Functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecule in antigen recognition by T cells in HLA-DR and DQ transgenic mice.

Studies in vitro have suggested that a species barrier exists in functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecules. However, whether mouse CD4+ T cells restricted by HLA class II molecules are generated in HLA class II transgenic mice and respond to peptide antigens across this barrier has remained unclear. In an analysis of T cell responses to synthetic peptides in mice transgenic for HLA-DR51 and -DQ6, we found that DR51 and DQ6 transgenic mice acquired significant T cell response to influenza hemagglutinin-derived peptide 307-319 (HA 307) and Streptococcus pyogenes M12 protein-derived peptide 347-397 (M6C2), respectively. Inhibition studies with several monoclonal antibodies showed that transgenic HLA class II molecules presented these peptides to mouse CD4+ T cells. Furthermore, T cell lines specific for HA 307 or M6C2 obtained from the transgenic mice could respond to the peptide in the context of relevant HLA class II molecules expressed on mouse L cell transfectants that lack the expression of mouse MHC class II. These findings indicate that interaction between HLA class II and mouse CD4 molecules is sufficient for provoking peptide-specific HLA class II-restricted T cell responses in HLA class II transgenic mice.

Comparative histopathological analysis between tenosynovitis and joint synovitis in rheumatoid arthritis.

AIMS: To clarify the histological and biological features of tenosynovitis accompanying rheumatoid arthritis (RA). METHODS AND RESULTS: Synovial tissue was obtained from the wrist joint and extensor tendon of the digits of six RA patients and the sections were examined by haematoxylin and eosin staining and immunohistochemical analysis. RA tenosynovitis exhibited the typical histological features of RA joint synovitis, including hyperplasia of the synovial lining and infiltration of leucocytes, largely CD4+ T cells and CD68+ macrophages. Notably, there was a significant correlation in the number of CD4+ T cells and CD68+ macrophages between the tenosynovium and joint synovium in each individual. Real-time reverse transcriptase-polymerase chain reaction analysis revealed similar mRNA expression patterns of various inflammatory mediators in tenosynovitis and joint synovitis. It was also observed that synovial fibroblasts isolated from the tenosynovium behaved in a manner similar to those isolated from the joint synovium with regard to proliferation and the production of inflammatory mediators. CONCLUSIONS: The histopathological features of RA tenosynovitis were indistinguishable from those of joint synovitis. Therefore, it is suggested that the ongoing inflammation is driven by similar mechanisms in the tenosynovium and joint synovium and that RA is probably a tissue-specific disease which targets systemic synovial tissues.

Osteopontin mRNA is expressed by smooth muscle-derived foam cells in human atherosclerotic lesions of the aorta.

Osteopontin is a phosphorylated, sialic acid-rich, noncollagenous bone matrix protein containing the Arg-Gly-Asp-Ser amino acid sequence responsible for cell adhesion. The protein strongly binds to hydroxyapatite and play an important role in calcification. Expression of osteopontin mRNA was analyzed in human aortic atherosclerotic lesion by Northern blot hybridization, as well as by in situ hybridization. The expression of osteopontin mRNA was detected in 24 out of 25 samples of aorta obtained from 17 autopsy cases, but not in one normal aortic sample. The magnitude of expression was proportional to the stage of atherosclerosis. In situ hybridization revealed that the cells expressing osteopontin mRNA were detected in the wall surrounding atheroma and closely associated with calcification. They were morphologically identified as foam cells and immunohistologically positive with HHF35, appearing to be derived from smooth muscle cells. These findings have suggested that smooth muscle cell-derived foam cells express osteopontin mRNA and play an important role in calcification of the atherosclerotic lesions.

Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase From Porcine Endothelial Cells by a CRISPR System.

BACKGROUND: We attempted to knock out the expression of Hanganutziu-Deicher (H-D) antigens through the use of a CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9 system for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). METHODS: Plasmids expressing hCas9 and sgRNA for pCMAH were prepared by ligating oligos into the BbsI site of pX330. The N-terminal and C-terminal EGFP coding regions overlapping 482 bp were PCR-amplified and placed under a ubiquitous CAG promoter. The approximately 400-bp genomic fragments containing the sgRNA target sequence of pCMAH were placed into the multi-cloning sites flanked by the EGFP fragments. The pCAG-EGxxFP-target was mixed with pX330 with/without the sgRNA sequences and then introduced into HEK293T cells. RESULTS: Four oligos and primers, gSO1, gSO3, gSO4, and gSO8, were nominated from 8 candidates. Among them, gSO1 showed the best efficiency. Pig endothelial cells (PECs) from an α-Gal knockout pig were then used to examine the changes in the expression of the H-D antigen by the knockout of the CMAH genome by the pX330-gS01. CONCLUSIONS: Changes in the expression of the H-D antigen in the PECs with the CRISPR (gS01) were clear in comparison with those in the parental cells, on the basis of FACS analysis data. The expression of the H-D antigen can be knocked out by use of the CRISPR system for pCMAH, thus confirming that this system is a very convenient system for producing knockout pigs.

Clinicopathologic characteristics of elderly patients with persistent ST segment elevation and inverted T waves: evidence of insidious or healed myocarditis?

OBJECTIVES: We sought to clarify the clinicopathologic characteristics of insidious or healed myocarditis in the elderly. BACKGROUND: Myocarditis is the cause of unexplained congestive heart failure and dilated cardiomyopathy. However, acute myocarditis of the Fiedler type is rare, and the incidence and implication of insidious or healed myocarditis in the elderly are not yet known. METHODS: In an autopsy study of 3,000 patients aged > or = 60 years, there were 12 (0.4%) with insidious or healed myocarditis, showing extensive and circumferential fibrosis and scattered lymphocytic infiltration of both ventricular walls without acute necrosis of the myocardial fibers. RESULTS: Unexplained congestive heart failure was found in seven cases. In all cases, electrocardiography had demonstrated upward elevation of the ST segment and inverted T waves for durations ranging from 1 month to 12.7 years (mean 5.7 years). Mean (+/- SD) heart weight was 338 +/- 81 g (range 220 to 470). In nine cases, fibrous lesions, which were scattered but extensive and circumferential, were located in the subepicardial and middle layers of the left ventricle. In the remaining three cases, the fibrous lesions were located predominantly in the subepicardial and middle layers, but the subendocardial layer was also locally involved. Fibrous lesions of the right ventricle were predominant in the subepicardial layer and involved the subendocardial layer in four cases. Scattered lymphocytic infiltration was found in the fibrous lesions. CONCLUSIONS: In more than half of the aged cases with insidious or healed myocarditis, unexplained congestive heart failure was also present. Fibrous lesions due to myocarditis were located predominantly in the subepicardial and middle layers and led to persistent upward elevation of the ST segment and inverted T waves.

Paroxysmal atrial fibrillation: high frequency of embolic brain infarction in elderly autopsy patients.

We examined brains clinicopathologically from 54 consecutive paroxysmal atrial fibrillation (PAF) patients aged 70 years or older and compared them with those of 59 age-matched controls. Symptomatic cerebral infarctions were present in 29 PAF patients (53.7%) and in 13 controls (22.0%) (p < 0.001). Symptomatic brain infarction was 2.4 times more common in PAF cases than in PAF-free controls; approximately 60% of the infarctions in the PAF cases were judged to be cardioembolic in origin. PAF in the elderly can be an important cause for cardiogenic cerebral embolism.

Embolic brain infarction in nonrheumatic atrial fibrillation: a clinicopathologic study in the elderly.

Although CT studies have addressed symptomatic and asymptomatic cerebral infarctions in nonrheumatic atrial fibrillation (NRAF), pathologic verification of the results is lacking. The purpose of this study was to assess the frequency, location, and extent of symptomatic and asymptomatic brain infarction in autopsy specimens from elderly patients with NRAF. We examined autopsy specimens from 136 consecutive NRAF patients 70 years of age or older who received no anticoagulant therapy during their lifetime and compared them with 231 age-matched control subjects with similar health histories except for the absence of NRAF. Symptomatic cerebral infarctions were present in 82 (60.3%) NRAF patients and in 55 (23.8%) control subjects (p < 0.0001). Of symptomatic cerebral infarctions, cardioembolic infarction was present in 53 (64.6%) NRAF patients and in two (3.6%) of the control subjects (p < 0.0001), atherothrombotic infarction in 13 (15.9%) NRAF patients versus 36 (65.5%) control subjects (p < 0.0001), and lacunar infarction in four (4.9%) NRAF patients versus 12 (21.8%) control subjects (p < 0.01). Stroke-related death occurred in 34 (25.0%) NRAF patients and in 18 (7.8%) control subjects (p < 0.0002). Symptomatic cerebral infarction was generally accompanied by asymptomatic infarctions in both NRAF patients and control subjects. Asymptomatic cortical infarctions were more common in NRAF patients, but asymptomatic infarctions in the white matter or deep structures were more common in control subjects. In this autopsy series of individuals over 70 years of age, symptomatic brain infarction was 2.5 times more common in NRAF patients than in NRAF-free control subjects; two-thirds of the infarctions in the NRAF cases were judged to be cardioembolic in origin. Most asymptomatic cerebral infarctions in the NRAF patients were located in the cortices.

Correlation of telomere lengths in normal and cancers tissue in the large bowel.

The hypothesis that telomeres in colorectal cancer cells exhibit age-related shortening, as in normal cells of the colorectal epithelium, was tested with samples of non-cancerous mucosa and cancer tissue from 124 patients (aged 29-97 years). Shortening with aging could be demonstrated for both normal and cancer tissues; regression analysis showed rates for length reduction of 44 and 50 base pair/year, respectively. Straight, essentially parallel, lines were obtained for the two cases, normal tissue values being about 2 kilobase pairs (kbp) higher, with a significant correlation between data at the individual patient level.

Primary undifferentiated small cell carcinoma of the esophagus.

We histologically examined undifferentiated small cell carcinoma of the esophagus from 21 patients and used immunohistochemical methods for detection of chromogranin A and p53, bcl-2, and Rb oncoproteins. Nine (43%) of the 21 carcinomas consisted solely of undifferentiated cells, but heterogeneous components of in situ or invasive squamous cell carcinoma or mucoepidermoid carcinoma were observed in the other 12 (57%) tumors. Squamous cell carcinoma in situ was observed in the mucosa adjacent to the main tumor in 7 (50%) of the 14 resected esophageal specimens. An admixture of invasive squamous cell carcinoma and undifferentiated carcinoma was observed in 4 (19%) of the 21 tumors, and mucoepidermoid carcinoma was noted in one case. Chromogranin A staining yielded a positive reaction in two (10%) undifferentiated components but was negative in all heterogeneous components. Multiple sites of p53 immunopositivity were seen in the undifferentiated component of 17 (81%) of the 21 tumors, as well as in the in situ or invasive squamous cell carcinoma or mucoepidermoid carcinoma components of 9 (75%) of 12 tumors. Seven (33%) of the 21 tumors showed positive bcl-2 immunoreactivity in the small cell component, but all of the heterogeneous components were negative. Rb protein immunoreactivity was observed in the small cell component of one (5%) case and in 9 (75%) of the 12 heterogeneous components. Six (86%) of the seven in situ squamous cell carcinoma components were positive for Rb protein. Eighteen (86%) of the 21 patients died within 24 months of diagnosis. Two patients (10%) who survived for more than 24 months had received chemotherapy.

Telomere shortening in gastric carcinoma with aging despite telomerase activation.

In the present study, we analyzed both telomere length and telomerase activity in surgical and autopsy samples of non-neoplastic mucosa and carcinomas of the stomach. Telomere length, determined by Southern blot analysis, demonstrated progressive shortening with age in non-neoplastic gastric mucosal specimens from 38 human subjects aged between 0 and 99 years, with an average annual loss rate of 46 base pairs (bp). The mean (+/- SD) telomere length in 21 gastric carcinomas was 7.0 +/- 1.6 x 10(3) base pairs (1.6 kbp). In 20 (95%) of the 21 subjects, the values were smaller than those in the nonneoplastic gastric mucosa (mean shortening 1.8 kbp), although a strong correlation was observed for the paired data (r = 0.69, P = 0.0004). Similarly, telomere lengths in carcinomas were shorter than those for intestinal metaplasia (a mean difference of 1.1 kbp). Telomerase activity, estimated using the telomeric repeat amplification protocol assay, was positive in 18 (86%) of the 21 gastric carcinomas, without significant differences among the three histological types (well, moderately, and poorly differentiated adenocarcinomas) or with sex or age. The results suggest that telomere length and possibly shortening rates vary with the individual, and that examination of both non-neoplastic mucosa and tumors is necessary to improve our understanding of the significance of telomerase in neoplasia.

Acute idiopathic interstitial myocarditis: case report with special reference to morphological characteristics of giant cells.

Necropsy findings of an acute fatal case of idiopathic interstitial myocarditis were reported. The patient was a 33 year old housewife who had acute cardiac failure on the sixteenth day after the onset of the disease. Necropsy showed important pathological changes confined to the heart. Both ventricles were affected by confluent granulomas with an ill defined patchy appearance. Histologically these lesions consisted of round cells, histiocytes, eosinophils and myogenic giant cells. The findings were compatible with those of interstitial myocarditis associated with a proliferation of giant cells. Both atriums were also affected to a minor extent, detectable only by histological examination. Electron microscopy and cytochemistry showed that most giant cells noted in the lesion showed myofibrils and primary lysosomes in the cytoplasm. Giant cells were positive for myoglobin. Though the macrophage origin of the giant cell in this disorder has been emphasised in a recent report, these cytological results suggest that giant cells observed in the cardiac granulomatous lesions of this case were mainly myogenic in origin.

Telomere shortening with aging in human liver.

Progressive telomere shortening with aging was studied in the normal liver tissue of 94 human subjects aged between 0 and 101 years old to determine the rate of telomere loss in 1 year. Telomere length demonstrated accelerated shortening with reduction of 55 base pairs (bp) per year. The mean telomere length in five neonates was 12.9 +/- 2.6 kilobase pairs (kbp), and that in one centenarian was 8.3 kbp. Mean telomere lengths by age group were 13.2 +/- 2.0 kbp (< or = 8 years; 10 subjects), 7.8 +/- 1.9 kbp (40-79 years; 29 subjects), and 7.5 +/- 2.0 kbp (> or = 80 years; 53 subjects), with reduction thus appearing to show slowing on the attainment of middle age. The difference of mean telomere lengths for two groups with or without advanced malignancies of other than liver origin was not significant in the older two groups. Despite the slow turnover of liver tissue, the overall reduction rate of telomere length decrease in 1 year was almost the same as that of digestive tract mucosa, with its very rapid renewal.

Incidence and pathogenesis of villous tumors of the gallbladder, and their relation to cancer.

The aim of this study was to investigate the incidence and pathogenesis of villous tumors of the gallbladder, and their relation to cancer. Five hundred and thirty-three cases of cholecystectomy and 1300 randomly selected autopsy cases, mainly elderly individuals, were investigated. Gallbladders were fixed in 10% formalin after operation or at autopsy, followed by macroscopic study. In cases of villous tumors, the entire gallbladder was cut into 5-mm-thick serial sections, embedded in paraffin, cut to 4-microns, stained with hematoxylin and eosin (H&E), and histologically studied. To investigate cancer-associated antigens, i.e., carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9, deparaffinized sections were examined by the peroxidase-antiperoxidase (PAP) immunohistochemical technique with anti-CEA and anti-CA 19-9 antibodies. Villous tumor was found in two resected cases (0.38%) and in one autopsy case (0.08%). Histologically, one of the villous tumors consisted mainly of a proliferation of lining epithelia; the other two consisted mainly of a proliferation of glands. In all three cases, the patients had had accompanying chronic or acute inflammation and two were accompanied by cholecystolithiasis, which made us aware of the importance of inflammation or trauma from stones in the pathogenesis of such neoplasms. Although no apparent cancerous epithelium was observed in any of these tumors by studying H&E specimens, moderate structural and cellular atypism was found in one of them. The atypical epithelium in this case was positively stained for CEA and CA 19-9. It was concluded that villous tumor should be considered to be a premalignant lesion.

Mucin-producing adenoma associated with pancreas divisum and hepatic hilar carcinoma: an autopsy case.

We present the autopsy case of an 82-year-old Japanese woman with a mucin-producing adenoma accompanied by pancreas divisum and a hepatic hilar carcinoma. She had suffered from a cholangiocellular carcinoma at the hepatic hilus for 2 months, which was treated with radiation and chemotherapy. She did not complain of any abdominal pain. Obstructive jaundice deteriorated despite percutaneous transhepatic bile duct drainage, and she died of hepatic insufficiency. At autopsy, a hepatic tumor was confirmed to have caused severe obstructive jaundice. Histological examinations showed moderately to poorly differentiated cholangiocellular adenocarcinoma with squamous metaplasia, probably due to radiation. A yellowish mucinous tumor was found in the head of the pancreas near the minor papilla. It consisted of multiple rice-sized cystic lesions with thin septa. Although it had no capsule, its margin was clear. Neither a wide opening of the major or minor papilla nor mucous drainage was observed. Gross examinations revealed unfused pancreatic ducts. The slightly dilatated dorsal duct and a branch of the mildly dilatated ventral duct showed tumor involvement. Histological examinations showed mild atypia of the epithelia, and this pancreatic tumor was diagnosed as branch duct-type mucin-producing adenoma with postradiation dysplasia. The combination of a mucin-producing tumor and pancreas divisum is rare, and this is only the third reported case.

Multiple colorectal cancers in the elderly: a retrospective study of both surgical and autopsy cases.

BACKGROUND: Multiple primary cancers are not rare events in the large intestine, and account for approximately 5-7% of patients with colorectal cancer (CRC). There are few reports demonstrating clinicopathologic features of multiple CRCs in the elderly. METHODS: We clinicopathologically investigated 947 surgical patients and 362 autopsy samples from patients aged 65 years or more with CRC, including 81 surgical and 34 autopsy cases of multiple CRCs. We compared the data in the very old group (age > or = 85 years) with those of the younger age groups, i.e., a young-old group (65-74 years) and a middle-old group (75-84 years). RESULTS: The proportion of multiple CRCs was 8.6% (81/947) in the surgical patients and 9.4% (34/362) in the autopsy cases, with no significant difference among the three age groups. Similar site distributions and sex ratios, indicating proximal shift and female predominance with advancing age, were found in multiple and single CRCs, except for autopsy cases with multiple CRCs. Multiple CRCs in nonadjacent segments of the large intestine accounted for 11% (9/81) in surgical cases and 35% (12/34) in autopsy cases. In autopsy cases, the incidence of extracolorectal malignancies in patients with a single CRC was 22% (17/76) in the young-old group, 27% (39/147) in the middle-old group, and 35% (37/105) in the very old group, whereas the incidences in patients with multiple CRCs were 25% (1/4), 11% (2/18), and 50% (6/12), respectively. Regarding the organs with extracolorectal malignancies, the stomach (29%) was most frequent, followed by lung (14%), hematopoietic system (12%), and pancreatobiliary system (10%). CONCLUSIONS: These results indicate that the incidence of multiple CRCs in elderly patients with CRC is approximately 8%-10%, with no age-related difference, while extracolorectal malignancies increase with advancing age.

Hepatocellular carcinoma arising in an elderly male with primary biliary cirrhosis.

We report the case of an elderly male with asymptomatic primary biliary cirrhosis (PBC) who developed a hepatocellular carcinoma (HCC). The 89-year-old man, who was otherwise healthy, was admitted for investigation of mild hepatic dysfunction, which had been detected during a routine physical check-up. Serum chemistry, positive anti-mitochondrial antibody (M2) and liver biopsy results led to a diagnosis of PBC. Three years later, at age 92, computed tomography (CT) and ultrasound scans of his abdomen revealed a large hepatic tumour, which was confirmed on liver biopsy to be HCC. The tumour ruptured 3 months after diagnosis and the patient was successfully stabilized by coil embolization of his right hepatic artery. We believe that, to date, this is the oldest reported patient to have had interventional radiology for the management of HCC.

Preclinical primary central nervous system lymphoma.

We report an autopsy case of preclinical primary central nervous system (CNS) lymphoma. The case was an 89-year-old female who died of rupture of a thoracic aortic aneurysm. No neurological abnormalities were found throughout our clinical observation. Serum anti-human T-cell leukemia virus type 1 and anti-human immunodeficiency virus were negative. Grossly, the brain was normal except for a small solid tumor, 6 by 6 by 2.5 mm, in the lateral ventricular wall at the left hippocampal fimbria, and multiple smaller disseminated nodules. Histological examination revealed multiple microscopic disseminated foci throughout the brain, including the cerebral subcortical white matter, basal nucleus, thalamus, brainstem and cerebellum. Histological classification is of lymphoblastic type non-Hodgkin's lymphoma of high-grade malignancy. Lymphoma cells are positive for leukocyte common antigen and L-26, indicating a B cell phenotype. In situ hybridization with Epstein-Barr viral probes is negative. It is concluded that this case may represent a relatively early preclinical stage of multifocal type of primary CNS lymphoma.

Three-dimensional organization of the hepatic microvasculature in hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant systemic fibrovascular dysplasia. Although hepatic vascular shunts are often observed in HHT, the responsible pathological mechanism is unknown. This issue was addressed by performing a 3-dimensional reconstruction study of the hepatic microvasculature of an HHT-involved liver in a 79-year-old woman. Clinical observation revealed high-output congestive heart failure and hepatic encephalopathy due to arteriovenous and portovenous shunts, respectively. Angiography revealed tortuous dilation of hepatic arterial branches and intrahepatic arteriovenous shunts. The 3-dimensional analysis of the autopsy liver revealed focal sinusoidal ectasia, arteriovenous shunts through abnormal direct communications between arterioles and ectatic sinusoids, and portovenous shunts due to frequent and large communications between portal veins and ectatic sinusoids. Type 1 HHT was suggested by the lack of endoglin immunoreactivity in the liver. The 3-dimensional reconstruction study of hepatic microvasculature was successful in identifying the pathological changes responsible for the intrahepatic shunts in HHT.

Effects of bacterial endotoxin on ciliary activity in the tubotympanum.

The discovery of endotoxin in effusion of otitis media with effusion (OME) has suggested the possibility that bacterial endotoxin may be involved in the pathogenesis and development of OME. In this study, we investigated the direct effects of lipopolysaccharide (LPS), possessing the major part of endotoxin activity, on the ciliary activity in the tubotympanum. The study shows that LPS deteriorates ciliary activity in a dose-response fashion and that even low levels can, with extended exposure, cause dysfunction of cilia. It can be postulated that endotoxin in middle ear effusions aggravates the condition of mucociliary dysfunction thus leading to chronic OME.

Acute effects of sulfur dioxide exposure on the middle ear mucosa.

A variety of atmospheric pollutants are known to depress mucociliary function in the respiratory system. Since the mucociliary function in the middle ear is similar, and the middle ear may be invaded by atmospheric pollutants, we decided to investigate the possible contribution of sulfur dioxide to middle ear effusion. Guinea pigs were exposed for 24 hours to 300 ppm of sulfur dioxide or air. Immediately after exposure, ciliary activity and epithelial structure were examined close to the tympanic orifice (proximal site) and more distal to it (distal site). In the animals exposed to sulfur dioxide, no effusion was found in the tympanic cavity. Ciliary activity was reduced only in the distal site. Electron microscopy demonstrated hypersecretion in the proximal site and severe pathologic changes in the distal site. Although the normally functioning cilia in the proximal site may prevent retention of surplus secretions in the ear, sulfur dioxide may promote middle ear effusion when combined with other detrimental factors, because it stimulates mucus secretion in the proximal site and impairs ciliary function in the distal site.