Preparing monolayers of non-adherent mammalian cells

A simple method is described for preparing monolayers of non-adherent cells, using concanavalin A to bind the cells to wells of plastic microtest plates. The method was used successfully with all 202 human cell types tested, which included 23 tissue culture lines, 165 fresh specimens of all major histological types of leukemia and lymphoma, 20 fresh myelomas, 2 fresh thymomas, normal spleen and lymph node cells, fractionated T lymphocytes and B lymphocytes from peripheral blood, and cultured fetal amniotic cells. All cell types attached firmly, and were not detached by subsequent vigorous washing. In contrast, attempted attachment of cells in serum free medium, or with poly-L-lysine or glutaraldehyde, was ineffective with many cell types. We used the monolayers as target cells for antibodies to cell surface antigens, utilizing immune rosetting or complement-mediated cytotoxicity. This procedure should simplify most assays involving non-adherent target cells.     

Identification of high risk patients with severe, but asymptomatic aortic stenosis

Whether asymptomatic patients with severe aortic stenosis benefit from surgery remains unclear. We report our data recently published in the New England Journal of Medicine on the natural history of this disease and predictors of outcome.     

TRIO amplification and abundant mRNA expression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer

Studies by comparative genome hybridization have suggested that 5p amplification is related to tumor progression in urinary bladder cancer. In this study seven genes (TAS2R, ADCY2, DNAH5, CTNND2, TRIO, ANKH, and MYO10) located to 5p15.31-5p15.1 were analyzed by fluorescence in situ hybridization using a tissue microarray containing samples from tumors and cell lines with known 5p amplification by comparative genome hybridization. Amplification frequency was highest for TRIO, which maps to 5p15.2 and encodes a protein with a putative role in cell-cycle regulation. To further investigate the role of TRIO amplification in bladder cancer, a tissue microarray containing samples from 2317 bladder tumors was used for fluorescence in situ hybridization analysis. TRIO amplification was strongly associated with invasive tumor phenotype, high tumor grade, and rapid tumor cell proliferation (Ki67 LI) (P < 0.0001 each). Only 7 of 456 pTaG1/G2 tumors (1.5%) but 62 of 485 pT1-4 carcinomas (12.8%) had TRIO amplification. TRIO amplification was not associated with poor prognosis. Using a frozen bladder tumor tissue microarray RNA in situ hybridization confirmed that TRIO is up-regulated in amplified tumors. It is concluded that TRIO up-regulation through amplification has a potential role in bladder cancer progression.     

Pulmonary response to silica or titanium dioxide: inflammatory cells, alveolar macrophage-derived cytokines, and histopathology

We investigated the effects of silica (SiO2) and titanium dioxide (TiO2) on the pulmonary recruitment of inflammatory cells and the ability of alveolar macrophages (AMs) to release the pro-inflammatory cytokines, interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF). Rats were intratracheally instilled with 5 to 100 mg/kg of the materials, and bronchoalveolar lavage cell populations and AM cytokine release were characterized on days 1, 7, 14, and 28. Both dusts elicited dose-related increases in neutrophils, lymphocytes, and AMs; however, this response was more pronounced and persistent with SiO2. SiO2 at greater than or equal to 50 mg/kg increased AM release of IL-1 and TNF at all time points; lower SiO2 doses had either a transient or no effect on AM-derived cytokines. TiO2 did not result in AM IL-1 release and increased TNF release transiently at doses greater than or equal to 50 mg/kg. Both dusts primed AMs to release increased levels of IL-1 and TNF upon in vitro stimulation with lipopolysaccharide. Histopathology (day 28) demonstrated dose-related interstitial inflammation associated with SiO2 exposure, an effect that was less severe with TiO2. SiO2 doses of greater than or equal to 50 mg/kg elicited a granulomatous response. Development of granulomatous inflammation only at SiO2 doses for which persistent AM IL-1 release occurred suggests involvement of this cytokine in the formation of SiO2-induced granulomas. The ability of SiO2 to activate AM release of IL-1 and TNF in a more pronounced and persistent manner than TiO2 is likely responsible, at least in part, for the greater inflammation and pneumotoxicity associated with SiO2.     

Microglia are more susceptible than macrophages to apoptosis in the central nervous system in experimental autoimmune encephalomyelitis through a mechanism not involving Fas (CD95)

Morphological studies have shown that macrophages and microglia undergo apoptosis in the central nervous system (CNS) in acute experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. To assess the relative levels of macrophage and microglial apoptosis, and the molecular mechanisms involved in this process, we used three-colour flow cytometry to identify CD45lowCD11b/c+ microglial cells and CD45highCD11b/c+ macrophages in the inflammatory cells isolated from the spinal cords of Lewis rats 13 days after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Simultaneously, we analyzed the DNA content of these cell populations to assess the proportions of cells undergoing apoptosis and in different stages of the cell cycle or examined their expression of three apoptosis- regulating proteins, i.e. Fas (CD95), Fas ligand (FasL) and Bcl-2. Microglia were highly vulnerable to apoptosis and were over-represented in the apoptotic population. Macrophages were less susceptible to apoptosis than microglia and underwent mitosis more frequently than microglia. The different susceptibilities of microglia and macrophages to apoptosis did not appear to be due to variations in Fas, FasL or Bcl- 2 expression, as the proportions of microglia and macrophages expressing these proteins were similar, and were relatively high. Furthermore, in contrast to T cell apoptosis, apoptosis of microglia/macrophages did not occur more frequently in cells expressing Fas or FasL, or less frequently in cells expressing Bcl-2. These results indicate that the apoptosis of microglia and CNS macrophages in EAE is not mediated through the Fas pathway, and that Bcl-2 expression does not protect them from apoptosis. Expression of FasL by macrophages and microglia may contribute to the pathogenesis and immunoregulation of EAE through interactions with Fas+ oligodendrocytes and Fas+ T cells. The high level of microglial apoptosis in EAE indicates that microglial apoptosis may be an important homeostatic mechanism for controlling the number of microglia in the CNS following microglial activation and proliferation.      

The formation of B-lymphocyte colonies in agar contained in glass capillaries.

Optimal conditions were established for a micro method for the production of colonies of B lymphocytes from mouse spleen cells cultured in agar in glass capillaries, in the presence of bacterial lipopolysaccharide (LPS). Besides LPS the cultures require 5 X 10(-5) M mercaptoethanol and 20% horse serum for optimal colony growth. Foetal calf serum and heat-inactivated horse or foetal calf serum were found to be inferior. An agar gel strength of 0.3% was best for colony counting. A sigmoid curve was obtained when the number of colonies formed was related to the seeded cell density suggesting that some kind of cell to cell co-operation is essential for colony formation. The daily kinetics of colony growth were followed by microscopic colony counting and photometric capillary scanning with integration of the signal areas. Both methods indicated that colony growth had ceased by day 6. The combination of both methods gave the most realistic picture of B-lymphocyte colony development.     

Cytoprotection by somatostatin of normal and malignant clonogenic cells against the in vitro cytotoxicity of bischloroethylnitrosourea (BCNU)

The various pharmacological effects of somatostatin may be explained by the hypothesis that the paracrine peptide, by "stabilizing" cell membranes, inhibits the secretion of hormones as well as protects other cells (vascular endothelium, parenchyma) from different lesions (vasculo-, organo-, cytoprotection). This hypothesis was tested in vitro, using bischloroethyl-nitrosourea (BCNU)-intoxicated stem cells of normal mouse granulopoiesis and of the L 1210 leukemia. Clonogenic mouse bone marrow and L 1210 cells were grown in agar-containing glass capillaries. Using these colony assays and a ID90 of BCNU, cyclic somatostatin influenced the BCNU-cytotoxicity neither at simultaneous nor at subsequent application. However, when given 2 h prior to BCNU, the inhibition of colony growth was almost totally abolished. This cytoprotective effect was seen with normal granulopoietic as well as with leukemic cells. The effect did not show up, if the inactive linear somatostatin was used. N-acetyl-cysteine, a SH-compound applied as a chemoprotective adjunct, did not reveal a cytoprotective effect under identical experimental conditions, either. The results were discussed in view of common efforts to reduce the toxicity of cancer chemotherapy.     

A DROP-IN Gamma Probe for Robot-assisted Radioguided Surgery of Lymph Nodes During Radical Prostatectomy

BackgroundThe DROP-IN gamma probe was introduced to overcome the restricted manoeuvrability of traditional laparoscopic gamma probes. Through enhanced manoeuvrability and surgical autonomy, the DROP-IN promotes the implementation of radioguided surgery in the robotic setting.ObjectiveTo confirm the utility and safety profile of the DROP-IN gamma probe and to perform a comparison with the traditional laparoscopic gamma probe and fluorescence guidance.Design, setting, and participantsTwenty-five prostate cancer patients were scheduled for a robot-assisted sentinel lymph node (SN) procedure, extended pelvic lymph node dissection, and prostatectomy at a single European centre.Surgical procedureAfter intraprostatic injection of indocyanine green (ICG)-^99mTc-nanocolloid (n = 12) or ^99mTc-nanocolloid + ICG (n = 13), SN locations were defined using preoperative imaging. Surgical excision of SNs was performed under image guidance using the DROP-IN gamma probe, the traditional laparoscopic gamma probe, and fluorescence imaging.MeasurementsIntraoperative SN detection was assessed for the different modalities and related to anatomical locations. Patient follow-up was included (a median of 18 mo).Results and limitationsOverall, 47 SNs were pursued in vivo by the DROP-IN gamma probe, of which 100% were identified. No adverse events related to its use were observed. In vivo fluorescence imaging identified 91% of these SNs. The laparoscopic gamma probe identified only 76% of these SNs, where the detection inaccuracies appeared to be related to specific anatomical regions.ConclusionsOwing to improved manoeuvrability, the DROP-IN probe yielded improved SN detection rates compared with the traditional gamma probe and fluorescence imaging. These findings underline that the DROP-IN technology provides a valuable tool for radioguided surgery in the robotic setting.Patient summaryRadioguided robot-assisted surgery with the novel DROP-IN gamma probe is feasible and safe. It enables more efficient intraoperative identification of sentinel lymph nodes than can be achieved with a traditional laparoscopic gamma probe. The use of the DROP-IN probe in combination with fluorescence imaging allows for a complementary optical confirmation of node localisations.