Examination of toxicity and collagen linearity after the administration of the protein cross-linker genipin in equine tendon and dermis: a pilot study.

OBJECTIVE: Collagen cross-linking is an attractive therapeutic route aimed at supplementing natural collagen stabilisation. In this study the toxicity of the cross-linker genipin (GP) was examined in avascular (tendon) and vascular (dermis) tissue. METHODS: High doses of GP were injected intratendinously into three yearling horses and evaluated at various time points up to 30 days. A second group of three yearlings were injected into the dermis and evaluated at various time points up to 1 year. Metrics used included lameness, circumferential swelling, ultrasound evaluation, microscopic morphology, collagen production and systemic effect on blood parameters. RESULTS: The tendon injection sites exhibited mild lameness and swelling with no apparent systemic toxicity or stabilisation defects. Treated tendons exhibited increased linear collagen microscopically. Dermal injections showed similar results, with mild swelling at the injection site. Microscopic morphology resulted in a decrease in dermal collagen at 30 days post-injection. Dermis injected at the high dose of 355 mmol/L examined 1 year post-treatment appeared similar to the untreated biopsies; however, there was an increase in mature collagen. CONCLUSION: GP injection appeared to be well tolerated, with transient lameness and mild circumferential swelling when injected into the tendon and local tissue swelling when injected into the dermis. No systemic hypersensitivities or toxicities were observed. Microscopically, GP resulted in increased linear collagen in tendons at 30 days post-injection and overall increased collagen in dermal tissue when evaluated 1 year post-injection.

Claudin 2 mRNA and protein are present in human keratinocytes and may be regulated by all-trans-retinoic acid.

Tight junctions are composed of claudins, occludins, junctional adhesion molecules and plaque proteins. Claudin 2 protein forms a cation-selective channel which confers increased permeability in renal epithelial cells and in the intestine where its expression is restricted to the leaky epithelium. Immunohistochemical staining revealed claudin 2 staining in the granular layer of adult epidermis. Analysis of Western blots revealed bands corresponding to the molecular weight of claudin 2 in Madin-Darby canine kidney II cells, human kidney, adult skin and neonatal keratinocytes. Reverse transcriptase polymerase chain reaction on mRNA and cDNA sequence analysis found a 99% sequence homology between our cDNA and human claudin 2 (NIH BLAST sequence). Further, we show that all-trans-retinoic acid increases the expression of claudin 2 in keratinocytes in a dose-dependent manner. The discovery of claudin 2 transcript and protein in the skin could be of importance in epidermal differentiation, barrier function and pathological conditions.

Histochemical and morphological identification of Kupffer cells activated by cisplatin.

Cisplatin is a potent anti-cancer agent which has been shown to activate Kupffer cells. These activated macrophages demonstrate an increase in extensions, lysosomes, and peroxisomes increasing their anti-tumor activity. Wistar rats were treated with cisplatin (9 mg/kg) and sections of liver were excised for light and electron microscopic analysis at 1, 6, 15, and thirty days post treatment. Non-specific esterase staining was used to differentiate Kupffer cells using light microscopy, morphologic criteria were used for TEM analysis. Liver sections taken 6 days post treatment showed the greatest number of activated macrophages, with the highest degree of activation. Interaction between natural killer cells and Kupffer cells was only seen 6 days post treatment. These results show that cisplatin's ability to enhance the immune system requires several days post treatment to reach maximum potency.