L-DOPA produced nitric oxide in the vitreous and caused greater vasodilation in the choroid and the ciliary body of melanotic rats than in those of amelanotic rats.

The nitrogen cycle initiates direct reduction of N2 to NH3 by enzymatic reactions. We hypothesize that L-dihydroxyphenylalanine (L-DOPA), a catecholamine, could be a source of nitric oxide (NO). In order to determine whether L-DOPA generates NO and induces any biological change in the eye, we measured the generation of NO in vitro and in vivo, and investigated the histopathological changes caused by injection of L-DOPA into the vitreous of rats. We also hypothesized that melanin granules may affect the generation of NO during the metabolism of L-DOPA, since L-DOPA is a precursor of melanin in the brain and the eye. Therefore, we compared the effects of L-DOPA on the generation of NO between amelanotic and melanotic rats. NO was measured as diffusion currents by NO electrodes. In vitro, various concentrations of L-DOPA (5, 29.9, 79.4, 152.7, and 249 microM) were added to the medium. The inhibition of NO generation by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was tested. In vivo, NO generation in the vitreous of rats was measured and the eyes were enucleated under anesthesia after L-DOPA injection. The ocular tissues were subjected to histological examination. NO was produced from L-DOPA in a dose-dependent manner and was scavenged by carboxy-PTIO in vitro. NO in the vitreous of melanotic rats was generated from L-DOPA. Histological examination with hematoxylin-eosin staining revealed vasodilation in the ciliary vessels and the choroid after L-DOPA injection. Both effects were greater in melanotic rats than in amelanotic rats. The vasodilation may be attributable to NO as well as to superoxides, which can be regulated by the existence of melanin.

Melanin granules prevent the cytotoxic effects of L-DOPA on retinal pigment epithelial cells in vitro by regulation of NO and superoxide radicals.

Inasmuch as the nitrogen cycle elicits the direct reduction of N2 to NH3 through enzymatic reactions, and inasmuch as L-DOPA (L-dihydroxyphentlalamine), a catecholamine, can be a source of nitric oxide (NO), it is possible that melanin granules in the eye affect the generation of NO, which causes damage to the retinal pigment epithelial (RPE) cells during the oxidation of L-DOPA. In order to confirm this possibility, we analyzed the correlations of NO generation, cell growth, and superoxide dismutase (SOD) activities in two types (melanotic and amelanotic) of bovine RPE cells following exposure to L-DOPA. NO generation from L-DOPA was determined using an NO detector that is reliant on redox currents. The concentration of NO was measured in terms of diffusion currents run between a working electrode and a counter electrode, both being set in culture medium placed in a Petri dish. For the assays, L-DOPA was added to the medium at various concentrations (5, 29.9, 79.4, 152.7 or 249 microM), and 6 min after addition, an NO-trapping agent 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was also added. The melanotic and amelanotic types of RPE cells were cultured separately in medium with L-DOPA under an atmosphere containing 20, 10 or 5% oxygen. Cell numbers were counted using a Coulter counter, and SOD activities were determined following incubation for 24, 48 or 72 hr using a modification of the luminol assay. The results obtained indicated that: (a) NO was produced from L-DOPA in a concentration-dependent manner and was trapped quantitatively by carboxy-PTIO; (b) the generation of NO was inhibited more markedly in the melanotic cell line than in the amelanotic one, suggesting an increased tolerance to L-DOPA-derived cytotoxicity in the former; and (c) the SOD activities were more affected by oxygen concentration in the melanotic cells than in the amelanotic ones. From these results, it is concluded that melanin granules in RPE cells have a role in preventing the cytotoxicity derived from L-DOPA and in regulating the generation of NO and superoxide radicals.

Telomerase activity of needle-biopsied liver samples: its usefulness for diagnosis and judgement of efficacy of treatment of small hepatocellular carcinoma.

BACKGROUND/AIMS: High values for telomerase activity in malignant tumors have been reported. The clinical usefulness of measurements of telomerase activity as a diagnostic tool and to evaluate treatment efficacy in small hepatocellular carcinoma was investigated. METHODS: We investigated 22 patients (26 nodules) with intrahepatic abnormal nodules < or =20 mm in size determined by abdominal ultrasound. All underwent needle biopsies of tumorous nodules and extranodular regions of the liver by ultrasound guidance for histopathological diagnosis and measurement of telomerase activity by the fluorescence-based telomeric repeat amplification protocol. Re-biopsy of the same nodule was performed 1 week after percutaneous ethanol injection therapy to measure telomerase activity in 10 patients (10 nodules) found to have hepatocellular carcinoma. Liver-biopsied samples from 30 patients with chronic hepatitis C were used as a control. RESULTS: Telomerase activity increased with statistical significance stepwise: chronic hepatitis (n=30, mean: 0.00 U) extranodular regions (pre-cirrhosis or cirrhosis, n=22, mean: 1.80 U), atypical hyperplasia (borderline or premalignant lesions, n= 15, mean: 7.02 U) and low-grade malignant hepatocellular carcinoma (n=11, mean: 31.96 U) (p<0.0001 by the Kruskal-Wallis test). Percutaneous ethanol injection therapy resulted in loss (0.00 U) of telomerase activity in 9 nodules and persistence in 1 nodule. CONCLUSIONS: Measurement of telomerase activity appeared useful for diagnosis of intrahepatic abnormal nodules and assessment of the efficacy of percutaneous ethanol injection therapy and may be used as an alternative diagnostic method, especially when pathohistological discrimination between atypical hyperplasia and well-differentiated hepatocellular carcinoma is difficult.

[The association between autoantibodies to enzyme and diseases--with special reference to antibodies to pyruvate dehydrogenase complex (PDH)].

A serological feature of primary biliary cirrhosis (PBC) is the presence of high-titer antimitochondrial autoantibodies (AMA) in patient sera. Five different target mitochondrial autoantigens recognized by sera from PBC patients have been identified as subunits of the 2-oxoacid dehydrogenase complex (2-OADC), of which pyruvate dehydrogenase complex-E2 (PDH-E2) is the most prominent antigenic component. Extensive molecular and immunological studies in PBC such as cloning of mitochondrial autoantigens, mapping of both T and B cell epitopes and immunohistochemical studies have provided valuable reagents in the understanding of immunopathogenesis of PBC. We mapped the epitope recognized by AMA specific to 2-oxoglutarate dehydrogenase complex-E2 (OGDC-E2) in patients with PBC using full-length rat OGDC-E2 cDNA and a series of expression clones spanning the entire molecule. It appears that the epitope is dependent on conformation and includes the lipoic acid-binding region. Furthermore we have taken advantage of the antigenic mapping studies of PDC-E2, OGDC-E2 and branched chain 2-oxoacid dehydrogenase complex-E2 (BCOADC-E2) subunits and designed a hybrid clone, pML-MIT3, that expressed three different immunodominant epitopes. Our results indicate that an immunoassay using recombinant, cloned autoantigen is a powerful and very specific method for detecting AMA in PBC.

[A statistical study of ocular complications of herpes zoster ophthalmicus and its prolongation factors].

A statistical study was carried out on 218 patients (107 male and 111 female) with herpes zoster ophthalmicus (HZO) who visited our clinic from April 1985 to March 1992. The incidence of ocular complications (OCs) and the rate of prolonged OCs (over 6 months) were also studied with the factors that were considered to have affected them. There were 114 patients who showed OCs out of 218 HZO patients (52.3%). The incidence of OCs was higher among the patients with nose eruption than among those without (p < 0.001), and lower among the patients with acyclovir treatment in advance than among those without (p < 0.001). The rate of prolonged OCs was higher among the patients over 50 years old than among those under 50 years old (p < 0.05), higher among the patients with nose eruption than among those without (p < 0.05), and lower among the patients with acyclovir treatment in advance than among those without (p < 0.01). Those with nose eruption had a high rate of prolonged iritis. Severe visual acuity loss remained in 3 cases (2.6%): 2 cases of corneal opacity in the center and 1 case of neurouveitis. Two patients suffered from high intraocular pressure complicated with prolonged iritis.

[Acute lymphocytic leukemia having surface phenotype of CD 2 and NKH-1 with rapid clinical course].

Abnormal expansion of large granular lymphocytes (LGLs) was observed in peripheral blood and bone marrow in a 28-year-old man. He had general lymphadenopathy and splenomegaly. Surface phenotypical analysis of LGLs showed that these LGLs express CD 2, Ia and NKH-1 but not express CD 3, CD 4, CD 8 and Leu 7. Cytochemical analysis of these LGLs revealed positive acid phosphatase and beta-glucuronidase reaction but negative alpha-naphthyl acetate esterase reaction. These LGLs showed very weak NK activity against only MOLT-4 but showed no cytotoxic activity against K 562. An beta-receptor gene rearrangement of human T-cell receptor was not found by Southern blot analysis. Rapid and fetal clinical course with the results of theses analytical studies showed that this case is highly suggestive of acute leukemia of LGLs which is committed to NK cell lineage.

Enzyme-linked immunosorbent assay using isolated (U) small nuclear ribonucleoprotein polypeptides as antigens to investigate the clinical significance of autoantibodies to these polypeptides.

In order to investigate the clinical significance of autoantibodies to individual U small nuclear ribonucleoprotein (snRNP) polypeptides, an enzyme-linked immunosorbent assay (ELISA) using isolated 68K, A, B/B', and D polypeptides from purified U1 snRNP was developed. The ELISA levels of IgG antibodies were positively correlated with results of immunoblotting and hemagglutination. In patients positive for antibodies to ribonucleoprotein, IgG anti-68K reactivity was associated with active mixed connective tissue disease, and in particular with myositis and esophageal hypomotility. IgG B/B' and D polypeptide reactivities were associated with systemic lupus erythematosus and renal disorder. Raynaud's phenomenon was infrequent in patients with high IgG B/B' and D polypeptide reactivities. Pleuritis/pericarditis was associated with the IgG B/B' polypeptide reactivities. In longitudinal studies, ELISA levels of IgG antibodies against these polypeptides changed in parallel with disease activity.

Aberration of monokine production and monocyte subset in patients with systemic lupus erythematosus.

We measured the production of interleukin 1 (IL-1) and prostaglandin by adherent cells from patients with systemic lupus erythematosus (SLE). Compared to normal subjects, IL-1 production in the patients was lower but prostaglandin E1 production was higher. Furthermore, examination of monocyte subsets in patients with SLE using monoclonal anti-monocyte/granulocyte antibodies disclosed abnormal expression of membrane antigens on monocytes. It is speculated that aberration of monocyte function is related to impaired monokine production and that membrane antigens play a role in immunodysregulation in patients with SLE.

Safety and efficacy of hepatitis B vaccine in hospital personnel.

Clinical trials of the hepatitis B vaccine developed in Japan were performed by the phase I, phase II and phase III studies. It was found that this vaccine did not only contained HBV itself but also other pathogens. It was ascertained to be adequately safe, since no major side effect was induced. After inoculation of the vaccine 3 times into 195 hospital employees, an HBs antibody seroconversion rate of 91.8% (97.0% in females and 86.3% in males) was obtained. In recipients aged more than 50 years, the rate was significantly lower. Although 8.2% of the recipients were non-responders to the vaccination, there was no significant correlation between the strength of the HBs antibody response and the lymphocyte subsets in the peripheral blood. It was observed, however, that among the non-responders, the incidence of low OKT4+/OKT8+ cell ratios tended to be somewhat increased. In follow-up studies of the antibody, majority of subjects, in whom the antibody level at 1 month after the 3 inoculations was below 50 RIA units, demonstrated negative conversion within 1 or 2 years. Namely, their value were reduced to below the minimal protective antibody value for prophylaxis of HBV infection.

Impaired B cell proliferation by Staphylococcus aureus Cowan 1 in patients with systemic lupus erythematosus.

We examined the proliferative response to Staphylococcus aureus Cowan 1 (SAC) by enriched peripheral blood B cells from patients with systemic lupus erythematosus (SLE). Responses of B cells from patients with active and inactive SLE were significantly lower than those of B cells from normal individuals. Hyporesponsiveness to SAC was not observed in healthy family members of SLE patients. This hyporesponsiveness did not correlate with prednisolone therapy and could not be attributed to serum factors; it did correlate with the presence of suppressor monocytes. However, we could not exclude the possibility of enhanced sensitivity of SLE B lymphocytes to suppressive signals delivered by the monocytes.