Toshikazu Gondo

Distribution of Chlamydia pneumoniae Infection in the Atherosclerotic Carotid Artery

BACKGROUND AND PURPOSE Chlamydia pneumoniae infection has recently become noteworthy in relation to atherosclerosis. We investigated by immunohistochemistry the distribution of C pneumoniae infection in the atherosclerotic carotid artery. METHODS Twenty carotid atherosclerotic lesions that were resected during carotid endarterectomy were investigated. Parallel sections were stained immunohistochemically with monoclonal antibodies for a C pneumoniae-specific antigen, macrophages, and smooth muscle cells. RESULTS Immunoreactivity for the C pneumoniae-specific antigen was observed in 11 of 20 specimens (55%), and intense immunoreactivity was observed in 7 of 20 (35%). C pneumoniae infection was observed in endothelial cells, macrophages and in smooth muscle cells that had migrated into the atheromatous plaque, as well as in smooth muscle cells and small arteries in the media underlying the atheromatous plaques. C pneumoniae infection was most prominently observed in smooth muscle cells. The severity of the infection as demonstrated by immunohistochemistry was not significantly related to general risk factors for atherosclerosis. CONCLUSIONS C pneumoniae widely infects endothelial cells, macrophages, and smooth muscle cells in the atherosclerotic carotid artery. The results of the present study can help us to understand how C pneumoniae infection contributes to the progression of carotid atherosclerosis.

Ultrastructural evidence for intracellular formation of amyloid fibrils in macrophages

Early amyloid deposition in the spleen was studied by immunoelectron microscopy following the administration of rapid amyloid-inducing agents to mice. Two days after the injection of an amyloidenhancing factor and casein solution, a small amount of amyloid material was observed at the border of the white pulp and the marginal zone (perifollicular area) and also within the white pulp. At this stage, amyloid fibrils were seen mainly in an extracellular distribution along the cytoplasmic processes of reticular cells and also in the cytoplasmic invaginations of macrophages. By immunoelectron microscopy, gold particles labelled fibrillar structures in lysosome-derived organelles in some macrophages as well as dense bodies consisted of a homogeneous, granular matrix not having any recognizable fibrillar structures. Similar immunolabelled organelles were also observed in the amyloid resorption stage, although, at that stage, they commonly contained other phagocytized materials as well. From these findings, we suggest that at least some amyloid fibrils are polymerized in the cytoplasm of the macrophages by the proteolytic cleavage of previously pinocytized serum amyloid A protein (SAA).

Useful polyclonal antibodies against synthetic peptides corresponding to immunoglobulin light chain constant region for immunohistochemical detection of immunoglobulin light chain amyloidosis

For the immunohistochemical detection of immunoglobulin (Ig) light chain amyloidosis on formalin‐fixed, paraffin‐embedded tissue sections, we prepared polyclonal antibodies against synthetic peptides corresponding to positions 118–134 of Ig λ light chain and positions 116–133 of Ig κ light chain. Nineteen cases of systemic Ig λ light chain amyloidosis (Aλ amyloidosis), 10 cases of systemic Ig κ light chain amyloidosis (Aκ amyloidosis), one case of immunohistochemically unclassified systemic amyloidosis and five cases of localized Aλ amyloidosis were tested with these antibodies. Anti‐λ (118–134) antiserum and the affinity‐purified antibody both reacted with 18 of the 19 cases of systemic Aλ amyloidosis and all cases of localized Aλ amyloidosis, although the immunoexpression was somewhat variable in intensity in different areas within the same specimen in both systemic and localized amyloidosis. The signal intensities in plasma cells and serum reacted for anti‐λ (118–134) antiserum were weaker than signals obtained with commercially available anti‐Ig λ light chain antibodies. Anti‐κ (116–133) antiserum and the affinity‐purified antibody reacted with nine of the 10 cases of systemic Aκ amyloidosis. We conclude that these antibodies against synthetic peptides corresponding to the Ig light chain constant region are useful for the classification of amyloidosis on formalin‐fixed, paraffin‐embedded tissue sections.

Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix

The previously isolated cDNA encoding human adenylate kinase (AK) isozyme 3 was recently renamed AK4. Consequently, human AK3 cDNA remains to be identified and we have little information about the functional relationship between human AK3 and AK4. In pursuit of the physiological roles of both the AK3 and AK4 proteins, we first isolated an authentic human AK3 cDNA and compared their expression. Nucleotide sequencing revealed that the cDNA encoded a 227-amino-acid protein, with a deduced molecular mass of 25.6 kDa, that shares greater homology with the AK3 cDNAs isolated from bovine and rat than that from human. We named the isolated cDNA AK3. Northern-blot analysis revealed that AK3 mRNA was present in all tissues examined, and was highly expressed in heart, skeletal muscle and liver, moderately expressed in pancreas and kidney, and weakly expressed in placenta, brain and lung. On the other hand, we found that human AK4 mRNA was highly expressed in kidney, moderately expressed in heart and liver and weakly expressed in brain. Western-blot analysis demonstrated expression profiles of AK3 and AK4 that were similar to their mRNA expression patterns in each tissue. Over expression of AK3, but not AK4, in both Escherichia coli CV2, a temperature-sensitive AK mutant, and a human embryonic kidney-derived cell line, HEK-293, not only produced significant GTP:AMP phosphotransferase (AK3) activity, but also complemented the CV2 cells at 42 degrees C. Subcellular and submitochondrial fractionation analysis demonstrated that both AK3 and AK4 are localized in the mitochondrial matrix.

Usefulness of endoscopic ultrasound-guided fine-needle aspiration biopsy for the diagnosis of pancreatic cancer.

BackgroundEndoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS-FNAB) has come into widespread use, mainly in Western countries, as an efficient and safe method for the cytologic or histologic diagnosis of pancreatic cancer. However, it still has received relatively little attention in Japan. To evaluate the clinical status of EUS-FNAB in Japan, we retrospectively analyzed the results with regard to the ability of EUS-FNAB to diagnose pancreatic cancer, as well as its safety.MethodsA total of 52 patients (37 male, 15 female; mean age, 62.5 years; range, 33–85 years) with focal pancreatic lesions underwent EUS-FNAB at our group of hospitals in one region of Japan. Final diagnosis was confirmed by histologic examination of surgical specimens or clinical follow-up.ResultsThe final diagnoses were malignant tumors in 32 patients and benign ones in 20. Insertion of the needle into the lesion was successful in 50 of the 52 patients (96.2%). Adequate specimens were obtained by EUS-FNAB from 47 of the 50 pancreatic lesions (94.0%). With five false-negative and no false-positive results, the accuracy, sensitivity, specificity, and positive and negative predictive values were 89.4%, 82.1%, 100%, 100%, and 79.2%, respectively. No complications occurred.ConclusionsEUS-FNAB is an efficient and safe method for the histologic diagnosis of pancreatic cancer. It should be considered as one of the indispensable modalities for the histological diagnosis of pancreatic cancer in Japan, as it is in Western countries.

Tumor HLA-DR expression linked to early intrahepatic recurrence of hepatocellular carcinoma

The outcome of patients with hepatocellular carcinoma (HCC) remains poor because of the high frequency of intrahepatic recurrence (IHR), particularly early IHR within 1 year of hepatectomy. To search for genes involved in early IHR, we performed DNA microarray analysis in a training set of 33 HCCs and selected 46 genes linked to early IHR from approximately 6,000 genes by means of a supervised learning method. Gene selection was validated by a false discovery rate of 0.37%. The 46 genes included many immune response‐related genes, which were all downregulated in HCCs with early IHR. Four of these genes (HLA‐DRA, HLA‐DRB1, HLA‐DG and HLA‐DQA), encoding MHC class II antigens, were coordinately downregulated in HCCs with early IHR compared to levels in HCCs with nonrecurrence. A cluster analysis reproduced expression patterns of the 4 MHC class II genes in 27 blinded HCC samples. To localize the major site of production of HLA‐DR protein in the tumor, we used 50 frozen specimens from 50 HCCs. Immunofluorescence staining showed that HLA‐DR protein levels in tumor cells, but not in stromal cells, were associated with the transcription levels of HLA‐DRA determined by both DNA microarray analysis and real‐time quantitative reverse transcription‐PCR. Univariate analysis showed that tumor HLA‐DR protein expression, pTNM stage and venous invasion were associated with early IHR. Multivariate analysis showed that tumor HLA‐DR protein expression was one of the independent risk factors for early IHR, suggesting HLA‐DR protein potential as a biomarker and a molecular target for therapeutic intervention.

Acceleration of murine AA amyloidosis by oral administration of amyloid fibrils extracted from different species.

We herein report that experimental murine amyloid A (AA) deposition is accelerated by oral administration of semipurified amyloid fibrils extracted from different species. Three groups of mice were treated with semipurified murine AA amyloid fibrils, semipurified bovine AA amyloid fibrils or semipurified human light chain‐derived (Aλ) amyloid fibrils for 10 days. After 3 weeks, each mouse was subjected to inflammatory stimulation by subcutaneous injection with a mixture of complete Freund’s adjuvant supplemented with Mycobacterium butyricum. The mice were killed on the third day after the inflammatory stimulation, and the spleen, liver, kidney and gastrointestinal tract were examined for amyloid deposits. Amyloid deposits were detected in 14 out of 15 mice treated with murine AA amyloid fibrils, 12 out of 15 mice treated with bovine AA amyloid fibrils and 11 out of 15 mice treated with human Aλ amyloid fibrils. No amyloid deposits were detected in control mice receiving the inflammatory stimulant alone or in amyloid fibril‐treated mice without inflammatory stimulation. Our results suggest that AA amyloid deposition is accelerated by oral administration of semipurified amyloid fibrils when there is a concurrent inflammatory stimulation.

Confocal observation of senile plaques in Alzheimer's disease: senile plaque morphology and relationship between senile plaques and astrocytes.

Senile plaques In the brains of Alzheimers disease (AD) were examined by confocal laser scanning microscopy (CLSM) with the following three findings. First, in sections stained with Congo red, the serial CLSM images of optical sections clearly revealed that a classic plaque is composed of a plaque core and a corona. Radially arranged process‐like structures, corresponding to bundles of amyloid fibrils, formed amyloid cores and stronger signals were detected in the center of some cores. Second, in sections stained with Congo red and anti‐gllal fibrillary acidic protein (GFAP), reactive astrocytes were found around the senile plaques and many astrocytlc processes surrounded the plaque cores and some processes had penetrated into them. Third, three‐dimensional reconstruction on classic plaque revealed that the surface of classic plaque showed a ‘coral‐like’ appearance.

In situ detection of oxidized n-3 polyunsaturated fatty acids in chronic hepatitis C: correlation with hepatic steatosis

BackgroundOxidative stress contributes to the pathogenesis of chronic hepatitis C. The aim of this study was to assess the peroxidation of n-3 polyunsaturated fatty acids (PUFAs) in the liver and its relation to hepatic steatosis in chronic hepatitis C.MethodsWe immunohistochemically detected malondialdehyde (MDA)-, 4-hydroxy-2-nonenal (HNE)-, and 4-hydroxy-2-hexenal (HHE)-protein adducts in liver biopsy specimens from 55 patients with chronic hepatitis C. Cells stained positively for HHE-protein adducts were quantified using computer-based image analysis. Fatty-acid composition was determined, by gas chromatography, for the noncancerous portions of resected livers, with or without steatosis, obtained from two patients with hepatitis C virus-associated hepatocellular carcinoma.ResultsThe detection rate of HHE-protein adducts (63.6%) was significantly higher than that of MDA-protein adducts (21.8%; P < 0.001) or HNE-protein adducts (29.1%; P < 0.001). Areas positively stained for HHE-protein adducts (HHE-positive areas) were significantly larger in 18 patients with steatosis (6.2 ± 3.6%) than in 17 patients without steatosis (3.4 ± 2.6%; P = 0.01). Resected liver tissue with steatosis showed a larger HHE-positive area (18.6%) and higher ratio of n-6 PUFA content to n-3 PUFA content (3 : 1) than liver tissue without steatosis (7.2%; 2 : 3). On multivariate analysis, the HHE-positive area (odds ratio, 1.55; 95% confidence interval [CI], 1.08–2.23; P = 0.019) was a factor associated with the presence of hepatic steatosis.ConclusionsHHE-protein adducts, which are a good marker for oxidative stress, are associated with steatosis in chronic hepatitis C.

Induction of Murine AA Amyloidosis by Various Homogeneous Amyloid Fibrils and Amyloid-like Synthetic Peptides

We investigated amyloid‐enhancing factor (AEF) activity of amyloid fibrils extracted from amyloid‐laden livers of mice, cow, cheetah, cat and swan. All amyloid fibrils were confirmed to be amyloid protein A (AA) by an immunohistochemical analysis. We found that these fibrils accelerated the deposition of amyloid in an experimental mouse model of AA amyloidosis. Furthermore, the degree of deposition was dependent on the concentration of fibrils. When we compared the minimal concentration of amyloid fibrils needed to induce deposition, we found that these fibrils showed different efficiencies. Murine amyloid fibril induced amyloid deposition more efficiently than cow, cat, cheetah or swan amyloid fibrils. These data suggest that amyloid deposition is preferentially induced by amyloid fibrils with the same primary sequence as the endogenous amyloid protein. We then analysed the AEF activity of synthetic peptides, synthesized corresponding to amino acids 1–15 of mouse SAA (mSAA), 2–15 of cow SAA (bSAA), 1–15 of cat SAA (cSAA), which was the same as cheetah, and the common amino acids 33–45 of these four SAA (aSAA). We found that mSAA, bSAA and cSAA formed amyloid‐like fibrils in morphology and showed similar AEF properties to those of native amyloid fibrils. Although aSAA also formed highly ordered amyloid‐like fibrils, it showed weaker AEF activity than the other synthetic fibrils. Our results indicate that amyloidosis is transmissible between species under certain conditions; however, the efficiency of amyloid deposition is species‐specific and appears to be related to the primary amino acid sequence, especially the N‐terminal segment of the amyloid protein.