip, and selected genes were confirmed by quantitative PCR and immunoblotting. Right ventricular systolic blood pressure and right ventricle to body weight ratio were elevated in the PTB and the hypoxic rats. Expression of the same 172 genes was altered in the chronic hypoxic and PTB rats. Thus, gene expression of enzymes participating in fatty acid oxidation and the glycerol channel were downregulated. mRNA expression of aquaporin 7 was downregulated, but this was not the case for the protein expression. In contrast, monoamine oxidase A and tissue transglutaminase were upregulated both at gene and protein levels. 11 genes (e.g. insulin-like growth factor binding protein) were upregulated in the PTB experiment and downregulated in the hypoxic experiment, and 3 genes (e.g. c-kit tyrosine kinase) were downregulated in the PTB and upregulated in the hypoxic experiment.

Conclusion/Significance

Pressure load of the right ventricle induces a marked shift in the gene expression, which in case of the metabolic genes appears compensated at the protein level, while both expression of genes and proteins of importance for myocardial function and remodelling are altered by the increased pressure load of the right ventricle. These findings imply that treatment of pulmonary hypertension should also aim at reducing right ventricular pressure.     

Enzyme-Linked Immunosorbent Assay Characterization of Basal Variation and Heritability of Systemic Microfibrillar-Associated Protein 4

Background

Microfibrillar-associated protein 4 (MFAP4) is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM), and variation in systemic MFAP4 (sMFAP4) has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing the basal sMFAP4 variability and the genetic contribution to the basal variation.

Methods

The sandwich ELISA was based on two monoclonal anti-MFAP4 antibodies and was optimized and calibrated with a standard of recombinant MFAP4. The importance of pre-analytical sample handling was evaluated regarding sample tube type, time, and temperature conditions. The mean value structure and variance structure was determined in a twin cohort including 1,417 Danish twins (age 18-67 years) by mixed-effect linear regression modeling.

Results

The practical working range of the sandwich ELISA was estimated to be 4-75 U/ml. The maximum intra- and inter-assay variation was estimated to be 8.7% and 6.6%, respectively. Sample handling and processing appeared to influence MFAP4 measurements only marginally. The average concentration of sMFAP4 in the serum was 18.9 ± 8.4 (SD) U/ml in the twin cohort (95% CI: 18.5-19.4, median sMFAP4 17.3 U/ml). The mean structure model was demonstrated to include waist-hip ratio, age, and cigarette smoking status in interactions with gender. A relatively low heritability of h² = 0.24 was found after applying a model including additive genetic factors and shared and non-shared environmental factors.

Conclusions

The described ELISA provides robust measures of the liver fibrosis marker sMFAP4. The low heritability and the relatively limited basal variation suggest that increased sMFAP4 reflects disease-induced processes.     

Molecular and functional characterization of mouse S5D-SRCRB: a new group B member of the scavenger receptor cysteine-rich superfamily

The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of ～100 aa, named scavenger receptor cysteine-rich (SRCR). Two types of SRCR domains (A or B) have been reported, which differ in the number of coding exons and intradomain cysteines. Although no unifying function has been reported for SRCR-SF members, recognition of pathogen-associated molecular patterns (PAMPs) was recently shown for some of them. In this article, we report the structural and functional characterization of mouse S5D-SRCRB, a new group B member of the SRCR-SF. The s5d-srcrb gene maps at mouse chromosome 7 and encompasses 14 exons extending over 15 kb. The longest cDNA sequence found is 4286 bp in length and encodes a mature protein of 1371 aa, with a predicted M(r) of 144.6 kDa. Using an episomal mammalian-expression system, a glycosylated soluble recombinant form >200 kDa was obtained and used as immunogen for the generation of specific rat mAbs. Subsequent immunohistochemical and real-time PCR analysis showed significant S5D-SRCRB expression in murine genitourinary and digestive tracts. S5D-SRCRB was shown to bind endogenous extracellular matrix proteins (laminin and galectin-1), as well as PAMPs present on Gram-positive and Gram-negative bacteria and fungi. PAMP binding by S5D-SRCRB induced microbial aggregation and subsequent inhibition of PAMP-induced cytokine release. These abilities suggest that S5D-SRCRB might play a role in the innate defense and homeostasis of certain specialized epithelial surfaces.     

Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum

Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.     

Binding between the integrin alphaXbeta2 (CD11c/CD18) and heparin

The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor alphaXbeta2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin alphaXbeta2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the alphaX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 microM affinity for the alphaX I domain. In studies of direct binding by the alphaX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the alphaXbeta2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation.