nan Kunio

Immunocytochemical co-localization of the proteasome in ubiquitinated structures in neurodegenerative diseases and the elderly.

To determine at the tissue level whether the proteasome (Ps), a unique nonlysosomal protease, is involved in the metabolism of ubiquitinated proteins, we examined for the first time the immunocytochemical localizations of both Ps and ubiquitin (Ub) in sections of various abnormal structures that are known to be ubiquitinated in various neurodegenerative diseases and in the elderly. Concomitant increases of Ps and Ub were observed at the sites of most dystrophic neurites in Alzheimer disease (AD) and parkinsonism-dementia complex on Guam (PDC) and in Lewy bodies in Parkinsons disease and diffuse Lewy body disease. but not in neurofibrillary tangles in AD or PDC, in filamentous inclusions within anterior horn cells in sporadic motor neuron disease, or in eosinophilic granules in the olivary nucleus of the elderly. These results at the tissue level indicated that Ps is involved in the metabolism of some, but not all, ubiquitinated proteins and structures in various neurodegenerative disorders. This suggests that the involvement of Ps in the metabolism of ubiquitinated structures differs in different cases and at different stages of disease. These results and our previous immunocytochemical studies of lysosomal cathepsin proteases suggest that both nonlysosomal and lysosomal systems are involved in the metabolism of various ubiquitinated proteins and that their involvements differ in different structures and at different stages of degeneration of the structures.

Abnormal distribution of cathepsin proteinases and endogenous inhibitors (cystatins) in the hippocampus of patients with Alzheimer's disease, parkinsonism-dementia complex on Guam, and senile dementia and in the aged

The immunolocalization of cathepsins B(CB), H and L and cystatinsα(Cα) andβ(Cβ) were examined in the hippocampus of cases of sporadic Alzheimers disease (12 cases), parkinsonism-dementia complex on Guam (eight cases), senile dementia of Alzheimer type (two cases), aged subjects with marked senile change (one case) and controls (12 cases, including six normal subjects). CB was lower in most nerve cells in patients than in controls, but markedly increased at the sites of intracellular neurofibrillary tangles (NFTs) and degenerative neurites and/or dendrites in and outside senile plaques (SPs), indicating its close involvement in the metabolisms of various proteins in NFT and SPs. Abundant Cα and Cβ were demonstrated in SP amyloid, suggesting that they are amyloid constituents or co-exist with amyloid. The present study indicated that CB, Cα and Cβ are closely involved in abnormal protein metabolism in NFTs and SP amyloid and suggested that degeneration or denaturation of intracellular proteins, including substrates for proteases and lysosomes, from some acquired cause, results in absolute and/or relative overload for these proteolytic systems, including their inhibitors. This results in incomplete and/or abnormal proteolysis related to NFT and/or amyloid formation.

Antibodies labeled with fluorescence-agent excitable by infrared rays

Abstract: Endoscopy is not significantly better than fiberscopy for the diagnosis of minute cancers of the digestive tract. However, labeling of these lesions with an agent that can be detected videoendoscopically, with subsequent computer processing of the electronic signals, should facilitate endoscopic diagnosis of microlesions. We developed an antibody labeled with an indocyanine green(ICG) derivative that has a specific fluorescence emission at 807 nm upon excitation at 768 nm. The physiochemical characteristics of this labeled antibody resemble those of ICG. The activity of the antibody is suitable for immunohistochemical staining, and the antibody fluoresces under infrared ray excitation. This antibody should prove useful for performing vital immunostaining for infrared endoscopy.

Basic studies on a labeled anti-mucin antibody detectable by infrared-fluorescence endoscopy

Background. We developed a fluorescent dye, indocyanine green (ICG)-sulfo-OSu, which was excited by infrared rays and conjugated to various antibodies. We attempted to clarify the staining patterns of anti-sulfomucin and anti-MUC1 antibodies in gastrointestinal cancer. We then evaluated the potential of the dye as a fluorescent label for antibodies specific to cancer, to be used as a diagnostic method for microcancer, with infrared fluorescence endoscopy. Methods. Paraffin sections of samples collected from 10 patients with esophageal cancer, 30 patients with gastric cancer, and 20 patients with colorectal cancer were immunohistologically stained using an anti-sulfomucin antibody and an anti-MUC1 antibody, and the staining patterns were examined. If a section had a high staining intensity, it was reacted with the ICG-suflo-OSu-labeled antibody and evaluated with infrared fluorescence imaging. Results. The staining patterns with the antibodies varied depending on the organs and the histological types and depth of the cancers, but the staining was generally good and the staining on the mucosal surface of cancer tissues was retained. Good images of cancer cells could be obtained by infrared fluorescence observation using the ICG-sulfo-OSu-labeled anti-MUC1 antibody. Conclusions. The anti-MUC1 antibody stained gastrointestinal cancer cells well, and nearly specific infrared fluorescence in cancer tissues was observed using the labeled anti-MUC1 antibody. The ICG-sulfo-OSu-labeled anti-MUC1 antibody has possible usefulness for the screening of cancer via infrared fluorescence endoscopy.

MIXED CONNECTIVE TISSUE DISEASE WITH FATAL PULMONARY HYPERTENSION

An autopsy case of mixed connective tissue disease (MCTD) with pulmonary hypertension is presented. A 34‐year‐old woman suffering from arthralgia, Raynauds phenomenon, and dyspnea of 6‐years duration was diagnosed as having MCTD on the basis of a high titer (1:160,000) of serum antibody to the ribonuclease‐sensitve component of extractable nuclear antigen. Examination of cardiac function revealed the complication of pulmonary hypertension. Autopsy revealed concentric intimal cellular proliferation of the small arteries and arterioles of both lungs. Typical plexiform lesions of these vessels were also observed. These findings coincide with those of plexogenic pulmonary angiopathy of primary pulmonary hypertension (PPH). This is the second autopsy case of MCTD with fatal pulmonary hypertension reported and our observations suggest that some cases with PPH who had immunological abnormalities but could not be classified as cases of classical collagen disease, may have been induced by MCTD.

A new infrared fluorescent-labeling agent and labeled antibody for diagnosing microcancers.

PURPOSEnWe have developed infrared fluorescent labeling agents and infrared-ray fluorescence endoscopes to establish a novel diagnostic technique. Since the fluorescence intensity of the initial labeled antibody (ICG-sulfo-OSu-labeled antibody) was not sufficient for practical use, we synthesized indocyanine green acylthiazolidinethione (ICG-ATT), which was expected to label various target molecules having amino groups efficiently.nnnMATERIALS AND METHODSnTo confirm imaging of infrared fluorescence intensity of ICG-ATT- and ICG-sulfo-OSu-labeled anti-MUC1 antibodies, cotton thread was soaked in various concentrations of the antibody solution in 0.1M PBS, and observed under the epi-illumination infrared fluorescence microscope. Localization and the intensity of infrared fluorescence and DAB coloring was compared in paraffin sections of human gastric mucosa.nnnRESULTSnIn the study of cotton threads, both labeled antibodies showed relatively clear infrared fluorescence, and significant difference was not observed between the two antibodies. ICG-ATT-labeled anti-MUC1 antibody produced stronger staining than that by ICG-sulfo-OSu-labeled antibody. Localization pattern of infrared fluorescent staining was in good agreement with that by the conventional method with oxidized DAB staining.nnnCONCLUSIONnICG-ATT is useful as a fluorescent-labeling agent for diagnosis of microcancers by infrared fluorescence endoscopes.

Prediction of prognosis in gallbladder carcinoma by mucin and p53 immunohistochemistry

Mucin core proteins are known to be present in various organs and are specifically expressed with carcinogenesis and closely associated with the prognoses of various malignant tumors in the digestive tract such as colorectal cancer. The present study evaluated correlations between mucin and p53 expression and prognosis of gallbladder cancer using surgically resected tissue specimens from 26 patients with gallbladder carcinoma surgically treated at our hospital. Immunohistochemical staining was performed using MUC1, MUC2, and p53 monoclonal antibody. The level of antigen expression in the lesion was classified into four stages: none(−), slight(+), moderate (++), and severe (+++). According to the UICC classification, histopathological grading, levels of T, N, and M factors, and tumor stages were compared with regard to the correlations with mucin and p53 expression. All cases were classified into two groups according to the results of mucin immunohistochemistry: group A (MUC1, ≥{++}; and MUC2, ≤+) and group B (MUC1, < ++; or MUC2, > +). Postoperative survival periods were compared between the two groups and p53-positive and -negative groups. Neither histological grading nor T factor correlated with mucin or p53 expression, respectively. Moreover, neither N factor nor M factor correlated with mucin or p53 expression. Furthermore, stage grouping did not correlate with mucin or p53 expression. However, when the correlation between the postoperative survival period and mucin expression was evaluated, the mean postoperative surgical period was significantly shorter in Group A than in Group B (1.02 years in Group A vs 2.92 years in Group B; P = 0.016). There was no relationship between postoperative survival period and p53 positivity. Mucin expression was independent of various tumor growth factors and clearly reflected the prognosis of gallbladder cancer. Because the relative malignancy of gallbladder cancer could be evaluated by examining the level of glycoprotein expression in tumor tissue, mucin could be a more important marker than p53 for predicting prognosis in gallbladder carcinoma using surgically resected tissue specimens.

Reflected illumination-type imaging system for the development of infrared fluorescence endoscopy

Fluorescent labeled monoclonal antibodies against carcinoembryonic antigen (CEA) may provide a specific label for lesions of the GI tract. We previously developed an indocyanine green (ICG) derivative (ICG N‐hydroxy sulfo succinimide ester; ICG‐sulfo‐Osu) as a fluorescent label that is excited by infrared rays and is suitable for vital immunohistochemical staining. We also previously developed a transmitted illumination‐type (transmitted type) fluorescence imaging system that uses infrared rays to detect ICG‐sulfo‐Osu. In this paper we describe a new reflected illumination‐type (reflected type) imaging system for infrared fluorescence endoscopy. We tested the efficacy of this system on sections of human esophagus and normal skeletal muscle stained with ICG‐sulfo‐Osu labeled anti‐epithelial membrane antigen (EMA) antibody, and on sections of human gastric cancer tissue stained with ICG‐sulfo‐Osu labeled anti‐CEA antibody. Infrared fluorescent images were obtained with both fluorescent antibodies, and results correlated well with oxidized DAB‐positive sites. Vital immunohistochemical staining of micro cancers should be detectable by exciting an ICG‐sulfo‐Osu labeled antibody specific to the tumor cells with infrared rays, using this reflected type imaging system.

Vital immunostaining of human gastric and colorectal cancers grafted into nude mice : a preclinical assessment of a potential adjunct to videoendoscopy

Abstract: Videoendoscopy has not significantly advanced diagnostic accuracy beyond that attainable by conventional fiberscopy, with respect to microcarcinomas of the digestive tract. We suspected that after the labeling of these lesions with an agent detectable by videoendoscope, digital processing of the images could facilitate endoscopic diagnosis of microcarcinomas. We have developed a novel antibody labeled with an indocyanine green (ICG) derivative that is evident by videoendoscope. However, the binding of such an exogenous antibody in vivo to tumor surfaces has not been described. In this preliminary study, after transplanting human gastric cancer or colorectal cancer into nude mice, we successfully bound the tumors in vivo with an anti-MUC1 mucin antibody, as subsequently confirmed by the performing of immunohistochemistry with a secondary antibody. The antibody labeled with an ICG derivative may therefore be clinically useful in detecting gastrointestinal microcarcinoma by videoendoscopy.

Usefulness of a hemoglobin index determined by electronic endoscopy in the diagnosis of helicobacter pylori gastritis

In patients with Helicobacter pylori infection, gastric endoscopy usually shows diffuse mucosal redness that disappears after successful H. pylori eradication. However, some infected patients do not show such redness. Therefore, disagreement continues concerning the need for gastric endoscopy in suspected H. pylori infection.