Ocular toxicity following topical application of anesthetic cream to the eyelid skin

BACKGROUND AND OBJECTIVE: The use of topical anesthetic cream in the periorbital region may be of clinical value. The potential for toxic effects from such use has not been studied in a controlled manner. This study was performed to evaluate the potential ocular toxicity of anesthetic cream topically applied to the eyelid in an animal model. MATERIALS AND METHODS: Ten rabbits underwent periorbital eutectic mixture of local anesthetics (EMLA) (2.5 percent lidocaine and 2.5 percent prilocaine) application and were observed for evidence of gross or microscopic ocular toxicity. Baseline external and anterior segment examinations were performed, including biomicroscopy and fluorescein staining, after which a standard quantity of EMLA cream (0.75 g) was applied along the upper eyelid and covered with an occlusive dressing. After 1 hour of treatment, the eyelid and anterior segment were examined for evidence of adverse reaction. The eyelids were excised and examined histopathologically. RESULTS: No significant adverse effects were noted on external lid and anterior segment examination. The histopathologic findings were within normal limits. CONCLUSIONS: This study suggests that external application of EMLA cream to the eyelid does not induce local toxicity in the rabbit model. The external application of EMLA cream may be safe in the periorbital region.     

7-Hydroxystaurosporine (UCN-01) induces apoptosis in human colon carcinoma and leukemia cells independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Persistent GAD 65 antibodies in longstanding IDDM are not associated with residual beta-cell function, neuropathy or HLA-DR status

Persistent humoral autoimmunity to the enzyme glutamic acid decarboxylase (GAD) has been described in a substantial proportion of patients with insulin-dependent diabetes mellitus (IDDM) of long duration. The source of the stimulus for this autoimmune reactivity is still unknown. Because the GAD 65 isoform is mainly expressed in pancreatic beta-cells and in the nervous system we investigated in the present study of the largest number of well characterized patients with longstanding IDDM (n = 105; median duration: 21 years; range: 10-46 years) the presence of autoantibodies to GAD 65 and their relationship to a residual C-peptide response or peripheral and autonomic neuropathy. Additionally we studied the HLA-DR status relative to GAD 65 antibodies in 86 out of the 105 individuals. One hundred healthy control subjects and 100 recent onset IDDM patients were also studied for GAD 65 antibodies. GAD 65 antibodies were detected in a radioligand-binding-assay with recombinant human GAD 65 and were present in 32% of the long-term diabetic patients, 82% of the recent onset IDDM patients and in 3% of the healthy control subjects. A preserved C-peptide response to i.v. glucagon (Hendriksen criteria) was observed in 23% of the long-term IDDM patients. Autonomic neuropathy and peripheral neuropathy was identified using criteria based on both symptoms and formal testing giving a frequency of 67% vs 79%. The HLA specific DR 4/X was observed in 47% and HLA-DR 3/X in 22% of the long-term IDDM patients. Patients who were heterozygous for DR3/DR4 were found in 23% of the cases. GAD 65 antibodies were significantly less frequent in the long-term IDDM patients compared to recent onset IDDM (p < 0.001), and diabetes duration showed a significant negative correlation with GAD 65 antibody index levels (r = 0.22, p < 0.01). Interestingly, GAD 65 antibodies were not significantly correlated either with residual beta-cell function or neuropathy and no particular HLA-DR status was associated with persistent GAD 65 antibodies. In conclusion neither residual beta-cell function nor diabetic neuropathy or a certain HLA-DR specificity are exclusively associated with persistent autoimmunity directed to GAD 65 in longstanding IDDM. The stimulus for the persistent humoral immune response and its significance for the disease process and its complications remain to be established.     

Activation of PKCalpha downstream from caspases during apoptosis induced by 7-hydroxystaurosporine or the topoisomerase inhibitors, camptothecin and etoposide, in human myeloid leukemia HL60 cells

We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKCalpha, betaI, betaII, delta, and zeta activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKCzeta and inhibited kinase activity of PKCbetaI, betaII, and delta. PKCalpha activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKCalpha activity during apoptosis in HL60 cells. However, CPT did not affect PKCbetaI, betaII and zeta, and activated PKCdelta. PKCalpha activation was not due to increased protein levels or proteolytic cleavage but was associated with PKCalpha autophosphorylation in vitro and increased phosphorylation in vivo. We also found that not only PKC delta but also PKC betaI was proteolytically activated in HL60 cells during apoptosis. The PKCalpha activation and hyperphosphorylation were abrogated by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKCdelta and betaI proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKCalpha and proteolytic activation for PKC delta and betaI.