Alteration of the basement membrane in human thyroid diseases: An immunohistochemical study of type IV collagen,laminin and heparan sulphate proteoglycan

Basement membrane (BM) alteration in thyroid diseases was examined by immunohistochemistry using antibodies for the three major BM proteins: type IV collagen, laminin and heparan sulphate proteoglycan. Linear epithelial BMs surrounding follicles accompanied by vascular BMs forming loops, similar to those seen in the normal thyroid, were observed in Graves' disease and adenomatous goitre. Hashimoto's thyroiditis showed scant epithelial BMs as a result of follicle destruction. In follicular adenomas, development of epithelial BMs seemed to be related to follicle formation; well-developed epithelial BMs were frequently seen in normo- or largefollicular type, whereas trabecular or solid types revealed scant or poorly developed epithelial BMs. Lumpy accumulation of BM proteins was detected in hyalinizing trabecular adenomas. Papillary carcinomas revealed two different types of papillae; one type contained both epithelial and vascular BMs, and the other had only vascular BMs. Epithelial BMs in invasive areas of papillary carcinoma were distributed in an irregular, interrupted manner, and were completely absent in many foci. Anaplastic carcinomas showed scant or a total loss of epithelial BMs. These results suggest that alterations of BM in thyroid diseases clearly reflect their architectural variations, presumably in connection with their function and/or biological behaviour.     

Cryofixation of basement membranes followed by freeze substitution or freeze drying demonstrates that they are composed of a tridimensional network of irregular cords

Since conventional chemical fixation may extract tissue components and thus alter structural organization, cryofixation was used to reexamine the ultrastructure of three thick basement membranes: lens capsule, Reichert's membrane, and Engelbreth-Holm-Swarm (EHS) tumor matrix, and two thin basement membranes, those of epididymis and seminiferous tubules. Cryofixation was achieved by slam freezing followed by either freeze substitution in dry acetone containing 1% osmium tetroxide and 0.05% uranyl acetate or freeze drying in a molecular distillation dryer. The results by both procedures demonstrate that thick basement membranes and the lamina densa of thin basement membranes are composed of a network of anastomosing strands referred to as cords. The cords vary in density and distinctiveness, but their thickness averages 3 to 5 nm in every tissue examined. The spaces separating the cords vary within wide limits, but their mean diameter is ～15 nm in every case. Two other common features are (1) the presence within the network of a few 1.5–3.0-nm-thick filaments and (2) 4.5-nm-wide sets of parallel lines referred to as double tracks. When these results are compared with those previously described after conventional fixation, no significant difference is observed in either the cord network or the associated filaments and “double tracks.” However, in the thin basement membranes processed by cryofixation, the lamina densa is in direct contact with epithelial cells, whereas, after conventional fixation, the lamina densa is separated from the epithelial cells by a pale layer referred to as lamina lucida or lamina rara. Immunogold labeling of three basement membranes after cryofixation and freeze substitution in acetone containing 0.3% glutaraldehyde yields strong reactions for laminin, type IV collagen, and heparan sulfate proteoglycan. Comparison with previous results indicates that conventional formaldehyde fixation adequately preserves laminin and type IV collagen but causes the loss of some proteoglycan. It is concluded that, except for this loss and the absence of lamina lucida in cryofixed thin basement membranes, the morphological and antigenic features obtained after cryofixation are similar to those observed in the past after conventional fixation. © 1993 Wiley-Liss, Inc.     

Basement membrane proteins and reticulin in a normal thymus and the thymus in myasthenia gravis

Summary The distribution of basement membrane (BM) proteins, laminin and type IV collagen were studied immunohistochemically in a series of 12 normal thymuses representing different age groups (0–52 years) and in 10 cases of myasthenia gravis (age 7–53 years). The staining pattern was compared with that of conventional reticulin staining. BM proteins were present at the capsule-parenchyma interface and scantily distributed in the medullary stroma, where they were closely associated with reticulin fibres. The extrathymic perivascular space was effectively vizualized by the staining of the BM's marginal to it. The fiber network present in this space stained with reticulin stain and, less continuously, in BM stainings. Lymph node like tissue with germinal centers was occasionally present in the perivascular spaces in normal thymuses and commonly in the myasthenia gravis cases, where the perivascular spaces were often dilated. The BM's of the perivascular space were mostly continuous in normal cases, but discontinuities were observed in cases of myasthenia gravis, especially in the spaces which were widely dilated. Immunohistochemical detection of BM proteins seems to be useful in the study of thymic structure, particularly in the demonstration of the characteristic changes of the perivascular space in myasthenia gravis. It is suggested that the reticulin fibres present in the medulla and in the perivascular space contain laminin and type IV collagen.     

Androgen receptor status in endocrine-paracrine cell types of the normal,hyperplastic, and neoplastic human prostate

Neuroendocrine differentiation is a frequent occurrence in common prostatic adenocarcinomas and may have prognostic implications in prostatic malignancies. In the present study, we used immunohistochemical double label methods to evaluate the nuclear androgen receptor (AR) status in endocrine-paracrine (EP) cells of normal, hyperplastic, and neoplastic prostate including tumours that recurred after hormonal and radiation therapy. In normal and hyperplastic glands, EP cells characterized by the panendocrine marker chromogranin A (Chr A) did not reveal AR-positivity. This may indicate that prostatic EP cells represent an androgen-indepenent cell population whose regulatory functions are not influenced by circulating androgens. Unequivocal co-expression of Chr A and AR was very rarely detected in subsets of endocrine differentiated tumour cells in treated and untreated specimens. The widespread absence of nuclear AR in neuroendocrine tumour cells suggests that this phenotype belongs to those cell clones in prostate cancer which are initially androgen-independent and refractory to hormonal therapy.This study was partially presented at the 82nd Annual Meeting of the United States and Canadian Academy of Patholoy, March 13–19, 1993.This study was supported by the Deutsche Forschungsgemeinschaft Bo-1018/1-5     

Immunohistochemical localization of myoepithelial cells and basement membrane in normal,benign and malignant human breast lesions

Summary Distributions of actin and type IV collagen were investigated immunohistochemically as markers for myoepithelial cells and basement membranes. Carnoy's and Methacarn-fixed, paraffin-embedded tissues from 103 human breast lesions from 103 patients were examined; 65 with carcinomas, 27 with mastopathies, 9 with fibroadenomas and 2 with phyllodes tumours. Fifty-five samples of the normal mammary gland tissue adjacent to tumours were also included for comparison. In normal breast and benign breast diseases, type IV collagen was identified around the mammary glandular cells and actin-positive cells were demonstrated to attach to basement membranes. In noninvasive carcinomas, type IV collagen was found as a continuous lining around a cell nest, while actin-positive cells were usually absent in ductal but quite numerous in lobular carcinomas. In invasive carcinomas, type IV collagen was fragmented or absent and actin-positive cells were very uncommon around the fragmentary basement membranes. These results suggest that the different distributions of myoepithelial cells and basement membrane material is useful in the differential diagnosis of surgical pathology of the breast.This work is supported in part by grant-in-aid for cancer research 62010025, from the Ministry of Education, Science and Culture, Japan     

Basement membrane proteins in salivary gland tumours

Summary Immunohistochemical localization of type IV collagen and laminin in normal salivary glands and in salivary gland tumours of various types was studied using rabbit antisera. In normal salivary glands, type IV collagen and laminin were co-localized in basement membranes surrounding acini, ducts, fat cells and peripheral nerves. In salivary gland tumours, three main patterns of co-expression of these basement membrane proteins were distinguished. Linear basement membrane-like staining was detected in duct-cell-derived benign salivary gland tumours and in acinic cell carcinomas. In invasive lesions, however, these basement membrane proteins were distributed in an irregular, interrupted manner, and in many cases they were completely absent. Both benign and malignant salivary gland tumours which have a prominent myoepithelial cell component display a particular deposition of basement membrane molecules adjacent to the modified myoepithelial cells, and at the margins of extracellular matrix deposits within these tumours.     

Nucleolar organizer regions in normal,hyperplastic and neoplastic parathyroid glands

Summary Using a one-stage silver staining technique, nucleolar organizer regions (AgNORs) were studied in paraffin sections of parathyroid glands (and in two lymph node metastases) from patients operated upon because of hyperparathyroidism or thyroid disease. The parathyroids were microscopically differentiated into normal, hyperplastic, adenomatous and carcinomatous glands. AgNORs were observed as distinct black dots of varying size and somewhat varying configuration in the nuclei of all glands. The mean number of AgNORs in the hyperplastic and adenomatous glands was not significantly different from that in the normal glands, whereas the carcinomatous glands exhibited significantly increased mean AgNOR number. No evidence was obtained for a role of AgNOR counting in the differentiation between normal and hyperplastic or adenomatous parathyroids, but the results suggest a potential role of enumeration of AgNORs in the discrimination between benign and malignant parathyroid neoplasms.     

Basement membrane composition of cartilage canals during development and ossification of the epiphysis

Background: Cartilage canals are perichondral invaginations of blood vessels and connective tissue that are found within the epiphyses of most mammalian long bones. Functionally, they provide a means of transport of nutrients to the hyaline cartilage, a mechanism for removal of metabolic wastes, and a conduit for stem cells that are capable of initiating and sustaining ossification of the chondroepiphysis. Morphological and biomolecular changes of the chondroepiphyses appear to potentiate vascular invasion and enable regional formation of secondary centers of ossification within the chondroepiphyses of developing bones. Methods: As both cell migration and vascular invasion are anchorage dependent processes, antibodies to laminin and Type IV collagen were used to assess compositional changes in the basement membrane of cartilage canals accompanying epiphyseal ossification. Results: Differences in chronological appearance, as well as, in distribution between the two components were noted in the chondroepiphysis. Laminin was distributed throughout the connective tissue of cartilage canal at all stages of developement, and not limited to an association with the vascular lumen. Type IV collagen was not Present during the initial perichondral invagination. Although staining for Type IV collagen was later acquired, its distribution was restricted to a discontinuous rimming of the periphery of the canal, and a diffuse presence within the intra-canalicular mesenchyme. Conclusions: Concurrent with chondrocyte hypertrophy and mineralization of the hyaline matrix, rapid changes in both the morphology of the vessel and distribution of the antibodies were detected. In addition to the presence of laminin at the interface of the endothelium and the hyaline matrix, a wide distribution within the connective tissue components of the newly ossifying matrix of epiphyseal bone could be detected. Type IV collagen remained closely associated with the lumens of the intra-canalicular vessels throughout the transition. Following ossification of the secondary center, staining for Type IV collagen could then be detected in the boneforming regions of transforming matrix as well, clearly delineating the individual vessels within the newly formed marrow spaces. This suggests that bone formation is intimately related to vessel staining for collagen type IV, and that acquired vessel competence is a facet of endochondral bone formation that results from provisional matrix changes. Furthermore, the data suggests that during bone formation under tension, basement membrane deposition can be demonstrated without an intermediary hyaline matrix hypertrophic chondrocyte phase. This data was interpreted to suggest that chondrocyte hypertrophy at the growth plate may be a reaction to vascular invasion, that in turn, stimulates adjacent chondrocyte proliferation. © 1995 Wiley-Liss, Inc.     

Immunohistochemical localization of laminin and type IV collagen in human cutaneous sensory nerve formations

We used immunohistochemical techniques and monoclonal antibodies to localize two basement membrane components (laminin and type IV collagen) in the nerves and sensory nerve formations, or corpuscles, supplying human digital skin. Furthermore, neurofilament proteins, S-100 protein and epithelial membrane antigen were studied in parallel. In dermal nerve trunks, immunostaining for laminin and type IV collagen was found to be co-localized in the perineurium and the Schwann cells, the stronger immunoreactivity being at the external surface of the cells. In the Meissner digital corpuscles, the immunoreactivity for laminin and type IV collagen was mainly observed underlying the cell surface of lamellar cells, while the cytoplasm was weakly immunolabelled or unlabelled. Finally, within Pacinian corpuscles co-localization of the two basement membrane molecules was encountered in the inner core, intermediate layer, outer core and capsule. Laminin and type IV collagen immunoreactivities were also found in blood vessels and sweat glands, apparently labelling basement membrane structures. The present results provide evidence for the presence of basement membrane in all periaxonic cells forming human cutaneous sensory nerve formations, and suggest that all of them are able to synthesize and release some basement membrane components, such as laminin and type IV collagen. The possible role of laminin in sensory nerve formations is discussed.     

Loss of laminin and type IV collagen in uterine luminal epithelial basement membranes during blastocyst lmplantation in the mouse

Background: Removal of the uterine luminal epithelium and its basement membrane is necessary for successful implantation of invasive blastocysts. Few reports, however, have specifically addressed the penetration and loss of the uterine luminal epithelial basement membrane (UEBM). We investigated the loss of UEBM by examining the distribution of laminin and type IV collagen. Methods: Blastocyst implantation sites were collected from mice on days 5,6, and 7 of pregnancy. Paraffin sections were prepared from these tissues and processed with standard immunoperoxidase techniques to reveal the distribution of laminin and type IV collagen. Results: On day 5 of pregnancy blastocysts were adherent to the uterine epithelium. The epithelium and UEBM were complete and uninterrupted. On day 6 the juxtaembryonic uterine epithelium was lost and focal discontinuities were seen along the UEBM. By 1200 hr the UEBM had receded to the region near the ectoplacental cone, but staining was reduced for both antigens over the entire region surrounded by decidual cells. This decreased staining of the UEBM occurred in areas not yet occupied by trophoblast cells. On day 7 the UEBM was lost over the entire embryonic half of the uterine lining, corresponding to the distribution of decidual cells. Conclusions: Progressive loss of the UEBM occurred in a consistent spatiotemporal pattern following the onset of blastocyst implantation. Diminished immunoreactivity of laminin and type IV collagen in the UEBM was closely correlated with the area occupied by decidualized endometrial stroma and occurred in areas not yet in contact with trophoblast cells. We conclude that decidual cells are instrumental in the removal of UEBM during early pregnancy. © 1995 Wiley-Liss, Inc.     

Ultrastructural localization of type IV collagen and laminin in the seven-day-old mouse embryo

Summary The localization of the basement membrane components type IV collagen and laminin was investigated in seven-day-old mouse embryos (NMRI) fixed with formaldehyde, using an immunoperoxidase technique. Posttreatment of the embryos with TBS (trishydroxymethylaminomethane buffered saline) buffer was prerequisite for restoration of the antigenicity after fixation. The localization of the peroxidase (PO) positive reaction after treatment with anti-type IV collagen and anti-laminin antibodies in the embryos has been compared with results obtained after fixating embryos with the addition of tannic acid. Tannic acid stained the basement membrane of the ectodermal cell layer, in particular the lamina densa. After immunostaining for type IV collagen and laminin, a strong PO-positive reaction in the lamina densa of the ectodermal basement membrane was observed.A basement membrane of the endodermal cell layer had not yet been formed at this developmental stage. In this region, which is where a basement membrane was to develop in later stages, a tannic acid positive material consisting of granules with a diameter of about 25 nm was found near the surface of the endoderm. Moreover, PO-positive patches were seen in this part of the embryo after staining for laminin as well as after staining for type IV collagen. These PO-positive patches were mainly localized in areas where mesodermal cells lay adjacent to the surface of the endodermal cell layer. No positive staining for type IV collagen and laminin was found in the cytoplasm of either cetodermal or endodermal cells.     

Effects of ACE inhibition on the expression of type IV collagen and laminin in renal glomeruli in experimental diabetes

In the present study, we examined electron microscopically and immunohistochemically the effects of perindopril, an angiotensin-converting enzyme inhibitor, on renal microangiopathy in streptozotocin-induced diabetes in rats. To investigate changes in glomerular basement membrane (GBM) and tubular basement membrane components, we immunohistochemically localized type IV collagen and laminin. Animals have been divided into three groups of eight adult male rats each. The first group was the non-diabetic control group. The second group consisted of untreated diabetic rats. The third group consisted of diabetic rats that were treated with perindopril for 6 weeks. Blood glucose levels and body weight were measured. Morphometric analysis of kidney tissue was performed using light and electron microscopy to quantify glomerular size and thickness of the GBM. Blood glucose levels in diabetic rats were significantly increased when compared with non-diabetic controls. Blood glucose levels were not affected by perindopril treatment. Untreated diabetic rats showed increased glomerular size, thickening of the GBM and an increase in mesangial matrix as compared with controls. Treatment with perindopril prevented effectively glomerular hypertrophy and thickening of the GBM. Significant increase in type IV collagen and laminin was found in thickened GBM and mesangial matrix in kidneys of untreated diabetic rats. In perindopril-treated diabetic rats, staining of type IV collagen and laminin was less strong when compared with untreated diabetic rats. In conclusion, our data suggest that perindopril treatment is effective in preventing renal lesions possibly by ameliorating the diabetes-induced increase in expression of type IV collagen and laminin.     

Keratin profiles in normal/hyperplastic prostates and prostate carcinoma

Summary Immunoreactivities in 25 cases of prostatic adenocarcinoma and 10 normal/hyperplastic prostates were investigated in methacarn-fixed, paraffin-embedded serial sections using a panel of nine anti-keratin monoclonal antibodies (mAbs); 34 E12, CK8.12, 312C8-1, CK4.62, RPN1165, RPN1162, 35H11, CK5, M20, and one of anti-actin mAb, HHF35. In normal/ hyperplastic prostates, RPN1162, 35H11, CK5 and M20 stained luminal cells without staining basal cells, and 34E12, CK8.12 and 312C8-1 stained basal cells but not luminal cells. Other mAbs, CK4.62 and RPN1165, stained basal cells as well as luminal cells. All of the mAbs labelling luminal cells stained cancer cells with variable frequencies in a manner unrelated to the grade of tumour differentiation. Of the prostate cancer cases 92% were scored positive with M20, 84% with 35H11, 80% with CK5, 68% with CK4.62, 60% with RPN1165 and 4% with RPN1162. However, basal cell-specific keratins labelled with 34E12, CK8.12 and 312C8-1 were totally negative in the cancer cells. HHF35 showed no labelling in normal, hyperplastic or neoplastic epithelial cells of the prostate. Our findings indicate that the major part of the cells of prostatic adenocarcinomas have keratin phenotypes similar to luminal cells but not basal cells, and that no myoepithelial differentiation can be detected in epithelial cell of the prostate. Thus, mAbs for keratins facilitate the identification of epithelial cell phenotypes in normal, benign and malignant conditions of the prostate.     

Expression of KAI1 in paraffin-embedded normal, hyperplastic and neoplastic prostate and prostate carcinoma cell lines

Expression of KAI1, a tumor metastasis suppressor gene, was studied with different fixatives in frozen and paraffin-embedded sections of human and rat prostate carcinoma cell lines and human prostate lesions by Immunohisto-chemistry. Immunoreactivity of the membrane antigen in cell lines was associated with known expression levels in these lines and the fixative used. Formalin and paraformaldehyde helped maintain the immunoreactivity of cells. In human prostate, frozen sections revealed diffuse reactivity of the antigen in normal and neoplastic tissues while paraffin-embedded tissues usually showed focal reactivity, although more than 50% of cases with normal epithelium and adenocarcinomas were reactive. In some cases, pretreatment with trypsln enhanced immunoreactivity. Benign prostatic hyperplasia (BPH) showed the most intense diffuse immunoreactivity, which suggested enhanced expression. Prostatic intraepithelial neoplasia (PIN) also often expressed high levels of KAI1. Three of five metastases were reactive but two primaries and their metastases were not. Lymphocytes in primary carcinomas and lymphocytes and germinal center cells in lymph nodes were immunoreactive, while adjacent primary or metastatic prostate adenocarcinoma epithelium was not immunoreactive. Although paraffin-embedded human tissues were not optimal for determining levels of expression of KAI1, they did show immunoreactivity that could have prognostic value and showed the specific cytoplasmlc localization of the protein in cells.     

Expression of type VII collagen,the major anchoring fibril component,in normal and neoplastic human nervous system

The distribution of type VII collagen was examined in the normal human nervous system, in brain tumour biopsies and in glioma cell lines by immunohistochemistry and western blotting. In normal tissue, positivity was observed beneath choroid plexus epithelial cells and around pineal gland and pituitary gland cell nests, while other brain regions and peripheral nerves were negative. Expression was preserved in most related tumours (choroid plexus papilloma, pineoblastoma, pituitary adenoma). Scattered abnormal vessels showed neoexpression of type VII collagen in about half of the astrocytic and ependymal tumours. Glioma cells in situ were consistently negative for type VII collagen, where-as the glioblastoma cell lines were positive. Our results suggest that anchoring fibrils or at least epitopes of their major structural component are present in normal and pathological cerebral structures, indicating a unique distribution of type VII collagen in the nervous system.     

KIT expression in fetal, normal adult, and neoplastic renal tissues

BACKGROUND: KIT is a transmembrane tyrosine kinase receptor, expressed in high amounts in various normal cells. In addition, c-kit mutation or activation is a major pathogenetic event in certain tumours (such as gastrointestinal stromal tumours). There are only limited data in the literature on the expression of KIT in normal and neoplastic renal tissues. AIMS: To investigate KIT expression in normal and neoplastic renal tissues. METHODS: KIT expression was evaluated by means of immunohistochemistry in paraffin wax embedded sections from 67 tissue samples. RESULTS: Eight of eight fetal kidneys, and 10 of 10 normal adult kidneys revealed cytoplasmic staining of renal tubules. The three cases of renal dysplasia studied expressed KIT in their normal and aberrant tubules. Two of 13 conventional renal cell carcinomas (RCCs), two of seven papillary type RCCs, four of seven chromophobe type RCCs, none of six nephroblastomas, seven of seven oncocytomas, two of two mesoblastic nephromas, and two of four angiomyolipomas were positive. CONCLUSION: KIT is expressed in normal fetal and adult renal tubules, and in a subset of renal tumours. The expression of KIT in these renal tumours may prove to have diagnostic relevance and/or therapeutic implications.     

Differential basement membrane composition in multiple epithelioid haemangioendotheliomas of liver and lung

We report a case of epithelioid haemangioendothelioma involving both lung and liver. The tumour cells were positive for factor-VIII-related antigen. Immunohistochemical analysis of various basement membrane components in tumour tissue of lung and liver showed striking differences. In the liver tumour there was selective expression of collagen IV, with minimal and focal amounts of laminin and basement membrane-associated heparan sulphate proteoglycan. In the lung tumour nodules, in contrast, all these basement membrane components were present. These patterns of basement membrane expression closely resemble those of normal liver and lung basement membrane respectively. We suggest that this provides evidence that epithelioid haemangioendothelioma arises from local endothelial cell proliferation and that it supports the assumption of a multicentric rather than metastatic origin when multiple tumour deposits are found.     

Widespread distribution of nuclear androgen receptors in the basal cell layer of the normal and hyperplastic human prostate

Summary The role of the basal cell layer in organogenesis, epithelial renewal and development of benign prostatic disorders is largely unknown. The objective of the present study was to investigate whether or not basal cells express the nuclear androgen receptor (AR). Computer-assisted image analyses of immunohistochemical double stainings were performed to localize AR and basal-cell-specific cytokeratins in identical sections. The results showed that the basal cells express nuclear AR widely in normal and hyperplastic conditions. When compared with the staining intensities detected in secretory luminal cells, the receptor was most frequently expressed at lower levels in basal cells, which may exhibit strong AR immunoreactivity focally. Basal cells with increased AR expression were most frequently detected in hyperplastic lesions including post-atrophic and atypical hyperplasia. The presence of nuclear receptors for both androgens and oestrogens or progestins in basal cells may indicate that these cells are targets of the hormonal imbalance which has frequently been implicated in the aetiology of benign prostatic hyperplasia.This study is dedicated to Professor Dhom on the occasion of his 70th birthday     

DNA-flow-cytometric measurements on the normal,atrophic, hyperplastic and neoplastic human endometrium

Summary DNA distribution patterns and the fractions of the cell cycle phases were determined by means of flow-through cytometry in 87 samples of normal, atrophic, hyperplastic and carcinomatous human endometrium. The S-phase fractions vary during the normal menstrual cycle between 1 and 3% and reach a periovulatory maximum between 4.4 and 4.7%. Atrophic endometrium and regressive-glandular cystic hyperplasia have little DNA synthesis (1.01% and 1.68% S-phase fractions respectively). Proliferating glandular cystic hyperplasia reveals 3.38% S-phase fraction, whereas adenomatous hyperplasia has an increased number of DNA-synthesizing cells (4.81%). The well-differentiated endometrial carcinoma shows no cytophotometrically detectable differences in comparison to adenomatous hyperplasia. All endometrial samples except for poorly differentiated endometrial carcinoma showed a diploid to tetraploid DNA distribution pattern. The poorly differentiated endometrial carcinoma displays two different types: one rapidly growing diploid-tetraploid tumor with 8.0 to 9.6% S-phase fractions, and another type with stemline deviations, polyploid nuclei and less pronounced synthetic activity.Dedicated to Professor Volker Becker at the occasion of his 60th anniversary