High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function.

Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signalling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through pre-pubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of tumour necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signalling of the testis in health and disease.

Sevoflurane preserves the endothelial glycocalyx against ischaemia-reperfusion injury.

BACKGROUND: Healthy vascular endothelium is coated by the glycocalyx, important in multiple endothelial functions, but destroyed by ischaemia-reperfusion. The impact of volatile anaesthetics on this fragile structure has not been investigated. We evaluated the effect of cardiac pre- and post-conditioning with sevoflurane on integrity of the endothelial glycocalyx in conjunction with coronary vascular function. METHODS: Isolated guinea pig hearts perfused with Krebs-Henseleit buffer underwent 20 min stopped-flow ischaemia (37 degrees C), either without or with 1 MAC sevoflurane. This was applied for 15 min before, for 20 min after, or both before and after ischaemia. Transudate was collected for assessing coronary net fluid extravasation and histamine release by mast cells. Coronary release of syndecan-1 and heparan sulphate was measured. In additional experiments with and without continuous sevoflurane, cathepsin B and tryptase beta-like protease activity were measured in effluent. Hearts were perfusion-fixed to visualize the endothelial glycocalyx. RESULTS: Ischaemia led to a significant (P<0.05) increase by 70% in transudate formation during reperfusion only in hearts without sevoflurane. This was accompanied by significant (P<0.05) increases in heparan sulphate (four-fold) and syndecan release (6.5-fold), with electron microscopy revealing massive degradation of glycocalyx. After ischaemia, histamine was released into transudate, and cathepsin B activity increased in effluent (P<0.05). Sevoflurane application attenuated all these changes, except for histamine release. CONCLUSIONS: Sevoflurane protects the endothelial glycocalyx from ischaemia-reperfusion-induced degradation, with both preconditioning and rapid post-conditioning being successful. The mechanism seems to involve attenuation of lysosomal cathepsin B release and to be independent from tissue mast cell degranulation.

Effectiveness of ozone against endodontopathogenic microorganisms in a root canal biofilm model.

AIM: To assess the antimicrobial efficacy of aqueous (1.25-20 microg mL(-1)) and gaseous ozone (1-53 g m(-3)) as an alternative antiseptic against endodontic pathogens in suspension and a biofilm model. METHODOLOGY: Enterococcus faecalis, Candida albicans, Peptostreptococcus micros and Pseudomonas aeruginosa were grown in planctonic culture or in mono-species biofilms in root canals for 3 weeks. Cultures were exposed to ozone, sodium hypochlorite (NaOCl; 5.25%, 2.25%), chlorhexidine digluconate (CHX; 2%), hydrogen peroxide (H(2)O(2); 3%) and phosphate buffered saline (control) for 1 min and the remaining colony forming units counted. Ozone gas was applied to the biofilms in two experimental settings, resembling canal areas either difficult (setting 1) or easy (setting 2) to reach. Time-course experiments up to 10 min were included. To compare the tested samples, data were analysed by one-way anova. RESULTS: Concentrations of gaseous ozone down to 1 g m(-3) almost and aqueous ozone down to 5 microg mL(-1) completely eliminated the suspended microorganisms as did NaOCl and CHX. Hydrogen peroxide and lower aqueous ozone concentrations were less effective. Aqueous and gaseous ozone were dose- and strain-dependently effective against the biofilm microorganisms. Total elimination was achieved by high-concentrated ozone gas (setting 2) and by NaOCl after 1 min or a lower gas concentration (4 g m(-3)) after at least 2.5 min. High-concentrated aqueous ozone (20 microg mL(-1)) and CHX almost completely eliminated the biofilm cells, whilst H(2)O(2) was less effective. CONCLUSION: High-concentrated gaseous and aqueous ozone was dose-, strain- and time-dependently effective against the tested microorganisms in suspension and the biofilm test model.

Insights from knock-out models concerning postischemic release of TNFalpha from isolated mouse hearts.

The inflammatory cytokine tumor necrosis factor alpha (TNFalpha) is controversially discussed in ischemia/reperfusion damage of the heart. Purpose of this study was to elucidate cellular sources of TNFalpha and parameters which possibly influence its release in the heart following ischemia. Isolated hearts of mice were subjected to 15 min of global ischemia and 90 min of reperfusion. We employed hearts of various mice knock-out strains (interleukin-6(-/-), matrix metalloprotease-7(-/-), mast-cell deficient WBB6F1-Kit(W)/Kit(W-v), TNF-R1(-/-)) and wildtype mice, the latter perfused without and with infusion of cycloheximide or TNFalpha-cleaving-enzyme inhibitor (TAPI-2). Normoxic control hearts showed basal release of TNFalpha during the whole experiment. Immunohistology identified cardiac mast cells, macrophages and endothelial cells as main sources. TNFalpha release was stimulated during postischemic reperfusion, occurring in a two-peak pattern: directly after ischemia (0-10 min) and again after 60-90 min. The first peak mainly reflects tissue washout of TNFalpha accumulated during ischemia. The second, protracted peak arose continuously from the basal level and was abolished by protein synthesis inhibitor cycloheximide. Both properties are characteristic for de novo synthesis of TNFalpha, e.g., in cardiac muscle cells. However, immunohistological staining for TNFalpha failed in cardiomyocytes after 90 min of reperfusion. In contrast to hearts of TNF-R1(-/-) and Kit(W/W-v)-mice, those of IL-6(-/-) and MMP-7(-/-) mice lacked the late TNFalpha peak. TAPI did not suppress release of TNFalpha. While autostimulation via TNF-R1 also does not seem obligatory and mast cell can be ignored as source of the second peak, IL-6 may support de novo synthesis of TNFalpha. Additionally, TNFalpha release may essentially involve cleavage of membrane bound TNFalpha by MMP-7.

Intracoronary formation and retention of micro aggregates of leukocytes and platelets contribute to postischemic myocardial dysfunction.

OBJECTIVE: Cardiac pump function and coronary regulation can be impaired after short-term ischemia. Recent studies with platelets (P) and neutrophils (PMN) yielded contradicting results about the "cellular" contribution to reperfusion injury. METHODS: Isolated guinea pig hearts performing pressure-volume work were employed, external heart work (EHW), aortic flow (AF), coronary flow (CF) and heart rate (HR) serving as parameters of cardiac function. After global ischemia, human blood cells were given as bolus (1 min) during reperfusion (intracoronary hematocrit 7%). Expression of specific adhesion molecules (P: CD62P, CD41; PMN: integrin CD11b) was measured on cells before and after coronary passage (FACS analysis). RESULTS: Postischemic recovery of pump function was significantly reduced in hearts with blood cell application (EHW: -cells 54 +/- 14%, +cells 41 +/-12%, p <0.05). Coronary response to bradykinin and reactive hyperemia were not effected. The blood-cell dependent functional loss was partly reduced by blocking CD18 (anti-CD 18) and completely abrogated by blockage of CD41 (lamifiban). The expression of CD11b on PMN and monocytes (M) and CD62P on platelets was significantly reduced in the coronary effluent and a significant decrease of CD41 on leukocytes occurred during coronary passage after ischemia. Increases in CD41 on PMN in the presence of lamifiban demasked intracoronary formation of micro aggregates (P/PMN). These micro aggregates were visualized by light microscopy. Electron microscopy revealed no significant microvascular plugging. CONCLUSION: 1) A specifically blood-cell induced loss of myocardial pump function has been demonstrated after short-term ischemia. 2) CD41 (= GpIIbIIIa) on P is responsible for this cardiac reperfusion damage. 3) The effect is causally linked to the formation of micro aggregates between PMN and P, but seems attenuated in the presence of erythrocytes as compared to effects reported from experiments in which PMN and P were applied singly or co-perfused. 4) Intracoronary retention of PMN, M and platelet-leukocyte micro aggregates seems to be transient, as adherence was not confirmed by electron microscopy.

[Chlamydial and human heat shock protein 60 homologues in acute coronary syndromes. (Auto-)immune reactions as a link between infection and atherosclerosis]. / Chlamydiales und humanes Hitzeschockprotein 60 bei akutem Koronarsyndrom. Antikörpervermittelte (Auto-)Immunreaktionen als Link zwischen Infektion und Arteriosklerose.

Recent studies provide evidence that infectious agents play a causal role in the pathogenesis of atherosclerosis. In this respect, a chronic persistent Chlamydia pneumoniae infection, indicated by the presence of chlamydial heat shock protein 60 (cHSP 60), is of central interest. Both cHSP60 and endogenous human (h) HSP60 are upregulated under stress conditions in intimal cells and serve as a target for cross-reactive cytotoxic HSP-serum-antibodies. Therefore, the present study evaluates the expressions of both HSP60 homologues in advanced human coronary lesions and a correlation between intimal tissuebound protein and serum antibodies (Ab) to HSP65. Coronary atherectomy specimens retrieved from 114 primary target lesions of patients with acute coronary syndrome (ACS; n=46) or stable angina (SA; n=68) were assessed immunohistochemically for the presence of cHSP60 and hHSP60. Chronic persistency of Chlamydia pneumoniae was additionally examined by transmission electron microscopy. Blood samples from30 patients were tested for anti-Chlamydia pneumoniae-IgG/IgA- and anti-HSP65-Ab titers and for serum CRP levels. Coronary plaques revealed immunoreactive cHSP60 in 47% and hHSP60 in 57% of the lesions colocalized within macrophages/foam cells. Chlamydia in foam cells most often presented ultrastructural patterns that pointed to the persistency of the pathogen. Intact, non-atherosclerotic vessels showed no signals. Mean expressions were 3.1% for cHSP60 and 3.3% for hHSP60. As a central finding, the expression of both HSP homologues was significantly (each p<0.001) higher in ACS lesions compared to SA lesions (cHSP60: 6.2 vs 1.0%, and hHSP60: 7.2 vs 0.7%). Moreover, we found positive correlations between both determinants in ACS and SA lesions (r=0.41, r=0.37; p<0.01). Most interestingly, cHSP60 revealed no relationship with anti-Chlamydia pneumoniae-IgG/IgA titers, whereas expression of cHSP60 as well as that of hHSP60 correlated with anti-HSP65-Ab titers (r=0.50, p<0.01, and r=0.42, p<0.05, respectively).cHSP60 and hHSP60 colocalize within coronary primary atheroma, most prevalent in lesions associated with ACS. For the first time, our data demonstrate a significant correlation between the intimal expression of these HSP60 homologues and serum HSP65 antibodies, thereby suggesting that humoral immune reactions to bacterial and human HSPs may play an important role in coronary atherosclerosis and plaque instability.

Cell proliferation, apoptosis, and expression of Bcl-2 and Bax in non-lactating human breast epithelium in relation to the menstrual cycle and reproductive history.

Cell proliferation, apoptosis, and the expression of Bcl-2 and Bax were investigated in breast tissue of healthy premenopausal women in order to study the effect of the menstrual cycle and reproductive history on the cell turnover in the non-lactating mammary gland epithelium. Immunohistochemistry was used to detect the proliferation-associated antigen Ki-67, as well as Bcl-2 and Bax. Apoptotic cells were identified by enzymatic labelling of fragmentized DNA (TUNEL-technique) and morphologic analysis. Consistent with published data, the proliferative activity and the frequency of apoptotic events as detected by morphologic analysis was higher in the luteal than in the follicular phase of the menstrual cycle. Parity, lactation, and age correlated with lower proliferative activity, whereas the frequency of apoptosis was not significantly influenced by the reproductive history. Staining patterns for Bax and Bcl-2 showed characteristic changes due to the menstrual cycle with a maximum of immunoreactivity for Bcl-2 in the follicular phase and for Bax in the luteal phase. However, there was no statistically significant association between Bcl-2/Bax immunoreactivity and menstrual cycle or reproductive parameters. We conclude that other molecular pathways than the Bax/Bcl-2 antagonism may additionally be involved in the regulation of apoptotic cell death in the breast epithelium. Knowledge of the entire complexity of apoptosis regulation is necessary to understand the observed effects of parity and lactation on mammary epithelial biology, and possibly to be able to influence pathological processes caused by an imbalance between cell renewal and elimination.

[Neointimal hyperplasia by luminal cell recruitment and not be transmural migration. The role of Bcl-2 and HSP47 after balloon angioplasty]. / Neointimale Hyperplasie durch luminale Zellrekrutierung und nicht durch transmurale Migration. Die Rolle von Bcl-2 und HSP47 nach Ballonangioplastie.

Restenosis post angioplasty remains the major limitation of several therapeutic interventions including stent implantation. This explains the ongoing interest in its basic pathogenic mechanisms and factors. The aim of the present study was to assess the localization and maximal expression of Bcl-2, a central antiapoptotic protooncogene, and of heat shock protein 47 (HSP47), a marker of early collagen synthesis, in the context with hyperplastic neointima formation as well as concomitant transmural remodeling processes following angioplasty. 0, 4, 24 and 48 hours, 4, 7 and 14 days post balloon traumatization by use of a rat carotid artery model, specific vascular wall compartments were evaluated concerning area, cell density as well as Bcl-2 and HSP47 expression by immunohistochemistry and morphometry, supplemented by electron microscopy (TEM). Neointimal cell accumulation was detected 4 days post angioplasty, characterized by luminal cells adherent to the internal elastic lamina, associated with maximal Bcl-2 and HSP47 expression amounting to 49% and 41%, respectively. With ongoing neointimal formation, a luminal prevalence of both key determinants and a decreasing expression in basal neointimal areas were found. In the media, a temporally reduced cell density was observed significant at 48 hours post trauma. Constitutive HSP47 expression of the media was constant during the entire observation period, whereas sparse Bcl-2 signalling was induced post angioplasty maximal on day 2 with 3% and on day 14 with 5%. The adventitia demonstrated a transient structural separation between day 4 and 7, exhibiting an inner layer with sparse cellularity and an outer layer with extremely high cell density as well as pronounced neovascularization. In this outer adventitia layer, a high frequency of signals for both Bcl-2 and HSP47 were observed amounting to 29% and 57%, respectively. Complementary TEM analysis gave no evidence of transmural migratory events propagated by adventitial cells and thereby supports early neointimal formation by luminal cell recruitment and marked co-expression of anti-apoptotic Bcl-2 and matrix-generating HSP47 as important survival factors. Clinical implications of these findings may be seen in the integration of proapoptotic substances with temporal efficacy in order to prevent restenosis, e.g., by use of coated stents.

Cutaneous eccrine glands of the foot pads of the rock hyrax (Procavia capensis, Hyracoidea, Mammalia).

In order to find correlations between skin gland morphology and specific ethological features, the cutaneous glands of the foot pads of Procavia capensis were studied by histological and various histochemical methods and by electron microscopy. In the foot pads, abundant specific eccrine skin glands occur, which consist of coiled tubular secretory portions and coiled ducts. The wall of the secretory part is composed of cuboidal glandular cells and myoepithelial cells. Among the glandular cells two types occur: clear and dark cells. Clear cells have numerous mitochondria and form a basal labyrinth, indicating fluid transport. Dark cells, which stain strongly with periodic acid-Schiff, contain a highly developed perinuclear Golgi apparatus, large amounts of rough endoplasmic reticulum and many secretory granules indicating production of glycoproteins. Cytokeratin (CK) 19 was found in secretory compartments and ducts, CK14 only in duct cells. Single cells of the secretory coils and ducts may be stained with antibodies against antimicrobial peptides. Some glandular cells contain proliferating cell nuclear antigen-positive nuclei especially in the ducts indicating an increased cell proliferation. Terminal transferase (TdT)-mediated d-UTP nick-end labeling-positive nuclei can be detected predominantly in the secretory coils and rarely in the transitional portions between ducts and end pieces. We suppose that proliferating cells migrate from the ducts to the secretory coils. The secretory product of the eccrine cutaneous glands seems to improve the traction between the foot pads of these animals and the steep and smooth rock formations among which they live.

[Chlamydia pneumoniae and cytomegalovirus in degenerative aortic valve stenoses]. / Chlamydia pneumoniae und Zytomegalie-Virus in degenerativen Aortenklappenstenosen.

Recent evidence suggests a causal relationship between inflammatory as well as infectious pathomechanisms and valvular degeneration. Based on the concept of chronic Chlamydia pneumoniae and cytomegalovirus (CMV) infections, and of variable stressors working on valvular microecology, the present study sought to assess the presence of the specific chlamydial heat shock protein (cHSP) 60, of CMV, of macrophages and of the human homologue hHSP60. Serial sections of high-grade degenerated native (n = 16) and prosthetic (n = 6) aortic valves were analyzed by immunohistochemistry for the presence of these determinants. Degenerated aortic valves revealed prevalence of Chlamydia pneumoniae in 41% (10 of 22) and CMV in 73% (16 of 22), while immunoreactive hHSP60 was present in 64% (14 of 22) and CD68 in 86% (19 of 22). Chlamydial HSP60, CMV and hHSP60 were predominantly found in valvular fibrosa; CMV showed a second predilection site at the ventricular luminal border. Both microorganisms revealed a strong correlation between each other (r = 0.73; p < 0.001) as well as with hHSP60 (cHSP60: r = 0.74; p < 0.001; CMV: r = 0.80; p < 0.001). Macrophage infiltration correlated with cHSP60 (r = 0.78; r < 0.001), CMV (r = 0.78; r < 0.001) and hHSP60 (r = 0.56; r = 0.007). Of note, the frequency of cHSP60, CMV and CD68 signaling was increased more than 5-fold in prosthetic valves compared to native valves (p = 0.017, p = 0.002 and p = 0.005). In summary, valvular infections of Chlamydia pneumoniae and of cytomegalovirus are frequently seen in degenerated aortic valves, irrespective of native or prosthetic origin. Colocalization of both HSP60 homologues and cytomegalovirus within macrophages in valvular fibrosa points to regional stressor effects that might be at least partly attributable to chronic persistent pathogen burden and molecular mimicry.

Microscopic anatomy of the eye of the deep-diving Antarctic Weddell seal (Leptonychotes weddellii).

The microscopic anatomy of the eye of the Weddell seal was studied with various light and electron microscopic methods with a view to correlating morphological findings with the biology of this seal which is adapted to the extremes of the Antarctic environment and to extreme diving excursions into the lightless depths of the sea. In the retina an area centralis was found but no fovea centralis. The densely packed photoreceptors consist exclusively of highly differentiated rods, which in primates detect light at low intensity but have rather poor image discrimination. The ganglion cells are relatively scarce, suggesting a high degree of convergence of the light-sensitive cells on the ganglion cells. The pigment epithelium is almost devoid of pigment granules. The extensive tapetum lucidum is about 400-500 microm thick and is composed of about 30 layers of specialized cells. The cornea is 650 (center) to 800-900 (periphery) microm thick. Its structure and glycosaminoglycan histochemistry correspond to that of other mammals. The iridocorneal angle is unusually deep and pervaded by an elaborate trabecular meshwork, which together with a complex canal of Schlemm can be correlated with the ability to absorb large amounts of fluid. The ciliary muscle and its antagonist, the membrane of Bruch, are poorly developed, suggesting relatively poor abilities of accommodation. The combination of a well-developed tapetum lucidum, an unpigmented pigment epithelium, well-developed rods, and a high number of rods converging on only few ganglion cells is obviously an adaptation to an extreme light sensitivity, enabling the animals to make use of the little light available in the deep sea.

Rhesus monkey (Macaca mulatta) mucosal antimicrobial peptides are close homologues of human molecules.

One component of host defense at mucosal surfaces appears to be epithelium-derived antimicrobial peptides. Molecules of the defensin and cathelicidin families have been studied in several species, including human and mouse. We describe in this report the identification and characterization of rhesus monkey homologues of human mucosal antimicrobial peptides. Using reverse transcriptase PCR methodology, we cloned the cDNAs of rhesus monkey beta-defensin 1 and 2 (rhBD-1 and rhBD-2) and rhesus monkey LL-37/CAP-18 (rhLL-37/rhCAP-18). The predicted amino acid sequences showed a high degree of homology to the human molecules. The expression of the monkey antimicrobial peptides was analyzed using immunohistochemistry with three polyclonal antibodies to the human molecules. As in humans, rhesus monkey antimicrobial peptides are expressed in epithelia of various organs. The present study demonstrates that beta-defensins and cathelicidins of rhesus monkeys are close homologues to the human molecules and indicate that nonhuman primates represent valid model organisms to study innate immune functions.

Histo-physiology of the scent-marking glands of the penile pad, anal pouch, and the forefoot in the aardwolf (Proteles cristatus).

The scentmarking glands of the anal pouch, penile pad, and the forefoot of the aardwolf (Proteles cristatus) were studied by histological, histochemical, immunohistochemical methods, and by electron microscopy. The morphological observations are correlated with eco-ethological aspects of this nocturnal animal. In all studied regions there was a superficial layer of holocrine sebaceous glands and a deeper layer of apocrine scent glands; these two types of glands apparently function in concert. Only in the forefoot were additional tubular glands, resembling eccrine sweat glands found, which may improve the frictional capacities of the paw, while apocrine and holocrine glands serve scent-marking functions of the forefoot. Penile pad and anal pouch are exclusively scent marking organs. The secretion modus of the apocrine glands is both via exocytosis and apocrine mechanism. Homogeneous apical, secretory granules, which contain glycoproteinaceous material, represent evidence for exocytosis. In the anal pouch, additional variably sized granules contain endogenous pigments which are probably responsible for the brownish coloration of the secretory product of the male animals. Variable heights of the glandular cells, frequent apical tall protrusions as well as pinched-off pieces of cytoplasm in the glandular tubules support the concept of an apocrine secretion in the scent glands. The immunohistochemical staining pattern of actin points to the involvement of actin filaments in the pinching-off process of the apical cell protrusion, which does not contain any cell organelles. The variable actin staining patterns suggest a dynamic process during which actin filaments form a ring or sheet at the basis of the pinching-off bleb. Proliferative and apoptotic phenomena show no preference for active and inactive glandular cells suggesting that replacement of cells occurs independently of the functional status of the glands.

Immunohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland epithelium.

The expression of the apoptosis-related proteins Bcl-2 and Bax was investigated by immunohistochemistry in the normal non-lactating human mammary gland in relation to cell proliferation and apoptosis. In order to characterize individual Bax/Bcl-2-immunoreactive cells, the epithelial markers cytokeratin 14 and 19 and the macrophage marker CD 68 were used. Secretory-like differentiation of epithelial cells was characterized by histochemistry and lectin staining of surface glycoconjugates. Cell proliferation was exclusively found in glandular epithelial cells with broad contact to the ductular lumen, whereas nuclei with apoptosis-related DNA fragmentation were seen predominantly in basally located glandular epithelial cells and in myoepithelial cells. Weak immunoreactivity for Bcl-2 and Bax was present throughout all epithelia, suggesting a balance between pro- and antiapoptotic effects in the majority of epithelial cells. However, specific cells showed a strong staining for Bax or Bcl-2. The strongly Bcl-2-immunoreactive epithelial cells were not identical with proliferating cells, but they resembled them in configuration and in the luminal intraepithelial position. In contrast, the strongly Bax-positive epithelial cells had no or only a narrow contact to the ductular lumen. The different patterns of Bax/Bcl-2 immunoreactivity in specific glandular epithelial cells suggest that there are also different grades of susceptibility towards apoptotic stimuli in individual glandular epithelial cells. We conclude that specific Bax/Bcl-2 expression patterns could reflect particular cell differentiation states, and that the strongly Bcl-2-positive cells in part could represent epithelial stem cells.

[Polyvinylpyrrolidone deposits in the pleura]. / Polyvinylpyrrolidon-Ablagerungen in der Pleura.

We report on the case of a 45-years old woman with repeated pleural effusions lasting for 2 years. She had a history of breast carcinoma 6 years ago, which was treated by breast amputation and radiation. After repeated histological and cytological examinations no tumorous compromise of the pleura or other cause for the effusions could be found. A pleural resection was performed. On the histological and electron microscopical examination deposits of a foreign substance were found, which was identified as polyvinylpyrrolidone. This substance was obviously introduced in the pleural cavity by the attempts to treat the effusions by pleurodesis with Diclofenac and Tetracycline (Vibravenös). The diagnosis is suggested by the histological findings, but it must be confirmed by anamnesis, also for PVP deposits in other organs.

[Persistence of Chlamydia pneumoniae in coronary plaque tissue. A contribution to infection and immune hypothesis in unstable angina pectoris]. / Persistenz von Chlamydia pneumoniae in koronarem Plaquegewebe. Ein Beitrag zur Infektions- und Immunhypothese bei instabiler Angina pectoris.

BACKGROUND AND OBJECTIVE: There is an increasing number of pointers towards a causative connection between Chlamydia pneumoniae and atherosclerosis. But the pathogenetic mechanism and intimal structures that are involved remain unclear. Starting with the hypothesis of a chronic infection, as demonstrated by the presence of the chlamydial stress (heat-shock) protein 60 (HSP 60), the presence and localization of these bacterial products in coronary atheromas was investigated. PATIENTS AND METHODS: Coronary atheroma tissue from primary stenoses in 42 patients (36 men, 6 women, mean age 60.2 +/- 7.3 years) was studied immunohistochemically in the course of a retrospective analysis for chlamydial HSP 60. The findings in clinically acute coronary syndrome (Braunwald's classification) present in 27 patients were compared with those in 15 patients with acute angina and evaluated in relation to expression and site of predilection. RESULTS: An immune reaction to chlamydial HSP 60 was demonstrated in 27 of 42 atheromas (64%). Intact, non-atherosclerotic vessels, such as the mammary artery and sphenous vein, showed no such signals. Chlamydial HSP 60 was localized in maximally 23% of all plaque cells, mostly in macrophages/foam cells, more rarely in smooth muscle cells. Chlamydia in foam cells most often revealed ultrastructural patterns that pointed to the persistence of the pathogen. Sites of predilection of chlamydial HSP were predominantly foam cell areas and cell-poor regions, more rarely inflammatory infiltrates and areas of rupture. When comparing both types of lesion, signals for chlamydial HSP 60 were present in 21 of the 27 atheromas (78%) with unstable angina or acute myocardial infarction, but in only 6 of the 15 atheromas (40%) with stable angina. Within the group with the acute coronary syndrome, the prevalence of chronic chlamydial infection was independent of a previous myocardial infarction. CONCLUSIONS: Chlamydial HSP 60 can often be demonstrated in primary coronary stenosis of symptomatic patients. It is most frequently found in macrophages/foam cells and is highly prevalent in the acute coronary syndrome. In-situ findings suggest a pathogenetically relevant role of chronic persistent infection of Chlamydia pneumoniae in unstable coronary plaques.

Three-dimensional in vitro cocultivation of lung carcinoma cells with human bronchial organ culture as a model for bronchial carcinoma.

We describe the development of a three-dimensional in vitro organ culture model for bronchial carcinoma using bronchial mucosa organ cultures and three different human non-small cell lung cancer cell lines. During precultivation, bronchial fragments obtained as biopsies during routine bronchoscopy had regenerated a complete epithelial covering with a well-preserved organotypic architecture around a nucleus consisting of connective tissue. To create cocultures, different types of confrontation between tumor cells and organ cultures were applied. Histologic light microscopy and scanning electron microscopy were used in analysis. When tumor cells were confronted with completely epithelialized organ cultures, they showed a low incidence of attachment. When organ cultures were wounded before confrontation, tumor cells always attached to the wounded side and showed a progressive invasion into the stromal tissue. Measurements of the penetration depth of tumor cells into the organ cultures after different incubation times permitted the quantitative evaluation of invasion. Histologic studies revealed well-differentiated normal epithelium in spite of long culture periods. Histologic features of the tumors were those of an invasive undifferentiated carcinoma and showed marked similarities to the situation in vivo. The coculture model permits internal controls because it contains both normal human epithelium and human tumor cells in the same organotypic culture. Therefore it offers opportunities for various in vitro investigations on therapeutic and diagnostic modalities of lung cancer, as indicated in this paper by an example of photodynamic procedures with 5-aminolevulinic acid.

Role of smooth muscle cell death in advanced coronary primary lesions: implications for plaque instability.

OBJECTIVE: Instability of coronary atheroma leads to the onset of acute coronary syndromes including myocardial infarction and death, as well as to the progression of the arteriosclerotic disease. As yet, the underlying factors and mechanisms causing plaque rupture are not completely understood. Since a low content of smooth muscle cells (SMCs) apparently plays a key role, the question points to the events leading to the loss of intimal SMCs. METHODS: We compared coronary atherectomy specimens from 25 patients with unstable angina to those from 25 patients with stable angina. Transmission electron microscopy was used to identify intimal cell population, to detect stage and cell type of apoptosis, and to differentiate between apoptosis and necrosis. RESULTS: Plaques associated with unstable angina contained more macrophages/lymphocytes and significantly less SMCs (P = 0.01), compared with stable angina plaques. Specific cell death forms, apoptosis and necrosis, were present in all coronary atheroma. As key findings, both the proportion of SMCs undergoing apoptosis and the frequency of cytoplasmic remnants of apoptotic SMCs (matrix vesicles) were significantly increased in unstable versus stable angina lesions (P = 0.002 and P = 0.002). In addition, cellular necrosis was more frequent in the first coronary atheroma group (P = 0.02). Positive correlations were found between the frequency of apoptotic cells and necrosis (r = 0.41, P = 0.04), and that of matrix vesicles and necrosis (r = 0.63, P = 0.001) only in plaques with unstable angina, but not in those with stable angina. CONCLUSIONS: Our data demonstrate that high cell death due to apoptosis and necrosis is a basic in situ feature found in advanced coronary primary lesions associated with unstable angina, possibly explaining their low density of (viable) SMCs. Thus, antagonization of intimal cell death should be considered in order to stabilize the intimal plaque texture of coronary atheroma with the ultimate goal to prevent plaque rupture.

[Chlamydia pneumoniae in coronary plaques: Increased detection with acute coronary syndrome]. / Chlamydia pneumoniae in koronarem Plaquegewebe: Vermehrter Nachweis bei akutem Koronarsyndrom.

BACKGROUND AND OBJECTIVE: There is seroepidemiologic and experimental evidence for a link between Chlamydia (C.) pneumoniae and arteriosclerosis. However, the clinical importance and the pathogenic pathways implicated remain unclear. In the present study, we sought to evaluate the presence and the location of C. pneumoniae in coronary atheroma, as well as a potential prevalence with unstable versus stable angina. PATIENTS AND METHODS: Retrospectively, coronary plaque material of primary lesions from 51 consecutive patients (44 men, 7 women, mean age 59.6 +/- 9.4 years) was examined for the presence of C. pneumoniae by use of immuno-histochemistry and transmission electron microscopy. The findings associated with clinically acute coronary syndrome according to Braunwald's classification (n = 31) were compared to those with stable angina (n = 20) and regarded for potential relations to characteristic intimal features. RESULTS: Immunoreaction for C. pneumoniae was found in 32 of 51 (63%) coronary plaques. Signals (% prevalence of specific intimal features) were present with necrotic areas (40%), sparse cellularity (40%), neo-vascularization (29%), thrombi (20%), ruptured plaque areas (19%), and fields rich in foam cells and calcifications (13%). Intimal hyperplasia and inflammatory infiltrates showed no signals. As the central finding in this report, C. pneumoniae immunoreaction was more frequently (P < 0.001) found in 26 of 31 (84%) lesions associated with unstable angina or acute myocardial infarction, compared to 6 of 20 (30%) lesions with stable angina (P < 0.001). Intact vessels devoid of arteriosclerotic disease, such as mammarial arteries and saphenous veins, were without C. pneumoniae signals (negative controls). Ultrastructurally, chlamydial elementary bodies were found in foam cells and phagocytosing macrophages, also in fragmented extracellular matrix adjacent to apoptotic and necrotic intimal cells. CONCLUSIONS: Chlamydiae pneumoniae were detected in 32 of 51 (63%) coronary primary lesions of symptomatic patients. Most importantly, there was a highly significant prevalence of lesions associated with acute coronary syndrome. Predilection sites of C. pneumoniae were areas that revealed small healing activity and (or) propensity to plaque rupture. The present in situ findings indicate a pathogenic role of Chlamydiae pneumoniae in human (coronary) plaque rupture.