A simple technique for viewing cultured cells from the side by light microscopy

Summary Cultured cells are grown on flexible plastic substrates (e.g., Formvar, polycarbonate, Teflon) which can be folded to form a cell-lined edge. Once folded, the substrate is incubated in growth medium within a simple viewing chamber constructed from a glass slide and a cover slip. By examining the folded edge with a light microscope, one can image, with good resolution, large numbers of cell profiles.     

The synovium and synovial fluid in multicentric reticulohistiocytosis--a light microscopic, electron microscopic and cytochemical analysis of one case

The synovium and synovial fluid have been studied in a patient with multicentric reticulohistiocytosis. Previously unreported histochemical, immunocytochemical and ultrahistochemical findings are presented and their relevance to the aetiology and pathogenesis are discussed. The synovial fluid analysis in this disease is characteristic and may be helpful in its early diagnosis.     

A chemotactic inhibitor in synovial fluid.

Synovial fluid was found to contain an inhibitor of neutrophil chemotaxis. The activity of this inhibitor was masked in native synovial fluid, but could be detected in fluid in which complement had been deactivated by mild heating. The inhibitor was most effective against the chemotactic activity of zymosan-activated serum (C5ades arg). It had little effect when N-formyl-methionyl-leucyl-phenylalanine served as chemoattractant. Inhibition was not the result of a direct effect on the neutrophils, since incubation of cells with synovial fluid did not alter their chemotactic response. The inhibitory activity was destroyed by boiling the synovial fluid or treating it with trypsin, suggesting that it is a protein (or proteins); it was not affected by hyaluronidase treatment. Gel filtration revealed that the inhibitor was present in native as well as decomplemented synovial fluid, and that its molecular weight was in the vicinity of 25,000. It is proposed that this inhibitory activity plays a role in the regulation of the inflammatory response in joints.     

Membrane N-acetylglucosamine: expression by cells in rheumatoid synovial fluid, and by pre-cultured monocytes

After 21-48 h in culture, 2-8% of human peripheral blood monocytes strongly express terminal N-acetylglucosamine (GlcNAc) on their membranes. This can be detected with a monoclonal antibody selected for binding to asialo-agalacto-fetuin, and is eliminated by incubating the cells in pure N-acetylglucosaminidase. Expression of GlcNAc is transient, and can no longer be detected by day 4. These cells are a subset of macrophages since they are positive for non-specific esterase and stained by the monoclonal antibody EBM 11. GlcNAc-positive cells showing double staining with monoclonal antibodies UCHM1 and RFD7 were detected. Their numbers were not influenced by the addition of GM-CSF, IFN-gamma, 1,25-(OH)2 cholecalciferol or indomethacin. Macrophages which give membrane staining for terminal GlcNAc were also found in rheumatoid synovial fluid, and in synovial tissue, though in the peripheral blood their frequency was the same in samples from normal donors and from patients with rheumatoid arthritis. Immunoblots of 24-48-h monocyte cultures or of fresh synovial fluid cells using the anti-GlcNAc monoclonal, show the anticipated agalactosyl IgG heavy chains, and an additional band of 70-80kDa.     

Modulation of phospholipase A2 activity in human synovial fluid by cations

Cell-free, Ca²⁺ dependent phospholipase A₂ activity (PLA₂) was measured in human synovial fluid of patients with various kinds of arthritis using [1–¹⁴C] oleate-labelled autoclaved Escherichia coli as substrate. PLA₂ activity at pH 7.0 and with 5 mM added Ca²⁺ was stimulated and then inhibited in a dose-dependent fashion by NaCl; maximal stimulation of 8.8 fold was found at 150 mM Na⁺. Similar effects were obtained with K⁺ Li⁺ and Ru⁺. In the absence of added Na⁺, PLA₂ activity was maximal with 25 mM Ca²⁺ (145 nmols/hr/mg), but in the presence of 150 mM Na⁺, activity was maximal with 4 mM Ca²⁺ (415 nmols/hr/mg). PLA₂ activity was optimal between pH 6.5–8.0 in presence of 150 mM Na⁺¹ and 4 mM Ca²⁺. There was no significant difference between PLA₂ activity in synovial fluids from rhematoid and other types of arthritis. Neutral active, Ca²⁺-dependent PLA₂ activity in acid extracts of human platelets, plasma, polymorphonuclear leukocytes and synovial fluid varied in response to added Na⁺. In presence of 150 mM added Na⁺ and 5 mM PLA₂ activity in human synovial fluid was inhibited by all multivalent cations tested. In the absence of Na⁺, Cu²⁺ and Mg²⁺ stimulated PLA₂ activity in a dose dependent fashion; whereas, Fe²⁺, Fe³⁺ and Al³⁺ were inhibitory. The extent of stimulation by Mg²⁺ was inversely related to the concentration of added Ca²⁺.     

Membrane N-acetylglucosamine: expression by cells in rheumatoid synovial fluid,and by pre-cultured monocytes.

After 21-48 h in culture, 2-8% of human peripheral blood monocytes strongly express terminal N-acetylglucosamine (GlcNAc) on their membranes. This can be detected with a monoclonal antibody selected for binding to asialo-agalacto-fetuin, and is eliminated by incubating the cells in pure N-acetylglucosaminidase. Expression of GlcNAc is transient, and can no longer be detected by day 4. These cells are a subset of macrophages since they are positive for non-specific esterase and stained by the monoclonal antibody EBM 11. GlcNAc-positive cells showing double staining with monoclonal antibodies UCHM1 and RFD7 were detected. Their numbers were not influenced by the addition of GM-CSF, IFN-gamma, 1,25-(OH)2 cholecalciferol or indomethacin. Macrophages which give membrane staining for terminal GlcNAc were also found in rheumatoid synovial fluid, and in synovial tissue, though in the peripheral blood their frequency was the same in samples from normal donors and from patients with rheumatoid arthritis. Immunoblots of 24-48-h monocyte cultures or of fresh synovial fluid cells using the anti-GlcNAc monoclonal, show the anticipated agalactosyl IgG heavy chains, and an additional band of 70-80kDa.     

A teflon culture and embedding device for the study of cells by light and electron microscopy

A method is described which permits continuous light microscopic observation of cell cultures under sterile conditions. Moreover, cells or groups of cells selected in such cultures may be processed for electron microscopy, without disturbance of possible cellular interactions, in such a way that these selected cells may be observed in the electron microscope. The method is based on the use of a transparant Teflon film in culture chambers.     

Stimulation of osteoclast formation by inflammatory synovial fluid

Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP⁺ and VNR⁺ cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.     

Beta-thromboglobulin in inflammatory synovial fluid

Activated platelets release substances which potentially can contribute to joint lesions in inflammatory arthritides. To elucidate a possible participation of platelets in inflammatory joint reactions, the concentrations of the platelet proteinβ-thromboglobulin (β-TG) were measured in 90 inflammatory synovial fluids. Seven percent of the patients with rheumatoid arthritis and none of the patients with other inflammatory joint diseases (e.g., Reiter's disease, reactive or crystal arthritides) hadβ-TG concentrations in synovial fluid exceeding the upper normal range of plasma β-TG. The absent or very modest signs of local platelet activation were contrasted by the pronounced neutrophilic and monocytic activation, as assessed by the measurements of some granule proteins: lactoferrin, myeloperoxidase, lysozyme, and ferritin. No correlation was found between these inflammatory cell markers andβ-TG. A positive correlation (p < 0.001) was noted betweenβ-TG andβ-microglobulin, which appeared in particularly high amounts in rheumatoid arthritis. This correlation may reflect a disturbed permeability of synovial membrane for LMW proteins or a related activation of platelets and lymphocytes. The present results do not give any evidence of platelet activation playing a major role in proliferative or destructive processes in arthritis.     

Production of PAF-acether by synovial fluid neutrophils in rheumatoid arthritis

PAF-acether (PAF) is a pro-inflammatory phospholipid molecule potentially involved in the pathogenesis of arthritis. PAF and related metabolites have been isolated in the synovial fluid from patients with arthritis.The aim of this study was to determine PAF production by neutrophils isolated from synovial fluid and blood in patients with rheumatoid arthritis. Blood neutrophils from normal donors were also studied for their capacity to form PAF. Neutrophils were stimulated with the calcium ionophore A23187 (2µM) for 1 to 60min. PAF released in the medium and PAF associated to cells were measured.In synovial fluid neutrophils, PAF production began as soon as 1 min of stimulation (16.1 ± 6.3 pmol per 1 × 10⁶ cells) and reached a maximum at 20min: 29.2 ± 2.8 pmol per 1 × 10⁶ cells (mean ± SEM, n = 5). The amount of PAF released in the supernatant increased with the length of stimulation and was maximal after 30min (33.5%, percentage of released over total PAF, n = 5). After A23187 stimulation, similar amounts of PAF were produced by blood neutrophils from RA and control patients. However, neutrophils isolated from the joint had a lower capacity to produce PAF than blood neutrophils from the same patients.The present results demonstrate the synthesis and release of PAF by synovial fluid neutrophils. They suggest that neutrophils may be the source of PAF locally present in the joint. Newly synthetised PAF could participate in the amplification of the local inflammatory reaction.     

Diffusion of imipenem in synovial fluid

The diffusion of imipenem (IMP) in the knee joint was studied after a 1 g i.v. administration of Tienam over one hour. The synovial fluid was collected under anesthesia during arthroscopy carried out for mechanical lesions of the knee (meniscal lesions after ligamental injuries or sequelae after meniscectomy), in 3 groups of six patients at one, two, or three hours after the end of injection of IMP. The concentrations of IMP determined by high performance liquid chromatography (HPLC) were: 42.5, 20.1, 9.3 and 5.7 mg/l in the blood at T0, T1, T2 and T3 hr, respectively; 20.4, 13.0 and 7.9 mg/l in the synovial fluid at T1, T2 and T3, respectively. The decrease of IMP concentrations in the synovial fluid was 1,5 times as low as in serum. On account of its broad-spectrum antibacterial activity and our data, IMP could be used in perioperative prophylaxis of the knee joint surgery.     

A plating method for preparation of cells for culture and for observation by light or electron microscopy

A plating technique that makes use of small volumes of cell suspensions (1–10 μl) is described. Its purpose is to permit microscopic examination or propagation in culture of the entire contents of the microdroplet. Cells are allowed to settle by gravity on Millipore filters where they rapidly and firmly attach after 20 minutes of incubation at 37°C. Such cells can be used for long- or short-term cultures and can be processed for light microscopy and transmission or scanning electron microscopy. The method is particularly useful for preparing cell suspensions for scanning electron microscopy since it is possible to match light photomicrographs with scanning electron micrographs of identical cells.     

Comparative studies of the same adenocarcinoma cells,macrophages, and mesothelial cells by light microscopy,scanning electron microscopy,and transmission electron microscopy

The identification of cells in body cavities of cancer patients is sometimes difficult to make. In order to make a definite cytological diagnosis, we observed the same cells by using light microscopy (LM)-scanning electron microscopy (SEM)-transmission electron microscopy (TEM). In this study we first stained cells by the Papanicolaou method after fixation in 1% glutaraldehyde for LM, and then attempted to observe them successively by SEM-TEM after fixation in 1% paraformaldehyde and 1.25% O^s0⁴. Our method and procedures in examining successively one and the same cells in body cavity fluids by using LM, SEM, and TEM ensured accurate discrimination among adenocarcinoma cells, mesothelial cells, and macrophages. The results of this study suggest that LM-SEM-TEM may be of diagnostic value in distinguishing among mesothelial cells, macrophages, and adenocarcinoma cells. This method also succeeded in disclosing differences between the ultrastructure of the cell surfaces, and those of the cytoplasm, and of the nuclei It is desirable that LM-SEM-TEM observation can be introduced into various aspects in order to obtain an improvement in the diagnosis by cytologic examination, the judgment of therapeutical effects, drug selection, and prognostic presumption. Diagn Cytopathol 1994; 11:333–342. © 1994 Wiley-Liss, Inc.     

A predominant Th1 polarization is present in synovial fluid of end-stage osteoarthritic knee joints: analysis of peripheral blood,synovial fluid and synovial membrane

Thorough understanding of the complex pathophysiology of osteoarthritis (OA) is necessary in order to open new avenues for treatment. The aim of this study was to characterize the CD4⁺ T cell population and evaluate their activation and polarization status in OA joints. Fifty-five patients with end-stage knee OA (Kellgren–Lawrence grades III–IV) who underwent surgery for total knee arthroplasty (TKA) were enrolled into this study. Matched samples of synovial membrane (SM), synovial fluid (SF) and peripheral blood (PB) were analysed for CD3⁺CD4⁺CD8^– T cell subsets [T helper type 1 (Th1), Th2, Th17, regulatory T cells] and activation status (CD25, CD69, CD45RO, CD45RA, CD62L) by flow cytometry. Subset-specific cytokines were analysed by cytometric bead array (CBA). SM and SF samples showed a distinct infiltration pattern of CD4⁺ T cells. In comparison to PB, a higher amount of joint-derived T cells was polarized into CD3⁺CD4⁺CD8^– T cell subsets, with the most significant increase for proinflammatory Th1 cells in SF. CBA analysis revealed significantly increased immunomodulating cytokines [interferon (IFN)-γ, interleukin (IL)-2 and IL-10] in SF compared to PB. Whereas in PB only a small proportion of CD4⁺ T cells were activated, the majority of joint-derived CD4⁺ T cells can be characterized as activated effector memory cells (CD69⁺CD45RO⁺CD62L^–). End-stage OA knees are characterized by an increased CD4⁺ T cell polarization towards activated Th1 cells and cytokine secretion compared to PB. This local inflammation may contribute to disease aggravation and eventually perpetuate the disease process.     

Morphological analysis of interstitial cells in murine epididymis using light microscopy and transmission electron microscopy

Smooth muscle contraction of the epididymis plays an important role in sperm transport. Although PDGFRα-positive interstitial cells (PDGFRα (+) ICs) are thought to be involved in controlling smooth muscle movement via intercellular signaling, they have not yet been reported to date in the epididymis. Therefore, we aimed to investigate the morphological characteristics of PDGFRα (+) ICs in the interstitial space of the murine epididymis. Immunohistochemistry showed that PDGFRα (+) ICs co-labeled with CD34 (PDGFRα (+) CD34 (+) ICs were distributed in the interstitial space of the murine epididymis from the initial segment (IS) to the cauda of the epididymis. PDGFRα (+) ICs that were not co-labeled with CD34 (PDGFRα (+) CD34 (?) ICs) were observed just beneath the epithelium from the corpus to the cauda but not in the IS. Both types of PDGFRα (+) ICs were in close proximity to each other as well as the surrounding nerves and macrophages. In addition, PDGFRα (+) CD34 (?) ICs beneath the epithelium were also in close proximity to the basal cells. Using transmission electron microscopy, we identified ICs that possessed elongated and woven cellular processes and were in close proximity to each other, surrounding the cells in the interstitial space. In the murine epididymis, it is suggested that there are two subtypes of ICs that show different distribution patterns depending on the segment, which may reflect segmental differences in mechanisms of sperm transport, forming a cellular network by physical interactions in the murine epididymis.     

Functional deficiency of antigen-presenting cells in the synovial fluid of rheumatoid arthritis.

In our study of rheumatoid arthritis (RA) patients, we observed a decrease of tetanus toxoid antigen-presenting capacity of synovial fluid (SF) adherent cells to autologous T cells of either SF or peripheral blood. Additionally, we found a higher capacity of adherent synovial cells to stimulate autologous T-lymphocytes. Our results suggest that antigen-presenting cells of the SF of RA patients have defects that may play a role in defective presentation of antigens in joints and may account for other abnormal functions important in the pathogenesis of RA.     

Disappearance of hypotetraploid clones after short-term culture of leukemic cells. A case report

A case of M4-AML (acute monoblastic leukemia) is presented which was found to be hypotetraploid (79–80-? chromosomes per metaphase) with direct chromosome preparation while 24- and 48-hour cultures contained predominantly normal and some nonclonal hyperdiploid cells. This observation emphasizes the need for both direct and culture methods to disclose the true karyotype of leukemic cells.