Earl P. Benditt

A method for staining epoxy sections for light microscopy

A technique for staining sections of osmium-fixed, epoxy-embedded tissues for light microscopy is presented. The method employs aqueous toluidine blue at pH 11.1 and does not require prior removal of embedding medium. When stained with this technique and viewed with an oil immersion objective, the images are striking because of their great definition and resemblance to the images are striking because of their great definition and resemblance to of areas seen in the electron microscope; it also permits better utilization of the full resolving power of the light microscope.

A significant part of macrophage-derived growth factor consists of at least two forms of PDGF

The macrophage has been suggested to be responsible for the connective tissue cell proliferation that accompanies most chronic inflammatory responses. One of the secretory products of activated macrophages is MDGF, a growth factor (or factors) for fibroblasts, 3T3 cells, smooth muscle, and vascular endothelium. This report demonstrates that a significant portion of the mitogenic activity for 3T3 cells secreted by cultured human alveolar and peritoneal macrophages is due to a molecule (or molecules) similar to platelet-derived growth factor (PDGF). Two size classes (approximately 37,000-39,000 and 12,000-17,000 daltons) of mitogenically active PDGF-like molecules are detected by two criteria--antigenic similarity with PDGF and ability to compete with 125I-PDGF for high-affinity binding to the PDGF receptor. The presence of mRNA for the B chain of PDGF is demonstrated by Northern analysis, and de novo synthesis of these molecules by activated macrophages is shown by immunoprecipitation of 35S-labeled proteins with anti-PDGF IgG.

Structural-functional correlations in renal disease: Part II: The Correlations*

Abstract The relationship between functional abnormalities and structural changes in disease has been the focal point of pathological investigation since the eighteenth century. However, the simultaneous study of these two complementary facets in human disease required proper methods, now available in the excellent and discriminating renal function tests and the percutaneous renal biopsy. Although the functional tests have provided numerical data for analysis, the standard disease descriptions and classifications have not provided an assay in terms suitable for quantitative comparisons. In Part I of this paper, we have presented a method of recording observations defined in relation to the anatomic parts of the kidney and some elementary pathologic processes affecting these parts. The observations included first order estimates of severity and extent of the changes. In developing this schema the aim was to avoid, as far as possible, any a priori judgment of the kind of renal disease in conventional terms or its etiology. Given this anatomic assay, one could then proceed to compare the morbid structure with the morbid function. This communication presents the data on 70 patients in whom the biopsy material was evaluated as described in regard to changes in glomerular, tubular, vascular and interstitial compartments. This information was compared with the simultaneously measured glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and concentrating and acidifying capacities. The results indicated some clear, if unanticipated, relationships. The most interesting and perhaps unexpected finding was that regardless of the basic disease in the kidney, impaired renal function was most closely related to changes in the tubules and in the interstitium.

Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain.

Recent evidence has shown the presence of Chlamydia pneumoniae antigens and nucleic acid in coronary artery atheromas from autopsy patients in South Africa. In this study, the immunocytochemical technique was used to demonstrate C pneumoniae antigens in atheromas of the aorta in autopsy patients from retrospective aortic atherosclerosis studies at the University of Washington. The patients were 34 to 58 years old. Immunoperoxidase staining using Chlamydia-specific monoclonal antibodies showed one of four fatty streaks and six of 17 fibrous plaques were positive for C pneumoniae antigens; four control aortic tissues were negative. Two of the positive plaques were from the same patient. Double-label immunocytochemical staining using Chlamydia- and tissue type-specific monoclonal antibodies demonstrated the antigens in the cytoplasm of macrophages and smooth muscle cells in the atheromatous lesion. This study suggested a wider involvement of C pneumoniae organisms in atherosclerotic lesions of the arterial system than has previously been documented.

Proliferation in primary and restenotic coronary atherectomy tissue. Implications for antiproliferative therapy.

On the basis of animal models of arterial injury, smooth muscle cell proliferation has been posited as a dominant event in restenosis. Unfortunately, little is known about this proliferation in the human restenotic lesion. The purpose of this study was to determine the extent and time course of proliferation in primary and restenotic coronary atherectomy-derived tissue. Primary (n = 118) and restenotic (n = 100) coronary atherectomy specimens were obtained from 211 nonconsecutive patients. Immunocytochemistry for the proliferating cell nuclear antigen (PCNA) was used to gauge proliferation in the atherectomy specimens. The identity of PCNA-positive cells was then determined using immunohistochemical cell-specific markers. Eighty-two percent of primary specimens and 74% of restenotic specimens had no evidence of PCNA labeling. The majority of the remaining specimens had only a modest number of PCNA-positive cells per slide (typically < 50 cells per slide). In the restenotic specimens, PCNA labeling was detected over a wide time interval after the initial procedure (eg, 1 to 390 days), with no obvious proliferative peak. Cell-specific immunohistochemical markers identified primary and restenotic PCNA-positive cells as smooth muscle cells, macrophages, and endothelial cells. In conclusion, the findings were as follows: (1) Proliferation in primary and restenotic coronary atherectomy specimens, as indicated by PCNA labeling, occurs infrequently and at low levels. (2) The response to injury in existing animal models of angioplasty may follow a very different course of events from the clinical reality in human atherosclerotic coronary arteries and may help explain why current approaches to restenosis therapy have been ineffective.

Catecholamines and cardiomyopathy: The pathogenesis and potential importance of myofibrillar degeneration

Abstract Myofibrillar degeneration is a common form of cardiac injury observed frequently in human beings at autopsy and is produced readily in experimental animals. Administration of exogenous catecholamines such as norepinephrine or isoproterenol has been frequently used as an experimental means of inducing cardiac injury. The potential role of catecholamines in the pathogenesis of this cardiomyopathy becomes more significant with the demonstration that local can induce a similar cardiac lesion. Early, the injured myocardial cells show clumping and disorganization of the cardiac myofibrils. Later, degenerative changes in cell cytoplasm and mineralization of mitochondria are manifest. Such injured cells may either die and be removed by phagocytosis or may be repaired by synthesis of new myofibrils. In the later stages stromal condensation and fibrosis may become evident. This lesion in its acute form in humans and when sufficiently extensive can contribute to or cause death. Since repcated episodes potentially may produce myocadial fibrosis, this lesion is worthy of more serious consideration in the evaluation of human heart disease manifest by impaired cardiac function and interstitial fibrosis.

5-Hydroxytryptamine in Mast Cells.

Summary 5-Hydroxytryptamine as well as histamine and heparin was demonstrated to be present in purified mast cells from the peritoneal washings of rats. The material was identified by bioassay and chromatography. The concentration of 5-hydroxytryptamine found in isolated mast cells agreed with that found by indirect means in the mast cells of the subcutaneous areolar tissue of rats. Implications of this finding are briefly discussed.

The major proteins of human and monkey amyloid substance: Common properties including unusual N-terminal amino acid sequences

Amyloid substance is a complex proteinaceous material found in the tissues of patients with the disease amyloidosis. Recently we have presented evidence that there are two chemically distinct kinds of amyloid substance, one associated with the classical inflammation-related amyloidosis and another, frequently designated atypical or paramyloid, occurring with tumors such as multiple myeloma or without evident pre-existing disease [l] . The classical, or inflammation-associated, substance is distinguished by: a) instability to alkali of its characteristic Congo red binding capacity; b) its amino acid composition and c) the presence of a major protein constituent, amyloid protein A. The family of proteins comprising amyloid protein A has a molecular weight range of 6000-8000 and a characteristic amino acid composition [2] ; in addition, human amyloid protein A has the capacity to bind Congo red and exhibits the characteristic hyperchromism and spectral changes previously described for amyloid substance [ 1] . In this communication we compare the chromatographic and electrdphoretic properties, the amino acid composition and the 24 amino acid N-terminal sequence of the humanand monkeyderived protein A of amyloid substance.

Aortic endothelial cell replication. I. Effects of age and hypertension in the rat.

The daily rate of cell replication in the aortic endothelium of the normal rat falls from a maximum of 13% at birth to 0.1-0.3% at age 5-6 months. This residual rate of replication largely represents the turnover rate of the endothelium in normal animals. After renal hypertension of 2-3 weeks duration, however, the rate of replication rises 10-fold to an average value of 1.6%. This increase may represent an increase in turnover; however, it probably represent, at least in part, a proliferative response to cover the expanded luminal surface of the dilated vessel.

THE HISTOCHEMISTRY OF CONNECTIVE TISSUE: II. THE EFFECT OF PROTEINS ON THE SELECTIVE STAINING OF MUCOPOLYSACCHARIDES BY BASIC DYES

The basic dyes, particularly those which exhibit metachromasia, have been used extensively in histochemical studies of the connective tissue mucopolysaccharides. The specificity of the reaction depends upon the combination of dye with the acid groups of the polysaccharides. It is frequently assumed that this reaction occurs quantitatively. Thus in addition to the use of basic dyes to demonstrate the presence of mucopolysaccharides in tissues, the amount of dye bound under standard conditions has been considered to be a measure of the quantity of mucopolysaccharide present (1, 2). Furthermore the dye binding at different hydrogen ion concentrations has been interpreted as an indication of the dissociation characteristics of the acid groups of the stainable material and used to determine the so-called “pH-signature” of particular substances (3). It is the purpose of this paper to show that protein may modify the reaction between dye and polysaccharide by competing with the dye for the stainable groups. This may lead to a masking of the polysaccharide and, since the competition shows a pH dependence, to an alteration of the “pH signature.”