Practical use of the central venous access port for contrast-enhanced CT: comparison with peripheral intravenous access regarding enhancement and safety.

AIM: To evaluate the efficacy of using the central venous (CV) port compared with peripheral intravenous access for contrast-material injection for contrast enhancement during the portal venous phase. MATERIALS AND METHODS: Patients were divided into three groups: CV delay, CV routine, and peripheral access (PA) groups. Patients in the CV delay group underwent injection in the arm-down position with an additional delay, while those in the CV routine and PA groups underwent injections with the routine injection protocol for portal venous phase imaging. Contrast enhancement was evaluated by measuring the mean radiodensity (Hounsfield units) values for the aortic arch, abdominal aorta, inferior vena cava, portal vein, and spleen. The peak injection pressure was recorded and compared among the three groups. RESULTS: No complications related to power injection were observed during 119 contrast-material injections performed using the CV port device. The CV delay group showed significantly lower radiodensity values than the PA group (165.7 ± 20.1 versus 181 ± 19 HU [p<0.01] for the portal vein); however, no significant differences in mean radiodensity values were observed between the CV routine and PA groups (p>0.05). The median peak injection pressure was 73.5, 67, and 47 psi in the CV delay, CV routine, and PA groups, respectively (p<0.01). CONCLUSION: The CV port can be used for safe contrast-material injection while maintaining contrast enhancement on portal venous phase comparable to that achieved with peripheral intravenous access.

Class A scavenger receptor (CD204) attenuates hyperoxia-induced lung injury by reducing oxidative stress.

To clarify the role of macrophage class A scavenger receptors (SR-A, CD204) in oxidative lung injury, we examined lung tissue of SR-A deficient (SR-A(-/-)) and wild-type (SR-A(+/+)) mice in response to hyperoxic treatment. Protein levels of bronchoalveolar lavage fluid (BALF) and pulmonary oedema (wet : dry weight ratios) were higher in SR-A(-/-) mice than those in SR-A(+/+) mice. Cumulative survival was significantly decreased in SR-A(-/-) mice. However, there were no differences in BALF macrophage and neutrophil count between the two groups. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that messenger RNA (mRNA) levels of the inducible nitric oxide synthase (iNOS) were increased during hyperoxic injury, and this increase was more prominent in SR-A(-/-) mice. Expression levels of iNOS in alveolar macrophages after hyperoxia in vivo and in vitro were higher in SR-A(-/-) macrophages compared with SR-A(+/+) macrophages. Immunohistochemistry using anti-nitrotyrosine antibodies revealed distinctive oxidative stress in the injured lung in both groups, but it was more remarkable in the SR-A(-/-) mice. After hyperoxic treatment, pulmonary mRNA levels of tumour necrosis factor-alpha(TNF-alpha) were elevated more rapidly in SR-A(-/-) mice than in SR-A(+/+) mice. Together these results suggest that SR-A expression attenuates hyperoxia-induced lung injury by reducing macrophage activation.

Immunohistochemical distribution and quantitative biochemical detection of advanced glycation end products in fetal to adult rats and in rats with streptozotocin-induced diabetes.

SUMMARY: We used immunohistochemical methods and four monoclonal antibodies for specific molecular structures of advanced glycation end products (AGE)-6D12, KNH-30, 1F6, and 2A2-to examine localization of AGE in fetal, young, and adult rats, and rats with streptozotocin-induced diabetes. 6D12 recognized N(epsilon)-(carboxymethyl)lysine (CML); KNH-30, N(epsilon)-(carboxyethyl)lysine (CEL); and 1F6, fluorolink. The epitope of 2A2 is as yet unknown. Immunoreactivities for these monoclonal antibodies were found in various organs and tissues in postnatal and adult rats, and accumulation increased with aging. In the fetuses, AGE structures were detected at 10 fetal days, and their accumulation increased during ontogeny. Reversed-phase high-performance liquid chromatography revealed CML in fetuses at 13 fetal days and in lungs of 28-week-old rats. In various organs and tissues of fetal, young, and adult rats, CML, CEL, 2A2-positive AGE, and fluorolink accumulated, in that order, which suggests that the accumulation of CML, a nonfluorescent/noncross-linked AGE, occurs earlier than accumulation of fluorolink, a fluorescent/cross-linked AGE. In diabetic rats, hepatocytes, splenic macrophages, renal glomerular endothelial and mesangial cells, testicular Leydig cells, and erythrocytes showed excessive accumulation of AGE, leading to the pathologic changes characteristic of diabetes mellitus. For the induction of these changes, persistent hyperglycemia and hyperketonemia might be important for acceleration of intracellular AGE accumulation in diabetic rats. Thus, AGE accumulation in tissues and cells occurs not only during aging and in diabetes mellitus but also from an early stage of ontogeny.

Familial amyloidotic polyneuropathy (ATTR Val30Met) with widespread cerebral amyloid angiopathy and lethal cerebral hemorrhage.

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) with cerebral hemorrhage. A 38-year-old woman with a typical FAP pedigree started developing severe diarrhea and sensori-motor polyneuropathy at the age of 28 years; autonomic nervous system, heart and renal dysfunction manifested themselves in the following years. Genetic analysis revealed a single amino acid substitution at codon 30 of transthyretin (ATTR Val30Met). Ten years after her initial symptoms, the patient died of a sudden convulsive attack and respiratory failure. Autopsy revealed lethal cerebral hemorrhages and uremic lungs. Histochemical and immunohistochemical analyses revealed TTR-derived amyloid protein in every tissue examined, particularly in glomeruli and peripheral vessels. Severe meningo-cerebrovascular amyloidosis was also detected. Because uremia causes oxidative damage to the vascular system and amyloid formation is closely associated with oxidative stress, it is possible that uremic endothelial damage facilitated an unusual cerebral amyloid deposition. In typical FAP (ATTR Val30Met), cerebral amyloid angiopathy does not usually have clinical manifestations. However, cerebral amyloid angiopathy should be considered to explain FAP symptoms when some risk factors such as uremic vascular damage are accompanying features.

Oxidative injury is present in Purkinje cells in patients with olivopontocerebellar atrophy.

To verify the presence of lipid peroxidation products in spinocerebellar degeneration (SCD), the cerebella from eight patients with olivopontocerebellar atrophy (OPCA) and six non-OPCA patients were immunohistochemically investigated with 4-hydroxy-2-nonenal (HNE) antibody. On average, 84.6% of Purkinje cells were positively or strongly positively immunostained in OPCA patients while only 15.5% were positive in non-OPCA patients. Other cells in the molecular and granular layers showed no obvious immunoreactivity. These data suggest that a lipid peroxidation product is present in Purkinje cells of OPCA patients and that oxidative stress may play an important role in the degeneration process of SCD.

Familial amyloidotic polyneuropathy (ATTR Ser50Ile): the first autopsy case report.

We report an autopsy case of a pedigree of familial amyloidotic polyneuropathy (FAP) with a mutation of isoleucine-50 transthyretin (ATTR Ser50Ile). A 47-year-old man started developing severe diarrhea and weight loss at age 41 years, followed by urinary incontinence, autonomic-nervous-system abnormalities and serious heart failure; the diagnosis of FAP (ATTR Ser50Ile) was made on the basis of genetic, histochemical and immunohistochemical analysis. Six years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, perforation of the sigmoid colon and marked systemic amyloid deposition. The total amount of amyloid deposited in the heart was greatly increased and was much lower in the thyroid gland and kidneys compared with amyloid deposits in ordinary FAP (ATTR Val30Met). Amyloid deposition in peripheral vessel walls was prominent, particularly in lymphatics and veins. His elder sister, 54 years old, started to develop orthostatic hypotension at age 49 years, followed by dysesthesia, diarrhea and severe congestive heart failure. Endomyocardial biopsy revealed severe TTR-amyloid deposition; ultrastructural examination demonstrated that amyloid fibrils were deposited disproportionately and extended radially around microvessels.

Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine.

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.

A new diagnostic procedure to detect unknown transthyretin (TTR) mutations in familial amyloidotic polyneuropathy (FAP).

Two patients with amyloidosis caused by transthyretin (TTR) were investigated by immunohistopathologic, mass spectrometric, and molecular genetic methods. After confirming the immunoreactivity of TTR in the amyloid deposits using anti-TTR polyclonal antibody, a new method: centrifugal concentration and electrospray ionization mass spectrometry (ESI-MS) was employed to detect the variant TTR in the serum. Only 50 microl of the serum and 30 microl of the anti-TTR antibody were needed for the analysis. After incubation with the antibody, the samples were passed through a 1000 kDa cut off centrifugal concentrator to retain the antibody, thereafter, the filtrate was analyzed by ESI-MS. Several forms of normal and variant TTR were detected in the serum samples: unconjugated TTR, cysteine and cysteine-glycine conjugated TTR. In the patients, a variant form of TTR was detected with a 26.0 Da higher molecular weight than that of normal TTR. Single-strand conformation polymorphism (SSCP) and direct sequence analysis confirmed the presence of a one-base substitution situated at the codon 50 from AGT (Ser) to ATT (Ile) in both patients, that corresponded to the increased molecular weight of 26.0. The present diagnostic procedure demonstrates the usefulness of both ESI-MS and SSCP to screen for TTR related amyloidosis rapidly. Moreover, the DNA samples obtained from the band showing abnormal electrophoretic migration pattern in SSCP, facilitate the direct sequence analysis to detect the unknown mutation, and the observed shift in molecular weight of the variant TTR in ESI-MS confirms the base substitution.

Localization of human acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in macrophages and in various tissues.

To investigate the distribution of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in various human tissues, we examined tissues of autopsy cases immunohistochemically. ACAT-1 was demonstrated in macrophages, antigen-presenting cells, steroid hormone-producing cells, neurons, cardiomyocytes, smooth muscle cells, mesothelial cells, epithelial cells of the urinary tracts, thyroid follicles, renal tubules, pituitary, prostatic, and bronchial glands, alveolar and intestinal epithelial cells, pancreatic acinar cells, and hepatocytes. These findings showed that ACAT-1 is present in a variety of human tissues examined. The immunoreactivities are particularly prominent in the macrophages, steroid hormone-producing cells, followed by hepatocytes, and intestinal epithelia. In cultured human macrophages, immunoelectron microscopy revealed that ACAT-1 was located mainly in the tubular rough endoplasmic reticulum; immunoblot analysis showed that the ACAT-1 protein content did not change with or without cholesterol loading; however, on cholesterol loading, about 30 to 40% of the total immunoreactivity appeared in small-sized vesicles. These vesicles were also enriched in 78-kd glucose-regulated protein (GRP 78), a specific marker for the endoplasmic reticulum. Immunofluorescent microscopy demonstrated extensive colocalization of ACAT-1 and GRP 78 signals in both the tubular and vesicular endoplasmic reticulum before and after cholesterol loading. These results raise the possibility that foam cell formation may activate an endoplasmic reticulum vesiculation process, producing vesicles enriched in the ACAT-1 protein.

Diffuse brainstem glioma in a patient with Laurence-Moon-(Bardet-)Biedl syndrome.

An autopsy case of a patient with diffuse brainstem glioma associated with Laurence-Moon-(Bardet-)Biedl syndrome is described. The subject was a 25-year-old woman who had been suffering from mental retardation, pigmented retinopathy, obesity, hexadactyly, amenorrhea and renal cysts. She developed dizziness, headache and consequent consciousness disturbance. Magnetic resonance images disclosed marked swelling of the pons without contrast enhancement. By means of combined chemotherapy and radiation, she survived for 15 months. Histopathological diagnosis for postmortem specimens obtained from the brainstem was glioblastoma multiforme. No pathogenetic association between the syndrome and brainstem gliomas is known, and the literature contains no cases of patients with this coincidence.

Colonic enteric nervous system in patients with familial amyloidotic neuropathy.

The colonic enteric nervous system was investigated in autopsy specimens from 12 patients with familial amyloidotic neuropathy (FAP) and 9 controls. The infiltration of amyloid deposits in the enteric nervous system was studied by double staining for amyloid and nerve elements. The myenteric plexus was immunostained for protein gene product (PGP) 9.5, vasoactive intestinal peptide (VIP), substance P and nitric oxide synthase (NOS). The immunostained nerve elements were quantified by computerised image analysis. Double staining revealed that there was no amyloid infiltration in the ganglia, or in the nerve fibres in the colonic enteric nervous system of FAP patients. The relative volume density of PGP 9.5-immunoreactive nerve fibres in both the circular and the longitudinal muscle layers in FAP patients did not differ significantly from that of controls. The relative volume density of VIP-immunoreactive nerve fibres in the circular muscle layer was significantly decreased in FAP patients compared with controls, but not in the longitudinal layer. The number of VIP-immunoreactive neurons/mm2 myenteric ganglia was significantly decreased in FAP patients. There were no statistical differences in the relative volume density for substance P- and NOS-immunoreactive nerve fibres between FAP patients and controls, nor was there any difference between FAP patients and controls regarding the number of NOS- and substance P-immunoreactive neurons/mm2 myenteric ganglia. It is concluded that the colonic enteric nervous system as a whole is intact and is not damaged by amyloid infiltration. The present observation of a reduction of VIP-immunoreactive nerve fibres and neurons in myenteric plexus of FAP patients might be one of the factors that contribute to the motility disorders seen in FAP patients.

Expression of von Hippel-Lindau protein in normal and pathological human tissues.

To examine the localization of von Hippel-Lindau (VHL) protein in human tissues, we produced four novel monoclonal antibodies against human VHL protein. Western blot analysis revealed that two of these antibodies recognized the epitope in amino acid sequence 60-89 of the VHL protein and the others recognized sequences 54-60 and 189-213. In a wild-type VHL gene-transfected cell line, immunocytochemistry and immunoelectron microscopy demonstrated the intracytoplasmic localization of VHL protein, particularly in mitotic cells. In normal human tissues, VHL protein was detected immunohistochemically in epithelial cells covering the body surface and the alimentary, respiratory, and genitourinary tracts; in secretory epithelial cells of exocrine and endocrine organs; in parenchymal cells of visceral organs; in cardiomyocytes; in neurons in nervous tissue; in lymphocytes in lymphoid tissue; and in macrophages. In pathological specimens, VHL protein was expressed in VHL-related tumor, as well as in endothelial cells, fibroblasts, and pericytes, all of which are involved in active angiogenesis. These findings suggest that these monoclonal antibodies can be useful for various immunological assays and that the VHL protein plays fundamental roles in physiological and pathological situations, especially in neovascularization.

Differentiation of a cell line of human cervical argyrophil small cell carcinoma to macrophage lineage cells.

To investigate the origin of argyrophil small cell carcinoma (ASCC) of the uterine cervix, we examined the influence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP), a known differentiation inducer, on the characteristics of an ASCC cell line, TC-YIK, which has been shown to be a useful in vitro experimental model of ASCC. In TC-YIK cells after treatment with dB-cAMP, two specific antigenic markers of macrophages, CD14 and human leukocyte antigen-DR, were detected by flow cytometric analysis. In addition, interferon-gamma mRNA was detected by reverse transcription-polymerase chain reaction and interferon-gamma protein was detected by ELISA. More than 90% of the cells stained positive for alpha-naphthyl butyrate esterase, 1% of the cells showed phagocytotic activity against Micrococcus lysodeikticus, and 22% of the cells had M. lysodeikticus adsorbed on their surface. Furthermore, granulocyte-macrophage colony stimulating factor accelerated the proliferation of TC-YIK cells. These results indicate that dB-cAMP promotes differentiation of ASCC cells to macrophages. In contrast, less than 10% of the cells showed stellate morphology, suggesting differentiation to neuronal cells after treatment with dB-cAMP, as reported previously. Thus, TC-YIK cells have been shown to differentiate both into macrophage lineage cells and neuronal cells, suggesting that ASCC originates from undifferentiated stem cells.

Colonic endocrine cells in patients with familial amyloidotic polyneuropathy.

OBJECTIVE: To establish whether the endocrine cell number is affected in the colon in Japanese FAP patients. SETTING: Department of Medicine, Umeå University Hospital and Department of Internal Medicine and Pathology, University Hospital, Kumamoto, Japan. SUBJECTS: Autopsy colon tissue specimens from 11 FAP patients and nine controls as well as 12 control biopsy specimens were included in the study. MEASUREMENTS: Endocrine cells in the colon were detected by immunohistochemistry and quantified by computerized image analysis. RESULTS: The autopsy material showed a slight autolysis. Neither enteroglucagon nor pancreatic polypeptide positive cells could be detected in the autopsy material, but were present in biopsy material. There was no statistical difference between autopsy and biopsy specimens regarding the number of peptide YY (PYY), somatostatin and serotonin cells. No significant differences were noted in PYY, somatostatin and serotonin immunoreactive cells in FAP patients compared to autopsy controls, though PYY cells tended to be decreased and serotonin and somatostatin cells tended to be increased in FAP patients. CONCLUSION: The difference between the Swedish and Japanese patients in the endocrine cell content points to the possibility of involvement of other factors than the endocrine cell depletion of the colon might be involved in the pathogenesis of gastro-intestinal dysfunction in FAP. The tendency of PYY to decrease in Japanese FAP might contribute to the development of diarrhoea in these patients.

Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages.

The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions. Our results thus support the idea that ACAT-1 plays an important role in differentiating monocytes and in forming macrophage foam cells during the development of human atherosclerosis.

Familial amyloidotic polyneuropathy type I with extracellular superoxide dismutase mutation: a case report.

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) Type I with mutations in both transthyretin (TTR) and extracellular superoxide dismutase (EC-SOD). This patient started to develop peripheral neuropathy at age 25, followed by cardiac, renal, and autonomic nervous system failure due to massive amyloid deposition. Thirteen years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, multiple abscesses in the bile ducts and urinary tract, and more marked amyloid deposition than commonly seen in FAP. Amyloid deposition occurred in various organs and tissues, especially prominently around blood vessels and in interstitial tissues, and was demonstrated immunohistochemically to be composed of TTR but not amyloid A (AA) and not amyloid L (AL) proteins. The serum EC-SOD content of the patient was 10 fold higher than those seen often in other FAP patients and in healthy controls. Genetic analysis demonstrated the single amino acid substitutions in Val30Met TIR and Arg213Gly EC-SOD. Since these data suggest the dissociation of EC-SOD from the vascular wall, massive amyloid deposition in the present case may be related to increased oxidative stress in loco.

The very low- and intermediate-density lipoprotein fraction isolated from apolipoprotein E-knockout mice transforms macrophages to foam cells through an apolipoprotein E-independent pathway.

Apolipoprotein E (apoE)-knockout mice develop severe atherosclerosis associated with high levels of very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) in plasma. To investigate the atherogenic role of VLDL and IDL, the lipoprotein fraction containing both VLDL and IDL (apoEko-VLDL/IDL) was isolated from plasma of apoE-knockout mice by ultracentrifugation, and its interaction with macrophages was studied. When peritoneal macrophages obtained from apoE-knockout mice were incubated with apoEko-VLDL/IDL, the level of cellular cholesteryl esters (CE) increased with the concentration of apoEko-VLDL/IDL. The level of cellular cholesteryl [3H]oleate formed reached 15.1 nmol/mg of cell protein upon incubation with 50 microg/mL apoEko-VLDL/IDL for 18 h, which was an 8.4-fold increase over the corresponding level induced by low-density lipoprotein (LDL). The cellular CE mass was also significantly increased by apoEko-VLDL/IDL. Morphologically, after exposure to apoEko-VLDL/IDL, macrophages became strongly stained with Sudan black B. The total binding of [125I]apoEko-VLDL/IDL to macrophages was effectively replaced by more than 80% by an excess of the unlabeled ligand. Specific binding, calculated by subtracting the nonspecific binding from the total binding, exhibited a saturation pattern. Similar results were obtained with cell association and degradation experiments. In addition, the endocytic degradation of [125I]apoEko-VLDL/IDL was partially inhibited by LDL, whereas acetyl-LDL did not show any effect. These results indicated that apoEko-VLDL/IDL in its unmodified form produced significant CE accumulation in macrophages through a specific and apoE-independent pathway. This pathway may explain, in part, the mechanisms of foam cell formation in arterial walls and the subsequent development of atherosclerosis in apoE-knockout mice.

Molecular cloning, functional expression and tissue distribution of rat acyl-coenzyme A:cholesterol acyltransferase.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an enzyme catalyzing the intracellular formation of cholesteryl esters from free cholesterol and fatty acyl-CoA. In the present study, we cloned rat ACAT cDNA and determined its tissue distribution. Rat ACAT cDNA, having a coding region of 1635 bp with its deduced protein sequence of 545 amino acids and two typical motifs such as signature sequences and leucine heptad motif, showed 83, 92 and 90% identity with human, mouse, and hamster ACAT, respectively. Expression of rat ACAT cDNA in A293 cells and CHO cells resulted in a 3.0 to 3.5-fold increase in the enzyme activity. Among twelve tissues examined, ACAT activity was highest in adrenal followed by liver and intestine while that of aorta was extremely low. The mRNA level was also the highest in adrenal among four tissues examined. However, in contrast to its high ACAT activity, the liver mRNA level was extremely low (adrenal >> intestine > aorta >> liver). Consistent with mRNA levels, immunohistochemical analyses with a specific ACAT antibody detected significant ACAT signals in adrenal and intestine but a negligible signal in liver. These results indicate that adrenal most abundantly expresses ACAT in rat. Furthermore, rat liver showed a high ACAT activity but an extremely low ACAT mRNA and negligible immunohistochemical reactivity, suggesting the presence of a structurally different ACAT protein(s) in rat liver.

Endoscopic and pathological manifestations of the gastrointestinal tract in familial amyloidotic polyneuropathy type I (Met30).

OBJECTIVES: To evaluate the characteristic changes in the gastrointestinal tract in familial amyloidotic polyneuropathy (FAP) (Met30), both fibre gastroscopy and colonoscopy studies were performed in FAP (Met30) patients. Microscopic changes were also examined in autopsied and biopsied materials from patients with FAP, and compared with data from autopsied samples from patients with AL amyloidosis, and secondary amyloidosis patients. DESIGN: Endoscopic and histopathological study. SETTING: Kumamoto University Hospital, Kumamoto, Japan. SUBJECTS: Nine patients with FAP (Met30) underwent fibre gastroscopy and colonoscopy. Six autopsied and 23 biopsied gastrointestinal samples from FAP patients, four from autopsied amyloidosis (including two myeloma associated form), and two from autopsied secondary amyloidosis patients were examined for histopathological study. MAIN OUTCOME MEASURES: Fibre gastroscopy and colonoscopy were employed for macroscopic study. Congo red and H-E staining were performed for histopathological study. Macroscopic changes in the gastrointestinal tract and microscopic differences in the amyloid distribution pattern were compared between the different types of amyloidosis. RESULTS: Fibre gastroscopy and colonoscopy for nine FAP patients revealed that four showed a fine granular appearance in the duodenum, three showed lack of lustre, and two showed mucosal friability in the gastrointestinal tract; however, no macroscopic abnormality was observed in four other FAP patients. Histopathological examination of tissue from FAP patients revealed that, although a small amount of amyloid was recognized in the submucosa perivascular layer, a significant amount of amyloid was seen in and around the nerves of the gastrointestinal tract, but very little in Auerbach's nerve plexus. In total, the amount of deposited amyloid in the tissues was small compared with that in other types of systemic amyloidosis, such as AL and secondary amyloidosis. CONCLUSION: These results suggest that the major reason why FAP patients show such severe gastrointestinal symptoms, compared with other types of systemic amyloidosis, may be because of the deposition of a significant amount of amyloid in the nerves in the gastrointestinal tract.

Immunohistochemical distribution and subcellular localization of three distinct specific molecular structures of advanced glycation end products in human tissues.

Using three mouse anti-human monoclonal antibodies for advanced glycation end products (AGEs), 6D12, 1F6, and 2A2, we examined the immunohistochemical distribution and localization of AGEs in various organs and tissues obtained from nondiabetic autopsy or biopsy cases (men and women, 41 to 86 years of age). 6D12 recognizes Nepsilon-(carboxymethyl)lysine (CML), a nonfluorescent and non-cross-linked AGE structure, and 1F6 recognizes fluorolink, a fluorescent and cross-linked AGE structure. The epitope of 2A2 is unknown but is different from that of CML and fluorolink or other known AGE structures such as pyrraline, pentosidine, and crosslines. Immunohistochemistry with these monoclonal antibodies revealed the intra- and extracellular accumulation of AGEs in these organs and tissues. By double immunohistochemical staining with two of the three monoclonal antibodies in different combinations, positive reaction products for all three monoclonal antibodies were demonstrated in macrophages widely distributed in various organs and tissues; endothelial cells of endocardium, arteries, veins, and blood capillaries; mesenchymal cells; epithelial or parenchymal cells; blood cells; and extracellular matrix. This result indicates that these three different AGE-specific molecules are formed intracellularly and extracellularly. In some cell types, however, one or two of these specific molecules were not always found together, suggesting that the molecular structures of AGEs and their formation are heterogeneous. Immunoelectron microscopy demonstrated the localization of AGE-labeled immunogold particles in the nuclei, nuclear envelope, mitochondria, endoplasmic reticula, Golgi complexes, endocytic vesicles, lysosomal vacuoles or granules, secretory granules, cytosol, and cell membranes, as well as in the extracellular matrix. In addition, the double histochemical staining method for ceroid/lipofuscin and immunohistochemistry for AGEs demonstrated intralysosomal formation and accumulation of AGEs in ceroid/lipofuscin pigments. These results suggest that the extracellularly produced AGEs are taken up by receptors into the cells and accumulate in secondary lysosomes and that AGEs are formed intranuclearly and/or intracellularly, probably via different metabolic pathways.