New Grocott Stain without Using Chromic Acid.

We established a new "ecological" Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide.

Involvement of leukotriene B4 in itching in a mouse model of ocular allergy.

Itching of ocular allergy is alleviated but not completely relieved by H(1) histamine receptor antagonists, suggesting that histamine is not the sole itch mediator in ocular allergy. We investigated whether leukotriene B(4) (LTB(4)), a mediator of cutaneous itch, is involved in the itch of ocular allergy in mice. Mice were immunized by the repeated subcutaneous injections of ragweed pollen and alum into the caudal back, and given a subconjunctival injection of ragweed pollen extract into the palpebra for allergic challenge. Challenge with ragweed pollen extract markedly elicited ocular scratching in sensitized mice. The scratching was almost abolished by mast cell deficiency. The H(1) antagonist terfenadine partially inhibited scratching at a dose that almost completely suppressed plasma extravasation. Scratching was inhibited by the glucocorticoid betamethasone and the 5-lipoxygenase inhibitor zileuton at doses that inhibited the challenge-induced production of LTB(4). A subconjunctival injection of LTB(4) at doses 1/10,000 or less than that required for histamine elicited ocular scratching in naïve mice. The LTB(4) receptor antagonist ONO-4057 inhibited the ragweed pollen challenge-induced ocular scratching at doses that suppressed LTB(4)-induced ocular scratching. In addition to histamine, LTB(4) is involved in the ocular itching of pollen allergy. H(1) receptor antagonists with an inhibitory effect on the action and/or production of LTB(4) may have more potent anti-pruritic activity than selective H(1) antagonists.

Stimulation of corneal epithelial migration by a synthetic peptide (PHSRN) corresponding to the second cell-binding site of fibronectin.

PURPOSE: Fibronectin plays an important role in the migration of corneal epithelial cells in vivo. The Arg-Gly-Asp (RGD) sequence in the principal cell binding domain of fibronectin mediates the interaction of fibronectin with integrins, whereas the Pro-His-Ser-Arg-Asn (PHSRN) sequence of fibronectin is thought to modulate this interaction. The authors examined the effects of a PHSRN peptide on corneal epithelial migration in vitro and in vivo. METHODS: Epithelial migration in vitro was examined with the rabbit cornea in organ culture. The motility and phenotype of simian virus 40-transformed human corneal epithelial (HCE) cells were evaluated by time-lapse and immunofluorescence microscopy, respectively. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin was examined by immunoprecipitation and immunoblot analysis. The healing of rabbit corneal epithelial wounds induced by 1-heptanol was evaluated by fluorescein staining. RESULTS: The PHSRN peptide stimulated corneal epithelial migration in organ culture in a concentration-dependent manner, and it increased HCE cell motility in vitro. The peptide induced the accumulation of F-actin and the formation of focal adhesions at the leading edge of HCE cells. It also upregulated the tyrosine phosphorylation of FAK and paxillin in HCE cells, but it did not affect HCE cell proliferation or attachment to a fibronectin matrix. Administration of the PHSRN peptide in eye drops promoted corneal epithelial wound closure in vivo in a dose-dependent manner. None of these effects of the PHSRN peptide were induced by a control NRSHP peptide. CONCLUSIONS: The PHSRN peptide mimics many of the effects of fibronectin on corneal epithelial cells and may prove suitable as a substitute for fibronectin in the treatment of persistent corneal epithelial defects.