Glial cells contribute more to iron and aluminum accumulation but are more resistant to oxidative stress than neuronal cells.

Iron (Fe) and aluminum (Al) have been implicated in the pathogenesis of Alzheimer's disease (AD). In this study, we examined neuronal and glial cells to clarify which contributes most to metal accumulation after internalization through the transferrin-independent iron uptake (Tf-IU) systems in primary neuronal and glial predominant (NP and GP) cells from rat cerebral cortex, which affect the accumulation of transition metals in a variety of cultured cells. Al more significantly upregulated the Tf-IU activity in GP cells than in NP cells. GP cells were more resistant to Fe and Al exposure than NP cells. However, a chemiluminescence analysis specific for reactive oxygen species (ROS) showed that ROS levels in Fe- or Al-loaded NP cells were twice as high as in Fe- or Al-loaded GP cells. Northern blot analysis and gel retardation assay showed that the Al and Fe exposure taken up by the cells suppress Tf receptor mRNA expression to a greater extent in GP than NP cells, indicating that Al and Fe more markedly accumulate in glial than in neuronal cells. These results suggest that glial cells rather than neuronal cells contribute to the metal accumulation and are more resistant to oxidative stress caused by metals than neuronal cells. The present study may help to explain the pathogenesis of neurodegeneration in AD disorders caused by metal-generated oxidative stress.

IL-6 increases endothelial permeability in vitro.

The effect of interleukin 6 (IL-6) on endothelial permeability was examined by measuring fluorescein isothiocyanate-labeled albumin flux across an endothelial cell monolayer. Bovine vascular endothelial cells (BVEC) were cultured up to confluency on collagen-coated polycarbonate micropore filters and then the filters were mounted on modified Boyden chambers. Treatment of the BVEC with IL-6 at 100 ng/ml for 21 h caused a remarkable increase in the permeability of fluorescein isothiocyanate-labeled albumin across the endothelial monolayer. This effect of IL-6 was concentration dependent, in the range from 10-200 ng/ml of IL-6. The effect of IL-6 was also time dependent, the maximal level being reached at 21 h from the beginning of the treatment. This stimulatory effect of IL-6 on albumin clearance was completely abolished by the addition of anti-IL-6 antibody. Light microscopic observation of a cross-section of a monolayer showed that the IL-6-induced increase in the permeability was correlated with changes in cell shape and rearrangement of intracellular actin fibers. IL-6 did not show any cytotoxicity toward or growth inhibition of endothelial cells, even at more than 200 ng/ml. The enhancing effect of IL-6 on the increase in the permeability was reversible; when IL-6 was removed by a medium change and the cells were incubated for a further 24 h without IL-6, the permeability was restored to the control level. These results suggest that IL-6 can induce an increase in endothelial permeability in vitro by rearranging actin filaments and by changing the shape of endothelial cells.

Uveitis associated with human T-cell lymphotropic virus type I.

Seroepidemiologic, clinical, and virologic studies were performed to determine whether human T-cell lymphotropic virus type I was closely associated with uveitis in two hospitals. One hospital was in an endemic area of the virus (Miyakonojo, Miyazaki) and the other hospital was in a less endemic area (Kurume). In the endemic area, the seroprevalence of the virus in patients with uveitis without defined causes (35.4%, 62 of 175 patients) was significantly higher than that in patients with nonuveitic ocular diseases (16.1%, 42 of 261 patients), or in patients with uveitis with defined causes (10.3%, eight of 78 patients). The seroprevalence in younger patients (20 to 49 years of age) with uveitis without defined causes in the area was 44.8% (30 of 67 patients), whereas it was only 9.3% (ten of 107 patients) in the other two groups. A similar observation was recorded even in the less endemic area (Kurume). Because the seroprevalence of the virus in the general population is known to be low in younger patients and to increase with age, these findings were interpreted to indicate that the association of human T-cell lymphotropic virus type I with uveitis was significant. Most patients, particularly those aged 20 through 49 years, had an intermediate uveitis characterized by a moderate inflammation in the vitreous body accompanied by an iritis and retinal vasculitis. The ocular symptoms in the patients differed from those of other types of uveitis common in Japan (Behçet's disease, Vogt-Koyanagi-Harada's disease, and toxoplasmosis, for example).(ABSTRACT TRUNCATED AT 250 WORDS)

Human T lymphotropic virus type 1 uveitis after Graves' disease.

A distinct clinical entity of uveitis associated with human T lymphotropic virus type 1 (HTLV-I) has been reported previously. During the period between January 1989 and April 1992, 93 patients were observed with HTLV-I uveitis and a significant correlation was found between Graves' disease and HTLV-I uveitis. Sixteen of the 93 patients with HTLV-I uveitis (17.2%) had a previous history of Graves' disease. Fifteen patients were female (15/60, 25.0%) and one was male (1/33, 3.0%). Interestingly, uveitis occurred after the onset of Graves' disease in all cases. On the other hand, none of 222 patients with idiopathic uveitis who were seronegative to HTLV-I had a history of Graves' disease. Although the mechanisms by which HTLV-I causes the correlation between uveitis and Graves' disease are unknown, the present data suggest that immune mediated or autoimmune mechanisms are involved in HTLV-I uveitis.

Determination of organic acids in urine by capillary zone electrophoresis.

The simultaneous measurement of organic acids was studied using capillary electrophoresis with direct measurement of UV absorption at 185 nm. The organic acids studied were oxalic, formic, malonic, fumaric, succinic, alpha-ketoglutaric, citric, acetic, pyruvic, lactic, isovaleric and hippuric acid. They were separated in a fused-silica capillary (100 cm x 75 microm I.D.) filled with 50 mM borax buffer (pH 10.0) containing cationic surfactant as the electroosmotic flow modifier. The method was successfully applied to the determination of organic acids in urine in comparison with an organic acid analyser.

Determination of synthetic food dyes by capillary electrophoresis.

A method for the determination of synthetic tar dyes used as food additives using capillary electrophoresis with photodiode-array detection was investigated. The dyes Erythrosine (R-3), Phloxine (R-104), Rose Bengal (R-105), Acid Red (R-106), Amaranth (R-2), New Coccine (R-102) and Allura Red AC (R-40) were separated on a capillary column (50 cm x 75 microns I.D.) and identified from the absorbance spectra of each peak. The electrophoresis buffer used was a mixture of 25 mM sodium phosphate buffer and 25 mM sodium borate buffer (1:1) (pH 8.0) containing 10 mM sodium dodecyl sulfate (SDS). Substitution of beta-cyclodextrin for SDS in the electrolyte buffer was effective for the separation of R-2 and R-102. This modified method could be employed as an additional assay method for these two dyes.

Preclinical and clinical study of FK506 in uveitis.

Efficacy of a new immunosuppressive agent, FK506, in refractory uveitis was studied in 8 patients: 5 with Behcet's disease and 3 with idiopathic retinal vasculitis. The agent was given by oral administration every 12 hours. The previous therapy with systemic corticosteroids or immunosuppressive agents including cyclosporine failed to subside uveitis in these cases. During the observation period of 21.6 +/- 7.8 weeks (mean +/- SD) under FK506 at doses with 0.05, 0.1, 0.15 or 0.2 mg/kg/day, the visual acuity was increased in 44% of treated eyes, unchanged in 44% and decreased in 12%. The inflammatory activity in the ocular fundus was improved in 69% and unchanged in 6% of treated eyes. The effects of FK506 on uveitis by the criteria of improvement of visual acuity and uveitis activity was dose-dependent: 0.05 and 0.1 mg/kg/day were ineffective but 0.15 and 0.2 mg/kg/day were effective in most cases. One patient with Behcet's disease converted from cyclosporine developed moderate renal impairment in 4 weeks under FK506 and the therapy was discontinued in 8 weeks, though the uveitis activity as well as visual acuity was markedly improved. Other 7 cases had no side effect of FK506. Although the number of cases was small and observation period was short, the present clinical data indicate that FK506 is effective to treat refractory uveitis.

[Uveitis in human T-lymphotropic virus type I (HTLV-I) carriers--2. An analysis of clinical features].

The clinical features of human T-lymphotropic virus type (HTLV-I) uveitis, an idiopathic uveitis in HTLV-I seropositive patients, were analyzed in a hospital located in an area where HTLV-I was endemic (Miyakonojo, Miyazaki). A total of 61 patients (25 males and 31 females) with HTLV-I uveitis were the subjects of the analysis. Intermediate uveitis with moderate to severe vitreous opacities accompanied by mild iritis and retinal vasculitis was the most characteristic feature, and was observed in the majority of the patients (66% of all or 84% in young adult patients between 20 and 49 years of age). The uveitis affected one eye in 57% and both eyes in 43% of the patients, with subacute onset of floaters and foggy vision. The uveitis responded well to therapy with topical or systemic corticosteroids, but recurred in many cases. A significant number of patients (15% of all and 25% of female patients) had a previous history of Graves' disease with hyperthyroidism.

[Uveitis in human T-lymphotropic virus type I (HTLV-I) carriers--1. A seroepidemiological study].

A seroepidemiological study was carried out to find out if human T-lymphotropic virus type I (HTLV-I) was closely associated with uveitis in two hospitals: one in an area where HTLV-I was highly prevalent (Miyakonojo, Miyazaki) and the other in an area where HTLV-I was less prevalent (Kurume). In the highly prevalent HTLV-I area, the seroprevalence in patients with idiopathic uveitis (78/206, 37.9%) was significantly higher than in patients with non-uveitic ocular diseases (73/380, 19. 2%) or in patients with uveitis with defined etiologies (8/80, 10.0%). A more striking finding was that the seroprevalence in the younger group (20-49 years) with idiopathic uveitis in the area was 40/82 (48.8%), but only 9/114 (7.9%) in the other two reference groups. The odds of contracting idiopathic uveitis for HTLV-I infected persons was estimated at 11.0 in the younger group, and 2.0 in the older group. Similar observations were recorded even in the less prevalent area (Kurume). These data thus suggest that HTLV-I infection is a risk factor for idiopathic uveitis.

[Uveitis in human T-lymphotropic virus type I (HTLV-I) carriers--3. A molecular biological study].

The presence of proviral DNA of human T-lymphotropic virus type I (HTLV-I) in the inflammatory cells in the aqueous humor was examined by polymerase chain reaction. The proviral DNA was detected in all the tested patients (n = 9) with HTLV-I uveitis. On the other hand, the provirus was not detected in 2 out of 3 HTLV-I seropositive patients with other types of uveitis, i.e., Behçet's disease, toxoplasmosis and Vogt-Koyanagi-Harada's disease. None of the 11 seronegative patients with other types of uveitis or senile cataract had proviral DNA in the aqueous humor. Thus, the fact that HTLV-I-infected cells were present in the aqueous humor of all HTLV-I uveitis patients suggests significant involvement of HTLV-I in the pathophysiology of the uveitis.

Sensitive determination of anandamide in rat brain utilizing a coupled-column HPLC with fluorimetric detection.

A fluorimetric determination method for N-arachidonoylethanolamine (anandamide) was developed using a precolumn fluorescence derivatization followed by coupled-column high-performance liquid chromatography (HPLC). Anandamide extracted from the rat brain tissue was derivatized with 4-N-chloroformylmethyl-N-methylamino-7-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl), purified by a solid-phase extraction (Emporetrade mark), and assayed by the coupled-column HPLC. The HPLC consisted of phenyl (100 x 4.6 mm i.d. ) and octadecylsilica columns (250 x 4.6 mm i.d.), both connected by a six-port valve. The concentration of anandamide in rat brain was 3. 37 +/- 0.73 pmol/g with 6.47 and 3.57% of intra- and inter-day precisions, respectively. Using this method, we investigated the alteration of anandamide concentration in rat brain 30 min after administration of anandamide (2 mg/kg, i.p.) to rats pretreated with or without phenylmethylsulfonyl fluoride (PMSF; 30 mg/kg, i.p.), an inhibitor of amidohydrolase. In rats pretreated with PMSF, the brain concentration of anandamide was approx. 16-fold higher than that of rats without PMSF (p < 0.01).

Analysis of creatinine, vanilmandelic acid, homovanillic acid and uric acid in urine by micellar electrokinetic chromatography.

A simultaneous determination of vanilmandelic acid, homovanillic acid, creatinine and uric acid using capillary electrophoresis was investigated. The optimum conditions of buffer concentration, pH and surfactant concentration were studied, and high resolution was obtained using a 30 mM phosphate buffer (pH 7.0) containing 150 mM sodium dodecyl sulfate. The detection was by UV absorbance at 245 nm and the column was a fused-silica capillary of 67 cm x 75 microm I.D.. The determination of these metabolites in human urine was completed within 15 min without any interferences.

Whole body autoradiographic study on the distribution of 14C-D-serine administered intravenously to rats.

The distribution of radioactivities in rats following intravenous administration of 14C-D- or -L-serine was investigated by whole body autoradiography. The radioactivities were distributed throughout the whole body in both cases with the greatest amount being found in the pancreas. D- and L- Serine levels in the pancreas were determined by high-performance liquid chromatography with a chiral column which revealed, for the first time, the existence of D-serine in the rat pancreas (12.6 +/- 7.90 nmol/g wet tissue) together with a much higher concentration (924 +/- 116 nmol/g) of L-serine. The results suggested that exogenous D-serine of dietary origin contributed at least in part to the D-serine levels found in mammalian tissues. The accumulation of radioactivity in the kidney, especially in the corticomedullary area, even at 24 hr after administration of 14C-D-serine suggested a possible link between acute necrosis of the renal proximal tubules and the administration of a large dose of D-serine [Am J Pathol 77: 269-282 (1974)].

Influence of different types and levels of dietary lipids on liver microsomal mixed function oxidase system in rats.

It has been shown that dietary polyunsaturated fatty acids stimulate the liver microsomal mixed function oxidase system. The influence of different levels of dietary lard, soybean oil and sardine oil on the mixed function oxidase system was investigated in rats. The diet containing 5% sardine oil rich in eicosapentaenoic and docosahexaenoic acids stimulated the mixed function oxidase system, but the diet containing 5% lard in which lard consisted of 10.7% linolenic acid and 1.5% linolenic acid seemed unlikely to stimulate enough the mixed function oxidase system. On the other hand, no definite effects of large doses of dietary lipids, 25% in the diets, on the mixed function oxidase system were observed.

Characterization of rat LECAM-1 (L-selectin) by the use of monoclonal antibodies and evidence for the presence of soluble LECAM-1 in rat sera.

We have characterized the rat LECAM-1 (L-selectin) by the use of newly generated hamster anti-rat LECAM-1 monoclonal antibodies (mAb) (HRL1, HRL2, HRL3, HRL4), with respect to the biochemistry, cellular distribution and function, and developed an ELISA system to detect the soluble form of rat LECAM-1. In the rat, lymphocyte and neutrophil LECAM-1 have apparent molecular masses of 65 and 62 kDa, respectively, and differential glycosylation may account for the molecular heterogeneity. Readily detectable levels of LECAM-1 are expressed on peripheral blood lymphocytes and neutrophils, but not on thymocytes. Lymphocyte LECAM-1 is rapidly shed from the cell surface upon cell activation with PMA, but not with interleukin (IL)-8. In contrast, neutrophil LECAM-1 showed rapid shedding upon stimulation with phorbol 12-myristate 13-acetate (PMA) or IL-8. Concomitantly there is up-regulated expression of Mac-1 in PMA- and IL-8-stimulated neutrophils. Neutrophil rolling in mesenteric venules was significantly inhibited by administration of function-blocking anti-rat LECAM-1 mAb HRL3, but not by non-blocking HRL4, indicating that LECAM-1 plays a significant role in leukocyte rolling. Given that LECAM-1 is rapidly shed from the cell surface, we attempted to develop an ELISA system for detecting LECAM-1 is soluble form, and measured the levels in experimental autoimmune uveitis. The circulating levels of LECAM-1 increased from day 4, which preceded the appearance of clinical signs of uveitis and remained high until uveitis subsided, suggesting that soluble LECAM-1 is potentially a useful parameter to monitor certain types of inflammatory or immune disorders.

HTLV-I uveitis: a distinct clinical entity caused by HTLV-I.

Seroepidemiological, clinical and virological studies were carried out in an HTLV-I endemic area to find out if HTLV-I caused an intraocular inflammatory disorder, uveitis. The seroprevalence in patients with uveitis without defined etiologies (62/175, 35.4%) was significantly higher than that in patients with non-uveitic ocular diseases (42/261, 16.1%) or in patients with uveitis with defined etiologies (8/78, 10.3%). Moreover, the seroprevalence in young adults (20-49 years) with uveitis without defined etiologies was 30/67 (44.8%), whereas it was only 10/107 (9.3%) in the other two groups. The uveitis in HTLV-I carriers was characterized clinically by a moderate inflammation of the vitreous body accompanied by a mild iritis and retinal vasculitis. The proviral DNA of HTLV-I was detected by polymerase chain reaction from the inflammatory cells in the anterior chamber in 9 out of 9 seropositive patients with the uveitis, but not in any of the tested patients with other types of uveitis. These data, thus, indicate that HTLV-I causes a specific type of intraocular inflammation, uveitis.

Monoclonal antibodies against CD54 (ICAM-1) and CD11a (LFA-1) prevent and inhibit endotoxin-induced uveitis.

We studied the effect of monoclonal antibodies (mAbs) against CD54 (intercellular adhesion molecule-1; ICAM-1) and CD11a (lymphocyte function-associated antigen-1; LFA-1) on the prevention and treatment of endotoxin-induced uveitis (EIU). When treated at the time of endotoxin injection the mean number of inflammatory cells infiltrating the eye +/- S.E.M. on histologic sections was 469.2 +/- 51.9 for controls, 13.8 +/- 2.6 for rats receiving anti-ICAM-1 mAb (P < 0.0001), and 195.8 +/- 48.8 for rats receiving anti-LFA-1 mAb (P = 0.0003). When treated after the start of inflammatory disease, the mean number of infiltrating inflammatory cells +/- S.E.M. was 273.0 +/- 30.7 for controls, 6.4 +/- 1.7 for rats receiving anti-ICAM-1 mAb (P < 0.0001), and 54.2 +/- 7.6 for rats receiving anti-LFA-1 mAb (P < 0.0001). The mean number of cells per milliliter of aqueous humor +/- S.E.M. was 1867.6 +/- 321.8 for controls, 21.7 +/- 5.3 for rats receiving anti-ICAM-1 mAb (P < 0.0001), and 295.1 +/- 71.2 for rats receiving anti-LFA-1 mAb (P < 0.0001). MAbs against ICAM-1 and LFA-1 significantly inhibited the development of EIU and were effective in treating clinically evident ocular inflammatory disease.