Human monocyte-derived macrophages and dendritic cells as targets for biomaterial cytocompatibility studies using an improved in vitro culture system.

Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF-alpha release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF-alpha by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials.

In vitro effects of zirconia and alumina particles on human blood monocyte-derived macrophages: X-ray microanalysis and flow cytometric studies.

The cytocompatibility of two particulate bioceramics, zirconia and alumina, was studied using human blood monocytes driven to differentiate into mature macrophages with granulocyte macrophage-colony-stimulating factor. Changes in individual cell elemental composition, particularly sodium and potassium content, were assessed by X-ray microanalysis of ultrathin freeze-dried sections. Phagocytosis and respiratory burst of macrophages exposed to biomaterial for 7 days were analyzed under flow cytometry using uptake of fluorescent latex beads and 2'7'-dichlorofluorescien diacetate oxidation, respectively. Zirconia and alumina particles were found to decrease the intracellular potassium/sodium ratio (an index of cell vitality) significantly (p<.01) in 7-day-cultured macrophages compared to control cells cultured out of material. Phagocytosis of both ceramic particles by macrophages was followed by a concomitant decrease in cell phagocytic ability (27%) and a marked altered oxidative metabolism (>2 times reduced by zirconia and >5 times reduced by alumina). The present study clearly demonstrates that reduction of the phagocytic capacity of macrophages associated with altered oxidative metabolism caused by biomaterial particles is characterized by changes in intracellular elemental content. Thus, investigation of cellular homeostasis by electron probe microanalysis together with analysis of functional changes may improve estimation of biomaterial cytocompatibility.

Image cytometry detection of breast cancer cells with > 5C DNA content and minor DNA stemlines.

OBJECTIVE: To determine if the presence of cells having a DNA content > 5c and occurring at very low frequency is related to breast cancer outcome. STUDY DESIGN: Feulgen-stained imprints of fresh tumors used for routine standard DNA image cytometry were reanalyzed, with the aim of detecting hyperploid (> 5c) cells or minor stemlines. Specially adapted software was used. RESULTS: The new DNA analysis showed discordance of 47.3% with standard DNA cytometry. Minor stemline or rarely occurring 5c exceeding cells were found. These were not detected by the first DNA analysis. The presence of both DNA hyperploid cells occurring as rare events and a DNA hyperploid stemline was related to outcome. CONCLUSION: The detection of DNA hyperploid cells, even in very small numbers, appears essential to outcome, particularly in diploid or single DNA aneuploid breast cancers.

Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry.

There are two ways of measuring the cell proliferation. The first one consists of quantifying the number of cycling cells with the help of antibodies directed against cells either in G1, S, G2 or M phase. The second way is to assess the cell cycle duration by the quantification of AgNOR proteins. Measuring both the features on the same slide represents an attractive way to tackle the proliferating activity of a cell culture or a tumor. Here, we propose a MIB-1 and AgNOR double staining method especially adapted to image cytometry measurement using MIB-1 antibody coupled to FITC in order to avoid the thresholding problems encountered with such a multilabeling technique. We have applied this new method on a series of 39 breast cancer cases, with at least 4 years follow-up, in order to determine the prognosis significance of this measurement. MIB-1 alone is not linked to prognosis, while the global mean AgNOR area is significantly linked to prognosis in terms of development of visceral metastasis or death. However, the global mean AgNOR area is insufficient to determine the time limit of appearance of metastasis or relapse. Our results clearly demonstrate that a high mean AgNOR area within a cell population having a high MIB-1 index can discern tumors with a high metastatic potential. By multiplying AgNOR area by the percentage of MIB-1 positive cells we calculate the proliferative activity, P, which brings very important information concerning the time limit of relapse.

In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics.

The biocompatibility of two implantable materials, zirconia and alumina ceramics, was investigated in vitro using human osteoblast cell cultures. The viability of osteoblast cells with the materials was evaluated by the methylthiazole sulfate test that revealed an absence of any cytostatic or cytotoxic effect. Cell proliferation kinetic and total protein synthesis in osteoblasts with zirconia or alumina were similar to that observed in control cells cultured on glass coverslips. Light and scanning electron microscopic examinations showed an intimate contact between osteoblasts and the substrates; well-spread cells were observed on the surfaces of both materials. Adhesion ability and morphological characteristics were preserved in osteoblast cultures with these substrates. Moreover, immunohistochemical staining in osteoblasts with zirconia and alumina showed the capacity of these cells to elaborate the extracellular matrix composed of types I and V collagen, osteocalcin, osteonectin, bone sialoprotein, and cellular fibronectin. Finally, DNA image cytometry and interphase silver-nucleolar organizer regions quantification were applied as complementary biocompatibility tests to detect any changes in DNA synthesis and cell proliferation, respectively. The results showed that neither material altered cell ploidy or cell growth rate in accordance with the absence of any inducing effect on DNA synthesis or proliferation.

Expression of gelatinase A and its activator MT1-MMP in the inflammatory periprosthetic response to polyethylene.

Wear debris of polyethylene prosthetic components is known to induce a host granulomatous reaction which recruits numerous macrophages and multinucleated giant cells. By releasing cellular mediators of a nonspecific inflammatory reaction, activated phagocytic cells are thought to play a key role in osteolysis leading to aseptic loosening of the prosthesis. Matrix metalloproteinases (MMPs) have been implicated in this destructive process by their ability to degrade extracellular matrix components of bone and adjacent connective tissue. To investigate the roles of gelatinase A, its activator MT1-MMP, and the MMP inhibitors TIMP-1 and TIMP-2 in aseptic loosening of polyethylene prostheses, immunohistochemistry (IHC) and in situ hybridization (ISH) were performed on periprosthetic pseudosynovial interface tissues. Gelatinase A and MT1-MMP were strongly detected immunohistochemically in macrophages and multinucleated giant cells in contact with polyethylene wear debris. In contrast to MT1-MMP, gelatinase A mRNAs were not found in phagocytic cells but in surrounding fibroblasts, thereby suggesting cooperation between macrophages and fibroblasts in this process. While TIMP-1 was expressed essentially in hyperplastic pseudosynoviocytes as assessed by IHC and ISH, TIMP-2, MT1-MMP, and gelatinase A were colocalized in phagocytic cells. These data support the concept of progelatinase A activation involving a trimolecular complex (MT1-MMP-TIMP-2-gelatinase A) mechanism. Thus, this study demonstrated that gelatinase A and its activator might contribute to the aseptic loosening of polyethylene prostheses.

[Management of DNA banks: ethical concerns]. / Gestion des biothèques: analyse des enjeux éthiques.

With the creation of DNA banks, short and long-term studies can be conducted on the DNA of many individuals using stored cells and tissues. These studies allow an analysis of the pathophysiological impact of genetics and help define individual markers predictive of risk. Genome analysis is thus an important advance in medical science, providing essential information for establishing appropriate measures to slow disease development, limit severity or improve safe recovery. The use of genetic results may however have an adverse effect in certain situations if the genetic information collected were deviated from its purely medical purpose under the influence of social, occupational or economic factors. The aim of our study was to analyse the ethical challenges linked to the implementation of DNA banks in France, particularly to see how to maintain the concept of individual protection in biomedical research within the patient-physician relationship in the current context of legal and administrative regulations in France. In this study, we discuss a set of criteria which should be systematically evaluated in information collection and consent procedures prior to blood or tissue procurement for DNA bank purposes.

DNA image cytometry and Ag-NORs-staining application in biocompatibility studies on human osteoblast cells in vitro.

We report here the study of the biocompatibility of a bone graft material, the Pyrost, using a previously established in vitro model of human osteoblasts. The effect of this material on cell proliferation was evaluated by the MTS assay. Results indicated the absolute absence of cytotoxic or cytostatic effect of Pyrost on cultured osteoblasts. Viability rate was more than 90% in cells cultured with the material compared to the control. Morphological analysis, undertaken by scanning electron microscopy showed a good adhesion and a spreading of osteoblasts in contact with the material that was colonized by cultured cells. In the second part of this work, we have introduced two methods as complementary biocompatibility tests: DNA image cytometry and interphase Ag-NORs quantification. DNA content was measured in cells cultured with or without Pyrost for 3, 9, 15 and 30 days. The determination of DNA indicated that the majority of osteoblasts population was diploid without aneuploidy. The DNA index and cell distribution profile in DNA histograms were similar in all cell populations. The Ag-NORs amount was used as a parameter for cell kinetic evaluation. We have measured the Ag-NORs index like DNA quantification. The proliferation rate, evaluated by Ag-NORs counts in osteoblasts cultured with or without the material, was identical. However, a decrease in Ag-NORs index was observed from day 3 to day 15 of incubation. These results showed a satisfactory biocompatibility of the Pyrost in human osteoblasts culture. The material did not alter cell viability and had no inducing effect either on proliferation rate or on cell ploidy as demonstrated by DNA image cytometry and Ag-NORs proteins staining.

[Biomaterials in an osteo-articular environment. Report of 129 anatomoclinical cases]. / Biomatériaux en milieu ostéo-articulaire. A propos de 129 observations anatomocliniques.

Actually, there is a range of biomaterials which are synthetic or metallic (or the both). They are employed as prosthesis (biostability property) or as bone graft (bioresorbability property). To understand the interactions between cells and such materials, we studied with human bone cellular cultures the cytologycal, immunohistochemical, cytogenetical and ultrastructural aspects of biomaterials in cell cultures. This paper concerns bioceramics like Pyrost, coral, biosorb, oxbone and polymers like polyethylene and silicones. The aim of this work is to evaluate the efficiency of some biomaterials. We found that porosity is primordial to promote biodegradation of bone substitutes. In fact, the biomaterials is integrated and lead to an osteoconduction, an osteoformation and finally an osteoinduction. Our observations show the implant resorption and ossification occurring in the matrix which penetrate it.

[Evolution of the practice of sperm cryopreservation and fate of the preserved specimens : retrospective study of testicular cancer]. / Evolution de la pratique d'autoconservation de sperme et devenir des paillettes mises en garde: étude rétrospective à propos du cancer du testicule. Fédération Française des CECOS.

Testis cancer, chemotherapy and radiation can induce temporary or permanent infertility in men. Cryopreservation of spermatozoa seems an absolute prerequisite in such situation for male patients who are old enough to procreate. We propose a national retrospective study carried out in 17 CECOS. This survey demonstrates a regular increase for semen preservation in testis cancer and analyses the modalities of gametes preservation and their possible use. In view of this activity, we propose a discussion of the attitude of the various CECOS and of particular cases involving ethical issues.

[Carcinoid thymus tumor at an advanced age: diagnostic value of mediastinal needle biopsy with computerized tomography]. / Tumeur carcinoïde du thymus à un âge avancé: intérêt diagnostique de la ponction-biopsie médiastinaie sous tomodensitométrie.

Carcinoid tumour of the thymus is a rare neuroendocrine tumour particularly at an advanced age. The authors report a case of a mediastinal mass in a man aged 85, the mass had remained asymptomatic for a long time. It was decided to achieve a diagnosis because the tumour was causing local compression: a mediastinal needle biopsy under computerised tomographic control confirmed that this was a carcinoid tumour and a study of the biopsy material using an electron microscope showed neurosecretory granules. A sternotomy enabled the tumour to be excised but a post-operative Pseudomonas pneumonia led to the death of the patient. This case underlines the diagnostic place of mediastinal needle biopsy in the presence of a mediastinal tumour. The technique can be carried out under computerised tomography or ultrasonography and this can be associated with a study of the biopsy specimen using electron microscopy which enables the diagnosis to be made before any therapeutic decisions. The treatment of choice of a carcinoid tumour of the thymus is surgery which confirms the tumour limits and also its thymic origin. Tumour excision can be completed using radiotherapy or even chemotherapy.

[Lymphadenopathy of dermatosis in the course of drug hypersensitivity. Cytologic, immunohisto- and immunocytologic, ultrastructural aspects. Report of a case]. / Lymphadénopathie des dermatoses au cours d'une hypersensibilité médicamenteuse. Aspect cytologique, immunohisto et immunocytologique, ultrastructural. A propos d'une observation.

In addition to the morphological details obtained from the imprints, a simple immunocytological study allowed us to diagnose one case of a dermopathic lymphadenopathy simulating a T cell lymphoma, following a drug-induced erythrodermia. We were able to identify the increase of CD1a+ and Prot. S100+ cells on acetone fixed imprints. The histological, immunohistological and ultrastructural investigations confirmed the value of the cytological study and that the dendritic cells were Langerhans cells (Birbeck granules+). Most of them were considered as migrating from the dermal lesions.

Use of chromosome painting for marker chromosome identification in two children with congenital disorders.

Identification of supernumerary de novo marker chromosomes was considered up to now as difficult and sometimes impossible with classical cytogenetical banding methods. The determination of their chromosomal origin is now easier with fluorescent in situ hybridisation techniques and enables an exact correlation between chromosomal aberration and phenotypic features to be established. The authors describe the use of chromosome painting with chromosome 13 and 18 Whole library DNA probe for identification of supernumerary markers in tow patients with congenital disorders. Cytogenetic examination in the first cave revealed a mosaicism with a ring chromosome 13 but clinical findings were different from the classical "ring 13 syndrome', and chromosome painting revealed in an extra--dicentric 13 chromosome (mos : 47, XX, -13, +r (13) +dic (13) / 46, XX, r (13) / 45, XX, -13 / 48, XX, -13, +r (13), (12) dic (13) / 47, XX, -13, + (2) r (13), R-banding pattern on prometaphases and chromosome painting in the second case confirmed the marker to be a 18 p isochromosome (47, XX, +i (18p)). The feasibility and the usefulness of chromosome painting in ascertainment of the possible genetic significance of markers is discussed.

Three-dimensional co-localization of nucleolar argyrophilic components and DNA in cell nuclei by confocal microscopy.

Silver dots deposited specifically on proteins of the nucleolar organizer regions (Ag-NOR proteins) after a one-step silver staining technique were visualized in cells in culture, in cells in smears, and in tissue sections, with a scanning laser confocal microscope working in the reflectance mode. After specific labeling of DNA with the fluorescent dye chromomycin A3, DNA and silver dots could be observed either individually or simultaneously. Therefore, it was possible to study the three-dimensional organization of nucleolar silver-stained structures relative to DNA with a high X, Y, and Z resolution. Our results showed that the argyrophilic components are organized as a twisted necklace structure within interphase nucleoli of cells in culture. We also demonstrated a striking three-dimensional symmetric disposition of NORs within the two sets of chromosomes in telophase cells. Similar results were obtained for cells in smears, although their three-dimensional organization was somewhat disturbed due to air-drying. We also demonstrated that silver dots cannot be visualized in the reflectance mode within sections of paraffin-embedded tissues. However, their simultaneous demonstration in non-confocal transmitted light, together with that of DNA in confocal mode, appeared very useful to study their localization within nuclei and mitotic chromosomes.

Multiple critical smallest region of overlap in monosomy 16Q syndrome?

A 18 months old female child with a de novo 16q deletion is described. The clinical findings in this patient are similar to the phenotype first described by Fryns et al. (11) in 16q deletion. The present deletion involves the region 16q11.2-q12.2 suggesting a second critical smallest region of overlap (S.R.O.) more proximal to the centromere than the one previously located in 16q21.

Discrepancies of DNA content of various solid tumours before and after culture measured by image analysis. Comparison of cytogenetical data.

Parallel cytophotometric ploidy studies and cytogenetic analysis were performed on 15 various human solid tumours. The quantification of DNA by image analysis was carried out on cytological imprints of fresh tumours and on smears obtained after cell culture. The results obtained by both sets of calculations were compared with each other and with the cytogenetic results. 6 cases (40%) showed concordance between the 3 techniques. One case was aneuploid for both DNA image analysis measurements but the cytogenetic data showed only a diploid stem line. In 3 cases out of 15 (20%), smears DNA analysis and cytogenetic results were concordant: in 2 tumours, the culture step failed to preserve aneuploid stem lines that were present in the imprint analysis. In the third one, a minority tetraploid peak observed after culture was absent on the imprint slide. Concordance between imprints and cytogenetic data and discordance with smears' analysis was observed in 3 cases (20%). These 3 cases were diploid or near diploid but the DNA analysis on the smears after culture showed an aneuploid stem line in each case. The last 2 cases showed a total disagreement between the 3 techniques. By measuring the DNA content with an image analyser, the observer can ensure that only tumoral cells are taken into account. The present study revealed that cytogenetic data represent only about 60% of the population that is effectively present in the culture dish and that the cultured population represents only 47% of the population present on the fresh tumour imprint.

[Cell proliferation in tumor pathology]. / Prolifération cellulaire en pathologie tumorale.

There are many ways to analyse and quantify the proliferating activity of a tumor. The different technics are more and more specific of each phase of the cell cycle. This paper firstly describes the basis of each of these technics (mitosis count, DNA staining with various compounds fluorescent or not, incorporation of specific molecules during the phase of DNA synthesis, detection of proliferation antigens by immunological methods). Secondly, the interest of the proliferating fraction knowledge of a tumor is discussed, for various pathologies and according to the technic used.

Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears.

Silver staining of nucleoli reveals argyrophilic proteins associated with nucleolar organizer region (Ag-NOR) proteins. Argyrophilic components appear as dots about 1 micron in diameter dispersed throughout the nucleolus (Ag-NOR dots). The count of Ag-NOR dots is a useful index for improving the cancer diagnosis and determination of prognosis. Here we describe software developed on a medium-cost image analyzer in order to evaluate the mean area of NORs and their number relative to an internal reference, the number and areas of clusters of NORs and the area of the nucleus. Statistical analysis of the data was performed during counting. The first application concerned counting NOR dots during mitosis in cell imprints; those counts were 2.3, 15.3 and 55.56 for the metaphase, telophase and interphase, respectively (relative to unitary dots of metaphase cells). In the second application we demonstrated a significant difference in NOR numbers between two groups of prostatic cancers with good and poor prognoses (6.05 +/- 2.79 SD and 7.96 +/- 3.01, respectively; with Student's t test, = 1.999; P = .05).

[Nucleolar organizers in superficial bladder tumors. Prognostic analysis of 37 observations]. / Les organisateurs nucléolaires (NORs) dans les tumeurs superficielles de la vessie. Analyse pronostique à propos de 37 observations.

Nucleolar Organisers (NORs) are intranucleolar segments of DNA coding for ribosomal RNA. The agyrophilic proteins (AgNOR) associated with NORs allow them to be cytolabelled on paraffin sections. The number of NORs (NOR index) is correlated with cellular proliferation and has a diagnostic and prognostic value in neoplastic disease. The AgNOR method was analysed in a series of 37 superficial bladder tumours with different clinical courses. The NORs were counted in normal urothelium, on superficial tumours used to establish the diagnosis of the disease and on recurrent superficial tumours. The NOR index was 4.54 for normal urothelium, 5.89 for non-invasive superficial tumours, 7.33 for invasive superficial tumours, and 9.75 for invasive recurrences. These results demonstrate an increase in nucleolar argyrophilia with invasion and invasive potential of superficial bladder tumours which were initially homogeneous for stage and grade. The AfNOR method could constitute a new method of early histoprognostic evaluation for urothelial tumours.