Methotrexate selectively modulates TH1/TH2 balance in active rheumatoid arthritis patients.

OBJECTIVE: The mechanism by which low dose methotrexate (MTX, the gold standard treatment for rheumatoid arthritis) exerts its anti-inflammatory effect in rheumatoid arthritis (RA) patients is still debated. Lately, the MTX immunosuppressive effect has been related to apoptosis, especially in active RA patients, with ROS involvement. METHODS: In the present research we investigated MTX oxidative effect and its ability to modulate immune balance in active versus non-active RA patients. RESULTS: Our results show that MTX induces IL-10 secretion (a TH2 cytokine) and significantly reduces TH1 profile in Peripheral Mononuclear Cells (PMNC) derived from active RA patients (n=28). Additionally, we found that MTX modulates the immune status towards TH2 dominance by decreasing the IL-12R and the CXCR3 receptors typical for the TH1 population. Moreover, MTX was found to inhibit the production of nitric oxide (NO) in these patients, a phenomenon that might contribute to MTX action toward cytokine homeostasis. A significant correlation was found between MTX IL-10 induction and NO inhibition in active RA patients. CONCLUSION: Our data suggest that, in active RA patients, apoptosis induction by MTX may be primarily due to IL-10 production via modulation of oxidative stress, which may restore the critically important immune balance. These findings may contribute to determining which group of RA patients may better respond to MTX therapy.

An information-theoretical model for breast cancer detection.

OBJECTIVES: Formal diagnostic modeling is an important line of modern biological and medical research. The construction of a formal diagnostic model consists of two stages: first, the estimation of correlation between model parameters and the disease under consideration; and second, the construction of a diagnostic decision rule using these correlation estimates. A serious drawback of current diagnostic models is the absence of a unified mathematical methodological approach to implementing these two stages. The absence of a unified approach makes the theoretical/biomedical substantiation of diagnostic rules difficult and reduces the efficacy of actual diagnostic model application. METHODS: The present study constructs a formal model for breast cancer detection. The diagnostic model is based on information theory. Normalized mutual information is chosen as the measure of relevance between parameters and the patterns studied. The "nearest neighbor" rule is utilized for diagnosis, while the distance between elements is the weighted Hamming distance. The model concomitantly employs cellular fluorescence polarization as the quantitative input parameter and cell receptor expression as qualitative parameters. RESULTS: Twenty-four healthy individuals and 34 patients (not including the subjects analyzed for the model construction) were tested by the model. Twenty-three healthy subjects and 34 patients were correctly diagnosed. CONCLUSIONS: The proposed diagnostic model is an open one, i.e. it can accommodate new additional parameters, which may increase its effectiveness.

Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Phorbol ester and calcium act synergistically to enhance neurotransmitter release by brain neurons in culture.

Preincubation of intact fetal brain neurons in culture with the phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) in the presence of calcium, resulted in the enhancement of the depolarization-induced, Ca2+-dependent neurotransmitter release by the cells. This effect was due to a marked decrease in the concentration of extracellular Ca2+ required to provoke the release. The concentration of Ca2+ needed to produce half-maximal release shifted from approx 0.1 mM in the absence of TPA to 0.018 mM in its presence. This activity of TPA was concentration-dependent (half-maximal effect at 4 nM TPA) and was also dependent on the presence of calcium during the preincubation period. The TPA-induced enhancement of the stimulated release was also observed when Ca2+ entry into the depolarized cells was partially inhibited by Co2+. The results suggest that TPA acts synergistically with Ca2+ to activate neuronal component(s) involved in Ca2+-dependent neurosecretion.

Calcium-dependent protein phosphorylation and dephosphorylation in intact brain neurons in culture.

Preincubation of intact fetal rat brain neurons in culture with 32Pi results in the incorporation of 32Pi into about 20 specific proteins. Upon stimulation by electrical field stimulation or by K+-induced depolarization, highly significant calcium-dependent increase in phosphorylation of a protein of app. Mr 43 000 and decrease in phosphorylation of an app. Mr 55 000 protein occur. These changes can be attributed to the entry of Ca2+ into the cellular cytoplasm since they can occur upon selective permeabilization of the cell membrane to Ca2+ by the Ca2+-ionophore A23187 and are not observed upon stimulation of the cells in the presence of the Ca2+ channel blocker D-600. These data suggest that these phosphoproteins may be involved in the regulation of processes underlying neurotransmitter release.

Determination of cellular thiol levels in individual viable lymphocytes by means of fluorescence intensity and polarization.

Cellular thiol levels regulate lymphocyte proliferation and death and play a significant role in the immune response. Therefore, the ability to analyze the total protein and non-protein thiol compounds and their distribution among individual living lymphocytes is of great importance. A quantitative measurement of intracellular sulphydryl groups in living lymphocytes using the Cellscan mark F (CS-F) cytometer, in conjunction with the probe CMFDA, is described. This technique permits the detection, identification, and study of sub-populations and single cells in a sample of heterogeneous lymphocytes. The Cellscan apparatus is a laser based scanning cytometer incorporating a unique cell carrier which allows repeated, high-precision measurements of fluorescence intensity (FI) and fluorescence polarization (FP) to be made on intact individual living cells under controlled physiological conditions. The discernible effect of fluorophore molecules bound to thiols having a higher FP than free molecules was used to estimate their relative fractions in living lymphocytes. The results were more conspicuous when the ratio between FP measured at two wavelengths (FPR) of the fluorogenic molecules was used for analysis. In addition, the intracellular dynamic changes in the FI, FP and FPR of the fluorescent probe were also monitored. The cellular sulphydryl content of each lymphocyte within a population was recorded by the CS-F, and sub-populations or individual cells were classified according to their thiol levels and their metabolic rates. Changes in thiol concentration were observed following mitogenic activation of peripheral lymphocytes.

Concerted enhancement of calcium influx, neurotransmitter release and protein phosphorylation by a phorbol ester in cultured brain neurons.

We have recently shown that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate enhances the depolarization induced, calcium dependent release of [3H]dopamine from cultured brain neurons in the rat. In the present study the effects of 12-O-tetradecanoyl-phorbol-13-acetate on the kinetic parameters of depolarization induced calcium influx and on Ca2+ dependent neurotransmitter release and protein phosphorylation were investigated. Depolarization induced neurotransmitter release from the neurons occurs in two phases: an initial, fast release and a subsequent slow release. At low extracellular Ca2+, 12-O-tetradecanoyl-phorbol-13-acetate enhanced the quantity of fast release and in addition, increased the rate constant of the slow release. These effects mimicked the effects of increasing the extracellular Ca2+. Various phorbol derivatives known to activate the Ca2+ activated phospholipid dependent protein kinase (protein kinase C) were also able to enhance the stimulated release of [3H]dopamine from the neurons. 12-O-tetradecanoyl-phorbol-13-acetate induced the incorporation of 32Pi into a protein with an apparent molecular weight of 45,000 daltons regardless of depolarization or of the presence of Ca2+. In addition, 12-O-tetradecanoyl-phorbol-13-acetate induced in unstimulated neurons, Ca2+ dependent increase in the amount of 32Pi incorporated into a 43,000 dalton protein and decrease in the amount incorporated into a 55,000 dalton protein. These changes mimicked the Ca2+ dependent changes in protein phosphorylation which occur upon stimulation of the neurons. Kinetic studies of depolarization induced Ca2+ uptake by the neurons indicated that 12-O-tetradecanoyl-phorbol-13-acetate enhanced the maximal influx of Ca2+ through the voltage sensitive Ca2+ channels by 40%. The results indicate that 12-O-tetradecanoyl-phorbol-13-acetate acts primarily on the regulation of stimulated Ca2+ entry into the cells. Consequently neurotransmitter release at submaximal extracellular [Ca2+] is enhanced.

Detection of antimitochondrial antibodies: characterization by enzyme immunoassay and immunoblotting.

Mitochondrial antigens were purified from rat liver and characterized by immunoblotting. Sera from 19 well defined patients with primary biliary cirrhosis (PBC) reacted with two mitochondrial polypeptides of 68 Kd and 45 Kd. Antibodies to these antigens were not detected in any of the sera of patients with cirrhosis of the liver, chronic active hepatitis or other autoimmune diseases. The two polypeptides were derived from the soluble fraction of the mitochondrial matrix. An enzyme-linked immunosorbent assay (ELISA) employing these rat liver mitochondrial antigens is described. Positive results were obtained with all except one PBC sera (95%), five out of 47 patients with cirrhosis (11%), one out of 20 patients with chronic active hepatitis (5%), and two out of 19 patients with various autoimmune disorders (11%). The titers detected in PBC were markedly higher than those recorded in patients with other liver and autoimmune diseases. Strong correlation was found between immunoblotting and the ELISA in determining antimitochondrial antibodies. The ELISA presented is easily performed and seems to be a useful diagnostic tool for antimitochondrial antibodies in patients with PBC.

Studies on synaptic vesicles in mammalian brain characterization of highly purified synaptic vesicles from bovine cerebral cortex.

Synaptic vesicles have been isolated from bovine cerebral cortex by sequential differential and density gradient centrifugations followed by chromatography on a Sepharose 6B column. We have studied the morphology, enzymatic markers, neurotransmitter and ATP contents and protein composition of the vesicles. The specific contents of acetylcholine, gamma-aminobutyric acid, aspartate, glutamate and catecholamines were 4--8-fold higher in the vesicle fraction compared to the crude synaptosomal pellet. Electron micrographs of the vesicle preparation showed enrichment of vesicular material with an average diameter of 50 nm. The purity of the preparation was assessed by the very low activities of enzymatic markers of cellular membranes and cytosol components. Some Ca--Mg-activated ATPase activity was detected in the vesicle preparations, but its content relative to the neurotransmitters fell on chromatography, suggesting that this activity may be partially contributed by non-synaptic vesicle components, such as small microsomes. The isolated synaptic vesicles were solubilized with 1% sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. The major Coomassie blue stained bands observed with apparent molecular weights of 160,000 and 55,000 were enriched in parallel to the increase in purity of the preparation.

Calcium uptake and calcium-dependent phosphorylation during development of rat brain neurons in culture.

The emergence of the capacities for calcium uptake and calcium-regulated protein phosphorylation during the development of embryonic brain neurons in tissue culture was examined. In the maturing cells, the enhancement in 45Ca2+-uptake upon stimulation with high K+ increased by 3-4 fold during the second week in vitro, in parallel to an increase in the capacity for high K+-induced Ca2+-dependent release of prelabeled [3H]dopamine. The pattern of incorporation of [32Pi]phosphate into the major phosphoproteins in maturing cells under nonstimulating conditions also changed during cell development: the incorporation of 32Pi into two proteins of apparent molecular weights--55,000 and 43,000 dalton--increased, but decreased in a 45,000 dalton protein. Stimulation of mature cells (after 10-11 days in vitro) resulted in a Ca2+-dependent increase in the amount of 32Pi incorporated into the 43,000 dalton protein and a decrease in the amount incorporated into the 55,000 dalton protein. This calcium-regulated phosphorylation pattern was not observed until 6 days in vitro. Introduction of Ca2+ into the immature cells by means of the Ca2+ ionophore A23187 did not alter the phosphorylation pattern and did not cause neurotransmitter release. The amount of [35S]methionine incorporated into a 43,000 dalton protein which comigrated with the 43,000 dalton phosphoprotein also increased upon cell maturation. The results suggest that this phosphoprotein (which does not comigrate with nonphosphorylated actin on two-dimensional polyacrylamide gels) develops in the cells in parallel to the emerging processes of the stimulation-induced calcium entry and calcium-dependent neurosecretion.

The relationship between in vitro fertilization and naturally occurring antibodies: evidence for increased production of antiphospholipid autoantibodies.

OBJECTIVE: Assessment of possible effects of ovarian stimulation during in vitro fertilization (IVF) treatment cycles on circulating levels of antiphospholipid and antinuclear autoantibodies. DESIGN: The study was performed prospectively. Sera were obtained at three time points along IVF treatment cycle. Levels of autoantibodies directed against nuclear components, mitochondrial antigens, and phospholipids were determined using enzyme-linked immunosorbent assay. PATIENTS: Thirty-five patients, who underwent at least one previous IVF attempt, and 36 age- and sex-matched controls were analyzed. All participants were randomly selected. RESULTS: The mean levels of antiphospholipid (but not antinuclear) autoantibodies in sera from IVF-treated patients were found to be significantly higher than the corresponding values of the control group (for immunoglobulin [Ig]M isotype: anticardiolipin, antiphosphatidyl L-serine; for IgG isotype: anticardiolipin, antiphosphatidyl L-serine, and antiphosphatidylcholine; P less than 0.0001, assessed by Mann-Whitney test). The autoantibody levels remained more or less constant at different time points along the treatment cycle. No correlation with age and number of previous IVF cycles was demonstrated. CONCLUSIONS: Serum levels of antiphospholipid (but not antinuclear) autoantibodies increase after IVF treatment. Based on these preliminary data, it is not yet possible to estimate if the observed changes in autoantibody levels might have any future clinical influence on infertile patients undergoing IVF treatment.

Increased anti-Sm antibodies in schizophrenic patients and their families.

1. Autoantibodies in the Sm complex have become a useful serologic aid in the diagnosis of systemic lupus erythematosus (SLE) and have rarely been observed in other diseases. 2. A subset of SLE patients have a variety of psychiatric abnormalities, including schizophrenia. 3. The authors have recently observed that schizophrenic patients have a high incidence of autoantibodies suggesting that autoimmune phenomena may play a role in the pathogenesis of this disease. 4. In the present study the authors investigated multicase families with schizophrenia for the presence of anti-Sm antibodies and showed that these autoantibodies are elevated both in patients and in their healthy relatives. 5. An autoimmune process may be involved in the pathology of schizophrenia.

Antineutrophil cytoplasmic autoantibodies penetrate into human polymorphonuclear leukocytes and modify their apoptosis.

OBJECTIVE: The interaction of extracellular anti-neutrophil cytoplasmic autoantibodies (ANCA) with neutrophilic granules may play an important role in the pathogenesis of ANCA-related disorders. It has been confirmed that apoptosis is an essential trigger associated with translocation of the cytoplasmic granules to the cell surface, and with the expression of ANCA antigens. Since cell penetration by autoantibodies and apoptosis may be associated processes, we tested the hypothesis that penetration of ANCA-autoantibodies into polymorphonuclear leukocytes (PMNs) has an effect on apoptosis and thereby can influence surface antigen expression. METHODS: PMNs were isolated from the blood of healthy volunteers and incubated in the presence of anti-proteinase3 (PR3) enriched IgG or normal human IgG. For each period of incubation (40 minutes or 12 hours) we evaluated: 1) PMN morphology by light microscopy (LM) and transmission electron microscopy (TEM) for general estimation of the apoptotic process, and 2) ANCA binding to the target antigen by immunogold electron microscopy (IgEM). RESULTS: Both normal and anti-PR3 IgG penetrate PMNs. The labeled PR3-ANCA were localized on PR3 granules, regardless of the granules' location within the cell, and in the sites where the PMN destruction processes were most expressed. The destructive processes showed extensive apoptotic characteristics, in contrast to PMNs penetrated by normal IgG. CONCLUSION: PR3 ANCA penetrate PMNs and, via the interaction between PR3-ANCA and PR3-containing granule components, initiate a modification of the apoptotic process.

Reactivity of peripheral blood lymphocytes to oxidized low-density lipoprotein: a novel system to estimate atherosclerosis employing the Cellscan.

BACKGROUND: The assumption that atherosclerosis involves an autoimmune response to oxidized LDL (oxLDL) is based on the presence of immunocompetent cells and immunoglobulin deposition in the atherosclerotic lesions by successful immunomodulation of the atherosclerotic process and by inhibition of experimental atherosclerosis by antioxidants. The Cellscan system is a multiparameter laser-based static cytometer that enables repeated monitoring of the fluorescence intensity (FI) and polarization (FP) of individual living cells. Analysis of intracellular fluorescein fluorescence polarization (IFFP) has previously been used to define activated lymphocyte population. HYPOTHESIS: In this study, the Cellscan apparatus has been used to monitor cellular response to oxLDL in patients with atherosclerosis and in controls. METHODS: The FI and FP of fluorescein diacetate (FDA)-labeled peripheral lymphocytes were measured following exposure to oxLDL in vitro. Using cluster analysis we were able to identify subpopulations of cells that were characterized by their FI and FP. Forty-two subjects were studied: 22 patients with severe coronary heart disease and 22 control individuals, either healthy or with other diseases. RESULTS: Fluorescence intensity of fluorescein-labeled peripheral blood lymphocytes (PBL) was markedly decreased upon exposure to high doses (> 25 micrograms/ml) of oxLDL concurrently with an increase in FP. A specific and dose-dependent reduction in FP of the high-intensity cell subpopulations, accompanied by higher FI, was evident in patients with ischemic heart disease upon exposure to low doses of oxLDL (up to 25 micrograms/ml). Maximal depolarization was shown upon triggering with 2 micrograms/ml oxLDL. The polarization ratio (the mean polarization value of the specific cell population with and without activation) obtained for patients' lymphocytes was significantly lower (p < 0.01) than that of the control group (0.936 +/- 0.05 and 1.028 +/- 0.055, respectively). CONCLUSION: These data suggest that PBL from patients with active ischemic heart disease show an increased reactivity to oxLDL. A 73% positivity rate was found for ischemic heart disease patients compared with 5% in the control subjects. One of the future prospects of this study might be the advent of a simple and rapid noninvasive test that could assess the extent of atherosclerosis, and possibly even the response to therapy, by monitoring the reactivity of PBL to oxLDL.

Concurrent inactivation of calcium dependent phosphorylation and neurotransmitter release in cultured rat brain neurons.

Depolarization of cultured rat brain neurons preloaded with 3H-dopamine provokes a transient (t 1/2 = 9.6 sec), Ca(2+)-dependent release of the labeled neurotransmitter from cells. In parallel, the amount of 32Pi incorporated into a protein of apparent molecular weight of 43,000 increased whereas the phosphorylation of a protein with an apparent molecular weight of 55,000 daltons decreased. The time course of the change in phosphorylation pattern was examined. The depolarization-induced phosphorylation of the 45,000 protein and dephosphorylation of the 55,000 dalton protein consisted of an initial, rapidly terminating phase (t 1/2 = 5 sec), and of a slow, Ca(2+)-independent phosphorylation of both proteins which persisted during maintained depolarization. The depolarization-evoked changes in the neuronal protein phosphorylation were dependent on the extracellular Ca2+ concentration (half saturation at 0.4-0.5 mM Ca2+). These data indicate that the entry of Ca2+ into the depolarized cells induces rapid phosphorylation-dephosphorylation activities. These processes terminate within 10 sec, concurrently with the depression of neurotransmitter release.

Donut-shaped chambers for analysis of biochemical processes at the cellular and subcellular levels.

In order to study cell-cell variation with respect to enzymatic activity, individual live cell analysis should be complemented by measurement of single cell content in a biomimetic environment on a cellular scale arrangement. This is a challenging endeavor due to the small volume of a single cell, the low number of target molecules and cell motility. Micro-arrayed donut-shaped chambers (DSCs) of femtoliter (fL), picoliter (pL), and nanoliter (nL) volumes have been developed and produced for the analysis of biochemical reaction at the molecular, cellular and multicellular levels, respectively. DSCs are micro-arrayed, miniature vessels, in which each chamber acts as an individual isolated reaction compartment. Individual live cells can settle in the pL and nL DSCs, share the same space and be monitored under the microscope in a noninvasive, time-resolved manner. Following cell lysis and chamber sealing, invasive kinetic measurement based on cell content is achieved for the same individual cells. The fL chambers are used for the analysis of the same enzyme reaction at the molecular level. The various DSCs were used in this proof-of-principle work to analyze the reaction of intracellular esterase in both primary and cell line immune cell populations. These unique DSC arrays are easy to manufacture and offer an inexpensive and simple operating system for biochemical reaction measurement of numerous single cells used in various practical applications.

Distinct effects of anti-tumor necrosis factor combined therapy on TH1/TH2 balance in rheumatoid arthritis patients.

The immune balance in patients with rheumatoid arthritis (RA), a disease characterized by TH1 dominance, treated by the preferred combined anti-tumor necrosis factor (anti-TNF) and methotrexate (MTX) therapy was evaluated by assessing the chemokine and cytokine receptors as well as apoptosis induction. A meta-analysis of combined therapy by TNF blockers and MTX in 15 RA patients, MTX monotherapy in 20 RA patients, and 11 diagnosed but untreated RA patients was performed by assessing several immune markers in the whole lymphocyte population, as well as in specific CD4 cells, by both flow cytometry and image analysis. A significant downregulation of CXCR3 and IL-12 receptors (both TH1 markers) and a significant increase in the chemokine receptor CCR4 and, to a lesser extent, IL-4R (both TH2 markers) were found; a particularly marked increase was found in patients treated by combined therapy. This phenomenon was pronounced in CD4 cells and was accompanied by a high proportion of apoptotic cells. The therapeutic effect of MTX and TNF blockers may be due to apoptosis induction in lymphocytes infiltrating from the inflammation site and restoring the TH1/TH2 balance.