Claire E. Lewis

ISLET AMYLOID FORMED FROM DIABETES-ASSOCIATED PEPTIDE MAY BE PATHOGENIC IN TYPE-2 DIABETES

Pancreatic islet amyloid deposits were found in 22 of 24 type-2 diabetic subjects (aged 48-68 years) and were not present in 10 age-matched controls. A novel peptide, 37 aminoacids long, termed diabetes-associated peptide (DAP), has been identified in amyloid-containing pancreatic extracts from 3 type-2 diabetic patients but not in extracts from 6 non-diabetic subjects. DAP has major homology with calcitonin-gene related peptide (CGRP) and the islet amyloid of all 22 diabetics showed CGRP immunoreactivity. The immunoreactivity was inhibited by preabsorption of three different CGRP antisera either with CGRP carboxyterminal peptide 28-37 or with extracted DAP. Both diabetic and non-diabetic subjects had CGRP/DAP immunoreactivity in islet B-cells. Electron microscopy of islets containing amyloid indicated fibrillar amyloid between the endocrine cells and capillaries, usually penetrating into deep invaginations of the plasma membrane of the B-cells. These results suggest that islet amyloid contains DAP, which may originate from B-cells. Accumulation of amyloid in islets is likely to impair islet function and may be a causal factor in the development of type-2 diabetes.

Current methods for assaying angiogenesis in vitro and in vivo

Angiogenesis, the development of new blood vessels from an existing vasculature, is essential in normal developmental processes and in numerous pathologies, including diabetic retinopathy, psoriasis and tumour growth and metastases. One of the problems faced by angiogenesis researchers has been the difficulty of finding suitable methods for assessing the effects of regulators of the angiogenic response. The ideal assay would be reliable, technically straightforward, easily quantifiable and, most importantly, physiologically relevant. Here, we review the advantages and limitations of the principal assays in use, including those for the proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as sponge implantation, corneal, chamber, zebrafish, chick chorioallantoic membrane (CAM) and tumour angiogenesis models.

Cytokine regulation of angiogenesis in breast cancer: the role of tumor-associated macrophages.

Studies over the past 20 years have established that the development of new capillaries from an existing vascular network (a process called angiogenesis) is an essential component of tumor growth. Malignant tumors do not grow beyond 2–3 mm3 in size unless they stimulate the formation of new blood vessels and thus provide a route for the increased inflow of nutrients and oxygen and outflow of waste products. Tumor angiogenesis also provides an essential exit route for metastasizing tumor cells from the tumor to the bloodstream. Indeed, extensive neovascularization is a poor prognostic factor in several forms of human cancer. Angiogenesis is a complex, multistep process driven by many local signals within the tumor. This involves the degradation of the extracellular matrix around a local venule after the release of collagenases and proteases, the proliferation and migration of capillary endothelial cells, and their differentiation into functioning capillaries. Cytokines produced by various cell types present within the microenvironment of solid tumors form a complex, dynamic network in which they have multiple effects on tumor progression. Herein we review our work on the presence, and possible regulatory influence on tumor angiogenesis, of a number of these cytokines within invasive breast carcinomas. We have combined immunocyto‐chemistry with a single cell cytokine release assay called the reverse hemolytic plaque assay to investigate the cellular sources of the key angiogenic cytokines, vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor‐α. Tumor‐associated macrophages in the stromal compartment of these tumors and/or malignant epithelial cells were seen to be a major producer cell for these cytokines, whereas tumor necrosis factor‐α receptors were expressed by leukocytes, malignant cells, and endothelial cells in tumor blood vessels. J. Leukoc. Biol 57: 747–751; 1995.

Secretion of epidermal growth factor by macrophages associated with breast carcinoma

By means of a cytokine release assay we have shown that in cell populations derived from primary breast carcinoma, epidermal growth factor (EGF) is secreted by cells with the characteristic morphological and immunophenotypic profile of activated macrophages (positive for CD68, CD16, CD25). EGF secretion was observed in 11 (31%) of 35 primary tumours. Secretion of EGF by normal or malignant epithelial cells was not observed. We found no association between EGF secretion by the primary tumour and recognised clinical indices of prognosis.

Calcitonin gene-related peptide and somatostatin inhibit insulin release from individual rat B cells

Hormone secretion from single, rat pancreatic B cells was visualised by a reverse haemolytic plaque assay for C-peptide. Quantitative analysis of the size and number of haemolytic plaques indicated that exposure to 3, 5, 10 and 20 mM glucose resulted in a dose-dependent increase in both the magnitude of C-peptide, and thus, insulin release by individual B cells and the recruitment of activity secreting B cells. Somatostatin and calcitonin gene-related peptide, fragment 28-37 (CGRP28-37) were shown to inhibit glucose-stimulated insulin release as assessed by the size of individual plaques and the number of recruited B cells, and hence to reduce the total area of plaques formed. In the presence of 15 mM glucose, a dose-dependent effect of CGRP28-37 on the secretion of insulin was observed, with the size of plaques formed by individual B cells reduced at concentrations of CGRP28-37 between 10(-5) and 10(-11) M. Thus, both somatostatin and CGRP28-37 can act directly on individual B cells to inhibit their secretory response to increasing levels of glucose. We suggest that these peptides which can be immunolocalised in islet cells may have a role in the regulation of insulin secretion.

DIABETES-ASSOCIATED PEPTIDE

SIR,-Severe hypoglycaemia is a feared complication of diabetes mellitus. Signs of hypoglycaemia are essential to recognise a fall in blood glucose. Dr Heller and colleagues (Aug 15, p 359) assessed hypoglycaemic warning symptoms and plasma adrenaline levels during insulin-induced hypoglycaemia in normal subjects and insulin-dependent diabetic patients. They found no increase in finger tremor and sweating in patients with hypoglycaemia

Cytokine Release by Single, Immunophenotyped Human Cells: Use of the Reverse Hemolytic Plaque Assay

Over the past few years, numerous studies have focused oti the role of cytokines in physiological and pathological processes. This has meant that our understanding of cytokine biology has progressed at a rapid pace. A complex, multifaceted picture has emerged of the events regulating the intracellular production., release and biological activity of these soluble mediators. Cytokines have been shown to be proteins which are rapidly induced and only transiently stored in producer cells before release. In addition, some cytokines have been shown to bind to surface sites on producer as well as target cells, and/or to be secreted in association with carrier proteins, competitive inhibitors, or soluble receptors, all of which could potentially interfere with or block the bioactivity of the cytokine molecule itself (for reviews, see Hamblin 1988, Arai et al. 1990). These ftndings have fuelled speculation concerning the interpretation of data obtained using conventional methods to analyze cytokine production, and prompted careful attention to the selection of appropriate and meaningful techniques for their accurate measurement.

Detecting cytokine production at the single-cell level

Cytokines are versatile mediators of intercellular communication. Their functional diversity has aroused considerable interest and prompted the rapid development of a number of techniques for their detection and measurement. However, conventional cytokine assays measure only their bulk release by large numbers of cells and give no indication of the identity or frequency of producer cells. Here, the advantages and disadvantages of a relatively new approach to detect cytokine production by single cells are reviewed.

Measurement of cytokine release by human cells A quantitative analysis at the single cell level using the reverse haemolytic plaque assay

The reverse haemolytic plaque assay has been adapted to detect and measure the release of such cytokines as interleukin-1, -2 and -6, GM colony-stimulating factor or interferon-gamma by individual human cells derived from either peripheral blood or enzymatically dispersed breast carcinomas. Since each of these peptides is released by more than one cell type, this in vitro assay has been coupled with immunocytochemistry to identify the particular cell type(s) contributing to the release of each cytokine. This technique is useful in (i) obviating the need for purification of a given cell type prior to estimating cytokine release, and (ii) evaluating quantitative differences in secretion amongst cells of a particular type. Such a method has the additional advantage over most alternative methods applied at the single cell level in that the cells remain viable at the end of the assay and can be used in further studies. This assay thus provides a powerful new tool in the investigation of the role of cytokines in both the normal modulation of the immune system and the development of such diseases as neoplasia.

Modelling Macrophage Infiltration into Avascular Tumours

In this paper a mathematical model that describes macrophage infiltration into avascular tumours is presented. The qualitative accuracy of the model is assessed by comparing numerical results with independent experimental data that describe the infiltration of macrophages into two types of spheroids: chemoattractant-producing (hepa-1) and chemoattractant-deficient (or C4) spheroids. A combination of analytical and numerical techniques are used to show how the infiltration pattern depends on the motility mechanisms involved (i.e. random motion and chemotaxis) and to explain the observed differences in macrophage infiltration into the hepa-1 and C4 spheroids. Model predictions are generated to show how the spheroids size and spatial structure and the ability of its constituent cells influence macrophage infiltration. For example, chemoattractant-producing spheroids are shown to recruit larger numbers of macrophages than chemoattractant-deficient spheroids of the same size and spatial structure. The biological implications of these results are also discussed briefly.