Kinetics of Langerhans cell chimerism in the skin of dogs following 2 Gy TBI allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Langerhans cells (LC) are bone marrow-derived cells in the skin. The LC donor/recipient chimerism is assumed to influence the incidence and severity of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). In nonmyeloablative (NM) HSCT the appearance of acute GVHD is delayed when compared with myeloablative conditioning. Therefore, we examined the development of LC chimerism in a NM canine HSCT model. METHODS: 2 Gy conditioned dogs received bone marrow from dog leukocyte antigen identical littermates. Skin biopsies were obtained pre- and post-transplant. LC isolation was performed by immunomagnetic separation and chimerism analysis by PCR analyzing variable-number-of-tandem-repeat markers with subsequent capillary electrophoresis. RESULTS: All dogs engrafted. Compared to peripheral blood chimerism the development of LC chimerism was delayed (earliest at day +56). None of the dogs achieved complete donor LC chimerism, although two dogs manifested a 100 % donor chimerism in peripheral blood at days +91 and +77. Of interest, one dog remained LC chimeric despite loss of donor chimerism in the peripheral blood cells. CONCLUSION: Our study indicates that LC donor chimerism correlates with chimerism development in the peripheral blood but occurs delayed following NM-HSCT.

Beneficial effect of the bioflavonoid quercetin on cholecystokinin-induced mitochondrial dysfunction in isolated rat pancreatic acinar cells.

The pathogenesis of acute pancreatitis (AP) is still poorly understood. Thus, a reliable pharmacological therapy is currently lacking. In recent years, an impairment of the energy metabolism of pancreatic acinar cells, caused by Ca(2+)-mediated depolarization of the inner mitochondrial membrane and a decreased ATP supply, has been implicated as an important pathological event. In this study, we investigated whether quercetin exerts protection against mitochondrial dysfunction. Following treatment with or without quercetin, rat pancreatic acinar cells were stimulated with supramaximal cholecystokinin-8 (CCK). CCK caused a decrease in the mitochondrial membrane potential (MMP) and ATP concentration, whereas the mitochondrial dehydrogenase activity was significantly increased. Quercetin treatment before CCK application exerted no protection on MMP but increased ATP to a normal level, leading to a continuous decrease in the dehydrogenase activity. The protective effect of quercetin on mitochondrial function was accompanied by a reduction in CCK-induced changes to the cell membrane. Concerning the molecular mechanism underlying the protective effect of quercetin, an increased AMP/ATP ratio suggests that the AMP-activated protein kinase system may be activated. In addition, quercetin strongly inhibited CCK-induced trypsin activity. The results indicate that the use of quercetin may be a therapeutic strategy for reducing the severity of AP.

Multi-walled carbon nanotubes induce oxidative stress and apoptosis in human lung cancer cell line-A549.

Multi-walled carbon-nanotubes (MWCNTs)-induced apoptotic changes were studied in human lung epithelium cell line-A549. Non-cytotoxic doses of MWCNTs were identified using tetrazolium bromide salt (MTT) and lactate dehydrogenase (LDH) release assays. Cells were exposed to MWCNTs (0.5-100 µg/ml) for 6-72 h. Internalization and characterization of CNTs was performed by electron microscopy. Apoptotic changes were estimated by nuclear condensation, DNA laddering, and confirmed by expression of associated markers: p(53), p(21WAF1/CIP1), Bax, Bcl(2) and activated caspase-3. MWCNTs induced the production of reactive oxygen species and malondialdehyde along with significant decrease in the activity of catalase and glutathione. MWCNTs-induced ROS generation was found not to be associated with the mitochondrial activity. In general, the changes were significant at 10 and 50 µg/ml only. Results indicate the involvement of oxidative stress and apoptosis in A549 cells exposed to MWCNTs. Our studies provide insights of the mechanisms involved in MWCNTs-induced apoptosis at cellular level.

Calpain-mediated breakdown of cytoskeletal proteins contributes to cholecystokinin-induced damage of rat pancreatic acini.

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 microM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 microM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins alphaII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, mu- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of alphaII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Inhibitory effect of the lectin wheat germ agglutinin (WGA) on the proliferation of AR42J cells.

The rat pancreatic acinar tumour cell line AR42J is a widely used model to study the secretion, proliferation and differentiation of cells under the influence of hormones. These so-called amphicrine cells synthesize and secrete digestive enzymes as well as neuroendocrine peptides. They possess both subtypes of the highly glycosylated cholecystokinin (CCK) receptor which are important for the regulation of secretion and for cell growth. AR42J cells extrude CCK and gastrin-like hormone peptides and have the ability of an autostimulation (autocrine loop). The lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) bind to the glycosylated sites of these CCK receptors with the effect inhibiting CCK binding and thus inhibiting the CCK-induced Ca2+ release and alpha-amylase secretion. The so-called trophic hormones CCK and gastrin stimulate the secretion and proliferation of AR42J cells within the autocrine loop via autostimulation of their CCK receptors. In preceding papers, we described the inhibitory effect of WGA on the binding of 125I-CCK-8s to the CCK-A and -B receptors and the subsequent enzyme secretion of AR42J cells. In the present work, we studied the influence of the lectins WGA, UEA-I and galectin-1, as well as of the lectin-like enzyme alpha-amylase, on the proliferation of AR42J cells and prevention of autostimulation. The proliferation inhibition of the growth fraction was measured by estimation of the S-phase fraction by DNA flow cytometry. Whereas WGA inhibited the growth fraction significantly, UEA-I, human galectin-1 and human alpha-amylase had no significant effect. In transmission electron microscopy, we observed the accumulation of typical zymogen granules under the effect of WGA and a better differentiation of cells.

Chronic inflammation and pain inside the mandibular jaw and a 10-year forgotten amalgam filling in an alveolar cavity of an extracted molar tooth.

A 55-year-old woman, suffered from severe pain in her mandibular jaw for several years. A metallic artifact of about 2(3) mm was detected by a panorama radiography in an edentulous region with a surrounding inflammation in close contact to the canal of the mandibular nerve. Inflammated tissue with the central metallic inclusion was removed from the bone under local anesthesia and operation. Postoperatively, pain and missensitivity disappeared within 1 week. Although the patient had no macroscopically visible so-called amalgam tattoo, the metallic cube was identified as amalgam by the detection of mercury, silver, tin, copper, and zinc using energy dispersive X-ray analysis (EDX) in a scanning electron microscope (SEM). Nevertheless, brown to black pigments in the connective tissue matrix and inside histiocytes, fibroblasts, and multinucleated foreign giant cells of the surrounding inflammatory tissue were observed by light and electron microscopy. However, the elemental analysis by EDX in SEM or by electron energy loss spectroscopy in transmission electron microscope detected only silver, tin, and sulfur but no mercury in these precipitates and in the residual bodies of phagocytes. The presented case demonstrates a seldom complication of amalgam deposition in the tissue. The authors assume that the chronic pain results from a forgotten amalgam filling inside an alveole after extraction of a molar tooth, causing a chronic inflammation by resolving mercury and other toxic elements out of the metallic artifact.

Nonsecretory multiple myeloma with amyloidosis. A case report and review of the literature.

We report a case concerning a 49-year-old female patient with thoracic pain. X-rays showed a single osteolytic lesion on the right seventh rib. The excision of the rib disclosed a plasmocytic plasmocytoma with extensive amyloidosis. Serum and urine protein electrophoresis were both negative for monoclonal gammopathy. Bone marrow biopsy showed that 80% of the marrow had been replaced by plasma cells. A diagnosis of nonsecretory multiple myeloma was made. Immunohistochemistry revealed amyloid light (AL) amyloidosis of kappa-light chain origin. The relationship between nonsecretory multiple myeloma and amyloidosis is discussed.

Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat.

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.

Inhibitory effect of the lectin wheat germ agglutinin on the binding of 125I-CCK-8s to the CCK-A and -B receptors of AR42J cells.

INTRODUCTION: Cholecystokinin (CCK) is a peptide hormone and plays a major role both in the regulation of pancreatic enzyme secretion and growth of the gastrointestinal tract. The pancreatic CCK receptors are highly glycosylated membrane proteins that are able to bind plant lectins such as wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I). AIM AND METHODOLOGY: In preceding papers, we demonstrated an inhibition of CCK-8s induced Ca2+ signaling and secretion of rat pancreatic acini and AR42J cells by the lectins WGA and UEA-I (Pancreas 2001;23:368-374). Here we studied the influence of WGA, UEA-I, and 22 other lectins on 125I-CCK-8s binding on AR42J cells. A binding assay was used with 125I-CCK-8s and dexamethasone-stimulated AR42J cells, bearing CCK-A as well as CCK-B receptors. RESULTS: WGA inhibits 125I-CCK-8s binding in a dose-dependent manner. The binding is affected at concentrations of WGA >1 microg/mL. The EC50 for inhibition is 8 microg/mL. At a concentration of 25 microg/mL, WGA inhibits the hormone binding 70%. This inhibition can be abolished by the specific sugars for WGA N,N',N"-triacetylchitotriose and N-acetylglucosamine, but not by N-acetylneuraminic acid. UEA-I diminished hormone binding but without significance, although UEA-I binds to the fucose residues of receptor glycosylations. All other 22 lectins tested here were ineffective. CONCLUSION: The blockage of CCK receptors by WGA explains the inhibition of CCK-8s induced Ca2+ signaling and the secretion of pancreatic acinar cells and AR42J cells. Although the inhibitory effect of WGA is in agreement with the findings of Santer et al, the results with UEA-I are in contrast to those of Santer et al (1990), who described a strong increase in 125I-CCK-8s binding to isolated crude rat pancreatic cell membranes in the presence of UEA-I.

Role of PGE2 in the development of pancreatic injury induced by chronic alcohol feeding in rats.

BACKGROUND: Eicosanoids are known to modulate inflammation. Moreover, some studies report that endogenous prostaglandin E(2) (PGE(2)) protects the pancreas against injury. Therefore, we investigated its role in a rat model of chronic alcohol consumption. METHODS: Rats were fed with 20% ethanol and a corn oil-supplemented diet using the interrupted alcohol feeding regimen (EI). Controls received water instead of ethanol (WI) or uninterruptedly ethanol (EU). After 13 mo, pancreas tissue was investigated morphologically, immunohistochemically and biochemically. RESULTS: Pancreatic tissue was more severely injured in EI than in WI and EU (p < 0.05). Fibrogenesis (alpha-smooth muscle actin-positive cells, collagen types I and III) was increased in EI compared to WI (p < 0.05). In EI, mast cell numbers were increased, compared to WI, but decreased, compared to EU (p < 0.05). EI showed decreased PGE(2) and malondialdehyde contents compared to EU (p < 0.05) and decreased glutathione concentrations compared to WI (p < 0.05). PGE(2) content and fibrogenesis were inversely correlated in EU. The same correlation was detectable as a trend in all alcohol-fed rats. CONCLUSION: The decrease in PGE(2) together with the increase in tissue damage and the inverse correlation between PGE(2) and fibrogenesis led us to suggest that endogenous PGE(2) plays a protective role in alcohol-induced injury in the pancreas.

Copper accumulation in actinomyces druses during endometritis after long-term use of an intrauterine contraceptive device.

A 32-year-old woman carried a copper intrauterine contraceptive device (IUCD) or intrauterine pessar (IAP) for more than 5 years. She had acyclic menstrual bleedings and underwent a corpus abrasio after explantation of IUCD. The histological study of paraffin sections showed an actinomycotic endometritis with brown to black deposits in or around typical actinomyces druses, but there was no carcinoma. The electron microscopic study of these accumulations by electron energy loss spectroscopy (EELS) in TEM demonstrated copper deposits in the shell and matrix of these druses as well as inside the bacteria. With scanning electron microscopy (SEM) and Energy Dispersive X-Ray Analysis (EDX), the electron-dense accumulations revealed high signals for copper and sulfur, but also of phosphorus and oxygen in a lower extent. This copper accumulation is discussed as an active uptake and concentration by these actinomyces bacteria.

Evidence that ultrafine titanium dioxide induces micronuclei and apoptosis in Syrian hamster embryo fibroblasts.

Inhaled ultrafine titanium dioxide (UF-TiO2) particles cause pronounced pulmonary inflammation, in contrast to fine TiO2. Previous studies provide evidence for the production of reactive oxygen species by alveolar macrophages, after overloading with UF-TiO2 particles and cytotoxicity of UF-TiO2 in rat lung alveolar macrophages. UF-TiO2 also causes pulmonary fibrosis and lung tumors in rats. UF-TiO2 particles are photogenotoxic, but in general, information on the genotoxicity of UF-TiO2 is still limited. We studied the potential of UF-TiO2 (particle size less than or equal to 20 nm) and fine TiO2 (particle size > 200 nm) to induce chromosomal changes, which can be monitored by the formation of micronuclei (MN) in Syrian hamster embryo (SHE) cells. We also analyzed UF-TiO2-treated cells for apoptosis induction. The MN assay revealed a significant increase in MN induction (p less than or equal to 0.05) in SHE cells after treatment with UF-TiO2 (1.0 micro g/cm2) for 12 hr (mean, 24.5 MN/1,000 cells), 24 hr (mean, 31.13 MN/1,000 cells), 48 hr (mean, 30.8 MN/1,000 cells), 66 hr (mean, 31.2 MN/1,000 cells), and 72 hr (mean, 31.3 MN/1,000 cells). Bisbenzimide staining of the fixed cells revealed typical apoptotic structures (apoptotic bodies), and the apoptosis-specific "DNA ladder pattern" resulting from internucleosomal cleavage was identified by gel electrophoresis. Furthermore, transmission electron microscopy of the exposed cells revealed the typical chromatin compaction of apoptosis.

Detection of gold particles in the neck skin after lightning stroke with evaporation of an ornamental chain.

A German couple was struck by lightning. Both patients survived this event. Whereas the husband was unconscious for only a few minutes, his wife fell into coma for 24 h. The lightning stroke entered the body of the woman behind the left ear and left it at the left shoe. The stroke caused a partial evaporation of a gold ornamental chain on the neck, resulting in a tattoo of the neck skin. A biopsy of the skin 6 months after the event showed the accumulation of gold particles of different size in the dermis down to the subcutaneous fatty tissue. In semithin sections, histiocytes, multinucleated foreign giant cells, and fibroblasts were visible with uptaken metallic particles. In transmission electron microscopy, gold globules of up to 30 microm in diameter were visible outside the cells in the collageneous matrix of the connective tissue besides smaller metallic particles up to 5 nm inside lysosomes and residual bodies of phagocytic cells. Four different kinds of gold particles could be differentiated: globules, granular irregular particles, tubules, and tanglelike tracks. In scanning electron microscopy, gold particles were demonstrated by backscatter detection in the connective tissue of subcutis, where the EDX elemental analysis showed strong signals of aurum (Au), copper (Cu), and argentum (Ag). The detected metals were quantified by AAS as 70% gold, 21% silver, and 9% copper, which demonstrates the composition of gold alloy of the neck chain of the patient. Tanglelike tracks and elongated gold deposits represent crystals of gold salts, as detected by electron diffraction and polarization microscopy. Attempts to remove the gold particles from the skin to remove the tattoo should not be undertaken because the gold is deep and widespread.

Hereditary hemochromatosis of a young girl: detection of early iron deposition in liver cell lysosomes using transmission electron microscopy and electron energy loss spectroscopy.

A 14-year-old girl demonstrated increased iron concentration and transferrin saturation, suggesting iron overload of unknown origin. Liver biopsy showed no fibrosis or hepatocytic atrophia. Nevertheless, Prussian blue reaction for histochemical detection of iron demonstrated very weak positive granules in a few hepatocytes on the periphery of hepatic lobules in close connection to bile capillaries. This very early stage of hemochromatosis was confirmed by TEM and EELS for iron accumulation inside hepatocytic lysosomes and residual bodies. Such siderosomes were scarce in number and iron content, compared to a case of manifested hemochromatosis and liver cirrhosis (Jonas L, Fulda G, Salemeh T, et al. Ultrastruct Pathol. 2001; 25: 111-118.). Liver iron concentration as measured by inductively coupled plasma-mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) yielded 2.005 mg/g tissue dry weight, which was considered not significantly increased. In the absence of known causes for secondary iron overload, the early diagnosis was evidenced by genotyping, revealed homozygosity for the HFE gene C282Y mutation, demonstrating the presence of hereditary hemochromatosis.

Expression and regulation of calpain in rat pancreatic acinar cells.

INTRODUCTION: Calpains, cytosolic Ca(2+)-dependent cysteine proteases, are expressed in a variety of mammalian cells and have been found to participate in stimulus-secretion coupling in platelets and alveolar cells. AIMS: In pancreatic acinar cells, expression of calpains and their role in the secretory process have not yet been elucidated. Both subjects, therefore, were examined in the current study. METHODOLOGY: mu-calpain and m-calpain were detected immunochemically. Calpain activation was measured by fluorescence spectrophotometry and single-cell fluorometry using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Amylase secretion and cell damage, characterized by lactate dehydrogenase release, were measured by colorimetric assays. RESULTS: Immunochemistry revealed cytoplasmic localization of both calpain isoforms. Immediately after increasing the cytosolic Ca(2+) concentration with ionomycin, a marked dose-dependent protease activation and cellular damage were observed. Inhibition of ionomycin-mediated enzyme activation through preincubation of cells with Ca(2+)-free medium, BAPTA-AM, or Z-Leu-Leu-Tyr-CHN(2) significantly reduced cell injury. Cholecystokinin (100 pM) also induced proteolytic activity, preceding cholecystokinin-stimulated amylase secretion. Protease activity and amylase release were significantly inhibited by Z-Leu-Leu-Tyr-CHN(2 ) retreatment. CONCLUSION: Calpains are expressed in pancreatic acinar cells and may participate in stimulus-secretion coupling. In addition, our study indicates that pathologic calpain activation may contribute to Ca(2+)-mediated acinar cell damage.

Expression of the cholecystokinin 2-receptor in normal human thyroid gland and medullary thyroid carcinoma.

OBJECTIVE: The cholecystokinin(2)-receptor (CCK(2)R) promotes secretion and cell growth induced by its ligands cholecystokinin (CCK) and gastrin. The receptor has recently been shown to be expressed in human medullary thyroid carcinomas (MTCs). The objective of this study was to analyze CCK(2)R expression in MTC samples of different tumor stages as well as in non-malignant thyroid tissues. DESIGN AND METHODS: Using RT-PCR we investigated 19 MTC samples and TT-cells (a human MTC cell line), as well as samples of normal thyroid. In addition, we performed immunohistochemistry using calcitonin- and CCK(2)R-specific antibodies on MTCs and samples of C-cell hyperplasia. RESULTS: We demonstrate for the first time that CCK(2)R is expressed not only in MTCs but in all samples of normal thyroid tissue. Using immunohistochemistry the receptor could be localized on calcitonin-secreting C-cells. The highest incidence of CCK(2)R expression in MTCs was observed in early-tumor stages, whereas CCK(2)R could not be detected in advanced or metastasized tumors. CONCLUSIONS: The expression of CCK(2)R in C-cells suggests a physiological function for gastrin and/or CCK in the regulation of calcitonin release, presumably related to bone and calcium metabolism. Moreover, these ligands might act as growth factors in MTCs. Efforts in the development of CCK(2)R scintigraphy for the detection of MTC lesions might have to consider a lower incidence of the receptor in advanced tumor stages.