Dorfin localizes to the ubiquitylated inclusions in Parkinson's disease,dementia with Lewy bodies,multiple system atrophy,and amyotrophic lateral sclerosis

In many neurodegenerative diseases, the cytopathological hallmark is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. Lewy bodies in Parkinson's disease and dementia with Lewy bodies disease, glial cell inclusions in multiple system atrophy, and hyaline inclusions in amyotrophic lateral sclerosis (ALS) are representative of these inclusions. The elucidation of the components of these inclusions and the mechanisms underlying inclusion formation is important in uncovering the pathogenesis of these disorders. We hypothesized that Dorfin, a perinuclearly located E3 ubiquitin ligase, participates in the formation of ubiquitylated inclusions in a wide range of neurodegenerative diseases. Here, we report that affinity-purified anti-Dorfin antibody labeled ubiquitylated inclusions of Parkinson's disease, dementia with Lewy bodies disease, multiple system atrophy, and sporadic and familial ALS. A double-immunofluorescence study revealed that Dorfin shows a distribution pattern parallel to that of ubiquitin. Furthermore, by a filter trap assay, we detected that Dorfin is present in the ubiquitylated high-molecular weight structures derived from these diseases. These results suggest that Dorfin plays a crucial role in the formation of ubiquitylated inclusions of alpha-synucleinopathy and ALS. However, because we failed to show the direct binding of alpha-synuclein with Dorfin, future investigations into the binding partner(s) of Dorfin will be needed to deepen our understanding of the pathophysiology of alpha-synucleinopathy and ALS.     

Immunohistochemical expression of MAM-3 and MAM-6 antigens in salivary gland tumours

Summary MAM-3 and MAM-6 antigens of human milk fat globule membrane were detected immunohistochemically in 93 cases of salivary gland tumours as well as in normal glands. The antigens were visualized in 10% formalin-fixed paraffin sections. MAM-3 (MoAbs 115G3, 67D11) antigen was distributed in intercalated and striated duct cells of the normal salivary glands, and in luminal tumour cells and squamous metaplastic cells of pleomorphic adenomas. In pleomorphic adenomas the frequency of positive staining with MoAb 67D11 (54/67; 80.6%) was higher than that with MoAb 115G3 (36/67; 53.7%). MAM-6 (MoAbs 115D8, 115F5) antigen was expressed in luminal and lateral borders of serous acinar cells and ductal of the normal glands, and also in luminal borders of tubulo-ductal and glandular structures of salivary gland tumours. Ductal basal cells were characterized by existence of positive staining for MAM-6 antigen, in adenolymphomas MAM-6 antigen was restricted to the basal tumour cells. Some mucous cells of mucoepidermoid tumours were stained specifically with MoAb 115G3, and epidermoid cells of mucoepidermoid carcinomas manifested MAM-6 antigen staining. Immunohistochemical localization of MAM-6 antigen resembled that of epithelial membrane antigen (EMA) detected with MoAb.     

An immunodominant haptenic epitope of carbamazepine detected in serum from patients given long-term treatment with carbamazepine without allergic reaction

An anticarbamazepine antibody was detected in the serum of a patient with severe carbamazepine-induced serum sickness. We found that the patient's T cells and IgG antibody recognized an epitope which appeared in subjects showing an allergic reaction, as well as that in subjects who showed no allergic reaction, after long-term carbamazepine therapy. These results show that an anti-carbamazepine immune response does not occur in the majority of subjects who undergo long-term carbamazepine therapy without developing allergic symptoms, although the immunodominant haptenic epitope of carbamazepine is present in their sera.     

Functional roles of MCP-1 in Propionibacterium acnes-induced, T cell-mediated pulmonary granulomatosis in rabbits

The immunological manifestation of granuloma formations in humans largely depends on the delayed-type hypersensitivity response. We investigated the involvement of monocyte chemoattractant protein-1 (MCP-1) in a rabbit model of T cell-mediated pulmonary granulomatosis. Intravenous injection of Propionibacterium acnes (P. acnes) into sensitized rabbits induced massive and diffuse pulmonary granulomas. Levels of MCP-1 in sera and bronchoalveolar lavage fluids (BALF) peaked before the granuloma formation reached the peak (on days 1 and 3 after challenge, respectively). Chemotactic activities toward monocytes and T cells in BALF were inhibited by anti-MCP-1 IgG by 80 and 36%, respectively. The phenotypic analysis of the migrating T cells revealed that activated and memory T cells rather than naive cells were preferentially attracted to the BALF. Administration of anti-MCP-1 antiserum inhibited the development of granuloma formation in both size and number, the numbers of infiltrating leukocytes in BALF, the expression of adhesion molecules on peripheral monocytes/T cells, and on macrophages/T cells in BALF, and the production of TNF-alpha in the lung. Anti-MCP-1 resulted in a trend toward decreased level of IL-1beta in the lung. The inhibition of the production of these cytokines appeared to be induced indirectly through the inhibition of the recruitment of macrophages that produce these cytokines. The results suggest important roles of MCP-1 in the development of granuloma formation in this model through the attraction and activation of specific types of cells.     

Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis

The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.     

Atrial natriuretic peptide in human urine

Summary A highly sensitive radioimmunoassay to measure atrial natriuretic peptide (ANP) concentration in urine has been established, and its clinical usefulness is presented. ANP in urine was stable at 4° C for several days and was easily measured by our radioimmunoassay. The average ANP excretion in 65 healthy persons was 25.0±1.4 ng/day (mean ± SEM) and the fractional excretion of ANP was 0.7±0.05%. In 14 patients with congestive heart failure, the average ANP excretion was 119.2±29.4 ng/day, which decreased to 53.3±11.0 after successful treatment.Abbreviations ANP atrial natriuretic peptide - hANP human atrial natriuretic peptide - RIA radioimmunoasay - NSB non specific bound - FE_ANP the fractional excretion of atrial natriuretic peptide - FE_Na the fractional excretion of sodium - SIADH the syngrome of inappropriate secretion of antidiuretic hormone     

Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis

Insulin-like growth factor-1 receptor (IGF-1R) has been implicated in regulation in tumor growth. The results of previous studies performed by radioimmunoassay are conflicting, and the prognostic significance of IGF-1R expression in primary breast cancer is still controversial. IGF-1R expression was evaluated in formalin-fixed, paraffin-embedded tissue of 210 primary breast cancer patients by using anti-IGF-1R antibody. The clinicopathologic variables and 5-year disease-free survival were studied, and their correlations between IGF-1R expressions were investigated. IGF-1R overexpression was observed in 43.8% of tumors. IGF-1R overexpression had no correlation with prognosis or with other clinicopathologic parameters, such as age, tumor size, nodal status, histologic grade, hormone receptor status, and human epidermal growth factor 2 status. Though its prognostic value in breast cancer is limited, immunohistochemical evaluation of IGF-1R by using this monoclonal antibody may be useful in translational research using archived material.     

Effects of granulocyte and granulocyte-macrophage colony-stimulating factors in a neutropenic murine model of trichosporonosis.

We produced disseminated trichosporonosis in a neutropenic murine model with Trichosporon asahii, which was identified by DNA relatedness analysis. We then assessed the efficacy of granulocyte colony-stimulating factor (G-CSF) (30 to 100 microg/kg of body weight per day) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.8 to 2 microg/kg x day). The administration of G-CSF either before or after infection improved the survival rate from less than 25% up to 100% (P < 0.05). The effects of G-CSF on organ clearance and histological examinations were most remarkable in the lungs. The levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage fluid (BALF) of neutropenic and G-CSF-pretreated mice were 60 +/- 6 ng/ml and 18 +/- 6 pg/ml, respectively, at 24 h after infection. Immunohistologically, alveolar macrophages proved to be the main source of TNF-alpha in BALF. GM-CSF increased neutrophil counts less significantly than did G-CSF and increased the lethality (P < 0.05) with a high level of TNF-alpha in BALF. Expecting to inhibit TNF-alpha, we administered anti-TNF-alpha intraperitoneally at the dose completely inhibiting TNF-alpha in plasma (2 x 10(4) U), but the TNF-alpha level in BALF and the lethality increased. Though the number of neutrophils at the early stage of infection appeared to be the most critical, the results suggest that other host defense mechanisms, such as TNF-alpha overproduction in the lungs, have an important role in the prognosis of trichosporonosis.     

Isolation of a lactose-binding protein with monocyte/macrophage chemotactic activity. Biological and physicochemical characteristics

BACKGROUND: We established a T cell line, STO-5, which constitutively produced monocyte/macrophage chemotactic activity via human T cell lymphoma-leukemia-virus-induced transformation of normal human T cells. METHODS: We isolated and purified a lactose-binding protein, MCF-pl5-L (MW of about 50 kD, pl of about 5) from a conditioned medium of STO-5. By using highly purified MCF-pl5-L, its biological and physicochemical properties were elucidated in comparison with C5a and MCP-1. RESULTS: MCF-pl5-L exhibited an evident dose-dependent monocyte chemotactic activity (MCA). MCF-pl5-L had no or little affinity for heparin unlike chemokines such as MCP-1. We further found that MCF-pl5-L exhibited potent chemotactic activity not only for monocytes but also for alveolar macrophages. In contrast, C5a and MCP-1 failed to show evident chemotactic activity for alveolar macrophages though they did show MCA. MCF-pl5-L failed to exhibit evident eosinophil and neutrophil chemotactic activities, indicating its chemotactic activity is selective for monocytes/macrophages. Regarding the biological functions of MCF-pl5-L other than MCA and chemotactic activity for alveolar macrophages, we found that MCF-pl5-L but not C5a and MCP-1 could prolong the life span of alveolar macrophages, probably by inhibiting apoptosis of macrophages, and stimulate the production of TNF-alpha from macrophages. CONCLUSIONS: These results suggest that MCF-pl5-L plays a role as an immune modulator for monocytes/macrophages in the site.