NorthernLights in slide-based cytometry and microscopy.

In recent years, slide-based cytometry has become a key technology for polychromatic cytometric investigations, and many efforts have been made to increase the number of measurable fluorochromes for multiparametric analysis. Sequential photobleaching of fluorochromes next to very photostable dyes is one approach for this technology. As the ALEXA dyes are known to be photostable as compared to the conventional fluorochromes FITC, PE (Riggs et al., Am J Pathol 1958;34:1081-1097), and APC, a differentiation within a fluorochrome pair is possible. Here, we have analyzed the newly available NorthernLights secondary antibodies for use in slide-based cytometry and microscopy. Currently, these fluorochrome-conjugates are now available with three distinct excitation- and emission maxima (NL493, NL557, NL637). Their spectral properties are similar to the frequently used fluorochromes FITC, PE, and APC and can, therefore, be used with most common excitation sources of cytometers or microscopes. As the NorthernLights are bright, resistant to photobleaching, stable in alcohols and xylene and of affordable price, these dyes are promising candidates for use with most laser- and HBO/XBA-based fluorescence microscopy-like techniques.

Characterization of functional domains of the tenascin-R (restrictin) polypeptide: cell attachment site, binding with F11, and enhancement of F11-mediated neurite outgrowth by tenascin-R.

The extracellular matrix glycoprotein tenascin-R (TN-R) is a multidomain protein implicated in neural cell adhesion. To analyze the structure-function relationship of the different domains of TN-R, several recombinant TN-R fragments were expressed in bacterial cells. Two distinct binding regions were localized on the TN-R polypeptide: a region binding the axon-associated immunoglobulin (Ig)-like F11 protein and a cell attachment site. The binding region of the glycosylphosphatidylinositol (GPI)-anchored F11 was allocated to the second and third fibronectin type III (FNIII)-like domain within TN-R. By using a mutant polypeptide of F11 containing only Ig-like domains, a direct interaction between the Ig-like domains of F11 and FNIII-like domains 2-3 of TN-R was demonstrated. The interaction of TN-R with F11 in in vitro cultures enhanced F11-mediated neurite outgrowth, suggesting that the combined action of F11 and TN-R might be of regulatory influence on axon extension. A cell attachment region was identified in the FNIII-like domain eight of TN-R by domain-specific antibodies and fusion constructs. This site is distinct from the F11 binding site within TN-R.

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), a neural member of the EGF family of differentiation factors, is implicated in neurite formation.

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline-rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.

The axonal recognition molecule F11 is a multifunctional protein: specific domains mediate interactions with Ng-CAM and restrictin.

F11 is a glycosyl phosphatidylinositol-anchored axonal surface glycoprotein belonging to a neural subgroup of the immunoglobulin superfamily. In this report, we demonstrate that the F11 protein displays three distinguishable activities: binding to the cell recognition molecule Ng-CAM, interaction with the extracellular matrix glycoprotein restrictin (RN), and a neurite outgrowth-promoting activity. By analyzing deletion mutants expressed in transfected COS cells, epitope mapping of monoclonal antibodies, and neurite outgrowth assays, we reveal that these activities can be localized to distinct regions within the F11 protein. The Ng-CAM-binding site resides in the first two immunoglobulin-like domains of F11, whereas the RN-binding site resides in the second or third domain. A neurite outgrowth-promoting activity of F11 characterized by in vitro culture of tectal cells is independent of F11-Ng-CAM and F11-RN binding.

Induction of axonal growth by heterophilic interactions between the cell surface recognition proteins F11 and Nr-CAM/Bravo.

F11 and Nr-CAM/Bravo are two axon-associated glycoproteins belonging to different subgroups of the immunoglobulin superfamily. In this report we have investigated the interaction of both proteins using neurite outgrowth and binding assays. Antibody blocking experiments demonstrate that neurite extension of tectal cells on immobilized F11 is mediated by Nr-CAM/Bravo. Binding studies further reveal a direct heterophilic interaction between F11 and Nr-CAM/Bravo. This activity can be mapped to the amino-terminal second or third immunoglobulin-like domain within F11 with domain-specific monoclonal antibodies and deletion mutant proteins expressed on COS cells. Furthermore, perturbation experiments with domain-specific monoclonal antibodies demonstrate that this region is required for adhesion and neurite extension.

Characterization of fibroblasts responsible for cartilage destruction in arthritis.

In the pathogenesis of rheumatoid arthritis (RA), synovial fibroblasts (SF) play a key role as they secrete distinct patterns of cytokines and express variable levels of costimulatory and adhesion molecules. The murine fibroblast cell line LS48 has been shown to be invasive in the cartilage destruction models in vivo and in vitro. The purpose of this study was to examine in detail the LS48 phenotype, to obtain a better understanding of the SF-mediated cartilage destruction in RA. The destructive fibroblasts line LS48 and the nondestructive 3T3 cells were cultured and characterized with slide-based and flow cytometry, using antibodies against several adhesion molecules, immunological acting molecules, and marker proteins. The invasive LS48 fibroblasts are characterized by significantly higher expression of adhesion molecules such as CD47 (IAP), CD51 (integrin alpha V), CD61 (GPIIIa), and CD147 (EMMPRIN), and immunological acting molecules such as CD40 (Bp50), CD55 (DAF), and TLR-2. The results from the slide-based and flow cytometry analyses were exactly the same, except for the selected CD147 and TLR-2. This study demonstrated that the destructive fibroblast cell line LS48 has the characteristics of RA SFs. The high expression of specific costimulatory and adhesion molecules underlines the aberrant phenotype of these cells when compared with noninvasive fibroblasts. Furthermore, slide-based and flow cytometry complement each other in fibroblast phenotyping. Overall, this study shows that LS48 is an excellent tool to gain a deeper understanding of SF in RA.

Pharmacodynamics of T-cell function for monitoring immunosuppression.

OBJECTIVES: Recent studies show that measuring pharmacodynamic (PD) effects offers a unique possibility to predict immunosuppression. Thus, in this study we have monitored the PD properties of immunosuppressants on diverse T-cell functions in heart transplant (HTx) recipients. MATERIALS: PDs and blood concentrations (PK) of three different basis-immunosuppressive drugs were studied: cyclosporin A (CsA); tacrolimus (TRL) and sirolimus (SRL). T-cell function was analysed by expression of proliferating cell nuclear antigen (PCNA) labelling, expression of cytokines (IL-2, IFN-gamma) and surface antigen (for example, CD25) by FACS analysis. RESULTS: In group I, at time points C0 and C2, increased CsA-PK significantly inhibited expression of IL-2, IFN-gamma, PCNA and CD25 (P < 0.05). Correlations (r(2)) at C2 between inhibition of T-cell functions (PD) with PK and with drug doses were: CsA-PK: 0.71-0.91 and CsA-dose: 0.73-0.87. In group II, increased TRL-PK over time did not further inhibit expression of CD25, but inhibited PCNA expression more on day 3, and IL-2 and IFN-gamma expression was significantly higher on days 2 and 3 compared to PD effects of CsA (P < 0.05). Blood SRL concentrations in C0 group III, increased on day 1 and remained stable at days 3 and 4. Expression of PCNA was not altered in the SRL-PK category, whereas expression of CD25 was higher and expression of cytokines was lower than PD effects of CsA. CONCLUSIONS: Our results show that PD effects on T-cell function can be used to monitor immunosuppression bringing potential to increase the efficacy and safety of immunosuppressive therapy after HTx.

Cytomics - importance of multimodal analysis of cell function and proliferation in oncology.

Cancer is a highly complex and heterogeneous disease involving a succession of genetic changes (frequently caused or accompanied by exogenous trauma), and resulting in a molecular phenotype that in turn results in a malignant specification. The development of malignancy has been described as a multistep process involving self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and finally tissue invasion and metastasis. The quantitative analysis of networking molecules within the cells might be applied to understand native-state tissue signalling biology, complex drug actions and dysfunctional signalling in transformed cells, that is, in cancer cells. High-content and high-throughput single-cell analysis can lead to systems biology and cytomics. The application of cytomics in cancer research and diagnostics is very broad, ranging from the better understanding of the tumour cell biology to the identification of residual tumour cells after treatment, to drug discovery. The ultimate goal is to pinpoint in detail these processes on the molecular, cellular and tissue level. A comprehensive knowledge of these will require tissue analysis, which is multiplex and functional; thus, vast amounts of data are being collected from current genomic and proteomic platforms for integration and interpretation as well as for new varieties of updated cytomics technology. This overview will briefly highlight the most important aspects of this continuously developing field.

Circulating endothelial progenitor cells are inversely correlated with the median oxygen tension in the tumor tissue of patients with cervical cancer.

Tumor hypoxia leads to adaptive responses in cancer cells, including an induction of vasculogenesis initiated by circulating endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs). The aim of the present study was to correlate the number of EPCs and CECs with the oxygenation of cervical cancer. Blood concentrations of EPCs were detected by FACS analysis with antibodies for CD34 and vascular endothelial growth factor receptor 2 (VEGFR2). CECs were evaluated by double staining for 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Di-LDL) and lectin in a cell culture assay. Ten patients with cervical cancer were compared with ten healthy volunteers. Intratumoral oxygen tension was assessed polarographically with the computerized Eppendorf histography system. Analysis of CEC numbers revealed no difference between patients and controls. However, patients had lower concentrations of CD34-positive hematopoietic stem cells (HSCs) but a significantly higher fraction of EPCs related to the number of HSCs (1.09% versus 0.53%). This fraction was significantly inversely correlated to the median oxygen tension (r = -0.74, p = 0.015). Our study shows for the first time a significant inverse correlation between the fraction of EPCs and intratumoral oxygen tension. We conclude that the fraction of EPCs should be further evaluated as a useful and convenient marker in the prediction of tumor tissue oxygenation.

Excess of malignancies in grandparents of children with malformations? Short communication.

In a prospective study, the occurrence of malignancies in children referred to genetic counseling for congenital malformations, in their sibs, parents and grandparents was registered in 120 families by means of personal interviews. One hundred-and-twenty age matched subjects, admitted for acute respiratory infections or trauma, served as controls. No difference in the occurrence of tumors or leukemias between the two groups was found when the values of patients, sibs, and parents were compared. At the same time, the grandparents of probands with malformations had had significantly more malignancies than the grandparents of the controls. This may be explained by the fact that grandparents lived beyond the age of the usual onset of common cancers and leukemias.

Quantification of cell-cycle distribution and mitotic index in Hydra by flow cytometry.

The applicability of flow cytometry (FCM) to analyse cell-cycle distribution and mitotic cells in Hydra oligactis and Hydra vulgaris is demonstrated. The freshwater polyps H. vulgaris and H. oligactis are well-accepted animal models for studying cell proliferation, regeneration and differentiation. Disintegrated animals were labelled for FCM analysis according to the method of Nuesse et al. [(1990) Flow cytometric analysis of G(1) and G(2)/M-phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements. Cytometry 11, 813]. Proliferation and regeneration experiments, in the absence or presence of the oligopeptide head activator, were quantified. Cell-cycle analysis of different parts of the animals shows low proliferation in the head region and high proliferation in the gastric and foot regions. Cell-cycle analysis of different parts of Hydra, comparison of H. oligactis and H. vulgaris, as well as pharmacological treatment, yielded results that are in agreement with prior microscopic analysis. Our results demonstrate that FCM is an appropriate technique for quantifying proliferation in this animal model. It can be used for basic research on development, regeneration and differentiation as well as for innovative drug investigation and toxicology studies.

Immunophenotyping of peripheral blood leukocytes by laser scanning cytometry.

Many clinical situations demand repeated analyses of blood parameters but permit only minimal amounts of peripheral blood to be taken, e.g., in neonates with low birth weight, during extensive operations of young children, or in patients with restricted bone marrow function. In these cases laser scanning cytometry is the ideal tool to determine the distribution of different leukocyte-subsets. The purpose of this protocol is to describe stepwise a new method of immunophenotyping by laser scanning cytometry. In this assay nuclear DNA is stained by 7-aminoactinomycin-D (7-AAD) and surface antigens are detected by direct three-colour immunofluorescence. For data acquisition, measurements are triggered on the 7-AAD-fluorescence. Data are obtained for forward scatter, green, orange, and long red fluorescence by excitation with the argon-laser, and for far red fluorescence by excitation with the helium-neon-laser. Using this protocol the amount of peripheral blood needed is minimised to 10 microl. Specimens can be stained a second time in a different way and analysed repeatedly and archived.

Pediatric cardiac surgery with cardiopulmonary bypass: pathways contributing to transient systemic immune suppression.

Cardiovascular surgery with cardiopulmonary bypass (CPB) can lead to postoperative complications like postpericardiotomy syndrome (PPS), capillary leak syndrome, or multiple organ failure. In children, PPS morbidity is up to 30%, and intra- and immediate postoperative mortality is up to 4%. For these complications, the CPB is made responsible. Its etiology is not yet clarified in detail, but is thought to be of immunologic origin. The exact knowledge of these reactions is crucial for the selection of treatment strategies. The immune response to CPB surgery in children comprises of a cascade of pro- and anti-inflammatory events. Proinflammatory responses are indicated by the release of interleukin (IL)-6 and IL-8, and the activation of alternative complement pathway. This reaction is mainly a response to surgical trauma and medication and only activation of the alternative complement pathway is CPB specific. Antiinflammatory response during CPB surgery is serologically indicated by the systemic release of the immunosuppressive cytokine IL-10 already before that of proinflammatory cytokines. CPB surgery induces population shifts of the leukocyte subsets, changes their degree of activation, and contributes to the phenotype of a peripheral immune suppression. Circulating neutrophils are selectively filtered and inactivated. T-helper (Th) cells shift transiently to the Th2 phenotype, indicating the prevalence for a humoral immune response. These alterations start immediately after the onset of the CPB. Increased immunosuppression may be involved in PPS development and may be linked to an allergic/atopic predisposition. A generalized model of the immune sequela to pediatric cardiovascular surgery with CPB is drawn. CPB induces a systemic transient anti-inflammatory response by elimination of activated cells, by compensatory reaction to local, systemically not observable, proinflammatory responses, by IL-10 release, by anesthetics and medication, and by leukocyte extravasation. The subsequent proinflammatory reaction is the reaction to surgical trauma modulating the anti-inflammatory reaction. Possible therapeutic consequences of these findings may include treatment strategies that modulate the anti-inflammatory response. More studies are needed to test this hypothesis.

Head-activator induced mitosis of NH15-CA2 cells requires calcium influx and hyperpolarization.

In NH15-CA2 cells head activator (HA) stimulates cell proliferation by acting in the G2/M transition. Cells in mitosis were analyzed by flow cytometry 2-4 h after HA application. HA in a dose-dependent manner stimulated mitosis. Mitosis was prevented by preincubation of cells with pertussis toxin identifying the HA receptor as being Gi-protein coupled. As second effect of HA, an increase in intracellular calcium concentration was observed. This increase in calcium concentration was abolished by inhibiting calcium influx from the extracellular space into NH15-CA2 cells either by chelating extracellular calcium with EGTA or by blocking calcium channels. The increase in intracellular calcium concentration led to an activation of calcium-dependent potassium channels. The higher potassium conductance resulted in hyperpolarization of NH15-CA2 cells. Blocking calcium channels with nickel chloride or potassium channels with tetraethylammonium chloride inhibited the effect of HA on cell proliferation. HA-induced mitosis was inhibited by charybdotoxin and apamin, but not by alpha-dendrotoxin confirming the notion that Ca(2+)-dependent potassium channels are involved in mediating the effect of HA on cell division.

New technologies for the human cytome project.

Cytomes or cell systems are composed of various kinds of single-cells and constitute the elementary building units of organs and organisms. Their individualised (cytomic) analysis overcomes the problem of averaged results from cell and tissue homogenates where molecular changes in low frequency cell populations may be hidden and wrongly interpreted. Analysis of the cytome is of pivotal importance in basic research for the understanding of cells and their interrelations in complex environments like tissues and in predictive medicine where it is a prerequisite for individualised preventive therapy. Analysis of molecular phenotypes requires instrumentation that on the one hand provides high-throughput measurement of individual cells and is on the other hand highly multiplexed, enabling the simultaneous acquisition of many parameters on the single cell level. Upcoming technology suitable to this task, such as slide based cytometry is available or under development. The realisation of cytomic technology is important for the realisation of the human cytome project.

Potential and challenges of a human cytome project.

BACKGROUND: The elucidation of the molecular pathways from the 20-40.000 genes of the sequenced human genome via investigation of genetic networks and molecular pathways up to the cellular and organismal phenotypes is highly complex and time consuming. STRATEGY AND GOALS: The proposed upside-down research strategy of a human cytome project accesses the expressed molecular cell phenotypes by differential screening, for example of diseased versus healthy, or undifferentiated versus differentiated cells to obtain information on disease or differentiation related molecular hotspots at the single cell level. The genome serves as inventory of the biomolecular capacities of organisms while the mechanisms of genome realisation are initially entirely bypassed. Detected molecular hotspots are further investigated by backward directed systems biology, including molecular pathway modelling to elucidate disease related molecular pathways. New drug targets may be identified to specifically influence such pathways. Differential screening provides, in addition, individualized disease course predictions for everyday medicine, in form of "predictive medicine by cytomics." The early recognition of future disease complications enables an immediate application of preventive therapies. This is likely to lower disease related irreversible tissue destruction and adverse drug reactions and will allow to individually optimize patient therapy. OUTLOOK: Immediate medical use, facilitated access to the detection of new drug targets, increased research speed and the stimulation for advanced technological developments represent major driving forces for the efforts to establish a human cytome project.

No effect of vanadate on the centromere separation sequence.

Alteration of the centromere separation sequence may lead to aneuploidy and may be an indicator of chromosome instability. The aim of this study was to examine whether this phenomenon could be influenced by a factor known to affect cell division. Human lymphocyte cultures were exposed to Na-vanadate in various concentrations and durations. As compared to controls, the inhibitory effect of vanadate manifested itself in a decrease of mitotic index values but the centromere separation sequences remained unchanged, i.e. chromosomes 2, 17-18 were the first, 1, 16 and the acrocentrics the last to separate in both the control and vanadate-treated cultures. The findings support the suggestion that the centromere separation sequence is hardly influenced by environmental factors but rather is a species specific, genetically determined phenomenon.

[Successful treatment of a right atrial thrombus secondary to central venous catheterization]. / Centrális vénás kanül szövödményeként kialakult jobb pitvari thrombus sikeres kezelése.

The authors report on the thrombolytic therapy in an infant born after 36 weeks of gestation weighing 2800 grams. She was repeatedly operated on for intestinal perforation and a right atrial thrombus developed as a complication of the central venous catheterisation. Complete dissolution of the thrombus was observed after 5 days of local urokinase infusion (initial dose: 3000 IU/kg/hr, gradually elevated up to 4500 IU/kg/hr) through an Epicutaneocava catheter positioned in the right atrium. Subsequently, prophylactic intravenous heparin infusion (100 IU/kg/hr), then subcutaneous low-molecular-weight heparin (2 x 100 AXa IU/kg/day) was given. No side effects of the treatment was observed. On follow-up examinations at the age of 5 months the infant proved to be healthy.

Cytometry in the brain: studying differentiation to diagnostic applications in brain disease and regeneration therapy.

During brain development, a population of uniform embryonic cells migrates and differentiates into a large number of neural phenotypes - origin of the enormous complexity of the adult nervous system. Processes of cell proliferation, differentiation and programmed death of no longer required cells, do not occur only during embryogenesis, but are also maintained during adulthood and are affected in neurodegenerative and neuropsychiatric disease states. As neurogenesis is an endogenous response to brain injury, visible as proliferation (of to this moment silent stem or progenitor cells), its further stimulation can present a treatment strategy in addition to stem cell transfer for cell regeneration therapy. Concise techniques for studying such events in vitro and in vivo permit understanding of underlying mechanisms. Detection of subtle physiological alterations in brain cell proliferation and neurogenesis can be explored, that occur during environmental stimulation, exercise and ageing. Here, we have collected achievements in the field of basic research on applications of cytometry, including automated imaging for quantification of morphological or fluorescence-based parameters in cell cultures, towards imaging of three-dimensional brain architecture together with DNA content and proliferation data. Multi-parameter and more recently in vivo flow cytometry procedures, have been developed for quantification of phenotypic diversity and cell processes that occur during brain development as well as in adulthood, with importance for therapeutic approaches.