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PubMed1991

A comparative study of quantitative stains for DNA in image cytometry.

In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide.

Analysis of Variance↗
PubMed1991

Imaging Ca2+ changes in individual oligodendrocytes attacked by T-cell perforin.

Cytosolic-free calcium changes in cultured rat oligodendrocytes attacked with murine T-cell perforin were measured using digital image processing of fura-2-loaded cells. Permeability to the membrane impermeant dye, propidium iodide, and the ability to retain fura-2 were also assessed. Oligodendrocyte response to perforin attack was heterogeneous, ranging from small, transient increases in cytosolic calcium to rapid cell death. Changes in cytosolic calcium occurred in all cells and about half of the cells also became permeable to propidium iodide. Only a minority of these cells proceeded to lysis, as evidenced by loss of fura-2. The cytosolic-free calcium rises visualized here in single cells could initiate non-lethal effects, thereby disturbing oligodendrocyte function and synthesis and maintenance of myelin.

Animals↗
PubMedDate not supplied

Indicators of Pneumocystis carinii viability in short-term cell culture.

Growth of P. carinii in culture has been difficult to document in the absence of reliable methods for distinguishing live from dead organisms. We studied three markers of cell function in P. carinii during the course of short-term cell culture, and correlated these with the number of P. carinii present in culture supernatants. The markers were glucan synthase activity, esterase activity and cell membrane integrity. The last two were assessed by double staining with fluorescein diacetate and propidium iodide followed by analysis of fluorescence using flow cytometry. The rise in P. carinii number after 5 to 7 days in culture was associated with increased glucan synthase activity. Flow cytometry analysis of day-6 P. carinii cultures confirmed that over 80% of the organisms catalyzed the conversion of fluorescein diacetate to fluorescein and excluded propidium iodide. The demonstration of three indices of metabolic activity in an expanding P. carinii population has confirmed the efficacy of a culture system as a means of sustaining the continued activity, albeit short-lived, of viable P. carinii.

Animals↗
PubMed1991

Recovery of cultured rat neonatal myocytes from hypercontracture after chemical hypoxia.

Cardiac myocytes exposed to anoxia hypercontract into a blebbed, rounded mass. Such hypercontraction is usually considered a manifestation of irreversible injury. Here, we studied functional recovery, long-term viability and ATP levels of cultured neonatal rat cardiac myocytes after metabolic inhibition with cyanide and 2-deoxyglucose, a model of 'chemical hypoxia' which mimics the ATP depletion and reductive stress of hypoxia. After addition of inhibitors, 5-day cultured myocytes ceased spontaneous contractions within 1-2 min, blebbed and hypercontracted after 35 min, and lost viability after 100 min as assessed by nuclear labelling with propidium iodide. 11-day cultured myocytes exhibited a similar progression of injury. When the metabolic inhibitors were removed, spontaneous contractions resumed after an average of 11 min in non-hypercontracted myocytes. In hypercontracted myocytes, weak contractions resumed after 35 min, regardless of the length of time spent in the hypercontracted state, but restoration of strong synchronous contractions took hours. Recovering cells remained viable and exhibited spontaneous contractions through 24 hours of observation, whereas contractility never returned in propidium iodide-labelled cells. ATP levels decreased rapidly after chemical hypoxia and were partially restored upon washout of inhibitors. ATP recovery was similar in non-hypercontracted and hypercontracted cells. Thus, contractile dysfunction during recovery in hypercontracted cells was not due to lack of regeneration of ATP. In conclusion, hypercontracture is not a manifestation of irreversible injury. Contractile dysfunction following recovery from hypercontracture observed here may represent 'in vitro stunning'.

Adenosine Triphosphate↗
PubMedDate not supplied

Improved preparation technique of cervical carcinoma for flow cytometric DNA analysis with tissue disintegration in hydrochloric acid.

An one-step procedure using a nuclear isolation medium containing propidium iodide has been found to be a suitable preparation technique for flow cytometric DNA analysis in breast cancer samples. In the case of cervical squamous carcinoma, a pretreatment with HCl seems to be a methodological improvement. One advantage with the HCl modification is that some "false" near-diploid cell populations are abolished. These "false" G0/G1 peaks may represent diploid nuclei with a different stainability for propidium iodide compared to normal diploid nuclei. The HCl treatment has, furthermore, the advantage of increasing the elution of nuclei (mean factor of 4.0), especially non-diploid nuclei from higher differentiated squamous carcinomas.

Cell Nucleus↗
PubMed1989

Improved detection of aneuploidy in malignant melanoma using multiparameter flow cytometry for S100 protein and DNA content.

DNA aneuploidy has been demonstrated to be an independent parameter of prognostic significance in malignant melanomas. In order to improve the detection of DNA aneuploidy in malignant melanomas, and to minimize diploid non-tumor cells in the sample, we developed a two-color staining strategy for S100 protein and DNA content in paraffin embedded samples. The ability to detect aneuploidy, defined as DNA ploidy index less than or equal to 0.90 or greater than or equal to 1.10, in 37 stage I malignant melanoma samples by flow cytometry analysis was significantly improved from 10.8% of cases using traditional one-color analysis for DNA content only (propidium iodide) to 32.4% of cases using two-color analysis for simultaneous measurement of both DNA (propidium iodide) and S100 protein (fluorescein conjugated antibody) (Chi-square with Yates' correction; p less than 0.05). The largest increase in sensitivity was found in level I and II melanomas less than or equal to 0.76 mm in thickness. In addition, we report the new observation that multiple S100 protein-positive subpopulations were found significantly more frequently in malignant melanomas (23/37 cases) than in compound melanocytic nevi (5/22 cases) (Chi-square with Yates' correction; p less than 0.01). These findings suggest that there is a previously unsuspected degree of tumor heterogeneity even in thin, presumably early, malignant melanomas.

Aneuploidy↗
PubMedDate not supplied

Rhodamine 123 and flow cytometry to monitor the cytotoxic actions of nucleoside analogues in nondividing human lymphocytes.

We used human lymphocytes from the peripheral blood of normal individuals to compare the cytolytic actions of 2-chloro-2'-deoxyadenosine (2-Cl-dAdo) and 9-beta-D-arabinofuranosylguanine (ara-G). Membrane integrity was ascertained by flow cytometric quantification of the cells' uptake of the fluorescent mitochondria-specific probe rhodamine 123. Addition of 10 microM ara-G to lymphocyte cultures led to a progressive decline in the number of cells that could assimilate rhodamine 123, and after 2 days exposure to drug less than 50% of the cells showed normal staining compared to untreated cells. 2-Cl-dAdo had similar cytotoxic effects at a 0.1 microM concentration. Under the same conditions trypan blue revealed only a 10-20% toxic effect by these drugs. Using rhodamine 123 in combination with the vital stain propidium iodide, we found that 90% of the cells with abnormal rhodamine 123 uptake also stained with propidium iodide. The changes in membrane permeability to fluorescent dyes correlated with the loss of lymphocyte ability to respond to the plant mitogen phytohemagglutinin. Our data indicate that rhodamine 123 is a more sensitive probe than trypan blue for ascertaining early loss of the membrane integrity of dying lymphocytes. Combined with flow cytometry, rhodamine 123 uptake represents a simple and rapid method for identifying the important biochemical events associated with the cytocidal and cytostatic effect of drugs against normal and leukemic cells.

Arabinonucleosides↗
PubMed1986

Leukoregulin-increased plasma membrane permeability and associated ionic fluxes.

The role of ion fluxes in the increased plasma membrane permeability of tumor cells exposed to the anticarcinogenic and tumor cell proliferation inhibitory lymphokine, leukoregulin, was examined by flow cytometic analysis of cell surface perturbations indicative of membrane destabilization. The pI 5.3 form of leukoregulin was isolated by ion exchange, isoelectric focusing, and molecular sizing chromatography of lymphokines from phytohemagglutinin-stimulated normal human lymphocytes. Membrane permeability increased within 5 min of leukoregulin exposure of tumor cells and was quantified by following the efflux of fluorescein or influx of propidium iodide. Membrane permeability increased in proportion to leukoregulin concentration. By 2 h, 0.25-30 units/ml induces a 10-90% change in K562 erythroleukemia cell permeability. Similar changes are effected by Ca2+ ionophores A23187 and X-537A but not by the Na+ ionophore monensin or the K+ ionophore valinomycin. The Ca2+ ionophores and the intracellular Ca2+ mobilizers ouabain and amphotericin B enhance, whereas calmodulin inhibits leukoregulin action. Ca2+ channel blockers nifedipine and verapamil and the Na+ and K+ ion transport inhibitors amiloride and atractyloside, respectively, neither alter membrane permeability nor influence leukoregulin activity. Further evidence for the role of increased Ca2+ flux in leukoregulin's action is provided by detection of increased intracellular free Ca2+ in leukoregulin-treated cells with the Ca2+-sensitive fluorescent probe 2-[2-bis(carboxymethyl)amino-5-methylphenoxy]methyl-6-methoxy-8-bis (carboxymethyl)aminoquinoline. Kinetic analysis of cell volume, forward and right angle light scatter, fluorescein efflux, and propidium iodide influx, moreover, reveals that the action of leukoregulin is unique. Membrane perturbations may be critical initial steps in the ability of leukoregulin to directly prevent the development of carcinogenesis as well as inhibit the continued proliferation of neoplastic cells.

Calcimycin↗
PubMed1977

Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei.

A one-step procedure for the preparation of nuclei for flow microfluorometric DNA analysis is described. The membranes of the cells were lysed by the non-ionic detergent Nonidet P40. Single-cell suspensions, and specimens of solid tissues obtained with fine-needle biopsy, could be prepared equally well as the nuclei of solid tissue cells were released separately. Lysis was performed in the staining solution containing either ethidium bromide or propidium iodide. Fluorescence due to fluorochrome binding to RNA, was abolished instantaneously by the presence of RNA-se, and fluorochrome binding to secondary binding sites in DNA was inhibited with NaCl. The preparation time was 10 min and the samples were stable for a minimum of 12 h. With the basic version of the method, usable, but not always optimal, results were obtained in all the cell types tested: four different mouse ascites tumors, leucocytes, bone-marrow, liver cells, human lymphomas, human carcinomas of the breast and lung, mouse mammary carcinoma and solid JB-1 tumor. The method was further optimized for the JB-1 ascites tumour. The resulting two modified techniques are described. Differences in the staining of leucocytes with the analogues ethidium bromide and propidium iodide were demonstrated.

Animals↗
PubMed1995

Apoptotic photoreceptor degeneration in experimental retinal detachment.

PURPOSE: To investigate the possibility that cell death in retinal detachment may occur by reactivation of apoptotic programmed cell death mechanisms. METHODS: Unilateral retinal detachments were created in adult cats using 0.25% sodium hyaluronate; detached and control retinas were studied at different intervals. Internucleosomal DNA fragmentation (one of the landmarks of apoptosis) was investigated in tissue sections with the TUNEL technique, which uses terminal transferase to label with biotinylated nucleotides the 3' ends of DNA fragments. Sections also were labeled with propidium iodide, which intensely stains pyknotic nuclei. In addition, one time point was selected for analysis with electron microscopy. RESULTS: TUNEL-positive (T+) and propidium iodide-positive (PI+) cells almost never were observed in retinas from control eyes, but they were abundant at defined time points after retinal detachment, appearing almost exclusively in the photoreceptor layer. Their frequency was particularly high 1 to 3 days after detachment but declined rapidly over the next several weeks. T+ cells were still present 28 days after retinal detachment. Electron microscopy also revealed evidence of apoptotic cells after retinal detachment. CONCLUSIONS: Results are consistent with the hypothesis that photoreceptor degeneration after retinal detachment occurs through apoptosis, usually associated with intrinsic, programmed cell death mechanisms. The detection of a rapid wave of photoreceptor degeneration seems to suggest that early therapeutic interventions might be recommended; agents capable of interfering with the apoptotic mechanism could have a role in the prevention of cell losses that represent a critical complication of retinal detachment.

Animals↗
PubMed1994

Induction of apoptosis by quercetin: involvement of heat shock protein.

Quercetin, a widely distributed bioflavonoid, inhibits the growth of tumor cells. The present study was designed to investigate the possible involvement of apoptosis and heat shock protein in the antitumor activity of quercetin. Treatment with quercetin of K562, Molt-4, Raji, and MCAS tumor cell lines resulted in morphological changes, including propidium iodide-stained condensed nuclei (intact or fragmented), condensation of nuclear chromatin, and nuclear fragmentation. Agarose gel electrophoresis of quercetin-treated tumor cells demonstrated a typical ladder-like pattern of DNA fragments. In addition, the hypodiploid DNA peak of propidium iodide-stained nuclei was revealed by flow cytometry. Quercetin induced apoptosis in cells at G1 and S in a dose- and time-dependent manner. The apoptosis-inducing activity of quercetin was enhanced by cycloheximide and actinomycin D. A nuclease inhibitor, aurintricarboxylic acid, inhibited quercetin-induced apoptosis, whereas deprivation of intracellular calcium by EGTA had no effect. 12-O-Tetradecanoylphorbol-13-acetate and H-7 did not affect the induction of apoptosis by quercetin. The synthesis of HSP70 was inhibited by quercetin when determined by immunocytochemistry, Western blot analysis, and Northern blot analysis. Quercetin-treated tumor cells were not induced to show aggregation of HSP70 in the nuclei and nucleolus in response to heat shock, resulting in apoptosis. By contrast, when tumor cells were first exposed to heat shock, no apoptosis was induced by quercetin. In addition, pretreatment of tumor cells with HSP70 antisense oligomer that specifically inhibited the synthesis of HSP70 enhanced the subsequent induction of apoptosis by quercetin. These results suggest that quercetin displays antitumor activity by triggering apoptosis and that HSP70 may affect quercetin-induced apoptosis.

Apoptosis↗
PubMed1994

Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis.

Mature resting mouse spleen B cells progress stochastically into apoptosis at a uniform rate over the first 16 h in vitro in 3 stages. In stage 1, early apoptotic B cells decreased the normal phospholipid packing of their plasma membranes, detected as increased binding of the lipophilic dye merocyanine 540, and also decreased in volume, detected as decreased forward scatter. In stage 2 there was abrupt internucleosomal cleavage of DNA, quantitated as hypodiploid nuclei by flow cytometry. Some stage 2 cells entered stage 3, where the plasma membrane became permeable to propidium iodide. B cells in later stages of this sequence retained the characteristics of earlier stages, whereas nonapoptotic B cells remained in their original state. Cycloheximide increased the progression of B cells through these three stages, whereas dextran sulfate inhibited stage 1 more effectively than stages 2 or 3. Increased orthogonal scatter also occurred late in some of the cells that had passed through stage 1, but did not correlate well with propidium iodide permeability. Fresh small dense spleen B cells contained 5% to 7% stage 1 cells but only about 1% stage 2 cells. Macrophages have been reported to destroy preferentially apoptotic thymocytes by recognizing plasma membrane alterations deriving from loose packing of phospholipid head groups. The recognition of stage 1 rather than stage 2 B cells by macrophages may help to keep the proportion of apoptotic cells low in vivo.

Animals↗
PubMed1993

Nitric oxide mediates Kupffer cell-induced reduction of mitochondrial energization in hepatoma cells: a comparison with oxidative burst.

The metabolic changes in rat hepatoma cell line, AH70 cells, after coculturing with Kupffer cells were visualized using a silicon-intensified target camera and subsequent processing with a computer-assisted digital imaging processor. In cocultured tumor cells, nonactivated Kupffer cells reduced mitochondrial energization as indicated by the decrease in the fluorescence intensity of rhodamine 123 (Rh123) and induced lipid peroxidation as shown by the dichlorofluorescein (DCF) activation. The reduction in Rh123 could be eliminated by addition of an analogue of L-arginine (NG-monomethyl-L-arginine), suggesting the involvement of nitric oxide (NO.) in the decrease in mitochondrial energization. Superoxide dismutase did not inhibit the reduction in Rh123 but significantly inhibited DCF activation. These findings indicate that the latter reaction was mediated by superoxide anion. Two h after the cells were cocultured, propidium iodide-positive, severely injured tumor cells significantly increased in number. This increase was significantly attenuated by addition of NG-monomethyl-L-arginine but not by superoxide dismutase, suggesting that NO. may be greatly involved in Kupffer cell-mediated injury of AH70 cells. In another set of experiments, the culture medium of Kupffer cells caused no significant alteration of Rh123, DCF, and propidium iodide-associated fluorescences in AH70 cells. In addition, ultrastructural observation revealed that the membrane-to-membrane attachment between Kupffer cells and tumor cells occurred within 30 min after coculturing. These results suggest that Kupffer cell-derived NO. release, triggered by the close contact with tumor cells, may induce damage to tumor cells via inhibition of mitochondrial energization.

Animals↗
PubMed1993

Ley specific antibody with potent anti-tumor activity is internalized and degraded in lysosomes.

BR96 is a monoclonal antibody (MAb) that recognizes many human carcinomas and can kill antigen-positive tumor cells in vitro. Using both gold and radiolabeled MAb, the distribution and cellular processing of BR96 during cytolysis has been determined. After a brief (< 3 minutes) MAb treatment, cells in suspension are stained by the nuclear viability dye propidium iodide. Whole MAb and F(ab')2 fragments are equally cytotoxic; monovalent F(ab) fragments, however, have no effect on dye uptake unless cross-linked with goat anti-mouse IgG. The level of toxicity is dependent on both MAb dose and on cell surface receptor density. Cell contact may regulate receptor expression. BR96 receptors are more abundant on cells migrating into the open areas of a scratch wounded confluent culture than on the adjacent contact-inhibited cells. BR96 can also inhibit the anchorage-independent growth of tumor cells in soft agar showing that its effects on propidium iodide staining are not due to transient changes in membrane permeability. Immunogold electron microscopy reveals that, after a 1-minute treatment, BR96 induces significant infolding of the plasma membrane and that internalized MAb is localized to these structures. Immediately thereafter, large cell surface and intracellular vesicles form, mitochondria are swollen, and membrane integrity is lost. Therefore, BR96 seems to cause morphological changes characteristic of necrosis rather than apoptosis. When bound to adherent carcinoma cells, BR96 is distributed uniformly on the apical surface of cells labeled at 4 C and is enriched at points of cell substratum contact. Upon warming of the cells to 37 C, BR96 localizes in small perinuclear clusters and the cell margin is now devoid of label. Immunogold electron microscopy reveals that BR96 undergoes receptor mediated internalization and is localized within the same coated pits, endosomes, and lysosomes as the transferrin receptor. Quantitative studies using iodinated BR96 show that after 6 hours of chase, a maximum of 53% of the radiolabel is located within the intracellular pool. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that 84% of this fraction is nondegraded. BR96 probably cycles between the medium and intracellular pools because the remainder of the radiolabel is in the medium as intact MAb. By 24 hours of chase, the intracellular fraction drops to 30%, while the remaining 70% is present in the culture medium, mostly as low molecular weight degradation products.

Antibodies, Monoclonal↗
PubMed1995

6-Coumarin diazonium salt: a specific affinity label of the Torpedo acetylcholinesterase peripheral site.

A 6-coumarin diazonium salt was synthesized and tested on Torpedo acetylcholinesterase as a site-directed irreversible probe for quaternary ammonium binding. The rate of the inactivation was examined as a function of time, inhibitor concentration, and pH, which allowed the determination of the dissociation and the rate constants of this efficient affinity labeling process. Protection experiments using tetramethylammonium, edrophonium, and propidium demonstrated that the labeling reaction occurred exclusively at the peripheral quaternary ammonium binding site of the enzyme. This result was confirmed by the modification of propidium binding at the peripheral site after inactivation reaction, as directly determined by fluorescence. Mutations of the likely labeled amino acid residues, Tyr70 and Tyr121, by histidine and phenylalanine indicated a predominant involvement of Tyr70 over Tyr121 in the coupling reaction.

Acetylcholinesterase↗
PubMed1996

Multiple fluorescence and reflectance simultaneous detection by confocal microscopy of HaeIII digested DNA sequences.

Human metaphase chromosomes were isolated and digested in situ with HaeIII restriction enzyme to detect cytosine and guanine-rich sequences (CpG islands), which are known to be associated with most of the mammalian genes. Digested DNA was reconstructed by in situ nick translation employing digoxigenin-labeled nucleotides. The DNA sequences were revealed by antibodies conjugated either with fluorescein isothiocyanate or 1-nm colloidal gold. DNA was counterstained with propidium iodide. A sensitive, high resolution method for visualizing three signals, simultaneously excited by a single argon laser line of 488 nm has been developed. The green fluorescence of fluorescein isothiocyanate was detected in combination with the red fluorescence of propidium iodide, and the third signal was imaged by employing the reflectance mode of the confocal microscope after silver enhancement of the gold beads. The high reflectance intensity, the accurate localization and the non-fading properties of colloidal gold made the reaction a valuable tool for the detection of antigens and, as a consequence, of specific DNA sequences in chromosome preparations. Overlaying of three signals allowed the simultaneous observation of distinct structures: total DNA, as well as fluorescein- and gold-labeled sequences after in situ nick translation, or total DNA and centromeric sequences of two different chromosome pairs (17 and X) after in situ hybridization. The use of HaeIII restriction enzyme that cut CpG islands combined with in situ nick translation identified the chromosome sites where active, inactive or housekeeping genes can be located. In chromosomes, the fluorescent reaction pattern showed large areas of labeling, while a more defined staining, often organized in spot pairs that resembled an R-like banding, was detected when the reflected mode was used. These results are confirmed by the observation that R-like bands actually are multiple symmetrical spots localized on sister chromatids. In addition, some chromosomes, and in particular 1 and 9, displayed a C-negative banding due to the negativity of the centromeric areas. Reflectance confocal scanning microscopy and in situ nick translation represent a powerful tool to study the in situ genome organization.

Chromosomes, Human↗
PubMed1997

Nucleic acid specificity of an acridine derivative permits its use for flow cytometric analysis of the cell cycle.

3-amino-6-methoxy-9-(2-hydroxyethylamine) acridine (AMHA) is an acridine derivative, which is easily excited in near ultraviolet and which emits a bright green fluorescence. The dye was preferentially incorporated into nucleic structures as attested by microscopic and cytometric analyses after RNase and DNase treatments. The affinity for RNA seemed low and similar to that observed for propidium iodide. AMHA was quickly accumulated in fixed cells, and in appropriate concentrations (10-50 microM) was a DNA- and RNA-specific dye. AMHA probably exhibits an adenine-thymine specificity, as suggested by its quenching after bromodeoxyuridine uptake: the fluorescence quenching was similar to that obtained for Hoechst 33258. After cell treatment by RNase and in the presence of MgCl2, AMHA staining allowed flow cytometric analysis of the cell-cycle distribution. The resulting histograms were similar to those obtained with propidium iodide (CV near 3.5%, and similar cell cycle distribution). Thus, AMHA is a suitable fluorescent dye for efficient analysis of the cell cycle by flow cytometry.

Aminoacridines↗
PubMed1997

[Clinical aspects, differential diagnosis and histogenesis of heterotopic ossification].

Investigations regarding proliferation behaviour, histogenesis and bone transformation were carried out on soft tissue samples with 133 heterotopic ossifications (HO), collected from the surgical material of the Institute of Pathology, Berufsgenossenschaftliche Kliniken Bergmannsheil, using methods of conventional histology, histochemistry, immunohistochemistry, electron microscopy and molecular biology, yielding the following results: 1. There is only a limited pathognomonic growth pattern for the variant of non-traumatic heterotopic ossification. Basically, the ossification foci may be divided into central, intermediate and peripheral zones. Expression intensities of the proliferation markers PCNA and MIB-1 show the proliferation behaviour in peripheral areas of osteogenesis to be similar to that found in autonomous osseous neoplasias. The phenotypical picture of zonal architecture found in HO can be seen by the simultaneous demonstration of variable image sequences of multifocally arising ectopic formations of new bone with varying degrees of maturation. 2. Vascular endothelium and pericytes are integrated into the formal histogenesis of heterotopic ossification and belong to osteoprogenitor cells as "stem cells" of heterotopic ossification. 3. The histochemically established expression pattern of alkaline phosphatasis is in good correlation with the degree of activity of the step-by-step development of heterotopic ossification. The intracellular demonstration of alkaline phosphatasis is an indicator for the transformation towards an osteogenic direction. In HO, the enzyme is a major characteristic of osteoprogenitor cells. 4. According to our immunohistochemical results, the proteoglycanes Decorin and PG 100-osteogenic matrix proteins-show a phase-like expression and are involved in osteoneogenesis. They are predominantly found in the area of mineralization. The proteoglycane 100 experiences a "modulation" and can be demonstrated almost selectively in osteoclasts in advanced stages of osteodevelopment. 5. Using in situ hybridization-with digoxigenin labeled cDNA probes- and a propidium iodide counterstaining a co-expression of collagene types I, II and III-mRNA could be demonstrated in samples of ossification. The expression pattern is similar to the collagen expression I. in early phases of embryonal bone development II. with callus proliferation and shows III. also similarities to chondral neoplasias. The results underline the reactive-neoplastic character of heterotopic ossification. 6. TGF-beta 1 mRNA shows a polytopic expression pattern with accumulation in areas of osteogenesis. TGF-beta 1 was found mostly in cartilage cells of heterotopic ossifications, as were collagene types I (alpha 1) and III (alpha 1) mRNAs. In sum, our in situ hybridization results underline the central part of cartilage cells in the ossification process in ectopic osteoneogenesis. This is also indicated by a phenotypical alteration of collagen expression as well as by an accumulation of TGF-beta 1 in chondral ossification areas. In situ hybridization propidium iodide counterstaining offers a reproducable image of morphological structures in the histological slide sample by means of fluorescence microscopy. 7. Phenotyping of osteroclasts in HO revealed their derivation from mature local macrophages. The immunohistochemical characterization with the demonstration of the vitronectin receptor classified the multinucleate giant cells of HO as original osteoclasts. Their origin is in accordance with the histogenetical concept of original bone. 8. Summarizing, the results characterize heterotopic ossification as a reactive proliferative and reversible neoplasia in chronically damaged soft tissue. From the formal pathogenetical point of view, relations could be established to orthotopic osteoneogenesis as well as to impaired proliferation kinetics, similar to osseous autonomous neoplastic lesions. (ABSTRACT TRUNCATED)

Biomarkers↗