Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinase-1 in the Retinal Pigment Epithelium and Interphotoreceptor Matrix: Vectorial Secretion and Regulation

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) play an essential role in both normal and pathological extracellular matrix degradation, and a TIMP has been associated with at least one type of retinal degeneration. We have studied expression of MMP-2 and TIMP-1 by zymography, immunocytochemistry, and immunoblotting in the retinal pigment epithelium (RPE) from normal, aged and diseased retinas. MMPs and TIMPs were found in the rat RPE, interphotoreceptor matrix (IPM), and in media conditioned by human and rat RPE in culture. In other polarized cells, MMPs and TIMP-2 are secreted vectorially towards the basal lamina. In the RPE, however, MMP-2 and TIMP-1 were secreted preferentially from the apical surface, the surface bordering the IPM. These findings provide new evidence that MMPs and TIMPs could play a role in the turnover of IPM components.Cell homogenates and conditioned media from RPE isolated from mutant Royal College of Surgeons (RCS) rats with inherited retinal dystrophy had similar amounts of MMP-2 and TIMP-1 as those from congenic control rats. The secretion of MMP-2 and TIMP-1 from RPE cell cultures isolated from young and aged human donors varied widely. However, with increasing cell passage number, secretion of MMPs and TIMPs from human RPE increased dramatically. Also, growing human RPE on bovine corneal endothelial cell-generated extracellular matrix instead of plastic reduced the secretion of both MMPs and TIMPs. These data suggest that the integrity of Bruch's membrane may serve to regulate RPE functions in MMP and TIMP secretion and that extracellular matrices contain signals that regulate MMP and TIMP synthesis and/or secretion by the RPE.     

神经肽Y对免疫细胞活性的影响

神经肽Y（NPY）是广泛分布于中枢神经系统和外周神经各部位的神经肽类物质。本实验观察NPY在体外对几种免疫细胞活性的直接作用。结果表明，NPY对小鼠T淋巴细胞丝裂原反应性和NK细胞的杀伤活性均无明显影响（P＞0．05），对巨噬细胞分泌溶菌酶有明显抑制作用（P＜0．05）；而对B淋巴细胞丝裂原反应性则有明显的促进作用（P＜0．05）。上述结果提示，NPY对部分免疫细胞功能的影响因细胞种类而异。     

Immunohistochemical differentiation of metastases of renal carcinomas versus other carcinomas with anti-γGT monoclonal antibody 138H11

Aims : Adenocarcinomas account for about 60% of metastatic cancers of unknown primary (CUP) site. In such a clinical CUP situation, histopathologists are challenged to differentiate renal cell carcinomas (RCC) from other adenocarcinomas with similar immunophenotypes, especially chemotherapeutically treatable mammary and ovarian carcinomas. Methods and results : Recently, we produced a monoclonal antibody (mAb), designated 138H11, against human gamma-glutamyltransferase (γGT), which stained over 98% primary clear cell and chromophilic RCC on frozen sections. The 138H11 epitope could not be stained using conventional techniques in most paraffin-embedded sections of the same origin, due to destruction by formalin fixation below the detection level. Here, we demonstrate that mAb 138H11 can specifically stain γGT in paraffin-embedded primary and metastatic RCC after enhancement with an ultrasensitive immunohistochemical method. We analysed a selected subgroup of adenocarcinomas with immunophenotypes which would not allow a differentiation from RCC in a CUP situation. We found a predominantly membranous expression of the 138H11 target antigen in 26/51 primary RCC and 15/34 metastatic RCC. In contrast, all 43/43 primary ovarian and bronchial carcinomas as well as 54/54 metastases of ovarian, mammary, bronchial and gastric carcinomas were negative for mAb 138H11. Conclusions : The data suggest that mAb 138H11 is useful for the immunohistochemical differentiation of RCC from other metastatic adenocarcinomas if the primary site of the tumour is not known.     

Reizhusten, Belastungsdyspnoe und zystische Lungenveränderungen bei einem 28jährigen Patienten

Zusammenfassung Eine neu aufgetretene Dyspnoesymptomatik und eine symmetrische, apikal betonte retikulonodul?re Zeichnungsvermehrung im R?ntgen-Thorax bei jungen rauchenden Erwachsenen müssen an das seltene Krankheitsbild der pulmonalen Histiocytosis X denken lassen. Klinischer Befund, laborchemische- und Lungenfunktionsuntersuchungen zeigen unspezifische Befunde. Neue radiologische Verfahren wie die hochaufl?sende Computertomographie (HRCT) leisten bei gezielter Indikationsstellung eine entscheidende differentialdiagnostische Hilfestellung. Dieser Fall verdeutlicht die M?glichkeit einer Diagnosesicherung durch transbronchiale Biopsien unter Verzicht auf offene Lungenbiopsien. Eine ambulante Diagnostik war m?glich, das h?here Risiko eines operativen Eingriffes konnte vermieden werden. Die Indikation zur Therapie ist nicht gesichert und wird daher durch den Grad der subjektiven bzw. funktionellen Einschr?nkung sowie den Verlauf bestimmt.     

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant

Macrophages, dendritic cells or B lymphocytes have been shownto play a major role in the presentation of soluble antigensto CD4⁺ T cells. In contrast, the capacity of these cells topresent particulate antigens such as bacterial or parasiticantigens to T cells remains controversial. To investigate thisquestion, well defined particulate antigens were prepared bycovalent linkage of proteins or peptides to 1 µm in diametersynthetic microspheres. The T cell immunogenicity of such particulateantigens was analyzed in vitro and in vivo. In vitro, a solubleprotein such as hen egg lysozyme (HEL) coupled to beads stimulateda strong proliferative T cell response of lymph node cells fromHEL-primed mice or of specific T cell hybridomas. HEL coupledto beads was presented to the specific T cell hybridomas bysplenocytes or by peritoneal macrophages, but not by lymphomaB cells. Immunization of mice with several different proteinantigens or with a synthetic peptide covalently linked to beadsinduced strong CD4⁺ T cell responses in the absence of adjuvant.The strong in vivo immunogenicity of proteins coupled to beadsdid not result from a non-specific adjuvant effect of beadssince covalent linkage of the antigen to beads was strictlyrequired to induce T cell responses in the absence of adjuvant.In vivo treatment by carrageenan showed that macrophages arerequired for the in vivo stimulation of T cell responses bythese particulate antigens. Thus, these results demonstratedthe role of phagocytic cells, especially macrophages, for invivo presentation of particulate antigens. These particulateantigens represent an interesting approach for the developmentof new vaccines, and for the in vivo analysis of the role ofvarious antigen presenting cells in T cell activation and differentiation.     

Distribution of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells

The cellular and regional distribution of glutathione (GSH) and GSH-related enzyme systems involved in cellular defense against reactive oxygen species and electrophilic xenobiotics in the nervous system has been extensively studied. However, little is known about the subcellular distribution of GSH systems in brain tissue and cultured neural cells. The present study investigates the distribution of mitochondrial and cytosolic GSH and GSH-related enzymes in cultured cerebellar astrocytes and granule cells, and compares them with levels in the adult rat cerebellum. Cytosolic GSH levels and cytosolic activities of glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in astrocytes were 57, 153, 245, and 92% higher than those found in granule cells, respectively. In contrast, granule cells contained significantly higher mitochondrial GSH levels than astrocytes. Granule cells also demonstrated comparable mitochondria/cytosolic concentrations of GSH and GR, GPX and GST activities to those observed in the cerebellar tissue, whereas ratios in astrocytes were markedly lower. Although in vitro treatments with 100 μM ethacrynic acid depleted both cytosolic and mitochondrial GSH in cultured astrocytes and granule cells in a time-dependent fashion, cellular GSH in granule cells was more resistant to the GSH-depleting agent than astrocytes. These results suggest that although GSH and GSH-related enzymes are abundant in cytosolic compartments of astrocytes, mitochondrial pools are relatively small. Since brain mitochondria are sites of significant hydrogen peroxide generation, the mitochondrial localization of GSH and its associated enzymes in neural cells provide important defenses against toxic oxygen species in the nervous system. Differences in subcellular distribution of GSH systems in individual neural cell types may provide a basis for selective cellular and/or subcellular expression of neurotoxicity.     

Expression of c-erbB-2 protein in keratinocytes of oral mucosal lichen planus and subsequent squamous cell carcinoma

Oral mucosal lichen planus (OMLP) is a well recognized mucosal disease with unknown etiology. Considerable controversy exists as to whether OMLP is intrinsically premalignant, or if the disorder facilitates the development of oral mucosal squamous cell carcinoma (OMSCC) by external factors. The aim of the present study was to investigate the expression of c-erbB-2 protein in the keratinocytes of initial biopsies of oral mucosal disorders diagnosed as OMLP with no evidence of epithelial dysplasia. and to compare the results with the expression of c-erbB-2 protein in subsequent biopsies obtained from the same patients. These results were compared with the findings from control groups (patients with dysplasia with no evidence of OMLP, patients with OMSCC with no evidence of OMLP and normal oral mucosa). The expression of the c-erbB-2 protein was evaluated by immunohistochemical staining of the gene product with the avidin-biotin-complex method using paraffin-embedded tissue sections. Five of the initial biopsies from patients with OMLP expressed the c-erhB-2 protein and one did not. None of the OMLP cases that subsequently showed evidence of dysplasia expressed the c-erhB-2 protein, and of the three OMSCC specimens from the patients with OMLP. two were negative and one expressed c-erbB-2 protein. The specimens from the control groups all expressed the c-erhB-2 protein. The results indicated the probability of the absence of c-erbB-2 staining being an indication of a potential for neoplastic transformation in OMLP with dysplastic changes.     

Granular cell tumours of the lower respiratory tract

Granular cell tumours rarely involve the lower respiratory tract. We report eight cases surgically resected at our institution. There were four females and four males, aged between 18 to 56 years (mean 40). One tumour associated with a peripheral lung adenocarcinoma was asymptomatic. The other lesions presented with obstructive pneumonitis (3 cases), haemoptysis (2), dyspnea (1) or cough (1). These tumours were tracheal (1) or bronchial (6) and one case was located in the lung parenchyma. Four cases were multicentric with associated lesions located in a bronchus (2), the oesophagus (1) or a mediastinal lymph node (1). All tumours, with the largest diameter ranging from 0.5–4.5 cm, were histologically invasive. The tumours were positive for S-100 protein, neuron specific enolase, KP1 (CD68) and vimentin. No tumour expressed desmin, keratin or p53 oncoprotein. Our study demonstrates that, in spite of marked anatomical and clinical polymorphism, the rare granular cell tumours of the lower respiratory tract have a constant histological appearance. Our observations confirm that large tumours (> 8–10 mm) usually extend beyond the tracheo-bronchial cartilages and, therefore, only surgical treatment may avoid recurrence.     

Research review: DNA polymerases as molecular markers of the regenerating capacity of hepatocytes

Hepatocyte regeneration has been widely investigated, with the mitotic index and the incorporation of [³H]thymidine being used as regeneration markers. We focused on the induction of DNA replication enzymes, particularly DNA polymerases (pol) α, δ, and ε. Using rat models, we have shown that the activity of pol α in crude liver extract well represents the regenerating capacity of hepatocytes. Using pol α as an indicator, we analyzed liver regeneration in rat models under various conditions: obstructive jaundice, external or internal biliary drainage, and the obstruction of portal vein branches. It has been revealed that the ligation of the common bile duct alone induces a certain amount of hepatocyte proliferation. It was striking that external biliary drainage suppressed regeneration capacity in cholestatic rat liver after partial hepatectomy. The strong regeneration in nonligated lobes induced by portal branch ligation was similar to the liver regeneration seen after partial hepatectomy with respect to the induction of DNA polymerases. Taken together, the aspects of DNA replication, particularly the induction of DNA polymerases, may contribute to shedding new light on the regeneration of human liver. This work was supported in part by a Grant-in-Aid for General Scientific Research and for Cancer Research from the Ministry of Education, Science and Culture, Japan, and by grants from the Uehara Memorial Foundation     

Effect of cortisone on cells at the bone-marrow interface

Summary A study of the association between the rate of proliferation of marrow fibroblast-like stromal cells (in vitro) and the rate of endosteal bone mineralization (EsMR) (in vivo) was undertaken in an osteopenic rat model. We report than 200 g male rats treated with cortisone acetate (5 mg/day for 7 days) exhibit decreases in marrow fibroblast colony-forming units (FCFU) and tetracycline-based measurements of EsMR at the level of the femoral midshaft. In cortisone-treated rats recovering for 1–3 weeks, the FCFU census and EsMR normalized during the first posttreatment week, remained at control levels after 2–3 weeks, and exhibited a relapse in the third week which signified only partial recovery. These changes were unrelated to patterns of body weight gain. The data indicate that the FCFU census can serve to index endosteal osteoblast vigor.