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.

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.

Breast carcinoma in women over the age of 85: distinct histological pattern and androgen, oestrogen, and progesterone receptor status.

AIMS: The pathogenesis of breast carcinoma in very elderly women is of interest, because oestrogen levels are likely to be extremely low during the development of the disease. In an effort to understand the pathogenesis of breast carcinoma in these women, this study was undertaken to compare the histological patterns and hormone receptor status of breast carcinomas arising in very elderly and younger women. METHODS AND RESULTS: Thirty-seven breast carcinomas from women over the age of 85 years at the time of their operation were examined histologically and compared with those from a large group of premenopausal women. The proportions of mucinous carcinoma and apocrine carcinoma were significantly greater in older women. The expression of steroid hormone receptors was studied immunohistochemically. Androgen receptor-positive carcinomas were significantly more frequent among older women, whereas progesterone receptor-positive carcinomas were significantly less frequent. There was no statistically significant difference in oestrogen receptor-alpha or -beta expression between the tumours from both groups. CONCLUSION: Breast carcinomas in women over the age of 85 years have a different morphological spectrum from carcinomas in younger age groups and may have different pathogenesis mechanisms that may be more dependent on androgen and androgen receptor interaction. Differences from the results of the other studies are discussed.

Argyrophilic grain disease mimicking temporal Pick's disease: a clinical, radiological, and pathological study of an autopsy case with a clinical course of 15 years.

This report concerns an autopsy case of argyrophilic grain disease (AGD) mimicking temporal Pick's disease. The patient was a Japanese woman without hereditary burden who was 89 years old at the time of death. She developed memory impairment and began wandering at the age of 74, followed by prominent character changes about 6 years after disease onset. A neurological examination 5 months before her death revealed poor rapport, unconcern, severe dementia, and double incontinence, without aphasia or muscle rigidity. Serial neuroradiological examination revealed progressive enlargement of the bilateral inferior horns of the lateral ventricle, reflecting progressive atrophy of the medial temporal lobes. Macroscopically, neuropathological examination showed circumscribed atrophy of the bilateral amygdalae, hippocampi, parahippocampal gyri, and lateral occipitotemporal gyri. Histologically, there was neuronal loss in the areas mentioned above, the caudate nucleus, putamen, thalamus, substantia nigra, and locus ceruleus, with ballooned neurons in the cerebral cortex and amygdala. Numerous argyrophilic grains with coiled bodies were present not only in the limbic system, but also in the affected cerebrum. Rare neurofibrillary changes were present in the limbic areas, consistent with Braak stage II, with no senile plaques. Based on these findings and a review of the literature, we note that AGD is clinicopathologically similar not only to mesolimbocortical dementia, but also to atypical senile dementia of Alzheimer type. This report may contribute to the elucidation of the clinicopathological hallmarks of AGD.

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.

Clinicopathological characteristics of left ventricular myocardium with transient asynergy: report of three cases.

The aim of the present retrospective study was to clarify the histopathologic substrates of left ventricular myocardium with transient asynergy due to acute ischemic insult in man. Three patients who had had prolonged chest pain, new abnormal Q waves and new ST segment elevation were studied. There was no significant elevation of serum creatine phosphokinase activity in two of the three patients. Echocardiograms on admission or the next day showed severe hypokinetic or akinetic motion and thinning of the anteroseptal and apical portions of the left ventricle and regional dilatation of the same portions. Disappearance of the abnormal Q waves, ST segment elevation resolution, and early T wave inversion were observed later. Complete improvement of the echocardiographic abnormalities was confirmed after a few weeks in all patients. Manifest ischemic lesions of subendocardial scars of the anteroseptal region of the left ventricle were detected in only one of the three cases by gross examination. However, on microscopic examination, islands of necrotic myocytes were interspersed with islands of viable cells throughout the jeopardized region in one case, although the scattered necrotic foci were restricted to the subendocardium and the trabeculae. Normal myocardium and subendocardial scars were observed in the other two cases. In conclusion, left ventricular myocardium with transient asynergy. detected clinically during acute ischemic attack, consists of normal myocardium or small ischemic lesions primarily in the subendocardium.

[Two elderly patients with sarcoidosis and Sjögren's syndrome].

Two elderly patients with sarcoidosis complicated with Sjögren's syndrome are described. Case 1: A 70-year-old woman was admitted due to dry eyes. Histological examination of a minor salivary gland specimen revealed lymphocytic infiltration, which was compatible with Sjögren's syndrome. Because uveitis was demonstrated, transbronchial lung biopsy (TBLB) was performed, to confirm a diagnosis of lung sarcoidosis. Histological examination of TBLB showed non-caseating granulomas compatible with sarcoidosis. Case 2: A 70-year-old woman was admitted due to dyspnea on exercise and blurred vision. Two years previously, Sjögren's syndrome was diagnosed because of a positive Shirmer test and positive SS-A antibody. Result of ophthalmic examination were compatible with uveitis. Histological examination of TBLB showed non-caseating granulomas compatible with sarcoidosis. Certain similarities between sarcoidosis and Sjögren's syndrome in terms of immunological aspects have attracted attention. In the present manuscript, a possible relationship between the two diseases as well as the characteristics of elderly sarcoidosis are discussed.

Rare complication of saccular aneurysm and histological dissection: an autopsy study of 5865 elderly cases.

The purpose of this study was to examine complicated lesions of saccular aneurysm and dissection. We investigated the ascending aortae in 5865 consecutive elderly autopsy cases, and 5 cases (0.085%) of aortic saccular aneurysms associated with dissection at the edges were selected. Their edges characteristically protruded like a shelf, which histologically consisted of an inner part of the dissected media. All patients were female and their mean age was 76.8 (67-89) years. The aneurysmal walls at the center showed severe fragmentation and disappearance of the elastic lamellae of the remaining outer media. Thus, partial sections of these lesions were not sufficient but cross-sections of the whole diseased regions including the edges were essential for diagnosis. One patient had 3 saccular aneurysms and an adjacent shallow depression, which we called "healed microscopic dissection". Histologically, it showed disappearance of the inner media and was replaced by fibrosis in continuation from the intima. This lesion showed no findings of intramural hemorrhage or thrombus, and thus it will differ from organized thrombi in the dissected false lumen. Another patient presented had an aneurysmal rupture which resulted in cardiac tamponade. This case implies the need for surgical treatment of this entity.

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.

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.

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.

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.

Pathologic characteristics of colorectal cancer in the elderly: a retrospective study of 947 surgical cases.

To determine the pathologic characteristics of colorectal cancer in the very old, a retrospective study of 947 consecutive Japanese patients aged > or =65 with 1,039 lesions were examined. Pathologic findings in the very old group (>85 years, n = 140) were compared with those in the younger groups; young-old group (65-74 years, n = 352) and middle-old group (75-84 years, n = 455). Although male:female ratio significantly decreased with advancing age, reaching 1:1.8 in the very old group, the relative odds of colorectal cancer in men were higher than that in women in all age groups. In the very old group, cancer of the proximal colon (proximal to the splenic flexure) accounted for 52% in women and 37% in men, being significantly higher than those in the younger groups. Proximal colonic cancers increased with advancing age in both genders. Higher proportions of poorly differentiated adenocarcinoma, mucinous carcinoma, cancer >5 cm in size, and protruding type cancer were present in the very old group, although these kinds of tumors typically occur in the proximal colon. The incidence of multiple cancers in the large intestine was not different among any age group (average, 8.6%). These results indicated that, even in the very old, colorectal cancers showed marked proximal excess, being explained by effect of both age and gender, and that the proximal shift may influence the proportion of histologic type and size of the tumor. These findings have important implications for screening and diagnosis of colorectal cancer in the elderly.

Fulminant hepatic failure in the elderly: a clinicopathological study of autopsy cases aged over 65 years.

The pathology of fulminant hepatic failure (FHF) in the elderly is little known because of its very low frequency. Thirteen autopsy cases, all above 65 years of age (mean +/- SD, 72 +/- 6 years), and 10 younger control cases, all below 40 years of age (30 +/- 7 years), were analyzed. The elderly group comprised 10 cases with subacute FHF and three cases with acute FHF, while the younger group comprised seven cases with subacute FHF and three cases with acute FHF. The most predominant pathological type in the elderly group was submassive hepatic necrosis (10 cases), followed by acute hepatitis with bridging hepatic necrosis (AH-BHN; two cases) and massive hepatic necrosis (one case). In two cases of submassive hepatic necrosis, hepatic regeneration seemed to be insufficient for the suggested history. The underlying diseases and terminal complications were significantly more frequent in the elderly group than in the younger group. In conclusion, the immune response in the elderly group is found to be strong enough to cause massive or submassive hepatic necrosis. However, impaired hepatic regeneration is occasionally observed in the elderly cases and AH-BHN is often lethal because of frequent underlying diseases and severe complications.

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.

Intracranial cavity volume can be accurately estimated from the weights of intracranial contents: confirmation by the dental plaster casting method.

Intracranial cavity volume is used to evaluate brain size relative to the intracranial space. This volume can theoretically be obtained from the weights and densities of the brain and surrounding cerebrospinal fluid (weight method). However, the accuracy of this method has not been examined. In this study, we examined the reliability of the weight method, by comparing the intracranial cavity volumes of 41 post-mortem cases obtained by the weight method (ICVw) with those obtained by a dental plaster casting method (ICVcast) which was shown to be unbiased. The ICVw was not significantly different from the ICV cast (P=0.49, paired t-test), and the standard error of difference was 18 ml (1.3% of ICVcast). These results show that the weight method is reliable, and applicable to routine autopsies.

Persistent infection of hepatitis C virus in the elderly: a clinical and quantitative pathological study of autopsy cases.

AIMS/BACKGROUND: The entire spectrum of persistent infection by hepatitis C virus (HCV) is still unknown. METHODS: A total of 37 autopsy cases, positive with second generation anti-HCV ELISA, were analyzed. The cases comprised 15 males and 22 females, the average ages being 76.5 in males and 81.1 in females. Twenty-one cases were found to be asymptomatic, while 16 were symptomatic. RESULTS: Based on two pathological parameters of hepatic fibrosis and inflammation, the cases were divided into five groups; group A (non-inflammatory group without significant fibrosis; 11 cases), group B (inflammatory group without significant fibrosis; 9 cases), group C (non-inflammatory group with significant fibrosis; 1 case), group D (inflammatory group with significant fibrosis; 11 cases) and group E (undetermined inflammatory index; 5 cases). All cases in group A and seven cases in group B were asymptomatic. Group A included 6 cases with normal liver. All cases in groups C and D were symptomatic. All examined cases of the inflammatory groups were positive for serum HCV-RNA. CONCLUSION: Anti-HCV-positive elderly autopsy patients include many asymptomatic cases. The two pathological parameters of hepatic fibrosis and inflammation can be used to divide the cases into five groups with each group being well correlated with clinical and virological features.

[Pathologic evaluation of the main cause of death in Japanese centenarians].

A total of 42 Japanese centenarians (9 males & 33 females) autopsied in Tokyo Metropolitan Geriatric Hospital during 22 years (1975-1996) were clinico-pathologically examined to determine details of the main cause of death. The main cause of death of the 42 cases were sepsis (16 cases), pneumonia (14 cases), suffocation (4 cases), heart failure (4 cases), cerebrovascular disorder (2 cases) and malnutrition (2 cases). Most pneumonias were caused aspiration of foreign bodies, and the origins of sepsis were pyelonephritis (7 cases), biliary tract infection (3 cases), necrotic lesions of the intestine due to ileus, ischemia and pseudomembranous colitis (3 cases) and indwelling vein catheter (3 cases). Malignant neoplasms were observed in 16 cases (38%), and 5 of them had 2 or 3 lesions. Thus, the total number of lesions of malignant neoplasms were 22, as follows; colonic cancer (36%), urinary bladder cancer (14%), lung adenocarcinoma (9%), gastric cancer (9%), malignant lymphoma (9%) and others. However, none of these malignant neoplasms were directly related with the cause of death. All 42 centenarians died not of simple "senile decay", but due to diseases.

Primary adenocarcinoma of the duodenum in the elderly: clinicopathological and immunohistochemical study of 17 cases.

Seventeen cases of primary duodenal adenocarcinoma occurring in the elderly (older than 65 years) were examined to clarify their clinicopathological features and biological behavior. The mean age was 77.4 years (range, 66-104), and there was no appreciable difference in the incidence between the sexes (female: male ratio, 8: 9). Thirteen patients had tumors located in the first portion of the duodenum, three in the second portion and one in the third portion. Grossly, there were three varieties of lesions: six polypoid, four flat-elevated and seven ulcerative-invasive. Sixteen cases showed well-differentiated adenocarcinoma, three of which were difficult to distinguish from adenoma, and one was poorly differentiated adenocarcinoma. Three of 16 tumors had poor differentiation in the invasive area, whereas mucosal lesions were well differentiated. Eight tumors had invaded the duodenal wall with occasional involvement of the pancreas. Immunohistochemistry demonstrated p53 protein overexpression in two intramucosal (22.2%) and five invasive (62.5%) cancers. In the intramucosal area the mean Ki-67-positive rate (PR) of the tumors with distant metastasis was significantly higher than that of the tumors without metastasis (46.0 vs 31.6%; P < 0.05), while there was no significant difference in the association between PR and gross feature or depth of the tumors. Clinical follow-up showed three of the five patients with invasive cancer died of carcinoma within 28 months. Compared with published data from other investigators, the results of the present study indicate a proximal shift of duodenal carcinoma in the elderly. Furthermore, it is concluded that invasive duodenal adenocarcinomas with high PR should be considered as potentially aggressive tumors, although their histology may indicate a high degree of differentiation.

The development of a pancreatic abscess, suppurative pylethrombosis, and multiple hepatic abscesses after a pancreatojejunostomy for chronic pancreatitis: report of a case.

We present herein an autopsy case of 63-year-old Japanese man who died as a result of pancreatic abscess, suppurative pylethrombosis, and multiple liver abscesses that had developed 10 years after a pancreato- and cystojejunostomy with side-to-side anastomosis for chronic pancreatitis. Even after this operation, the patient had continued to consume excessive amounts of alcohol. He had first experienced back pain with leukocytosis 9 years after the operation, which relapsed the following year. Despite percutaneous transhepatic gallbladder drainage, his icterus had deteriorated into hepatic insufficiency. Computed tomographic scans of the abdomen had disclosed multiple liver abscesses. At autopsy, a pancreatic abscess and suppurative pylethrombosis as well as multiple liver abscesses were found. There have been few reported cases of such lethal complications developing after a pancreato- and cystojejunostomy for chronic pancreatitis. As the consumption of alcohol would have exacerbated the chronic pancreatitis, such patients should be strongly advised to abstain from drinking alcohol.