Dynamics of M1 macrophages in oral mucosal lesions during the development of acute graft-versus-host disease in rats.

The role of macrophage infiltrates in oral mucosal acute graft-versus-host disease (AGVHD) remains unclear, although clinical studies suggest that macrophage infiltration correlates directly with the severity of AGVHD. In this study, we investigated the role of M1 macrophage infiltration in the oral mucosa of rats with AGVHD. Lewis rat spleen cells were injected into (Lewis × Brown Norway) F1 rats to induce systemic GVHD. Tongue samples were evaluated using histology, immunohistochemistry, dual immunofluorescence, real-time reverse transcription-polymerase chain reaction, Transwell migration assays and Stamper-Woodruff binding assays. At the onset of oral mucosal AGVHD, dual immunofluorescence and migration assays revealed that M1 macrophages had accumulated in the basement membrane (BM) region via the laminin/CD29 ß1 integrin pathway. Macrophage-secreted matrix metalloproteinase-2 was related to BM degradation. The adhesion of macrophages to the oral epithelium could be inhibited by pretreating macrophages with a CC chemokine receptor 2 (CCR2) antibody and/or pretreating lesion sections with monocyte chemoattractant protein-1 (MCP-1) antibody. Our data show that the migration and adhesion of M1 macrophages are associated with oral mucosal AGVHD, which is mediated in part by both laminin/CD29 ß 1 intern and MCP-1/CCR2 pathways. Therefore, our study provides additional support for the contribution of macrophage infiltrate to the development of oral mucosal AGVHD.

Alterations in PNA binding of keratinocytes in oral keratosis.

Changes in the expression of peanut lectin (PNA) were examined in keratinocytes of oral keratosis showing a mixture of hyperortho- and hyperparakeratinized epithelium. In the hyperorthokeratinized epithelium, which was reacted with anti-filaggrin antibody in both granular and cornified cells, PNA bound to the surface of keratinocytes from the spinous layer to the granular layer. Neither anti-filaggrin nor PNA reactions were detected in keratinocytes of the hyperparakeratinized epithelium. After neuraminidase pretreatment, however, PNA staining appeared in all cells, except cornified cells, of both hyperortho- and hyperparakeratinized epithelia. These findings suggest that PNA-binding epitopes in keratinocytes were modified by sialic acid during the hyperparakeratotic process of oral keratosis.

Peripheral ameloblastoma with potentially malignant features: report of a case with special regard to its keratin profile.

A peripheral ameloblastoma with atypical features occurring on the left maxillary alveolar ridge of 40-year-old man is described, along with an immunohistochemical profile of its cytokeratin (CK). The lesion apparently originated from the surface gingival epithelium. The tumor nests or strands were highly cellular with a variable degree of squamous differentiation and microcyst formation. Occasional mitotic figures and dystrophic calcification, both of which are not seen in conventional ameloblastomas, were also observed. The tumor infiltrated deep into the alveolar mucosa, including the periodontal ligament, and showed histological and topographical evidence of atypism, resulting in resorption of the underlying alveolar bone. On the CK immunohistochemistry, CK19 was demonstrated in all the types of neoplastic epithelia, including microcyst-forming cells, densely packed round or spindle cells within the tumor nests, cells with squamous metaplasia, and peripheral tall columnar cells. The CK immunohistochemical findings suggest the lesion's cell of odontogenic origin; they may reflect an immature phenotypic expression of cell differentiation in the odontogenic epithelia during the tumor growth in the gingival mucosa.

Telomerase activation and p53 mutations in urethane-induced A/J mouse lung tumor development.

The mouse telomerase holoenzyme, which synthesizes telomeric DNA de novo, is a ribonucleoprotein complex that includes the mouse telomerase RNA component (mTERC), mouse telomerase-associated protein (mTEP1) and mouse telomerase reverse transcriptase (mTERT). To determine the role of telomerase in urethane-induced lung tumorigenesis in A/J mice we examined telomerase activity and the expression of each telomerase subunit in 20 tumor samples, harvested at 16, 28, 40 and 50 weeks after urethane treatment. The telomeric repeat amplification protocol assay showed that statistically significant telomerase activation occurred both early and late in tumorigenesis. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNA expression levels of mTEP1 and mTERT were up-regulated during tumor progression, while mTERC expression was not significantly different between tumors and normal lung. We further examined mTEP1 protein expression in normal lung tissue and lung tumors; western blot analysis showed preferential expression of mTEP1 protein in lung tumors compared with normal lung and immunohistochemistry revealed that a majority of the adenoma cells were positively stained in the nucleus, whereas only a few of the adjacent normal alveolar cells were immunoreactive. In addition, we investigated DNAs of the 20 tumor samples by single strand conformation polymorphism and sequencing analyses to examine whether alterations of the p53 gene in exons 5-8 were associated with telomerase activity. Although we found one nonsense, two missense, two silent and one simultaneous double mutation at different codons in six late stage tumors, there was no apparent correlation between telomerase activity and p53 mutations. Collectively, these results suggest that mTEP1 as well as mTERT may be involved in the regulation of telomerase activity and that telomerase activation may contribute to lung tumorigenesis in A/J mice independently of p53 gene alterations.

Immunohistochemical detection of cytokeratin 18 and its neo-epitope in Warthin's tumor (adenolymphoma) of the parotid glands.

An immunohistochemical method using a monoclonal antibody M30 (MAb M30), which reacts with the product released by cleavage of cytokeratin 18 (CK18) by activated caspase, was used to investigate the presence and extent of apoptosis in 36 cases of Warthin's tumor (WT) of the parotid glands. The distribution of CK18 in WT was also determined and compared with that of the product detected by MAb M30. In WT, CK18 was observed mainly in the tumor cells of duct-like structures, but not in the cells of lymphatic tissues. Positive MAb M30 reaction products were found in luminal contents, duct-like structures and the cytoplasm of some macrophages in lymphatic areas near the duct-like structures in WT. These findings indicated that apoptotic cells are phagocytosed and eliminated as waste by macrophages. It is suggested that a mechanism which regulates the balance of proliferative activity and apoptosis may be closely linked to the growth of WT.

[Pathology of periodontal disease].

Periodontal diseases are usually divided into two categories, periapical and marginal periodontal diseases, according to the sites. The marginal periodontitis is common and causes loss of teeth in the adult. This begins as a marginal gingivitis, which usually progresses to destructive chronic periodontitis. The authors describe here the classification, pathology and various related factors of human adult periodontitis.

Immunohistochemical detection of cytokeratin 18 and its neo-epitope in human salivary glands and pleomorphic adenomas.

An immunohistochemical method using a monoclonal antibody M30 (MAb M30), which reacts with the product released by the cleavage of cytokeratin 18 (CK18) by activated caspase, was used to investigate the extent of apoptosis in human salivary glands and pleomorphic adenomas. The distribution of CK18 in the salivary glands and adenomas was also determined and compared with that of the product detected by MAb M30. CK18 was detected in the cytoplasm of serous acinar and ductal cells in normal human salivary glands. In pleomorphic adenomas, CK18 was observed mainly in the tumor cells of duct-like structures, but not in those of myxomatous or chondroid tissues. Positive MAb M30 reaction products were found in the cytoplasm of acinar cells in the restricted lobules of normal salivary glands and in the luminal contents of duct-like structures in pleomorphic adenomas. These results suggest that a mechanism which suppresses apoptosis may be linked to the growth of human pleomorphic adenomas.

Comparative image analysis of EGF immunoreaction in rat submandibular gland using 3,3'-diaminobenzidine with metal enhancer substrate.

We have shown the efficacy of image analysis using 3,3'-diaminobenzidine (DAB) with a metal enhancer substrate for demonstrating quantitative differences in the amount of epidermal growth factor (EGF) in the submandibular gland from normal, castrated, and testosterone propionate (TP) treated castrated rats. Immunohistochemical determination of EGF visualized by DAB-nickel reagent was performed with image analysis using a computed image analyzer system (ACAS 570). Immunohistochemistry for EGF disclosed positive staining in granular convoluted tubule cells in the tissue sections from each experimental group. Using the tools of a competent data program installed in the ACAS 570 software, we measured quantitative differences among the experimental glands examined. Castration was shown to elicit a significant reduction in the EGF-positive area and staining intensity, and administration of TP to the castrated animals restored these parameters to levels greater than those of normal rats. Our study demonstrates that a simple, inexpensive, commercially available metal enhancer substrate can be applied accurately to the computer assisted quantification of histochemical hormone-induction studies.

Use of the monoclonal antibody M30 for detecting HSG cell apoptosis.

An immunocytochemical method using a monoclonal antibody (MoAb), M30, which reacts with the product resulting from the cleavage of cytokeratin 18 by activated caspase, was applied to detect the apoptosis of human salivary gland tumor (HSG) cells induced by epigallocatechin gallate (EGCG), gallic acid (GA) and sodium ascorbate (SA). EGCG, GA and SA dose-dependently induced HSG cell death. Immunoreactive products were significantly observed in the cytoplasm of HSG cells after treatment with all these compounds. The reactions occurred with lower concentrations of these agents and after shorter treatment times, in comparison with DNA fragmentation detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. These results suggest that immunocytochemical staining with the MoAb M30 may be useful for detecting the apoptosis-inducing activities of various chemical compounds.

Intrahepatic cholangiocarcinoma associated with hepatolithiasis.

BACKGROUND/AIMS: Despite sporadic reports of cholangiocarcinoma (CC) associated with hepatolithiasis, this entity has not been widely studied. The purpose of this study was to clarify its clinical features and optimal management by studying the 10 patients we have encountered with this condition. METHODOLOGY: There were six women and four men, with a mean age of 61 years. The patients underwent anatomic hepatic resection (n = 5) or biliary drainage (n = 5). The clinical features and results of surgery were studied. RESULTS: The characteristic findings included tumor-related symptoms, irregular ductal stricture or obstruction, and hepatic lobar atrophy with a whitish mass. The tumor and stones were located in the same hepatic lobe. Eight patients had advanced CC with periductal tumor infiltration, while two had in situ carcinoma characterized by intraductal tumor growth, papillary adenocarcinoma, and mucin-hypersecretion. Seven patients died within 6 months after surgery, while the remaining three, including the two with in situ carcinomas and one with an involved node at the dissected hilum, are alive more than 4 years after anatomic hepatic resection. CONCLUSIONS: Recognition of the clinical features of CC associated with hepatolithiasis, which were clarified in this study, is important in treating patients with hepatolithiasis. An anatomic hepatic resection with hilar nodal dissection offers long-term survival in selected patients.

Fabrication of High Performance Laminated Polarization Splitters Consisting of Thick a-Si:H /SiO(x):H Multilayers.

We report fabrication techniques ofa-Si:H/SiO(x):H multilayers having ample thickness and flat layer boundaries for high performance laminated polarization splitters (LPS's). In the new fabrication process we used the following techniques to achieve low stress and high surface flatness: SiO(x):H film deposition by rf sputtering with a mixture of Ar/H(2), two-step deposition using rf bias sputtering, and elimination of surface roughness and defects by mechanical polishing. This process enabled deposition of a multilayer as thick as 265 mum while preserving layer boundaries as flat as 1 nm (rms). As a result, LPS's having low loss, a large aperture, and a long splitting distance were successfully obtained. The high optical performance is applicable to functional devices integrated into fibers or planar waveguides.

Glucose-loading thallium-201 myocardial SPECT.

UNLABELLED: This study was designed to evaluate the feasibility of assessing myocardial viability using glucose loading followed by 201Tl SPECT. METHODS: First, the effect of insulin on the kinetics of 201Tl uptake was evaluated in isolated perfused rat hearts. Second, glucose-loading 201Tl myocardial SPECT was performed in 13 nondiabetic patients with histories of anterior myocardial infarction. Thirty minutes before acquiring rest 201Tl SPECT, 20 g of glucose were intravenously injected into the fasting subjects. Thallium perfusion defects were compared to those of conventional rest-redistribution SPECT images obtained within a 2-wk interval. SPECT images were divided into 21 segments, and a defect score in 17 segments was calculated as a sum of the semiquantitative defect scores (0 = normal; 1 = mildly decreased uptake; 2 = severely decreased uptake; 3 = absence of uptake). RESULTS: Thallium-201 uptake in isolated hearts showed a significant increase of 26% after insulin loading. Eleven (24%) of 45 segments showing perfusion defects on the conventional rest SPECT images demonstrated 201Tl uptake on glucose-loading SPECT imaging. Defect scores decreased significantly on the glucose-loading SPECT images (9.9 +/- 2.2 in early images; mean +/- s.e.) compared with the conventional rest-redistribution SPECT images (12.6 +/- 6.9 in delayed images, p < 0.05). CONCLUSION: Glucose-loading SPECT represents a superior method for assessing myocardial viability using 201Tl.

[Central ocular hypotensive effect of noradrenaline].

We investigated the effects of topical and central administration of noradrenaline (NA) on the intraocular pressure (IOP) in pigmented rabbits. Unilateral instillation of NA (200 micrograms) produced no significant change in IOP. On the other hand, intracerebroventricular (ICV) administration of NA (0.01-1 micrograms) decreased IOP in both eyes in a dose-related fashion. The ocular hypotensive effect of NA was diminished by sympathectomy of the superior cervical sympathetic nerve or by ICV pretreatment with yohimbine hydrochloride 1-10 micrograms). These results suggest that the hypotensive effects of contrally administered NA might be mediated by alpha 2-adrenoceptor stimulation in the central nervous system.

Cerebral infarction due to a spontaneous tumor embolus from lung cancer.

A cerebral infarction of the left occipital lobe developed in a 65-year-old man with squamous cell carcinoma of the right lung. Neurological examinations and brain CT showed findings typical of ordinary infarction. Postmortem examination revealed a tumor embolus within the posterior cerebral artery. Such spontaneous tumor emboli large enough to obstruct a larger-sized artery are rare.

Humoral hypercalcemia of malignancy associated with parathyroid hormone-related protein producing transitional cell carcinoma of the ureter.

A 78-year old male with ureteral carcinoma manifesting hypercalcemia is reported. He was diagnosed as having ureteral carcinoma of the left side 2 years previously and was treated by nephrectomy with ureterovesicostomy. In October 1991, he was admitted for anorexia. A clinical examination revealed recurrence of the ureteral carcinoma with metastasis to the rectum and liver. His serum calcium level was elevated (13.9 mg/dl). In addition to rehydration and furosemide, treatment with eel-calcitonin and prednisolone failed to decrease his serum calcium level. Finally, he was administered mithramycin but he died 13 days later. He had no evidence of bone metastasis or hyperparathyroidism. Nephrogenic cAMP and urinary parathyroid hormone-related protein (PTHrP) were markedly elevated. Immunohistochemical study demonstrated expression of PTHrP in the tumor cells. Thus, the hypercalcemia was thought to be mediated by PTHrP secreted from the neoplastic tumor. Although there have been several reports of ureteral carcinoma associated with humoral hypercalcemia of malignancy, this is considered to be the first case associated with elevation of PTHrP.

Control of in vivo fate of albumin derivatives utilizing combined chemical modification.

Three types of bovine serum albumin (BSA) derivatives such as lactosylated BSA (LBSA), mannosylated BSA (Man-BSA), and cationized BSA (cBSA) were synthesized and their hepatic disposition characteristics in mice were evaluated by pharmacokinetic analysis. At lower doses (< or = 1 mg/kg), LBSA and Man-BSA were very rapidly eliminated from the blood circulation due to uptake by parenchymal and nonparenchymal cells of the liver, respectively, via receptor-mediated endocytosis (Nishikawa et al., 1992; Nishida et al., 1991a, b). These uptake processes were nonlinear and the apparent hepatic uptake clearances (CLliver) were decreased at administered doses higher than 1 mg/kg, e.g. 10, 20, and 100 mg/kg. The liver accumulation of cBSA was also nonlinear, but its binding and/or uptake capacity in the liver was larger than those of LBSA and Man-BSA; i.e., CLliver decreased at doses higher than 20 mg/kg. In the next step, we modified these BSA derivatives by attaching polyethylene glycol (PEG), a modifier known to reduce the hepatic uptake and increase plasma retention, to achieve precise control of the in vivo disposition characteristics of BSA derivatives. By conjugation with PEG having a molecular weight of 10 kDa, the CLliver values of LBSA, Man-BSA, and cBSA were decreasing to one-seventh, one-fortyfifth, and one-onehundredthirtieth, respectively. However, liver accumulation of PEG modified LBSA and Man-BSA at 24 h after i.v. injection was not significantly different from unmodified BSA derivatives. These results suggest that it is possible to control the hepatic uptake of protein drugs by a combination of introduction of charge or sugar moieties and PEG conjugation.

Immunohistochemical detection of sialyl Le(x) antigen on mucosal Langerhans cells of human oral mucosa following neuraminidase pretreatment.

Histochemical assessment of selected carbohydrate sequences on Langerhans cells of human oral mucosa was made by combined use of enzyme digestion and immunostaining with monoclonal antibodies against specific carbohydrate structures. In both frozen sections and epithelial sheets without the enzyme pretreatment, mucosal Langerhans cells, identified by positive staining with anti-CD1a and HLA-DR antibodies, did not express any carbohydrate antigens on their surface. In contrast, following neuraminidase pretreatment of both types of material, the fucosylated type 2 chain (Le(x)) became detectable on Langerhans cells, indicating that sialic acid is the terminal residue of this sequence. Other enzymes were ineffective in this apparent unmasking, and the staining patterns of the other related carbohydrate sequences (Le(y)+, Le(a), Le(b)) remained unaffected by pretreatment with any of the enzymes used. These findings suggest that the mucosal Langerhans cells possess a unique carbohydrate chain, the sialyl fucosylated type 2 sequence (sialyl Le(x) antigen).

Nematolysosomes (elongate lysosomes) in rat hepatocytes: their distribution, microtubule dependence, and role in endocytic transport pathway.

The distribution of lysosomes in rat hepatocytes was examined by three-dimensional electron microscopy combined with acid phosphatase (ACPase) cytochemistry. In the 2-microns-thick sections observed under 200- or 1000-kV TEM, it was apparent that ACPase activity localized on elongate lysosomes (we refer to them as nematolysosomes) with a diameter of 70-100 nm and lengths of several micrometers, as well as spherical lysosomes and trans-Golgi cisternae. Though most spherical lysosomes were located within the pericanalicular region, nematolysosomes were widely distributed throughout the hepatocytes. Typically, it appeared that the nematolysosomes elongated from the subsinusoidal region to the pericanalicular-Golgi complex area and they frequently formed a network at the cell periphery along the sinusoidal front. Furthermore, the formation of nematolysosomes was independent of new protein synthesis, but highly dependent on the integrity of microtubules. After a 6-8 h colchicine treatment, nematolysosomes were shrunk and/or fragmented, becoming roughly spherical lysosomes scattered throughout the cells. Nematolysosomes recovered their normal profiles after 24 h due to the reversible effect of the drug on microtubules. When the hepatocytes were exposed to horseradish peroxidase (HRP) in vitro or in vivo, HRP was quickly sequestered in nematolysosome-like structures via pinocytosis from the sinusoidal surface and transported to an area near the Golgi complex. These findings raise the possibility that the nematolysosomes engage in microtubule-dependent transport of macromolecules from the sinusoidal circulation to the Golgi complex area.