Expression of myelencephalon-specific protease in transient middle cerebral artery occlusion model of rat brain

Myelencephalon-specific protease (MSP) is one of the serine proteases and is expressed in the central nervous system of rats. Its function and alternation in brain injury have not yet been clarified. In this study, we investigated the expression of MSP after transient middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. In situ localization of MSP mRNA demonstrated a higher level in the corpus callosum and around the ischemic area from 12 h to 14 days after MCA reperfusion, with the peak of expression coming 3 days after reperfusion in both regions. Immunohistochemically, the expression of protein was found 1 day after reperfusion in the same brain region that was observed for mRNA. The peak was 7 days after reperfusion in both regions. Micro-autoradiography, immunostaining and double immunohistochemical labeling revealed the expression of MSP to be located mainly in the oligodendrocytes. The present results indicate that MSP may be related to the turnover of the myelin-associated proteins and the extracellular matrix proteins after transient MCAO. The activation of MSP may play a role in remodeling processes such as neurite outgrowth and remyelination.     

New monoclonal antibody (AIC) identifies interstitial cells of Cajal in the musculature of the mouse gastrointestinal tract

Interstitial cells of Cajal (ICC) are pacemaker cells for the spontaneous muscular contractions and neuromodulators that mediate neurotransmission from enteric neurons to smooth muscle cells in the gastrointestinal (GI) tract. They express c-Kit, and the antibody for c-Kit (especially ACK2) has been a useful tool for functional and morphological studies. ACK2, however, does not work on tissues fixed with paraformaldehyde, and not all ICC express c-Kit in human. Therefore, in order to find a new marker of ICC and/or new antibody resisting aldehyde fixation, we produced a new monoclonal antibody that identifies ICC and then investigated the properties of its antigen. Isolated ICC were used for immunization. Hybridomas fused with myeloma SP2 were screened by immunohistochemistry. ACK2 and each antibody were applied on serial sections, and the clone producing anti-ICC antibody (AIC) that stains ICC was established. The distribution of AIC immunopositive cells was examined in other organs and also GI muscles of W/Wv mice. The biochemical properties were studied using dot blot analysis. AIC recognized ICC; however, distribution of immunopositive cells in W/Wv mice and other organs was different from that of c-Kit. The immunoreactivity was stable for paraformaldehyde but was blocked by either Triton X-100 or SDS. In conclusion, new antibody AIC recognized ICC but the antigen was not c-Kit, which confirms the existence of good markers of ICC besides c-Kit. Although the antigen has not been isolated, AIC is suitable for morphological study and is useful for investigation of ICC in c-Kit mutants.     

Non-invasive methods of assessing the tear film

The interaction between the tear film and the ocular surface epithelium is crucial for the maintenance of ocular surface health; interference with this relationship may cause dry eye. Several diagnostic techniques have been developed to assess the tear film and diagnose dry eye but many of these tests are invasive and modify the parameter which they are designed to measure. Non-invasive or minimally invasive tests may overcome this problem and provide more reproducible and objective data. One test of this kind is meniscometry, which is particularly useful in assessing tear volume indirectly by measuring tear meniscus radius. The newly developed video-meniscometer, which enables calculation of the meniscus radius digitally, is useful for the diagnosis of tear-deficient dry eye. Video-meniscometry also has other applications, to the study of tear and eye drop turnover, determining the indication for punctal plugs and in demonstrating dysfunction of the tear meniscus. Interferometry of the tear film lipid layer is useful in screening and evaluating dry eye severity and in selecting dry eye candidates for punctal occlusion. It is also useful for analysing tear lipid layer pathophysiology more clearly, especially in combination with meniscometry. Meibometry is a minimally invasive technique to quantify the amount of meibomian lipid on the lid margin. Lipid is blotted onto a plastic tape and the change in optical density is used to calculate lipid uptake. Laser meibometry has increased the scope of this technique for the assessment of meibomian gland dysfunction; also, the delivery of lipids from the lid reservoir to the preocular tear film can be analysed using interferometry and laser meibometry. The present report reviews the application of these techniques to the study of tear film physiology and dry eye.     

New surgical methods for punctal occlusion of severe tear-deficient dry eye and its outcome

PURPOSE: Although several surgical methods for punctal occlusion have been reported, it is difficult to obtain complete punctal occlusion in all cases since recanalization of the punctum often occurs after the operation. In this report, new surgical procedures to obtain sufficient occlusion of punctum are introduced and the outcome of the present method is compared with that of previous methods. SUBJECTS AND METHODS: Subjects were 33 severe tear-deficient dry eye patients (4 males and 29 females; mean age: 54.3 yrs.) and they were considered as candidates for punctal occlusion of both upper and lower puncta. The previous surgical punctal occlusion method had been performed in 12 cases (group A: 10 females and 2 males) and the newly-developed method was applied to 21 cases (group B : 19 females and 2 males). Corneal epithelial damage(scored with AD classification; A : area, D : density, scored from 0 to 3) was compared before and after the treatment and the control-lability of dry eye with only an artificial tear preparation was compared between the previous and the new methods. The newly-developed surgical procedures include the following steps: (1) diathermy of the epithelium of the canaliculus using a newly-designed electric needle for diathermy, (2) full removal of the epithelium of the canaliculus using a hand motor drill, (3) cutting the punctum at two opposite points parallel to the lid margin, (4) suturing the punctum to close the cut punctum with 10-0 nylon or 8-0 absorbable sutures. RESULTS: In group A, corneal epithelial damage improved a little from A, D : 2.4, 2.1 to A, D: 1.8, 1.9 during an average of 7.2 months of alone follow-up, and only 2 of the 12 cases obtained control with eye drops. In group B, the corneal epithelial damage improved significantly from A, D : 2.1, 2.2 to A, D : 1.0, 1.3 during an average of 8.2 months of follow-up, and all cases obtained control with eye drops alone. CONCLUSION: The new surgical methods are regarded as a more complete way of performing surgical punctal occlusion compared to previous methods.     

Skp2 overexpression is a p27Kip1-independent predictor of poor prognosis in patients with biliary tract cancers

To better understand the pathogenesis of biliary tract carcinoma (BTC) and to increase the accuracy of predicting outcomes for patients with this disease, we performed molecular cytogenetic analyses of BTC cell lines and tumors to identify non-random amplification(s) and target gene(s) within the amplicons. Among several non random chromosomal aberrations detected in BTC cell lines by comparative genomic hybridization, gain/ampli-fication of DNA at 5p was the most frequently observed alteration. We assessed the copy number and expression status of the possible target gene SKP2 for 5p amplification in cell lines and 33 primary stage II or III tumors of BTC. SKP2 was amplified, and subsequently overexpressed in both cell lines and primary tumors of BTC. However, levels of Skp2 and p27Kip1 proteins were not correlated inversely. Heightened expression of Skp2 and reduced expression of p27Kip1 were both associated with a shorter disease-free and/or overall survival in univariate analyses. In multivariate regression analyses, Skp2 and p27Kip1 were independent predictive factors. Those results suggest that (a) overexpression of Skp2 through an amplification mechanism may contribute to the progression of BTC, (b) not only each molecule, but also the combination of Skp2 and p27Kip1, might be a useful predictor of the prognosis of BTC, and (c) molecular targets of Skp2 other than p27Kip1 may also be important factors in the pathogenesis of this disease.     

Herpes simplex keratitis after ophthalmic surgery

PURPOSE: We report 6 cases of herpes simplex keratitis after ophthalmic surgery, in eyes without clinical history of herpes simplex keratitis. CASES: These cases comprised 6 patients examined at our hospital between April 1992 and November 2001. Past operations were keratoplasty in 5 eyes and cataract surgery in 1 eye. Clinical findings and predisposing factors were evaluated retrospectively. The period between herpetic epithelial keratitis onset and ophthalmic surgery ranged from 1.5 to 79 months. Predisposing factors included corticosteroid therapy and operative wound. The herpetic epithelial lesions were dendritic ulcers in 2 eyes, geographic ulcer in 1 eye, and atypical epithelial lesions in 3 eyes; in all cases, herpes simplex virus (HSV)-DNA was detected by polymerase chain reaction (PCR) in tear fluid. All herpetic epithelial lesions healed with oral and topical acyclovir. CONCLUSIONS: When corticosteroids are used following ophthalmic surgery, physicians should be alert to the possibility of herpetic epithelial keratitis, even in patients with no clinical history of herpes simplex keratitis. PCR detection in tear fluid is helpful in diagnosing this disease.     

Thalidomide is anti-angiogenic in a xenograft model of neuroblastoma

Thalidomide has previously been shown to have anti-angiogenic properties. More recently, clinical efficacy of this agent has been demonstrated in multiple myeloma and prostate cancer. Neuroblastoma is the most frequent solid tumor of the abdomen of childhood, yet children with this disease frequently have metastases at presentation. Such patients have a very poor prognosis with current therapies. Thus, new approaches are needed. We have previously shown that VEGF antagonists can inhibit neoangiogenesis and tumor growth in experimental neuroblastoma. In this study, we investigated the anti-angiogenic and anti-tumor properties of thalidomide in a xenograft model of human neuroblastoma. Tumors were induced in athymic mice using the human neuroblastoma cell line NGP. Intraperitoneal thalidomide (100 mg/kg/dose) or vehicle was administered beginning one week after implantation, and animals euthanized at six weeks. Thalidomide treatment did not significantly alter tumor growth as compared with controls. However, thalidomide suppressed angiogenesis, as demonstrated both by fluorescein angiography and immunohistochemical staining, and induced apoptosis of endothelial cells in neuroblastoma xenografts. Quantification of microvessel density demonstrated a significant reduction of vasculature in treated tumors (p<0.004). Thalidomide induced co-option of host vasculature, an effect noted previously after VEGF blockade. This study demonstrates that thalidomide has anti-angiogenic properties in experimental neuroblastoma.     

Down-regulation of SKP2 induces apoptosis in lung-cancer cells

S-Phase kinase associated protein 2 (SKP2), an F-box protein constituting the substrate-recognition subunit of the SCF^SKP2 ubiquitin ligase complex, targets cell-cycle regulators, such as the cyclin-de-pendent kinase inhibitor p27^KIP1, for ubiquitin-mediated degradation. Our earlier studies indicated frequent amplification and over-expression of the SKP2 gene in primary small-cell lung cancers (SCLCs) and cell lines derived from this type of tumor, and showed that down-regulation of SKP2 expression by means of an antisense oligonucleotide inhibited the growth of SCLC cells in culture (Yokoi et al. Am J Pathol, 161, 207-216, 2002). The anti-sense effect was confirmed in two cell lines of non-small cell lung cancer (NSCLC) that also exhibited over-expression of the gene. In the work reported here, we examined the mechanism(s) responsible for antisense-mediated growth inhibition of SCLC- and NSCLC-derived cultures. SKP2 -antisense treatment not only suppressed DNA synthesis, as determined by [³H]thymidine incorporation, but also induced spontaneous apoptosis characterized by an increase in the sub-G1 population, fragmentation of nuclei, and activation of caspase-3. Our results suggest that since down-regulation of SKP2 appears to induce apoptosis in lung-cancer cells directly, targeting this molecule could represent a promising new therapeutic approach for this type of cancer, and possibly other tumors that over-express SKP2. (Cancer Sci 2003; 94: 344–349)