K. W. Schmid

Quantification of Plasminogen Activators and Their Inhibitors in the Aortic Vessel Wall in Relation to the Presence and Severity of Atherosclerotic Disease

Increased expression of plasminogen activator inhibitor-1 (PAI-1) has been demonstrated in the human atherosclerotic vessel wall and may contribute to the impaired plasma fibrinolytic capacity in patients at high risk of atherothrombotic events. In addition, the mural PA/plasmin system may have important pathobiologic functions during atherogenesis. We quantitatively analyzed PAs of the tissue type (TPA) and urokinase type (UPA), PAIs, and plasminogen in protein extracts from different layers of human aorta in relation to the presence and severity of atherosclerotic lesions. In comparison with normal control vessels, intimal and neointimal TPA concentrations were reduced in atherosclerotic aortas except in the necrotic core areas of advanced plaques, where TPA was mainly complexed to PAI-1 in extracellular matrix deposits. In the media, TPA antigen was higher in lesional segments and closely associated with smooth muscle cells. UPA antigen was increased in the intima of atherosclerotic lesions and colocalized with tissue-infiltrating macrophages and neointimal smooth muscle cells. By spectrophotometric assay, neither TPA nor UPA activity could be detected in intimal or medial extracts. PAI-1 concentrations increased significantly in the intima of atherosclerotic segments compared with adjacent uninvolved areas or control aortas. The immunohistochemical distribution of PAI-1 was similar to that observed for TPA. A large excess of PAI-1 over PA concentrations, particularly in the intimal layer, characterizes atherosclerotic lesions of the human aorta and suggests that PA action is locally confined and counterbalanced by enhanced PAI expression and accumulation.

Significance of metallothionein overexpression in human tumours

Metallothioneins (MTs) have been described in most vertebrate and invertebrate species as low molecular weight proteins with high cysteine content (, 30%), lack of aromatic residues and presence of 7–12 group IIb heavy metal atoms per molecule. Two major isoforms of MT (MT-I and MT-II), resolvable through ion-exchange chromatography, are distributed in most adult mammalian tissues. Recently, a further chargeseparable MT isoform (MT-0), and genes for two MT isoforms with restricted tissue distribution—MT-III (brain neurones) and MT-IV (stratified epithelia)— have been described. MT-I and MT-II isoforms are usually constitutively expressed at low basal levels, but are inducible by a variety of metals, hormones, inflammatory cytokines and xenobiotics. Induction of MTs appears to be important in detoxification of metals, zinc and copper homeostasis, and also protection against reactive oxygen species. Recently, MT-I and MT-II double null as well as MT overexpressing transgenic mice have been constructed to evaluate further their physiological and pathological significance. The interest in the role of MTs in tumours has emerged essentially through three distinct perspectives. The first relates to the possible association of MT deficient state with sensitivity to carcinogenic and anticarcinogenic effects of cadmium, spontaneous mutagenesis and anti-cancer drugs. The second pertains to the possible role of MT overexpression in resistance of tumours to anti-cancer therapy. More recently, immunocytochemically detectable MT overexpression has been described in a variety of human tumours, in relation to different stages of tumour development and progression. The mechanistic significance of the first two associations has been considered in a series of reviews relatively recently. The present article is therefore focused on evaluating the possible significance of MT overexpression in human tumours in association with clinicopathological indices of tumour progression.

Presence and possible significance of immunocytochemically demonstrable metallothionein over-expression in primary invasive ductal carcinoma of the breast

Metallothioneins (MTs) are ubiquitous low-molecular-weight proteins with a high affinity for heavy metal ions such as zinc, copper and cadmium. MT over-expression has been associated with resistance against anticancer drugs. In the present study we investigated 86 cases (45 cases of tumour category pT1 and 41 of category pT2) of routinely fixed and paraffin-embedded primary breast carcinomas immunohistochemically with a monoclonal antibody to an epitope of MT shared by its I and II isoforms. Immunohistochemically demonstrated MT over-expression was found in the invasive components of 7 of 32 pT1 and 17 of 28 pT2 invasive ductal carcinomas, whereas all 26 invasive lobular carcinomas gave weak or negative results. Fourteen of 17 pT2 and 2 of 7 pT1 invasive ductal carcinomas with MT over-expression developed metastases during follow-up with poor prognostic outcome. In contrast only 3 of 11 pT2 and none of the 25 pT1 cases without MT over-expression had a poor clinical course (P < 0.001). It is concluded that MT over-expression is associated with significantly poor prognosis particularly in pT2 invasive ductal breast carcinomas.

DIFFERENTIAL EXPRESSION OF ANNEXINS I, II AND IV IN HUMAN TISSUES : AN IMMUNOHISTOCHEMICAL STUDY

Abstract Annexins constitute a family of Ca2+- and phospholipid-binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. To elucidate a possible correlation of those functional proposals with the tissue distribution of annexins, we analysed immunohistochemically the expression of annexins I, II and IV in a broad variety of human tissues. Annexins I and II were chosen for this study since their functionally relevant N-terminal domains are structurally closely related, whilst annexin IV is structurally less related to the former two proteins. The study revealed distinct expression patterns of annexins I, II and IV throughout the body. Annexin I was found in leucocytes of peripheral blood, tissue macrophages and T-lymphocytes and in certain epithelial cells (respiratory and urinary system, superficial cells of non-keratinised squamous epithelium), annexin II in endothelial cells, myoepithelial cells and certain epithelial cells (mainly respiratory and urinary system), whereas annexin IV was almost exclusively found in epithelial cells. Epithelia of the upper respiratory system, Bowman’s capsule, urothelial cells, mesothelial cells, peripheral nerves, the choroid plexus, ependymal cells and pia mater and arachnoid of meninges generally strongly expressed all three annexins investigated. The characteristic expression in different tissues and the intracellular distribution indicates that the three annexins investigated are involved in aspects of differentiation and/or physiological functions specific to these tissues.

Murine leukocytes with ring-shaped nuclei include granulocytes, monocytes, and their precursors.

Leukocytes with ring‐shaped nuclei (ring cells) are present in bone marrow (BM; ~50% of BM cells), in peripheral blood (PB), and in inflammatory infiltrates of mice, but also in humans during myeloproliferative disorders. They are usually referred to as polymorphonuclear cells (PMN), but we demonstrate that they additionally encompass different types of mononuclear (MNC)‐like ring cells. PMN ring cells had constricted ring‐shaped nuclei with a wide cytoplasmic center and were sorted among the GR‐1high fraction. The MNC‐like ring cells belonged to the GR‐1low fraction. Their nuclei were not segmented and the cytoplasmic center of their nuclei was small. They were heterogeneous with one subgroup containing monocytes/macrophages according to ultrastructure, immunophenotype (BM8, F4/80, CD13, ER‐HR3), activity of unspecific esterase, and phagocytosis of Leishmania major. A second subgroup contained myeloic precursor cells as they proliferated (Ki67), expressed ER‐MP12, and showed on ultrastructure distribution patterns of peroxidase activity compatible with myelocytes, promyelocytes, or promonocytes. A third subgroup of cells had large, sometimes lobulated nuclei, was lineage markernegative/low (GR‐1, Mac‐1, B220 etc.), CD38−, but c‐kit+ and sca‐1+, and thus belonged to a close progeny of murine hematopoietic stem cells. In PB, ring cells encompassed mainly PMN, but also monocytes and cells with characteristics of both the granulocytic and monocytic lineage. Thus, ring cells comprise mature and precursor forms of myeloic cells. Their analysis revealed that in mice a clear distinction between the granulocytic and monocytic lineage beyond the GM‐CFU stage is not always feasible. J. Leukoc. Biol. 65: 217–231; 1999.

Complete reversion and prevention of rectal adenomas in colectomized patients with familial adenomatous polyposis by rectal low-dose sulindac maintenance treatment. Advantages of a low-dose nonsteroidal anti-inflammatory drug regimen in reversing adenomas exceeding 33 months.

PURPOSE: This nonrandomized, controlled Phase II pilot study aims at the lowest effective dose of rectally applied sulindac to achieve and maintain adenoma reversion in colectomized patients with familial adenomatous polyposis (FAP). METHODS: The study group (n = 15) underwent proctoscopic and laboratory follow-up for polyp reversion every 6 to 12 weeks. Polyp reversion was followed by dose reduction in predefined steps. Proliferating cell nuclear antigen/cyclin (PCNA) and KI-67 proliferation indices (PI) were performed by point counting. Prostaglandin (PG)E2 and PGF2α were quantified by time-resolved competitive fluorescence immunoassay. RESULTS: All patients responded to therapy within 6 to 24 weeks. Sixty and 87 percent of patients achieved complete adenoma reversion after 48 weeks at 53 and 67 mg of sulindac per day per patient on average, respectively. Reversion was evident compared with the control group. Dose reduction by one-sixth to one-eighth of the usual oral dose was significant (Manns trend test,P < 0.05). PCNA and KI-67 Pls of adenomatous and flat mucosa were significantly reduced (Wilcoxons test,P<0.05). Correlation of PCNA and KI-67 Pls indicate similar reaction of different tissue structures (Spearmans rank correlation test,P<0.01). Nonsteroidal anti-inflammatory drug-induced redifferentiation from high-grade to low-grade dysplasia occurred in all but two patients. Tissue-PGE2 levels were greatly reduced. Unwanted, curable side effects were rare (gastritis,n=2), and laboratory controls are within detection limits. CONCLUSIONS: Low-dose rectal sulindac maintenance therapy is highly effective in achieving complete adenoma reversion without relapse in 87 percent of patients after 33 months. Rectal FAP phenotype should be crucial for the surgical decision. Colectomy with ileorectal anastomosis and regular chemoprevention might proceed to be a promising alternative to pouch procedures. Chemoprevention with lower incidence of FAP-related tumorsviadysplasia reversion may be possible in the future.

Immunohistochemical Distribution and Serum Levels of the Ca2+ -Binding Proteins MRP8, MRP14 and Their Heterodimeric Form MRP8/14 in Crohn’s Disease

In previous histochemical studies the distribution of the two Ca(2+)-binding proteins MRP8 and MRP14 as well as their heterocomplex MRP8/14 has been demonstrated in different inflammatory diseases. Monoclonal antibodies against MRP8 and MRP14 and their heterodimer MRP8/14 (27E10 epitope) were used to investigate immunohistochemically the distribution of these proteins in routinely processed small and large bowel tissues from patients with Crohns disease (CD). Furthermore, we used a sandwich immunoassay to measure serum concentrations of MRPs in 62 patients were simultaneously assessed by the Crohns disease activity index (CDAI) and the severity activity index of Goebell (SAI). In our immunohistochemical study, MRP8, MRP14 and heterocomplex MRP8/14 were demonstrated in the majority of granulocytes and macrophages in active CD. Additionally, a strong complex MRP8/14 immunoreactivity was present in epithelial cells adjacent to ulcerative and fissuring lesions in the bowel. Serum MRP8/14 concentrations were significantly (p < 0.0001) increased in patients with active CD (CDAI > 150, SAI > 120). No correlations were found for level of MRP14 and MRP8 alone, respectively. The follow-up of individual patients with initially active CD showed a further increase in MRP8/14 levels during acute attacks of the inflammatory process. We suggest that our assay for MRP8/14 discriminates well between active and inactive CD and may have considerable potential in the analysis of clinical disease activity in CD patients. Our morphological results confirm the finding of increased MRP8/14 serum levels in patients with active CD.

Immunohistochemically demonstrated metallothionein expression in malignant melanoma.

Metallothioneins are ubiquitous proteins with a high affinity for heavy metal ions, e.g. zinc, copper and cadmium. Experimentally, metallothionein over‐expression in cell lines derived from a variety of cancers has been associated with resistance to anticancer drugs and irradiation therapy. Using a monoclonal antibody (E9) to metallothionein we investigated immunoreactive expression in routinely fixed and paraffin‐embedded tissue from 63 cases of malignant melanoma and 13 secondary deposits. Whereas a variety of cells in normal skin showed metallothionein expression, all forms of benign naevi studied were uniformly negative. In contrast 13/30 ‘thin’ (≤1.5 mm; 0.7 ± 0.4). 25 29 ‘thick’ malignant melanoma (> 1.5 mm; 5.5 ± 3.9) and 12/13 metastases were positive. Six patients with thin and 19 with thick melanoma with metallothionein expression died during a mean observation period of 6.4 ± 1.8 and 3.6 ± 2.5 years, respectively, their survival distribution function analyses giving statistically significant results for both the vertical tumour thickness (P < 0.0001) and metallothionein expression (P < 0.0001). These immunohistochemical results, based on routinely processed paraffin‐embedded tissue, suggest that metallothionein expression in malignant melanoma is significantly associated with progressive disease and might therefore be a useful prognostic indicator.

Different variants of frontotemporal dementia: a neuropathological and immunohistochemical study.

Abstract Histological and immunohistochemical findings in 20 cases of frontotemporal dementias – 8 cases of dementia of frontal lobe type (DFT), 7 cases of Pick’s disease (PD), and 5 cases of motor neuron disease with dementia (MND/D) – are presented. Common features of all three syndromes were: frontotemporal atrophy, involvement of subcortical nuclei, and swollen chromatolytic cells. Ubiquitin (Ub)-positive and tau-negative inclusions in cortical, hippocampal, and motor neurons were found in MND/D and DFT cases, suggesting a common pathogenesis of MND/D and DFT. MND/D showed the same cytoskeletal alterations in motor nuclei as MND without dementia: Bunina bodies and skein-like, Ub-positive inclusions. DFT differed from PD in the preponderance of histopathological changes in upper cortical layers, the sparseness of chromatolytic cells, and the absence of tau-positive Pick bodies (PBs). There were, however, two transitional cases showing Pick-type histology but no PBs, thus linking DFT and PD. PBs expressed chromogranin B and secretoneurin strongly, but chromogranin A only weakly. They were negative for the 70-kDa heat-shock protein, metallothionein, and glutathione-S-transferase.

Clinical and genomic influence of sulindac on rectal mucosa in familial adenomatous polyposis

PURPOSE: A study was performed to evaluate the antiproliferative effects of low doses of the nonsteroidal drug, sulindac, on adenomas and rectal mucosa in familial adenomatous polyposis and to analyze the influence on tumor-suppressor genes and on apoptosis. METHODS: This was a prospective, controlled, nonrandomized Phase II dose-finding study for sulindac. The study group (n=28) and control group (n=10) underwent colectomy and ileorectal anastomoses, with repeated proctoscopy with endoluminal ultrasound and biopsies every three months. Dose-reduction of sulindac according to adenoma reversion was predetermined. Proliferation marker, Ki-67 (MIB1 and 5), on frozen or paraffin sections evaluated the antiproliferative effects; mutant p21ras,pantropic p53, mutant p53, and anti -bcl-2 were performed as enzyme-linked immunosorbent assay procedures and/or immunohistochemistry on paraffin sections. RESULTS: All patients responded to sulindac after 24 weeks (at the latest). There was a significant reduction of adenomas and dose reduction to 67 mg/day after three years of therapy (Manns test for trend,P<0.001). Results consisted of 78 percent complete reversions, 22 percent partial reversions of adenomas at latest re-examination, and no influence on upper gastrointestinal tract adenomas. No influence was detected on repeated hemograms, liver, or renal function at high or low doses. There was a permanent antiproliferative effect (Ki-67) of low-dose sulindac, significant blocking ofrasmutation activation, and a significant difference of untreated and treated mucosa in mutant p53 content (Wilcoxons or Kruskal-Wallis each,P<0.05). Reverse correlation of anti-bcl-2 and p53 immunostaining on mucosa sections was an indication of adenoma relapse. CONCLUSIONS: Low-dose antiproliferative sulindac therapy is highly effective in adenoma reversion in familial adenomatous polyposis patients. Sulindac shows influence on tumor-suppressor genes and on apoptosis markers. An immunostaining correlation indicates adenoma relapse in flat microadenomas in advance of macroscopic appearance. Low-dose sulindac treatment may develop into an additive permanent therapy for colectomized familial adenomatous polyposis patients.