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C M van der Loos

Publications and source records attributed to C M van der Loos.

36 records · Page 2Linked to original sources
PubMed1994

Quantification in immunohistochemistry: the measurement of the ratios of collagen types I and II.

Quantitative techniques in immunohistochemistry are needed, but they are rarely applied because of doubtful reproducibility. We have developed a method for the detection of collagen types I and III in situ. The method applied was a two-step immuno-alkaline phosphatase technique with visualization of the end-product with Fast Red. The staining intensity was measured with a microdensitometer and the results expressed as ratios. The method yielded results that were unaffected by variations in tissue section thickness but which were proportionally related to time and antigen concentrations. Leiomyoma tissue, with a ratio of collagen types I and III of approximately 1.0, was used to establish the appropriate dilutions of the antibodies, thus assuring identical optical densities. By having the leiomyoma tissue sections incubated together with the heart tissue specimens, leiomyoma tissue was also helpful in correcting deviations from the 1.0 ratio. Accurate measurements of collagen type I/III ratios in normal human heart specimens were obtained with the present quantitative immunohistochemical technique.

Adolescent↗
PubMed1994

Reappraisal of in situ immunophenotypic analysis of psoriasis skin: interaction of activated HLA-DR+ immunocompetent cells and endothelial cells is a major feature of psoriatic lesions.

Psoriasis is an inflammatory skin disease of unknown aetiology. Many observations indicate that T cells play an important role in the pathogenesis of the disease. Upregulation of MHC class-II molecules on immunocompetent cells, endothelial cells and keratinocytes on lesional psoriatic skin has been regarded as a hallmark of the disease. However, there is some controversy in the literature regarding the cell types expressing class-II molecules and there is limited information about the presence of immune cells other than T cells and antigen presenting cells in the cellular infiltrates of psoriatic skin. We therefore reinvestigated the subject using immunocytochemical single and multiple staining techniques. In agreement with earlier reports, our studies showed that the cellular infiltrates in lesional skin consist largely of HLA-DR+/IL-2R+ T cells, HLA-DR+/CD1a+ Langerhans cells, and HLA-DR+/CD68+ macrophages. We found increased HLA-DR expression mostly on immuno-competent cells and endothelial cells, but no prominent HLA-DR expression on keratinocytes in lesional psoriatic skin. Upregulation of HLA-DR on endothelial cells and in mononuclear infiltrates was also evident in the non-lesional skin of psoriatic patients as compared with normal controls. B cells and natural killer cells were also found in the cellular infiltrates in lesional psoriatic skin. In spite of the presence of a large amount of activated T cells in the epidermis, we found that HLA-DR expression on keratinocytes was not a major feature of psoriatic skin.

Adult↗
PubMed1994

The neonatal heart has a relatively high content of total collagen and type I collagen, a condition that may explain the less compliant state.

OBJECTIVES: This study evaluated the extent of the collagen network in neonatal heart muscle and whether the type I/type III collagen ratio is the same as in the adult heart. BACKGROUND: The functional integrity and the stress-strain relation of heart muscle depends largely on the extracellular collagen matrix. The question therefore arises whether the altered compliance of the neonatal heart could relate to the developmental state of collagen and, in particular, the distribution of types I and III collagen. Type I collagen mainly provides rigidity and type III collagen elasticity. METHODS: Specimens from the left lateral wall of the left ventricle of human hearts (immature to full term, n = 23; 3 weeks to 12 years, n = 17) were used to determine the total collagen amount, using the hydroxyproline assay. Similar left ventricular specimens of human hearts (fetal to mature, n = 20; 2 months to 1.5 years, n = 6) were fixed in formalin, paraffin embedded and stained with Sirius red F3BA for total collagen. The ratio of total collagen to total protein was quantified spectrophotometrically. Frozen sections of left ventricular myocardium (immature to mature, n = 17; 4 months to 12 years, n = 10) were stained with antibodies raised against types I and III collagen. Antibody titration was done on human leiomyoma tissue with a known type I/type III collagen ratio. The endomysial collagen types were quantified using a spectrophotometer and expressed as a ratio. Adult human myocardium (n = 10) was used as reference. RESULTS: The study showed that the total amount of collagen increases with age. However, the ratio of total collagen to total protein and the ratio of type I to type III collagen were very high in hearts of the very young. During development, a gradual decrease occurred, with the total collagen/total protein ratio reaching normal levels at approximately 5 months after birth and the type I/type III collagen ratio stabilizing at a much later age. CONCLUSIONS: These findings suggest that the relative high content of collagen, related to the myocytes, and the high ratio of type I to type III collagen provide the substrate for a rigid, less compliant heart in neonates.

Cadaver↗
PubMed1994

Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology.

BACKGROUND: The study was designed to verify the concept of plaques "at risk" and whether inflammation could play a role in plaque rupture and thrombosis. METHODS AND RESULTS: In 20 patients who had died of acute myocardial infarction, the thrombosed coronary artery was identified and the site of plaque rupture was traced in serial sections. The cellular characteristics of the fibrous cap at the immediate site of rupture were analyzed and compared with the adjacent cap tissue by use of monoclonal antibodies reactive with macrophages, T lymphocytes, and smooth muscle cells. A deep intimal rupture, extending into the lipid core, was encountered in 12 plaques, whereas 8 had superficial erosions only. Ten atherosclerotic plaques had a distinctly attenuated fibrous cap covering a large atheroma, 7 showed a thick fibrocellular cap overlying a lipid pool, and 3 showed a fibrocellular lesion without a clear lipid core. Macrophages, and to a lesser extent T lymphocytes, were the dominant cells at the immediate site of either rupture or superficial erosion in each instance. These sites, moreover, were always characterized by abundant expression of HLA-DR antigens on both inflammatory cells and adjacent smooth muscle cells, suggesting an active inflammatory reaction. In terms of overall cellular composition of the ruptured plaques, the dominant cell types were macrophages and T cells in 11, smooth muscle cells in 3, and mixtures of both in 6. CONCLUSIONS: The underlying atherosclerotic plaque morphology in complicated coronary artery lesions causing acute myocardial infarction is heterogeneous with respect to both plaque architecture and cellular composition. However, the immediate site of plaque rupture or erosion is always marked by an inflammatory process. This suggests that inflammation plays a role in destabilizing the fibrous cap tissue and, thus, in enhancing the risk of coronary thrombosis.

Coronary Artery Disease↗
PubMed1994

Double epi-illumination microscopy with separate visualization of two antigens: a combination of epi-polarization for immunogold-silver staining and epi-fluorescence for alkaline phosphatase staining.

We present a method for an epi-illumination immunohistochemical double staining approach. The method combines the use of an immuno-alkaline phosphatase technique and the immunogold-silver technique, visualized with epifluorescence and epi-polarization illumination, respectively. Out of six tested alkaline phosphatase activity-revealing methods, only the reaction product obtained with the Becton Dickinson CAS Red kit showed an intense red fluorescence with a rhodamine filter set and no signal with epi-polarization illumination. The silver precipitate did not exhibit any signal with the rhodamine filter set. This allows separate observation and photographic recording of two antigens in one tissue section, an objective that cannot be achieved with conventional immunoenzyme double staining methods. The double epi-illumination approach presented is compatible with different immunoenzyme double staining protocols.

Alkaline Phosphatase↗
PubMed1994

Fibrous and lipid-rich atherosclerotic plaques are part of interchangeable morphologies related to inflammation: a concept.

BACKGROUND: The morphology of advanced atherosclerotic plaques varies from solid fibrous lesions, considered essentially stable, to lipid-rich lesions with large atheromas prone to rupture. The latter situation is often associated with large amounts of foam cells. An in-situ inflammatory process influenced by activated T cells and macrophages can be demonstrated in atherosclerotic plaques; however, the relationship between the inflammation and clinically relevant morphological types has not yet been investigated. A study of plaque morphology, focusing on the relationship between inflammatory cells, smooth muscle cells, and the collagen matrix, on the one hand, and the 'classic' plaque morphologies (fibrous versus lipid-rich), on the other, may shed light on this concept. MATERIALS AND METHODS: Immunocytochemical techniques were used in combination with connective tissue stains to study the topographic distribution of smooth muscle cells, collagen, and inflammatory cells in different morphologic types of advanced atherosclerotic plaques in aortic and carotid arteries obtained at autopsy. RESULTS: Lesions with an inconspicuous lipid core were defined as fibrous (n = 7). They contained a dense collagen matrix and the dominant cell type was the smooth muscle cell; lymphocytes and macrophages were sparse. Lesions with a large lipid core were defined as lipid-rich (n = 13). They contained a thin fibrous cap with a loosely arranged matrix dominated by macrophages and T cells. Most lesions (n = 21), however, had a morphology that ranged between fibrous and lipid-rich. The cellular components consisted either of mixed smooth muscle and inflammatory cells or of local distinct zones of inflammatory cells within a fibrous cap otherwise dominated by smooth muscle cells and collagen. Thus, zones of severe inflammation were invariably associated with dissolution of the connective tissue matrix and abundant human leukocyte antigen-DR expression on inflammatory cells and the remaining smooth muscle cells. CONCLUSION: Our observations support the concept that inflammatory mechanisms modulate plaque morphology, by promoting either synthesis or lysis of the fibrous cap. Our hypothesis is that fibrous and atheromatous lesions are essentially interchangeable.

Aortic Diseases↗
PubMed1993

Practical suggestions for successful immunoenzyme double-staining experiments.

Many methodologies exist to perform an immunoenzyme double staining. Hence, the practical problem arises as to which of these methods is optimal for one's own experimental design. A process of selection is described which is derived from our own practical experience. First, a general strategy is outlined for the handling of tissue sections to be used for multiple staining methods. Secondly, the selection of an appropriate immunoenzyme double-staining concept is made using a flow chart. Thereafter we give criteria for the definitive selection of an immunoenzyme double-staining protocol based on the characteristics of the tissue or cell type under study. Particular attention is given to the selection of appropriate detection systems, applying enzymes or gold particles, and good contrasting colour combinations. The problems of visualizing co-localization using immunoenzyme double staining are dealt with, and suggestions are made to adapt the method, if necessary, in order to optimize it.

Animals↗
PubMed1989

Atherosclerotic lesions in humans. In situ immunophenotypic analysis suggesting an immune mediated response.

The immunophenotypical features of the cellular infiltrates in different types of human atherosclerotic lesions, including diffuse intimal thickening as a potential but controversial precursor lesion, have been examined using monoclonal antibodies. Special emphasis is put on monocytes/macrophages, lymphocytes, and their possible interactions. Immuno-double staining techniques have been employed to study these aspects. T lymphocytes and macrophages were detected in diffuse intimal thickening, fatty streaks, and atheromatous plaques. In some lesions a predominance of suppressor/cytotoxic lymphocytes was found, whereas in other lesions mixtures of T suppressor/cytotoxic cells and T helper/inducer cells were found in ratios varying from 1:1 to 4:1. A substantial number of T cells and macrophages was considered to be immunoactivated because of the expression of HLA-DR and, to a lesser extent, of I12 receptor molecules. The activation was particularly evident at sites of close cell-to-cell contact between monocytes/macrophages and lymphocytes. These observations suggest that a specific in situ immune mediated hypersensitivity reaction is associated with the development of atherosclerosis.

Adult↗
PubMed1988

Use of commercially available monoclonal antibodies for immunoenzyme double staining.

An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green- and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.

Animals↗
PubMed1988

Ultrastructural localization of steroid sulphatase in cultured human fibroblasts by immunocytochemistry: a comparative study with lysosomal enzymes and the mannose 6-phosphate receptor.

Immunocytochemistry was used to study the subcellular localization of steroid sulphatase in cultured human fibroblasts. Ultra-thin cryosections were incubated with antibodies raised against steroid sulphatase purified from human placenta and immune complexes were visualized with gold probes as electron dense markers. Steroid sulphatase was found in rough endoplasmic reticulum, Golgi cisternae and in the trans-Golgi reticulum, where it co-distributes with lysosomal enzymes and the mannose 6-phosphate receptor. The enzyme was not detected in lysosomes. Steroid sulphatase was also found at the plasma membrane and in the endocytic pathway (i.e. coated pits, endosomes and multivesicular endosomes). These may be the sites where sulphated oestrogen precursors are hydrolysed. Also here, it co-localizes with lysosomal enzymes and the mannose 6-phosphate receptor. It is concluded that microsomal steroid sulphatase and lysosomal enzymes share several cellular compartments.

Carrier Proteins↗
PubMed1987

The skin immune system (SIS): distribution and immunophenotype of lymphocyte subpopulations in normal human skin.

The complexity of immune response-associated cells present in normal human skin was recently redefined as the skin immune system (SIS). In the present study, the exact immunophenotypes of lymphocyte subpopulations with their localizations in normal human skin were determined quantitatively. B cells were not found to be present in normal human skin. Lymphocytes were always of T-cell type, and 90% of these T cells were clustered in 1-3 rows around postcapillary venules of the papillary vascular plexus or adjacent to cutaneous appendages. In such perivascular localizations, they were found to differ from their circulating counterparts in three ways. First, skin perivascular cells were found to be approximately evenly distributed over CD4+ inducer and CD8+ suppressor-cytotoxic T-cell subsets (mean CD4/CD8 ratio: papillary layer 0.96, reticular layer 0.99). Second, within the category of CD4+ inducer T cells, most were phenotyped as CD4+, 4B4+ helper inducer T lymphocytes, whereas CD4+, 2H4+ suppressor inducer T lymphocytes were found to be relatively rare (less than 5%). Third, the majority of skin perivascular T cells were activated as they expressed HLA-DR and interleukin 2 receptors. Intraepidermal, directly subepidermal, and other ("free") lymphocytes were mostly of the CD8+ suppressor-cytotoxic T-cell subset but accounted for less than 10% of the total number of lymphocytes. Intraepidermally localized T cells accounted for less than 2% of the total number of lymphocytes present in normal skin. Our results indicate that preferential perivascular localization of activated T lymphocytes is the characteristic of normal human skin. This might be a reflection of continuous antigen recognition upon endothelial cell presentation and/or continuous T cell-mediated endothelial cell activation thereby inducing enhanced antigen clearing by the skin's endothelium.

B-Lymphocytes↗
PubMed1987

An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique.

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).

Alkaline Phosphatase↗
PubMed1984

Human placental steroid sulphatase--purification and monospecific antibody production in rabbits.

Human steroid sulphatase was purified 43-fold from placental microsomes using a four step procedure: solubilization with Miranol H2M, Bio-Gel A 1.5 m chromatography, column chromatofocusing and Sephadex G-75 chromatography. The purified enzyme that appeared electrophoretically homogeneous was used to immunize rabbits. Protein blotting demonstrated that the resulting antiserum mainly reacted with a polypeptide of 63 000 dalton, which is about the size of placental steroid sulphatase. The antiserum was freed from minor impurities by absorbing it to Sepharose 4B with immobilized antigens prepared from a steroid sulphatase deficient placenta.

Animals↗
PubMed1983

The nature of placental steroid sulphatase deficiency in man.

Human placental steroid sulphatase was partially purified from microsome suspensions of control and steroid sulphatase deficient placentae. After polyacrylamidegel electrophoresis, staining for protein and enzymatic activity revealed that steroid sulphatase from control placenta migrates at Rf = 0.44. In steroid sulphatase deficient microsomes no protein band and only a very faint sulphatase activity band could be detected at Rf = 0.44. Immunoelectrophoresis employing antisera raised against both steroid sulphatase preparations, only gave a protein precipitation line with sulphatase activity when using control placenta microsomes and the antiserum against steroid sulphatase from control placenta. All other placental microsomes-antisera combinations appeared to be negative. Our results strongly suggest that the nature of X-linked steroid sulphatase deficiency is a decreased amount of steroid sulphatase protein.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Biochemical investigation to the reliability of the histochemical demonstration of microsomal arylsulphatase activity in cryostat sections.

Polyacrylamide gel-electrophoresis was performed with an extract from cultivated skin fibroblasts. Arylsulphatase activity is measured and visualised using the biochemical substrate dehydroepiandrosterone sulphate and the histochemical substrate 6-bromo-2-naphthyl sulphate respectively. The histochemical substrate was hydrolysed at Rf = 0.49 and 0.58 while the biochemical substrate was hydrolysed only at 0.49. We conclude that two different microsomal arylsulphatases exist: a sulphatase able to hydrolyse steroid sulphatases (Rf = 0.49) and one unable to hydrolyse steroid sulphatases (RF = 0.58). In consequence if is recommended to carry out an electrophoresis experiment after the histochemical investigation, in order to discriminate between these two types of sulphatase.

Arylsulfatases↗
PubMed1979

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections.

The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study show that the enzyme histochemical technique--except for some limitations--is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.

Animals↗