Antiviral effect of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine on adenovirus

PURPOSE: Adenovirus is the most frequent causative virus of conjunctivitis in Japan. Recently (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (HPMPC) has been promoted as a new drug against adenoviral conjunctivitis. So we examined the antiviral activity of HPMPC against adenoviruses in vitro. METHOD: The antiviral activity of HPMPC against adenovirus (Ad) type 3, type 4, type 19, and type 37 isolated from conjunctivial scrapings in Japan and the prototype of adenovirus type 5 was examined by plaque reduction assay using A 549 cells in vitro. RESULTS: The 50% inhibitory dose (ID50) of HP-MPC was 3.50 (1.44-4.79) micrograms/ml for Ad type 3, 4.50 (4.17-4.92) micrograms/ml for Ad type 4, 2.11 (1.03-3.13) micrograms/ml for Ad type 5, 1.64 (1.40-2.02) micrograms/ml for Ad type 19, and 2.02 (1.17-2.73) micrograms/ml for type 37. The 50% cytotoxic dose of HPMPC for A 549 cells was 205 micrograms/ml by the deoxythimidine uptake inhibition test, and 537 micrograms/ml by the trypan blue exclusion inhibition test. CONCLUSIONS: HPMPC proved to be highly effective in inhibiting replication of adenoviruses at lower concentrations than the cytotoxic level in vitro.     

TGF-beta1 stimulates production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) by human corneal epithelial cells

Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of ocular surface diseases. This study investigated the regulated expression of gelatinases (MMP-2 and -9), collagenases (MMP-1 and -13) and stromelysins (MMP-3, -10, and -11) by TGF-beta1 in cultured human corneal epithelial cells. Primary human corneal epithelial cell cultures were grown to confluence and treated with different concentrations (0.1, 1.0, 10 ng ml(-1)) of TGF-beta1 in serum-free medium for 6-24 hr. Total RNA was isolated from cultured cells with or without TGF-beta1 treatment for 6 hr and subjected to semi-quantitative RT-PCR and Northern hybridization. Conditioned media were collected from cultures with or without TGF-beta1 treatment for 24 hr to evaluate the MMP production by ELISA and activity assays. Semi-quantitative RT-PCR revealed that the expressions of MMP-9, -1, -13, -3, -10 and -11 mRNA were up-regulated by TGF-beta1 in a concentration-dependent fashion, while MMP-2 and MMP-14 production did not change. Northern hybridization confirmed these findings. Gelatin zymography, MMP ELISA and activity assays showed concentration-dependent stimulated production and activity of MMP-9, -1, -13, -3 and -10 protein in the conditioned media of cultures treated for 24 hr with TGF-beta1. In conclusion, our results demonstrate that TGF-beta1 stimulates the expression and production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) in human corneal epithelial cells. These findings suggest that TGF-beta1 may play a role in the pathogenesis of MMP mediated ocular surface diseases, such as sterile corneal ulceration.     

Lack of retinal toxicity of the anticytomegalovirus drug (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine.

The drug (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (HPMPC) is an antiherpesvirus group compound with a higher potency and longer duration of action against human cytomegalovirus (CMV) than ganciclovir or foscarnet. Twenty eyes of ten New Zealand white rabbits received 0.1-ml injections of either normal saline or HPMPC at doses of 10, 50, 100, 300, or 1000 micrograms. The animals were killed on days 14 and 28. Toxicity was assessed by indirect ophthalmoscopy, electroretinography (ERG), and light and electron microscopy. Both a- and b-wave ERG findings and indirect ophthalmoscopic appearance of retinas in all groups were normal. Light and electron microscopy of perfusion-fixed retinal tissue revealed no morphologic changes at doses of 100 micrograms or lower. The pharmacokinetics of eight rabbits injected intravitreally with 100 micrograms of HPMPC showed a 24.4-hr half-life for the drug. These results indicate that HPMPC is not toxic to the rabbit retina at 500-1000-fold the dose that is effective in suppressing CMV infections. Doses of 100 micrograms also were injected into the vitreous of monkey eyes. Intravitreal injections of HPMPC may be efficacious in inhibiting CMV retinitis for longer dosing intervals than can be used with other anti-CMV compounds.     

Reversed needle pass clear-corneal or limbal incision suturing technique using the 3-throw (1-1-1) adjustable square knot

A single radial suture is required for a corneal or limbal incision that does not seal despite stromal hydration. In the traditional technique for placing this suture, the needle enters from the corneal side of the limbal incision and exits toward the scleral side and the suture is usually tied with a 3-1-1 surgical knot. We present an improved suturing technique in which the needle path is reversed. The needle enters on the scleral side of the limbal incision, exits on the corneal side toward the apex, and is tied with an adjustable 1-1-1 knot.     

(S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine in the therapy of thymidine kinase-positive and -deficient herpes simplex virus experimental keratitis.

The phosphonylmethoxyalkyl derivative, (S)-1-(3-hydroxy-2-phosphonyl methoxypropyl)cytosine (HPMPC), was evaluated for its efficacy in the topical treatment of experimental keratitis caused by thymidine kinase-positive (TK+) or thymidine kinase-deficient (TK-) herpes simplex virus type 1 (HSV-1) strains. The HPMPC 0.2% eyedrops were as effective as the reference compound, (E)-5-(2-bromovinyl)-2'-deoxyuridine, (BVDU) 0.2% eyedrops in stimulating the healing of epithelial disease caused by the HSV-1 TK+ strain. Both drugs achieved a significant (P less than 0.005) healing effect compared with placebo eyedrops. No significant differences were noted in the efficacy of HPMPC 0.2% eyedrops when instilled one, three, or nine times a day. In the treatment of keratitis caused by the HSV-1 TK- strain, 0.2% BVDU eyedrops were similar to placebo; 0.2% HPMPC eyedrops again had a brisk and significant healing effect (P less than 0.005).     

Matrix metalloproteinase (MMP-2, -9) and tissue inhibitor (TIMP-1, -2) activity in tear samples of pediatric type 1 diabetic patients

Background

The presence of matrix metalloproteinase (MMP-2, -9) and tissue inhibitor (TIMP-1, -2) activity in tear samples of pediatric type 1 diabetes mellitus (DM) patients and potential correlations with clinical parameters (Schirmer testing, glycosylated hemoglobin-HB_A1C) were investigated.

Methods

Tear samples from the right eyes of 27 type 1 DM patients and 17 healthy control subjects were included in this study. The MMP gelatinolytic activity was determined by gelatin zymography analysis using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), while MMP and TIMP concentrations (in ng/ml) were quantified in tears of type 1 diabetic patients and healthy controls, with the use of enzyme-linked immunosorbent assay (ELISA).

Results

MMP-9, TIMP-1, -2 levels, MMP-9/TIMP-1, and MMP-9/TIMP-2 ratios in the patient group were significantly elevated. There was a significant correlation between TIMP-2 and HB_A1C values, as well as between MMP-2 and MMP-9.

Conclusions

Significant correlations between TIMP-2 and HB_A1C and between Schirmer test results and HB_A1C were revealed. Significant increase in tear MMP and TIMP levels in pediatric type 1 diabetic patients may be suggestive of disease progression and localized pathologic remodelling. Further studies are required in order to ascertain whether MMPs and TIMPs could be employed as indicators of early disease progression.     

近视眼角膜上皮细胞EGF R、TGF-βRⅠ及IL-1RJ的表达

目的:检测近视患眼角膜上皮细胞表皮生长因子受体(EGF R)、转化生长因子βⅠ型受体(TGF-βRⅠ)和白细胞介素1Ⅰ型受体(IL-1RⅠ)的表达.方法:将PRK术中获取的角膜上皮细胞制成细胞涂片,ABC法检测.结果:EGF R、TGF-βRⅠ和IL-1RⅠ在全部标本表达,强度依次为EGF R＞IL-1RⅠ＞TGF-βRI.结论:近视患眼角膜上皮细胞表达此三种受体,推测PRK术后此三种受体参与角膜创面修复.     

Changes in the balance of the tissue inhibitor of matrix metalloproteinases (TIMPs)-1 and -3 may promote keratocyte apoptosis in keratoconus

Keratoconus is a disease in which the central cornea becomes thinned. This could result from corneal stromal cell apoptosis or be induced or perpetuated by the activation of matrix degrading enzymes, particularly members of the matrix metalloproteinase (MMP) family. In some circumstances, the MMP inhibitors TIMP-1 and TIMP-3 exhibit anti-apoptotic and pro-apoptotic properties, respectively. Because they potentially influence keratoconus progression, the effects of TIMP-1 and TIMP-3 on stromal cell viability were investigated. The TIMP-1 and TIMP-3 proteins were over-expressed in cultured corneal stromal cells by using the adenoviral vectors RAdTIMP-1 and RAdTIMP-3 and quantified by enzyme-linked immunosorbant assay (ELISA). Apoptotic cells were detected by TUNEL and caspase-3 activity. The anti-apoptotic effects of TIMP-1 were investigated by co-infecting it with RAdTIMP-1 and RAdTIMP-3 and by adding TIMP-1 protein to stromal cell cultures prior to infecting them with RAdTIMP-3. Immunohistochemistry was used to localise and determine relative numbers of apoptotic and TIMP producing stromal cells in sections of normal and keratoconic corneas. The results showed that over-expression of TIMP-3 induced apoptosis in corneal stromal cell cultures. Up-regulated TIMP-1 production or the addition of exogenous TIMP-1 protein prevented stromal cell overgrowth, changed stromal cell morphology and reduced the extent of TIMP-3 induced apoptosis. Localised relative concentrations of TIMP-1/TIMP-3 could thus determine whether these cells remain viable or become apoptotic. This may be relevant to the keratoconic condition since significantly more apoptotic cells were identified in the anterior stroma of keratoconic corneas than normal corneas and the majority of theTIMP-1 and TIMP-3 producing stromal cells were also located in this region.