Noninvasive delivery of siRNA into the epidermis by iontophoresis using an atopic dermatitis-like model rat.

Topical application of siRNA to the skin should be an effective treatment for serious skin disorders, such as atopic dermatitis. However, it is difficult to introduce hydrophilic macromolecules, including siRNA, into the skin by conventional methods. For efficient delivery of siRNA, we examined an iontophoretic technique, since it is suitable for the delivery of charged molecules. Naked siRNA effectively accumulated in the epidermis (and not in the dermis) after iontophoretic delivery. In contrast, siRNA did not penetrate tape-stripped skin by passive diffusion. In a rat model of atopic dermatitis, skin was sensitized with ovalbumin to stimulate IL-10 mRNA expression as observed in skin lesions. Iontophoretic delivery of anti-IL-10 siRNA significantly reduced (73%) the level of IL-10 mRNA. In conclusion, we successfully delivered naked siRNA into the epidermis and concomitantly suppressed the expression of an endogenous immuno-regulatory cytokine.

[Two cases of digitalis toxicity with reversible and severe decrease of visual acuity].

BACKGROUND: We performed electrophysiologic tests on two patients with digitalis toxicity who first had photophobia and xanthopsia and revealed reversible reduced visual acuity and binocular central scotoma. CASES: The patients were a 72-year-old male and a 54-year-old male who had symptoms of digitalis toxicity. FINDINGS: The corrected visual acuity was severely decreased during digitalis toxicity, 0.02 oculus dexter (OD) and 0.1 oculus sinister (OS) in case 1 and 0.04 OD and 0.2 OS in case 2. But visual acuity recovered as the blood levels of digitalis decreased to the normal level and the corrected visual acuity was 0.7 OD and 0.8 OS in case 1 and 0.8 OD and 0.9 OS in case 2. We recorded 30 Hz-flicker electroretinogram (ERG), single flash ERG, photopic ERG, and scotopic ERG when digitalis blood levels were elevated and normal. Decreased amplitudes of 30 Hz-flicker ERG and photopic ERG suggested that photoreceptor function was disturbed at digitalis toxicity and cone dysfunction was more severely disturbed than rod dysfunction. CONCLUSION: 30 Hz-flicker ERG, as well as electrocardiogram and digitalis blood level, is a relatively convenient and useful measure of digitalis toxicity. It is necessary consiler toxicity when severe visual dysfunction is observed in patients with digitalis therapy.

Do idebenone and vitamin therapy shorten the time to achieve visual recovery in Leber hereditary optic neuropathy?

OBJECTIVES: The authors investigated the effectiveness of idebenone combined with vitamin B2 and vitamin C in the treatment of patients with Leber hereditary optic neuropathy (LHON) in an early stage as compared with untreated patients with LHON. These agents may stimulate the formation of ATP. MATERIALS AND METHODS: For this retrospective study, the authors selected 28 outpatients with LHON from the Keio University Hospital. These patients were followed for 2 to 19 years from disease onset. They were divided into two groups: 14 untreated patients (11778 mutation in 10 patients, 3460 mutation in 2 patients, and 14484 mutation in 2 two patients); and 14 treated patients (11778 mutation in 11 patients, 3460 mutation in 1 patient, and 14484 mutation in 2 patients). The treated patients were administered medical treatment with idebenone, vitamin B2, and vitamin C for at least 1 year. The current study evaluated the following: 1) number of eyes with visual recovery > or = 0.3; 2) interval between the onset of LHON and the beginning of visual recovery; 3) interval between the onset of LHON and visual recovery to 0.3; and 4) interval between the beginning of medical treatment and the beginning of visual recovery in the treated subjects. RESULTS: There was no significant difference in the number of eyes with visual recovery > or = 0.3 in the two groups with the 3460, 11778, or 14484 mutation. Patients with visual recovery showed a fenestrated scotoma or a clearing of central vision. The mean interval between the onset of LHON and the beginning of visual recovery was significantly shorter in the treated group (11.1 months) than in the untreated group (17.4 months) (P = 0.03). The mean interval between the onset of LHON and visual recovery to 0.3 was significantly shorter in the treated group (17.6 months) than in the untreated group (34.4 months) (P = 0.01). The mean interval between the initiation of medical treatment to the beginning of visual recovery was 5.4 months. CONCLUSIONS: Results suggest that the administration of idebenone, vitamin B2, and vitamin C sped the recovery of vision in patients with LHON.

Retinal pigment epithelial cell-based gene therapy against hemoglobin toxicity.

To determine if overexpression of the human heme oxygenase (HO-1) protects retinal pigment (RPE) cells from hemoglobin toxicity, a human RPE cell line was infected by an adenoviral vector containing the HO-1 (Ad-HO-1) gene or transfected with a plasmid containing the cytomegalovirus promoter and HO-1 cDNA (pRc/CMV-HO-1) complexed to cationic liposomes. Phase contrast microscopy and acid phosphatase activity were examined to insure homogeneity of the cell line. Mitochondrial cytochrome and microsomal heme content were measured in both transduced and control cells. RPE cells were then challenged with hemoglobin and their viability estimated. We determined that cells transfected with Ad HO-1 overexpressed HO-1 compared to control cells: HO-1 mRNA levels were increased 3-fold within 3 days, decreasing in 7 days. In addition, we permanently transfected RPE cells with HO-1 gene. Transfected cell clones selected for neomycin resistance had elevated levels of HO activity 3-fold higher than control. Transfected cells exposed to hemoglobin had a survival rate of 93%; non-transfected cells had a 65-75% rate of survival. Transfected cells overexpressing HO-1 proved highly viable when challenged with hemoglobin. HO-1 appears to be an important component of the cellular anti-oxidant defense mechanisms against hemoglobin toxicity. However, the choice of transient or permanent expression of HO-1 against hemoglobin toxicity and hemorrhage needs to be further evaluated.

Two genes, COL4A3 and COL4A4 coding for the human alpha3(IV) and alpha4(IV) collagen chains are arranged head-to-head on chromosome 2q36.

We first isolated and characterized genomic DNA fragments that cover the 5' flanking sequences of COL4A3 and COL4A4 encoding the human basement membrane alpha3(IV) and alpha4(IV) collagen chains, respectively. Nucleotide sequence analysis indicated that the two genes are arranged head-to-head. To determine transcription start site for COL4A4 gene, we performed RACE and RNase protection assays, indicating that there are two alternative transcripts presumably derived from two different promoters. Interestingly, one transcription start site (from exon 1') of COL4A4 is only 5 bp away from the reported transcription start site of COL4A3, whereas the other transcript (from exon 1) starts 373 nucleotides downstream from the first one, generating the two kinds of transcripts that differ in the 5' UTR regions. Expression of these two transcripts appears tissue-specific; exon 1 transcript was expressed predominantly in epithelial cells, while exon 1' transcript showed rather ubiquitous and low expression. The nucleotide sequence of the promoter region is composed of dense CpG dinucleotides, GC boxes, CTC boxes and a CCAAT box but no TATA box. These results provide information to delineate the promoter activity for the tissue-specific expression of the six type IV collagen genes and basement membrane assembly in different tissues and organs.

Spectrum of pathogenic mitochondrial DNA mutations and clinical features in Japanese families with Leber's hereditary optic neuropathy.

PURPOSE: To investigate the incidence and clinical significance of primary or proposed secondary mitochondrial DNA (mtDNA) mutations in Japanese patients with Leber's hereditary optic neuropathy (LHON). METHODS: Blood samples from the 80 unrelated Japanese patients with bilateral optic atrophy were screened for primary LHON mutations. Patients found to have a primary LHON mutation were then tested for 9 proposed secondary LHON mutations. We investigated the association between these mutations and clinical characteristics. RESULTS: Primary mtDNA mutations were identified in 68 patients: at np 3460 in 3 (4%) of 68 patients, at np 11,778 in 59 patients (87%), and at np 14,484 in 6 patients (9%). We identified 5 secondary mtDNA mutations (at np 3394, 4216, 7444, 9438 or 13,708) in 10 (15%) of 68 LHON patients and 3 mutations (at np 3394, 4216 or 3708) in 6 (7%) of 90 healthy Japanese individuals. No patient was positive for more than one secondary mutation. The frequency of secondary mutations was similar in the 68 LHON patients and 90 controls. The clinical features of the Japanese patients with any of the 3 primary LHON mutations were similar to those of Caucasian patients, despite different mtDNA backgrounds in these populations. The percentage of patients with familial LHON harboring the 3460 or 14,484 mutations was lower in the Japanese population. CONCLUSIONS: Japanese patients with LHON exhibited a very high incidence (87%) of the 11,778 primary mutation. Most of the proposed secondary LHON mutations were rare in the Japanese population and they, except the 7444 mutation, may not influence the clinical features of LHON.

Collagen production by cultured human retinal pigment epithelial cells.

We investigated extracellular matrix produced by human retinal pigment epithelial cells (RPE) in vitro using electron microscopy and enzyme linked immunosorbent assay (ELISA). The thickness of the matrix under the cell layer was about 30 microns after 360 days of culture. It consisted mainly of fibrous and granular components. Type IV and V collagen were detected but type I and III were not detected by ELISA. It appeared that RPE can secrete type IV and V collagen and form a thick membrane which may cause proliferative vitreoretinopathy (PVR) by contraction. Control of RPE proliferation and secretion of extracellular matrix is indispensable for prevention of PVR.

High incidence of visual recovery among four Japanese patients with Leber's hereditary optic neuropathy with the 14484 mutation.

The 14484 mutation in the ND6 gene of mitochondrial DNA (mtDNA) is a genetic mutation associated with Leber's hereditary optic neuropathy (LHON) in Caucasian patients who show a high incidence of visual recovery. We evaluated four Japanese patients with LHON associated with the 14484 mutation who were negative for eight proposed secondary mutations. There was no family history of optic atrophy in three of the four patients. All four patients were initially diagnosed as having optic neuritis, either anterior (Cases 1 and 3) or retrobulbar (Cases 2 and 4), based upon their fundus findings and clinical history. Molecular genetic testing of mtDNA confirmed the diagnosis of LHON in all four patients. The three patients who experienced recovery had their vision return to 20/50 or better in both eyes. The patient who did not was a heavy consumer of alcohol and tobacco. These findings indicate that Japanese patients with the 14484 mutation have a visual prognosis similar to that of Caucasians with this mutation.

[Detection of mycobacterial DNA with polymerase chain reaction in eye discharge and gastric juices in a case of scleritis].

We report a case of mycobacterial scleritis in which prompt diagnosis was made by the detection of mycobacterial DNA with polymerase chain reaction (PCR) in eye discharge and gastric juices, when conventional tests were negative. A 77-year-old woman who had a past history of pulmonary tuberculosis visited the outpatient clinic of Tokai University Hospital complaining of pain in her right eye. She was diagnosed as having scleritis and uveitis. There were no indications of active tuberculosis. We examined the gastric juices, sputum, and eye discharge by microscopy, culture, and PCR for detection of mycobacterium. The results of microscopy and culture were negative, but with PCR we detected atypical mycobacterium in eye discharge and gastric juices. After oral treatment with antituberculosis agents, the patient's eye symptoms disappeared. Detecting mycobacterial DNA with PCR could be useful for early diagnosis of mycobacterial scleritis, so that treatment with antituberculosis agents could be started.

High incidence of pre-excitation syndrome in Japanese families with Leber's hereditary optic neuropathy.

Cardiac conduction abnormalities have been reported in families with Leber's hereditary optic neuropathy (LHON). The pre-excitation syndrome Wolff-Parkinson-White syndrome or Lown-Ganong-Levine syndrome, is reportedly common in Finns with LHON, being seen in 14 (9%) of the 163 individuals with mitochondrial DNA (mtDNA) mutations. While this syndrome is thought to be rare in other ethnic groups with LHON, the present study of 35 Japanese LHON families confirmed that it is also relatively common among Japanese families, being seen in 5 (8%) of the 63 individuals with mtDNA mutations. It remains to be determined whether the high incidence of the pre-excitation syndrome in Finnish and Japanese LHON families is due to a particular genetic composition of ethnic groups such as in Finland and in Japan, or only to a reporting bias.

[A histopathological study of corneal amyloidosis secondary to trichiasis].

We present a case of secondary corneal amyloidosis whose etiological mechanism was investigated by immunohistochemistry and electron microscopy. A 48-year-old woman had suffered from trichiasis in the right eye for 35 years, and developed secondary corneal amyloidosis, a phenomenon previously described but whose etiological mechanism has not been explained. Slitlamp examination of the cornea revealed a white excrescence with a diameter of 2 mm. The lesion was excised and examined by light and electron microscopy. Large deposits of an amorphous eosinophilic material were observed beneath the atrophic epithelium. Amyloid was detected in these deposits using Congo red stain, polarized light, and electron microscopy. Neither vascularization nor infiltration of inflammatory cells was observed. Immunohistochemical tests for protein AL, protein AA, prealbumin, beta 2-microglobulin and cytokeratin in paraffin sections were all negative. Characteristic findings were observed in the border zone between the basal cells and the deposits. Numerous digitiform cell processes and membrane-bound globular fragments of basal cells were seen in the superficial region of the deposits. The cell membrane of some globules was interrupted and the contents appeared to have been discharged into the stroma. These findings suggest that basal cells of the corneal epithelium provide an amyloid precursor on the stroma.

[Evaluation of risk factors of interferon-associated retinopathy in patients with type C chronic active hepatitis].

The risk factors of retinopathy associated with administration of interferon have not been fully clarified. We prospectively examined the retinal condition in 50 patients with type C chronic active hepatitis during alpha-interferon treatment. 43 patients (86%) were shown to have retinopathy during the course of interferon treatment, and were divided into three groups. Grades I, II and III were patients having a single episode of transient retinopathy with soft exudate or hemorrhage (34%), frequent episodes of retinopathy (42%), and exacerbating retinopathy requiring change or cessation of interferon treatment (10%), respectively. The patients with grade II and III were found to have the first retinal changes within 8 weeks after initiation of the interferon therapy. Early onset of retinopathy and presence of systemic disease such as diabetes mellitus or hypertension were risk factors for serious retinopathy with statistical significance. The grades of retinopathy were also well correlated with dosage and duration of interferon treatment. These results suggest that careful fundus examination is required up to 8 weeks after initiation of interferon treatment, especially for the patients with risk factors such as early onset of retinopathy, presence of systemic diseases, and large dosages and long duration of interferon therapy, in order to prevent serious ocular complications.

Histochemical demonstration of acid phosphatase in a human retinal pigment epithelial cell line.

The retinal pigment epithelium (RPE) has numerous lysosomes and shows the highest acid phosphatase activity among eye tissues. In an established cell line of human RPE (K-1034), acid phosphatase activity, as a lysosome marker was investigated histochemically to evaluate the maintenance of original RPE characteristics. Even after 100 passages, RPE cells showed remarkable enzyme activity, and had numerous electron-dense bodies which were confirmed to be lysosomes. These results show that this cell line can maintain one of the vital functions of the RPE and provide useful experimental material for functional studies of the human RPE.

Inhibition of corneal ulceration by tetrapeptidyl hydroxamic acid.

The inhibitory activity of a new peptidyl collagenase inhibitor, FN-439 or tetrapeptidyl hydroxamic acid (H2N-C6H4-CO-Gly-L-Pro-D-Leu-D-Ala-NHOH), was determined against vertebrate collagenases derived from human fibroblast, human polymorphonuclear leukocyte (PMN) and tadpole skin. In addition, the effect of FN-439 in inhibiting corneal ulceration was also investigated with alkali-burned rabbit corneas. FN-439 can block the active site of collagenase, and hydroxamic acid can chelate Zn2+ which is essential for collagenase activity. Furthermore, this compound contains D-amino acids to resist nonspecific host-derived degradative enzymes. In our experiments, corneal ulceration occurred in 5 of the 9 control eyes, but in none of the 9 eyes treated with FN-439 (P < 0.01). The only cellular elements observed at the ulcerated area were PMNs and monocytes. FN-439 appeared to act against PMN collagenase. In addition, we compared the change in the concentration of FN-439 (D-peptide) and the L-form of FN-439 (L-peptide) in aqueous humor aspirated from the rabbit eyes burned with alkali. After incubation for 3 hours, the concentration of the D-peptide was decreased by 3%, while that of the L-peptide was decreased by 60%. FN-439 may be useful for treating noninfectious corneal ulcers because of its potent activity (IC50 = 1 microM) and chemical and biological stabilities.

Mitochondrial induction of adult T cell leukemia derived factor (ADF/hTx) after oxidative stresses in retinal pigment epithelial cells.

PURPOSE: Adult T cell leukemia derived factor (ADF) is a human homologue of thioredoxin (hTx), which exhibits scavenging activity with reactive oxygen intermediates. In their previous study, the authors found that after transient retinal ischemia, the expression of thioredoxin in rat retinal pigment epithelium (RPE) layer increased markedly. The present investigation is to determine intracellular ADF localization in RPE after transient ischemia and in cultured human RPE cells after oxidative insult by H2O2. METHODS: The authors employed immunoelectron microscopy to examine ADF localization in RPE. Labeling density analysis was performed to supplement the main observation in the experiment of transient retinal ischemia. 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) assay was performed to verify the protective role of recombinant ADF (rADF) against H2O2. RESULTS: In immunogold electron microscopy, sparse ADF-positive labeling was seen in the cytosol and mitochondria in normal rat RPE and in untreated cultured RPE cells. After oxidative stress, it was concentrated in mitochondria in both groups. MTT assay proved that rADF protected cultured RPE from the toxicity of H2O2. CONCLUSIONS: This study shows the induction of ADF/hTx in mitochondria of RPE after oxidative stresses and its protective effect on cultured RPE exposed to H2O2. The data indicate the possibly important role of ADF/hTx in the protection of retinal cells from the oxidative stresses associated with retinal ischemic disease and probably with regular visual activity.

Re-evaluation of the colorimetric assay for cytidine deaminase activity.

This study re-evaluated the colorimetric assay for cytidine deaminase (CTD), and showed that the optimum conditions were pH 7.5, 37 degrees C, and up to 24 h. In addition, this method was found to require protein precipitation. Following these modifications, intra-assay and inter-assay coefficients of variation were below 5 per cent, indicating that the assay was highly reliable. CTD activity was determined in 282 serum samples from 206 normal pregnant women by the incubation of 100 microliters of serum and 400 microliters of 1.4 mmol/l cytidine substrate for 16 h at 37 degrees C. Following protein precipitation, the ammonia liberated during conversion was measured by a colorimetric procedure. The mean (+/- SD) CTD activity was 7.31 +/- 2.50 U at 3-12 weeks of gestation, 8.70 +/- 2.12 U at 13-24 weeks, 7.59 +/- 2.25 U at 25-36 weeks, and 7.29 +/- 2.16 U at 37-42 weeks. High levels of CTD activity were found in patients with abruptio placentae and amnionitis associated with intrauterine fetal death (IUFD). The increase in CTD activity was noted from 3 days to 1 week before the confirmation of IUFD. The placenta contains extremely high levels of CTD, but cord serum does not. Thus, the excessive elevation of CTD activity was probably derived from progressive placental damage. This modified CTD assay was concluded to be simple and reliable, and may perhaps be useful in detecting pregnancy disorders.

Morphologic and chromosomal study of a human retinal pigment epithelial cell line.

A spontaneously established cell line of human retinal pigment epithelium (RPE) was investigated morphologically and studied by chromosomal analysis. Even after the 100th passage, cultured cells maintained epithelioid morphology, and had numerous microvilli and membrane-bound small dense bodies which looked like lysosomes. Abundant rough-surfaced endoplasmic reticulum and mitochondria were observed in the cytoplasm, suggesting that these cells were metabolically active. The absence of pigmentation was noted from the 6th passage. In chromosomal analysis, a small number of 2nd passage cells showed karyotypic changes. At the 12th passage, a single cell line (44, 6-monosomy and Y-missing) was established. By the 100th passage, further karyotypic changes were noted (low tetraploid). The 6-monosomy might have played an important role in the establishment of this cell line. These results indicated that this cell line underwent some changes in chromosomal count and pigmentation but retained many original morphological characteristics. After further evaluation of its characteristics, this cell line, K-1034, could be useful experimental material in the study of the function of the human RPE.

Cytidine deaminase activity in abnormal pregnancy.

OBJECTIVE: To determine the usefulness of cytidine deaminase (CTD) activity in the prediction of abnormal pregnancy and the prognosis of fetal outcome. METHOD: The CTD activities in 367 pregnant women and 17 placentas and their cord sera were determined. The CTD activity was estimated to determine the amount of ammonia liberated during conversion of cytidine into uridine. The Kruskal-Wallis test and paired t-test were employed for statistical comparisons. RESULT: The placentas contained extremely high levels of CTD activity, but the cord serum did not. The mean value of CTD activity in abnormal pregnancy women was significantly higher than in normal pregnancy women. The two cases with a CTD activity of 40 U or more had the worst infant prognosis. CONCLUSION: The CTD activity in abnormal pregnancies was excessively elevated due to a damaged placenta under progressive deterioration. This CTD assay was simple and had predictive value for the prognosis of an infant of an abnormal pregnancy.

Superior altitudinal hemianopia and herpes zoster.

A healthy 41-year-old women had acute retrobulbar optic neuritis with superior altitudinal hemianopia four weeks after cutaneous herpes zoster. Her visual acuity decreased to 0.04 OD, and mild iritis was noticed. However, ophthalmoscopic examination and fluorescein angiography disclosed no remarkable change. She had a low amplitude during flash visual-evoked potential testing with almost normal latency time in the acute stage. Corticosteroid therapy was administered, and her visual acuity and visual-field defect rapidly improved. Complete recovery, including the pattern-reversal visual-evoked potential results, was obtained.

Urinary surge of human luteinizing hormone in infertile women.

An improved semiquantitative immunoassay method allows human luteinizing hormone (hLH) in urine to be detected from 2.5 to 640 mIU/ml has developed. With this method, the hLH surges in every 3 h urine samples while awake were measured in 8 normal and 12 infertile women. The result was that the urinary hLH surge in infertility was insufficient regardless of the high urinary E2 value. This result may play, at least in part, an etiologic role in infertility.