Alpha-synuclein-positive structures induced in leupeptin-infused rats.

Abnormal accumulation of alpha-synuclein is regarded as a key pathological step in a wide range of neurodegenerative processes, not only in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) but also in multiple-system atrophy (MSA). Nevertheless, the mechanism of alpha-synuclein accumulation remains unclear. Leupeptin, a protease inhibitor, has been known to cause various neuropathological changes in vivo resembling those of aging or neurodegenerative processes in the human brain, including the accumulation of neuronal processes and neuronal cytoskeletal abnormalities leading to neurofibrillary tangle (NFT)-like formations. In the present study, we administered leupeptin into the rat ventricle and found that alpha-synuclein-positive structures appeared widely in the neuronal tissue, mainly in neuronal processes of the fimbria and alveus. Immunoelectron microscopic study revealed that alpha-synuclein immunoreactivity was located in the swollen axons of the fimbria and alveus, especially in the dilated presynaptic terminals. In addition colocalization of alpha-synuclein with ubiquitin was rarely observed in confocal laser-scan image. This is the first report of experimentally induced in vivo accumulation of alpha-synuclein in non-transgenic rodent brain injected with a well-characterized protease inhibitor by an infusion pump. The present finding suggests that the local accumulation of alpha-synuclein might be induced by the impaired metabolism of alpha-synuclein, which are likely related to lysosomal or ubiquitin-independent proteasomal systems.

Inter--rater and test--retest reliability of the Japanese version of the subjective deficit syndrome scale.

The subjective deficit syndrome scale (SDSS) was established in 1990 by Jaeger et al. to assess systematically subjective experiences of schizophrenic patients. It was translated into Japanese in 1999 by this paper's second author (T.I.). In this study, the inter-rater reliability of this Japanese version (SDSS-J) was examined. The subjects studied were 13 schizophrenic patients (male 6, female 7; respective average ages at study entry and disease onset: 41 and 25 years), who were being followed up at the outpatients' service of the Department of Neuropsychiatry, Hyogo College of Medicine. Four psychiatrists attended together, the systematic interview with each subject to rate the SDSS-J and independently rated 19 items. The severity scales of the items assessed by these raters ranged from 0 to 4 for 12 items and from 0 to 3 for 7. The ANOVA ICC inter-rater reliability values for the 19 individual items ranged from 0.67 to 0.96. The ANOVA ICC test-retest reliability values achieved by two raters were also high overall, ranging from 0.72 to 1.00, except for one item (item 13) assessed by one rater. Our results suggest that the SDSS-J is a potentially useful rating scale for evaluating subjective experiences of schizophrenic subjects.

Comparative evaluation of the effect of antioxidants in the conservation of ram semen.

The aim of the present study was to develop a treatment supporting the membrane of ram spermatozoa. Semen of different ejaculates collected from breeding rams was mixed and samples of 10(9) sperm cells per ml and Tris-egg yolk extender were completed with the following antioxidants: alpha-tocopherol acetate (E), glutathione peroxidase (GP), Aromex (AR), resveratrol (R), resveratrol + vitamin E (RE), resveratrol + Aromex (RAR), resveratrol + GP (RGP). Peroxidation was evaluated by the analysis of malondialdehyde (MDA) during incubation for 30, 60 and 120 min at 37 degrees C as well as during a 24-h incubation at 5 degrees C. The success of preservation was checked in a 9-day-long period by observing the acrosomal defects and the motility of spermatozoa. Concentration of MDA was 4.06 nmol/10(9) spermatozoa in samples treated with 15 micrograms R while the control sample contained 69.79 nmol MDA per 10(9) spermatozoa after 24-h incubation. Following 30-, 60- and 120-min storage the concentration of MDA in control and R-treated samples was 25.89, 36.91, 49.57 and 3.69, 3.74, 3.74 nmol/10(9) spermatozoa, respectively. Moreover, a significantly higher proportion of motile sperm cells was observed in the treated than in the control samples. The frequency of acrosomal defects was lower in the treated groups than in the control. These results indicate that RAR treatment can improve the effects of ram semen preservation.

Familial dementia with Lewy bodies (DLB).

We report a rare familial case of dementia with Lewy bodies (DLB). The patient was a man who died at the age of 51. His parents were first cousins. Among three siblings, two were diagnosed as probable cases of DLB, and one was a possible case, according to the clinical diagnostic criteria of the consortium on DLB. Following the patient's autopsy, he was found to have had DLB without neurofibrillary tangles or senile plaques (pure form of diffuse Lewy body disease). His other siblings have been followed for more than ten years. Although these patients with familial DLB displayed clinical variability, all three siblings showed progressive dementia of early onset and progressive language disorder with paraphasia and difficulty in finding words. Psychotic features were also seen in the three siblings. The patient's sister showed compulsive behavior, and the other two siblings showed symptoms of parkinsonism. Neuropathologically, in addition to the usual neuropathology of DLB, the autopsy findings showed numerous small spheroids in the stratum pyramidale from the subiculum to CA1 of the hippocampus. Significant neuronal loss in CA2-3 of the hippocampus was detected. Axonal flow disturbance may be involved in the hippocampal formations of this incidence of familial DLB.

Immunophenotypic studies on atypical lymphocytes in psychiatric patients.

By using phase-contrast microscopy combined with a fluorescent staining technique, the frequency of blast-type atypical lymphocytes (BTALs) appearing in peripheral blood and the phenotypic expression of their surface antigens were studied in 24 patients with schizophrenia, 16 with mood disorder and 14 healthy controls. BTALs were classified as being stimulated or activated cells, morphologically characterized by their large size, dark cytoplasm, a hollow perinuclear containing a few granules and finely dispersed chromatin structures with a few evident nucleoli. A significantly higher number of BTALs were found in the schizophrenic patients compared with healthy control subjects or patients with mood disorder. Further, there was a significant difference in the frequency of BTALs between patients with mood disorder and healthy control subjects. No significant difference in the frequency of BTALs was found between the schizophrenic patients with and without medication. Immunostaining of BTALs revealed that these cells consisted of B, T and non-B, non-T cell subpopulations. Contrary to our expectations, the T cell was only one third of the BTAL population. HLA-DR and CD38 were expressed on most BTALs (> 70%), while CD25, an early activation marker of T cells was rarely found on BTALs (< 0.3%). The differences in activated lymphocyte populations which appeared as morphologically atypical in the circulation among some psychiatric patients and infectious or autoimmune diseases are discussed. This is the first report on populations of BTALs.

The severity of clinical symptoms in ragweed-allergic patients is related to the extent of ragweed-induced complement activation in their sera.

We have previously reported a correlation between the extent of ragweed allergen (RWA)-induced in vitro serum complement activation and the symptom scores registered daily during the ragweed (RW)-blooming season in RW-allergic patients. The present study was performed in 22 15-17-year-old RW-allergic adolescents. Serum samples were incubated with 100 micrograms/ml RWA, and the generation of different complement activation products was measured by ELISA or RIA. Symptom scores were registered for 4 weeks during the RW-blooming season. The patients were divided according to the extent (low or high) of the generation of complement activation products, and symptom scores registered in the two groups were compared by two-way ANOVA. Significantly higher symptom scores were obtained in the high than in the low complement activation group (P values: 0.049 for C1rC1sC1inh, 0.022 for C3bBbP, 0.015 for C5b-9, 0.0001 for C3a, and 0.0008 for C5a). Similar results were obtained at the measurement performed in the sera obtained from the same patients half a year before the season (P values: 0.022 for C3bBbP, and 0.005 for C5b-9). These findings indicate that complement activation induced by the allergen may enhance the clinical symptoms of RW allergy.

Synapse alteration in hippocampal CA3 field following entorhinal cortex lesion.

To model one aspect of the neurodegeneration observed in Alzheimer's disease and to investigate the synaptic alteration of the hippocampus associated with entorhinal cortex lesion, ibotenic acid was used to produce selective unilateral neuronal loss in rat entorhinal cortex. Immunohistological and microdensitometrical analyses confirmed ibotenic acid lesion of the entorhinal cortex after 3 months and showed a decrease of synaptophysin-immunoreactive substances in the stratum lucidum of the CA3 field. This study demonstrates that entorhinal cortex lesion can lead to synaptic alterations and cause damage to presynaptic terminals with projecting area in the disruption of the entorhinal cortex hippocampus relay passage.

Elevated Le(y) antigen expression on T-lymphocytes in schizophrenic patients.

Le(y) is a carbohydrate determinant of membrane glycoconjugates and is expressed in some tumor and embryonic cells. On T lymphocytes, it is known that human immunodeficiency virus (HIV)-infected human lymphocyte T-cell lines express elevated Le(y) antigen and AIDS patients show the highest Le(y) expression in T lymphocytes at lower CD4/CD8 ratio. Later, a comparative elevation of Le(y) expression on T-cell subsets has been noticed to be mainly present in patients with viral diseases, such as acute and chronic hepatitis, implying an association of the highest Le(y) expression with viral infection. We found that Le(y) antigen was most expressed in both CD4+ and CD8+ subsets of peripheral T lymphocytes in hospitalized schizophrenic patients. On the other hand, atypical lymphocytes with stimulated morphology are known to appear in the blood circulation of schizophrenic patients. Similar atypical lymphocytes have also been described in viral and autoimmune diseases. Two possibilities have been discussed: viral association in the pathology in some schizophrenic patients; and immunological abnormalities including environmental effects under hospitalization on immune status, since normal controls (staff in psychiatric hospitals) showed higher Le(y) expression than normal controls under non-psychiatric circumstances.

Differences in the complement activation induced by preformed and nascent immune complexes.

While preformed BSA-anti-BSA immune complexes (PIC) bind efficiently to human RBC after their interaction with human complement, nascent BSA-anti-BSA immune complexes (NIC) formed in the presence of complement do not bind to autologous RBC. The same results were obtained with tetanus toxoid-anti-tetanus toxoid PIC and NIC. In order to elucidate the causes of this marked difference between the RBC-binding properties of PIC and NIC, the profile of complement activation induced by them was compared using haemolytic assays and sensitive ELISA tests. BSA-anti-BSA NIC activated C1 more efficiently than PIC. This was reflected in a higher C4 content of the isolated NIC and higher C1 INH-C1s and lower C1q-fibronectin complex level in the NIC-treated serum as compared to the PIC-treated ones. Although isolated NIC contained more C3 than isolated PIC did, there was no significant difference in the AP activation. These findings suggest that the failure of NIC to bind to RBC is not due to a lack of C4-binding, or C3-binding and/or activation, but rather to the special structure of this type of complex.

Concentration of C1 inhibitor in sera of healthy blood donors as studied by immunoenzymatic assay.

One hundred and forty sera from healthy blood donors (age 19-30 years) were studied for the concentration of C1 inhibitor. The determinations were performed by enzyme-linked immunosorbent assay with the use of specific anti-C1 inhibitor antibodies fixed to the wells of a microplate and peroxidase labeled as the second layer. The geometric mean of the C1 inhibitor value was 0.25 g/l with a standard deviation of 0.09 and a standard error of 0.0077 g/l while the median and mode values were equal to 0.27 g/l. The technique is relatively simple and can be used for the screening of hereditary or acquired angioedema. The technique can be applied also for the study of population differences in the C1 inhibitor concentration and for the study of its synthesis in in vitro systems.

Effect of conditioned media of acute myeloid leukemia blast cells on complement synthesis by cultured human cells of monocyte and hepatocyte origin.

The effect of conditioned media of 3-day cultures of blast cells from peripheral blood of 5 patients with acute myeloid leukemia (CM-AML) was studied on the synthesis of C2, factor B (Bf) and C1 esterase inhibitor (C1-INH) by human monocyte-macrophage cultures and HepG2 hepatoma cell line. The level of C2 in the culture supernatants was measured by immune hemolysis, those of Bf and C1-INH by ELISA. CM-AML was added to the monocyte cultures on day 3 and replaced by culture fluid on day 6. Compared to the control cultures, CM-AML significantly increased C2 and Bf levels and slightly decreased C1-INH levels in the culture fluids on day 6. On day 9, Bf synthesis enhancement still could be observed but C2 and C1-INH levels did not significantly differ from those of the control. CM-AML significantly increased the synthesis of factor B by the HepG2 cells too. A strong correlation was found between the results of the Bf protein and RNA determinations, which means that the supernatants of AML blasts affect the gene expression of factor B at a pretranslational level. The selective complement synthesis modifying effect of CM-AML was not due to interferons (IFN) because neither IFN-alpha nor IFN-gamma could be detected in these conditioned media. The present findings indicate that the hypercomplementemia observed in AML patients can be due to unknown factor(s) produced by leukemic blast cells.

FN-C1q and C1 INH C1r-C1s complexes as indicators of complement activation in patients with chronic lymphocytic leukaemia.

We have previously found low levels of C1 and C4 INH in the sera of chronic lymphocytic leukaemia (CLL) patients. Hypocomplementaemia was supposed to be the consequence of a permanent activation of the classical pathway. We have compared the levels of C1 INH-C1rC1s and C1q-FN complexes in the sera of 95 CLL patients and 100 healthy controls, because these complexes are known to be formed in the early stage of classical pathway activation. A significant increase in the level of both types of complexes was found in sera of CLL patients as compared to the controls. These findings support the assumption that the classical complement pathway is activated in the patients with CLL.