Flow Cytofluorimetric Analysis Research Articles

Uptake Profiles of Human Serum Exosomes by Murine and Human Tumor Cells through Combined Use of Colloidal Nanoplasmonics and Flow Cytofluorimetric Analysis.

Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth.

Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis

BackgroundMyasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG.MethodsThe aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system.ResultsWe found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals.ConclusionOur data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis.

Alterations on peripheral B cell subsets following an acute uncomplicated clinical malaria infection in children

BackgroundThe effects of Plasmodium falciparum on B-cell homeostasis have not been well characterized. This study investigated whether an episode of acute malaria in young children results in changes in the peripheral B cell phenotype.MethodsUsing flow-cytofluorimetric analysis, the B cell phenotypes found in the peripheral blood of children aged 2–5 years were characterized during an episode of acute uncomplicated clinical malaria and four weeks post-recovery and in healthy age-matched controls.ResultsThere was a significant decrease in CD19+ B lymphocytes during acute malaria. Characterization of the CD19+ B cell subsets in the peripheral blood based on expression of IgD and CD38 revealed a significant decrease in the numbers of naive 1 CD38-IgD+ B cells while there was an increase in CD38+IgD- memory 3 B cells during acute malaria. Further analysis of the peripheral B cell phenotype also identified an expansion of transitional CD10+CD19+ B cells in children following an episode of acute malaria with up to 25% of total CD19+ B cell pool residing in this subset.ConclusionChildren experiencing an episode of acute uncomplicated clinical malaria experienced profound disturbances in B cell homeostasis.

Morphological and functional changes of mitochondria in apoptotic esophageal carcinoma cells induced by arsenic trioxide.

To demonstrate that mitochondrial morphological and functional changes are an important intermediate link in the course of apoptosis in esophageal carcinoma cells induced by As2O3. The esophageal carcinoma cell line SHEEC1, established in our laboratory, was cultured in 199 growth medium, supplemented with 100mL x L(-1) calf serum and 3 mol x L(-1)As2O3 (the same below). After 2, 4, 6, 12, 24 h of drug adding, the SHEEC1 cells were collected for light-and electron-microscopic examination. The mitochondria were labeled by Rhodamine fluorescence probe and the fluorescence intensity of the mitochondria was measured by flow cytometer and cytofluorimetric analysis. Further,the mitochondrial transmembrane potential (MTP, psim) change was also calculated. The mitochondrial morphological change after adding As2O3 could be divided into three stages. In the early-stage (2-6h) after adding As2O3, an adaptive proliferation of mitochondria appeared; in the mid-stage (6-12 h) a degenerative change was observed; and in the late-stage (12-24 h) the mitochondria swelled with outer membrane broken down and then cells death with apoptotic changes of nucleus. The functional change of the mitochondria indicated by fluorescent intensity, which reflected the MTP status of mitochondria, was in accordance with morphological change of the mitochondria. The fluorescent intensity increased at early-stage, declined in mid-stage and decreased to the lowest in the late-stage. 24 h after As2O3 adding, the cell nucleus showed typical apoptotic changes. Under the inducement of As2O3, the early apoptotic changes of SHEEC1 cells were the apparent morphological and functional changes of mitochondria, afterwards the nucleus changes followed. It is considered that changes of mitochondria are an important intermediate link in the course of apoptosis of esophageal carcinoma cells induced by As2O3.

The final maturation of at least some single-positive CD4(hi) thymocytes does not require T cell receptor-major histocompatibility complex contact.

The majority ( approximately 70%) of postselection CD4(+) single-positive (SP) thymocytes are CD8(lo)CD4(hi). These cells express very low levels of CD8, undetectable by flow cytofluorimetric (FCM) analysis, but sufficiently high to allow purification by panning. Unlike the fully mature CD8(-)CD4(hi) thymocytes, which account for the remaining approximately 30% of the SP CD4(+) thymocytes, CD8(lo)CD4(hi) cells are functionally immature and short-lived unless they receive an unidentified maturation signal from the thymus. In this study, we tested the hypothesis that this signal is provided by a T cell receptor (TCR)-major histocompatibility complex (MHC) class II interaction. Using intrathymic transfer, we show that the immature CD8(lo)CD4(hi) cells could complete their intrathymic maturation and populate the peripheral lymphoid organs in the absence of MHC class II (and class I) molecules. Furthermore, in mice devoid of class II (and class I) molecules, the progeny of CD8(lo)CD4(hi) cells was long-lived and functionally reactive to allogeneic class II molecules, although their numbers in the spleen and the mesenteric lymph node were approximately 40-50% lower than those in class II(+) mice 5 mo after transfer. Control experiments demonstrated that the surviving cells did not originate from the contaminating mature thymocytes. These results demonstrate that the final maturation, proliferation, and peripheral survival (up to 5 mo) of at least some postselection CD4(+) SP cells do not require the TCR-MHC class II interaction. They also indicate that the TCR-MHC class II interaction(s) required for the intrathymic development of long-lived CD4(+) SP cells occurs before the CD4(hi) SP stage of development.

Antigen presentation function of brain-derived dendriform cells depends on astrocyte help.

In mouse brain primary culture, supplementation with granulocyte macrophage colony-stimulating factor (GM-CSF) induces development of dendriform cells emerging on the astroglia monolayer. As revealed by flow cytofluorimetric analysis, >70% of isolated cells are CD11c(+) and express the dendritic cell (DC) marker 33D1. Additional expression of F4/80 and CD11b suggests a myeloid origin of these cells. The lymphoid DC marker CD8alpha is lacking while DEC-205 has been detected on approximately 10% of the cells. When freshly isolated, such brain-derived DC-like cells are excellent antigen-presenting cells (APC) but their functional capability is lost during subculture with GM-CSF. In contrast, their antigen presentation function remains stable in the presence of GM-CSF plus astrocytes or astrocyte-conditioned medium. The responsible astrocytic activity co-fractionates with macrophage colony-stimulating factor (M-CSF). Neutralization of the activity with anti-M-CSF antibody and substitution with recombinant M-CSF provide evidence that, in addition to GM-CSF, M-CSF is required to preserve the functional capability of these brain-derived APC. Responsiveness of the isolated cells to M-CSF is substantiated by the expression of c-fms/M-CSF receptor gene. Consistently, GM-CSF proves stimulatory for astrocytes by up-regulating their secretion of M-CSF. Furthermore, depletion or blocking of endogenous M-CSF in primary brain cell culture prevents the development of functionally active APC regardless of exogenous GM-CSF. In sum, these findings ascribe an immature DC phenotype to GM-CSF-grown myeloid brain cells and indicate a role for astrocytic M-CSF in maintaining their antigen presentation function.

Leukocyte Microparticles Stimulate Endothelial Cell Cytokine Release and Tissue Factor Induction in a JNK1 Signaling Pathway

A role of membrane microparticles (MP) released by vascular cells in endothelial cell (EC) activation was investigated. Flow cytofluorimetric analysis of blood samples from normal volunteers revealed the presence of an heterogeneous MP population, which increased by approximately 2-fold after inflammatory stimulation with the chemotactic peptide, N-formyl-Met-Leu-Phe (2,799 +/- 360 versus 5241 +/- 640, p < 0.001). Blood-derived MP stimulated release of EC cytokines interleukin (IL)-6 (377 +/- 68 pg/ml) and MCP-1 (1, 282 +/- 79) and up-regulated de novo expression of tissue factor on the EC surface. This was associated with generation of a factor Xa-dependent procoagulant response (2.28 +/- 0.56 nM factor Xa/min/10(4) cells), in a reaction inhibited by a monoclonal antibody to tissue factor. Fluorescent labeling with antibodies to platelet GPIbalpha or leukocyte lactoferrin demonstrated that circulating MP originated from both platelets and leukocytes. However, depletion of platelet MP with an antibody to GPIbalpha did not reduce EC IL-6 release, and, similarly, MP from thrombin-stimulated platelets did not induce IL-6 release from endothelium. EC stimulation with leukocyte MP did not result in activation of the transcription factor NF-kappaB and was not associated with tyrosine phosphorylation of extracellular signal-regulated protein kinase, ERK1. In contrast, leukocyte MP stimulated a sustained, time-dependent increased tyrosine phosphorylation of approximately 46-kDa c-Jun NH(2)-terminal kinase (JNK1) in EC. These findings demonstrate that circulating leukocyte MP are up-regulated by inflammatory stimulation in vivo and activate a stress signaling pathway in EC, leading to increased procoagulant and proinflammatory activity. This may provide an alternative mechanism of EC activation, potentially contributing to dysregulation of endothelial functions during vascular injury.

Identification of an important thyrotrophin binding site on the human thyrotrophin receptor using monoclonal antibodies

A thyrotrophin (TSH) binding site has been identified on the extracellular domain of the human thyrotrophin receptor (hTSHR) using monoclonal antibodies that recognise the native hTSHR. These antibodies were produced by immunising BALB/c mice with denatured recombinant material, selected by their reaction with recombinant hTSHR expressed on heterologous cell lines using flow cytofluorimetric analysis, and characterised by immunoblotting and immunoprecipitation. The epitopes the monoclonal antibodies recognise were determined using multiple overlapping synthetic peptides. All of the antibodies reacted with epitopes within the region 335–390; these epitopes must be accessible on the external surface of the native hTSHR. None of the antibodies stimulated cAMP production of recombinant hTSHR cell lines. The epitopes of two antibodies (residues 337–342 and 355–358) are in the small peptide thought to be removed by proteolytic processing of hTSHR. A further five different antibodies (determined from their variable region sequences) all reacted with residues 381–384 emphasising the immunogenicity of this region. The functional importance of residues 381–384 as a TSH binding site was shown by the fact that some of these monoclonal antibodies caused inhibition of radiolabelled TSH binding of 80–90% at 1 μg/ml and greater than 50% inhibition at 0.1 μg/ml (0.65 nM—i.e. comparable in effectiveness with TSH itself). Residues 381–384 may form part of the target regions recognised by inhibitory autoantibodies found in Graves’ disease.

Inhibition of telomerase activity during induction of HL-60 cells by retinoid Ro13-7410

Objective: To investigate the effects of Ro13-7410 on telomerase activity and cell cycle distribution. Methods: Telomerase activity of HL-60 cells induced by retinoid Ro13-7410 was detected by telomerase PCR-ELISA-kit. The cell cycle was analyzed by flow cytometry. Results: Telomerase activity declined gradually after 10−6 mol/L Ro13-7410 treatment, and the inhibition of telomerase activity at day 5 of treatment with Ro13-7410 was less effective than with Retinoid Acid (RA). DNA flow cytofluorimetric analysis revealed that Ro13-7410 caused partial cells arrest in the G2/M phase after 4-days treatment. Conclusion: Telomerase activity declined gradually and partial cells were arrested in the G2/M phase after Ro13-7410 treatment.

Studies on binding of HIV-1 p24 gag peptide to HLA-Cw3 + cells

Human major histocompatibility complex class I antigens, HLA-C, are expressed on the cell surface at approximately a tenfold lower level than HLA-A and -B. We hypothesized that the expression of HLA-C is limited by the quantity of high affinity peptides which bind to these molecules, thus allowing only a small fraction of HLA-C molecules to be transported and/or to remain stable on the cell surface. If this assumption is correct, then the addition of exogenous peptide should increase cell surface HLA-C expression. To verify the hypothesis, we pulsed lymphoblastoid cell line PAJ (HLA-Cw3 +) with synthetic HIV-1 p24 gag 145–152 peptide, known to be presented to T-lymphocytes by HLA-Cw3 molecule. PAJ (HLA-Cw3 +) cells bound approximately two times more of the peptide than HAJ (HLA-Cw3 −), and four times more than 500/C9 (HLA-Cw3 −) cells. Accordingly, overnight pulsing of PAJ cells with the p24 gag 145–152 peptide caused an increase in class I HLA expression detected on the cell surface by flow cytofluorimetric analysis with anti-HLA-B,C monoclonal antibodies but not by anti-HLA-A antibody. In contrast, HLA-Cw3 − cells treated in the same manner did not show any increase of HLA class I expression. Our data suggest that low concentration of high affinity peptides within the cell may be one of the factors limiting cell surface expression of HLA-C molecules.

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin‐mediated cell adhesion

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extra-cellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a “DNA ladder” on gel electrophoresis, by propidium iodine staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to β, integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interations by means of perturbing antibody against β, subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings incidate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death. Int. J. Cancer 70:688–698, 1997. © 1997 Wiley-Liss, Inc.

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin-mediated cell adhesion.

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extracellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a "DNA ladder" on gel electrophoresis, by propidium iodide staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to beta 1 integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interactions by means of perturbing antibody against beta 1 subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings indicate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death.

Tyramine-containing poly(4-nitrophenylacrylate) as iodinatable ligand carrier in biodistribution analysis.

Targeted label or drug delivery requires access to convenient carrier systems and methods for efficient ligand conjugation. The main purpose of this study is to design an iodinatable synthetic polymer, whose application in vivo in tumor-bearing mice is tested with several related carbohydrate ligands, namely ABH and Lewis blood group epitopes. Tyramine and aminopropyl derivatives of the synthetic oligosaccharides were attached to poly(4-nitrophenylacrylate). Following iodination, the biodistribution of the sugar-free and the substituted polymers was determined in tumor-bearing mice. Flow cytofluorimetric analysis assessed tumor cell binding of further ligand types to human tumor cells in vitro. Quantitative ligand incorporation was achieved under mild conditions. Whereas the ligand-free poly[N-(2-hydroxyethyl)acrylamide] (MW 30 kDa) showed preferential accumulation in kidney, neoglycopolymers were found in substantial amounts in liver, kidney or spleen. The nature of the carbohydrate structure quantitatively influenced the distribution pattern. Tumor cell binding of blood group determinants and three further ligand types revealed non-uniform intensity in labeling and percentage of positive cells even in comparison between lines with identical histogenetic origin. Carbohydrate-exposing poly[N-(2-hydroxyethyl)acrylamide] polymers with tyramine as an iodine acceptor distribute in mice with a profile which is quantitatively influenced by small structural variations of the ligand part. Further refinement of the ligand structure may increase the level of selectivity for organ and tumor accumulation.

EDTA-dependent pseudothrombocytopenia: a clinical and epidemiological study of 112 cases, with 10-year follow-up.

In the past 10 years, we have observed 112 cases of EDTA-dependent pseudothrombocytopenia (PTCP) due to in vitro platelet clumping at room temperature. 93 patients had antiplatelet antibodies (48 IgM, 30 IgG, 3 IgA, and 12 had two different isotypes concomitantly). In 20% of patients, the presence of IgM antibodies characteristically accompanied platelet agglutination also at 37 degrees C, and in citrated blood. The phenomenon was not age or sex related, nor was it associated with any particular pathology or use of specific drugs, and was present in both healthy subjects and patients with various diseases. Flow cytofluorimetric analysis of CD5-positive B cells, which are responsible for autoantibody production, did not demonstrate any changes in the percentage and absolute number of this lymphocyte subset. Average follow-up was 5 years (6 months-10 years); however, previous clinical records disclosed that PTCP was present for more than 15 years in four cases, and more than 20 years in three others, with no clinical manifestation of disease. This study confirms that EDTA-dependent PTCP is a phenomenon related to the presence of natural autoantibodies with antiplatelet activity, devoid of pathological significance. Its clinical interest resides in the need for its prompt and certain recognition in order to avoid unnecessary examinations and therapeutic interventions. The best and most rapid technique for obtaining accurate platelet counts in PTCP subjects is to collect and examine EDTA blood at 37 degrees C; however, clumping will still be present in about 20% of these cases, and even in citrated blood. To obviate this phenomenon, blood should be collected in ammonium oxalate, and platelets counted in a Burker chamber.

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span.

Comparing the properties of 'young' and senescent ('aged') O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a beta-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows: (1) Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA120 as well as Erythrina cristagalli and Erythrina corallodendron lectins specific for terminal beta-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation. (3) The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive N-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively. (4) Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages.

Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes

The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 · 10−8 to 2 · 10−7 M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.

The Impact of HIV-1 Infection on Phenotypic and Functional Parameters of Cellular Immunity in Chimpanzees

As a means of assessing the immunological impact of HIV infection in the chimpanzee, as well as the participation of the cellular components in the control of HIV infection in these animals, various aspects of cellular immunity were investigated in chronically HIV-infected chimpanzees. Eight HIV-1-infected chimpanzees were included in this study; two of them were infected for more than 5 years and six for nearly 3 years at the time of study. All of the chimpanzees received either 40 or 100 TCID50 of HTLV-IIIB. Circulating peripheral blood lymphocytes were studied by flow cytofluorimetric analysis in order to reveal possible alterations in the CD4:CD8 ratio, as well as in specific CD4+ and CD8+ cell subpopulations. Chronically infected chimpanzees did not present significant alterations in the percentage of CD4+ or CD8+ lymphocyte subsets. Interestingly, the CD8+/CD57+ cell subset was not detectable. The expression of markers for activation on circulating lymphocytes, usually higher in the HIV-infected patients, was not altered in infected animals. The functional aspects of specific anti-HIV-1 non-MHC and MHC-restricted cellular cytotoxic reactivities were also investigated. The results were compared with the findings in normal uninfected chimpanzees and in HIV-infected humans. Only one chimpanzee (881) developed a detectable, specific non-MHC-restricted anti-HIV-1- reactivity. Compared to that seen in humans, the ontogeny of this activity is delayed. Among the other infected chimpanzees, no specific anti-HIV cellular reactivities were detectable in the peripheral blood. In chimpanzees, HIV-1 infection evidently does not elicit the same strong cellular reactivity as that detected in infected patients. The absence of chronic cellular activation, despite continued viral replication, may highlight a key determinant in HIV-1-induced pathogenesis that is likewise absent in infected chimpanzees.

Cytokine Production in Virus-Infected Mice: A Single Cell Analysis

Antiviral activity of IFN-gamma and TNF has been demonstrated in vitro and in vivo. Recombinant vaccinia viruses that encode either of these cytokines are highly attenuated in vivo and, indeed, are no longer lethal when they are used to infect athymic nude mice. Thus, these cytokines are able to replace the requirement for CD8 T cells in immunodeficient mice. We believe that the antiviral properties of some cytokines are a central component in the physiological control of virus infection. In this report, we used flow cytofluorimetric analysis to simultaneously detect cytokine production and T cell phenotype. Importantly, this method allowed the analysis of cytokine production in cells freshly isolated from tissues. In contrast to other procedures, restimulation of the cells in vitro was not necessary. We demonstrate that CD8 IFN-gamma+ cells were predominantly induced early in the response to vaccinia virus infection and, at the cellular peak of the response, CD8 IFN-gamma+ and CD4 TNF+ T cells were present at equal frequencies. Hence, these findings support the concept that effector T cells produce cytokines that are known to be antiviral, and that these cytokines are an important component of effector T cell activity.

Cholestasis induces major histocompatibility complex class I expression in hepatocytes

The hepatic expression of major histocompatibility complex (MHC) antigens is normally limited. However, aberrant expression occurs in cholestatic diseases such as primary biliary cirrhosis. The aim of this work was to assess the effect of cholestasis itself on hepatocyte MHC expression and to determine if immunosuppressive drugs might modulate this expression. Liver fragments taken from six patients with extrahepatic cholestasis and eight control patients were analyzed for MHC expression by direct immunofluorescence. MHC class I expression by hepatocytes was present in six of six cholestatic patients and zero of eight control subjects. Hepatocytes did not express MHC class II in either group. In further studies, cholestasis was induced in rats by ligation-section of the bile duct. Five groups of rats were studied: control, 3-day cholestasis, 5-day cholestasis, 5-day cholestasis plus cyclosporine, and 5-day cholestasis plus corticosteroids. Hepatocyte MHC class I expression was detected by immunofluorescence in bile duct-ligated rats but not in control animals. Flow-cytofluorimetric analysis of isolated hepatocytes showed that the percentage of hepatocytes expressing MHC class I increased from day 0 (9.9%) to days 3 (50.2%) and 5 (82.9%); this hyperexpression was not modified by cyclosporine (79.7%) or corticosteroids (77.9%). The percentage of hepatocytes spontaneously expressing MHC class II was low (0.05%) and was not significantly modified by cholestasis or immunosuppressive drugs. Thus, a nonimmunological factor such as cholestasis is able to modulate MHC expression. The liver may become more vulnerable to immune destruction in the presence of cholestasis, and immunosuppressive treatment has no effect on this phenomenon.

Cholestasis induces major histocompatibility complex class I expression in hepatocytes

The hepatic expression of major histocompatibility complex (MHC) antigens is normally limited. However, aberrant expression occurs in cholestatic diseases such as primary biliary cirrhosis. The aim of this work was to assess the effect of cholestasis itself on hepatocyte MHC expression and to determine if immunosuppressive drugs might modulate this expression. Liver fragments taken from six patients with extrahepatic cholestasis and eight control patients were analyzed for MHC expression by direct immunofluorescence. MHC class I expression by hepatocytes was present in six of six cholestatic patients and zero of eight control subjects. Hepatocytes did not express MHC class II in either group. In further studies, cholestasis was induced in rats by ligation-section of the bile duct. Five groups of rats were studied: control, 3-day cholestasis, 5-day cholestasis, 5-day cholestasis plus cyclosporine, and 5-day cholestasis plus corticosteroids. Hepatocyte MHC class I expression was detected by immunofluorescence in bile duct-ligated rats but not in control animals. Flow-cytofluorimetric analysis of isolated hepatocytes showed that the percentage of hepatocytes expressing MHC class I increased from day 0 (9.9%) to days 3 (50.2%) and 5 (82.9%); this hyperexpression was not modified by cyclosporine (79.7%) or corticosteroids (77.9%). The percentage of hepatocytes spontaneously expressing MHC class II was low (0.05%) and was not significantly modified by cholestasis or immunosuppressive drugs. Thus, a nonimmunological factor such as cholestasis is able to modulate MHC expression. The liver may become more vulnerable to immune destruction in the presence of cholestasis, and immunosuppressive treatment has no effect on this phenomenon. (Gastroenterology 1992 Apr;102(4 Pt 1):1371-7)