Expression of functional TRPV1 receptor in primary culture of canine keratinocytes

The interest for the endovanilloid system and for transient receptor potential vanilloid 1 (TRPV1) is continuously increasing, due to their involvement in inflammation, nociception and pruritus. Even if TRPV1 enrolment was highlighted in both physiological and pathological conditions, some aspects remain unclear, mostly in veterinary medicine. This study aimed to verify the expression and functionality of TRPV1 in canine keratinocytes to investigate in vitro the role of TRPV1 in these cells that are involved in different cutaneous pathologies. Keratinocytes primary cultures were isolated from bioptical samples and cultivated. Binding assay (using 3 [H]-resiniferatoxin), displacement assay (in the presence of 1.2 nM 3 [H]-resiniferatoxin) and functional assays (in the presence of 1 μCi/45 Ca2+ ) with vanilloid agonists and antagonists, specifically addressed to TRPV1 receptor, were performed. Binding assay demonstrated the presence of measurable concentrations of TRPV1 (Bmax = 1,240 ± 120 fmol/mg protein; Kd = 0.01 ± 0.004 nM). Displacement assay highlighted the highest affinity for resiniferatoxin (RTX) and 5-iodo-resiniferatoxin (5-I-RTX), among agonists and antagonists, respectively. The same compounds results as the most potent in the functional assays. This study demonstrated the identification and the characterization of TRPV1 receptor in primary canine keratinocytes cultures. The results are promising for a clinical use, but further in vivo investigations are required

Structure–function relationships of the antigenicity of mycolic acids in tuberculosis patients

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid