In Vivo Methods for the Assessment of Topical Drug Bioavailability

This paper reviews some current methods for the in vivo assessment of local cutaneous bioavailability in humans after topical drug application. After an introduction discussing the importance of local drug bioavailability assessment and the limitations of model-based predictions, the focus turns to the relevance of experimental studies. The available techniques are then reviewed in detail, with particular emphasis on the tape stripping and microdialysis methodologies. Other less developed techniques, including the skin biopsy, suction blister, follicle removal and confocal Raman spectroscopy techniques are also described

The pervasive effects of recombinant Fasciola gigantica Ras-related protein Rab10 on the functions of goat peripheral blood mononuclear cells

Background: Fasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats.Methods: We cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0.Results: The FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-β) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway.Conclusions: rFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite

In Situ Inner Lumen Attachment of Heparin to Poly(ether sulfone) Hollow Fiber Membranes Used for Microdialysis Sampling

An in situ chemical surface modification method to attach heparin to the inner lumen of a single hollow fiber poly­(ether sulfone) (PES) membrane incorporated into a commercial microdialysis sampling device is described. The immobilization process uses gentle, room-temperature conditions with the enzyme laccase (E.C. 1.10.3.2) and 4-hydroxybenzoic acid (4HBA). The resulting functionalized inner membrane surface with a carboxylic acid functional groups allowed for (1-ethyl-3-(3-(dimethylamino)­propyl) carbodimide)/<i>N</i>-hydroxysuccinimide) EDC/NHS chemistry to attach heparin to the membrane surface. X-ray photoelectron spectroscopy measurements suggested successful attachment of 4HBA polymers and heparin onto the PES membrane. The microdialysis extraction efficiency after membrane surface modification was measured with model compounds fluorescein isothiocyanate (FITC)-labeled dextrans and lysozyme and the cytokines acidic fibroblast growth factor (aFGF) and CXCL1 (KC/GRO). This work demonstrates an in situ method to modify commercially available PES hollow fiber microdialysis membranes with amine or carboxylic acid functional groups

How minimally invasive is microdialysis sampling? A cautionary note for cytokine collection in human skin and other clinical studies.

It is common to refer to microdialysis as a minimally invasive procedure, likening it to insertion of an artificial capillary. While a comparison of this type allows the process to be easily visualized by those outside the field, it tends to provide a false impression of the localized perturbation of the tissue space that is caused by catheter insertion. With the increased acceptance of microdialysis sampling as a viable in vivo sampling method, many researchers have begun to use the technique to explore inflammatory and immune-mediated diseases in the skin and other organs. Unfortunately, many of the molecules of interest, particularly chemokines and cytokines, are known to be generated during the inflammatory response to wounding and the subsequent cellular events leading to wound repair. With more than 11,000 reports citing the use of microdialysis sampling, only a few researchers have sought to assess the tissue damage that is incurred by probe insertion. For this reason, caution is warranted when collecting these molecules and inferring a role for them in clinical disease states. This commentary seeks to remind the research community of the confounding effects that signaling molecules related to the wounding response will have on clinical studies. Proper controls must be incorporated into all studies in order to assess whether or not particular molecules are truly related to the disease state under investigation or have been generated as part of the tissue response to the wound incurred by microdialysis catheter implantation