Free fatty acid perturbation of transmembrane signaling in cytotoxic T lymphocytes

Transmembrane signaling in CTL is found to be extremely sensitive to short term exposure to long chain free fatty acids (FFA). Both alloantigen specific target cells and the lectin Con A were used to stimulate cloned murine CTL. This stimulation was monitored by changes in intracellular calcium concentrations ([Ca2+]i) using the fluorescence indicator fura-2. Treatment of the CTL cells with oleic acid (18:1) at concentrations corresponding to less than 10% (mol/mol) bound to the cell, completely inhibits target cell or Con A-mediated rise in [Ca2+]i. The inhibitory effect of oleic acid is observed within seconds of addition and the inhibition is completely reversed by treating cells with fatty acid free BSA. In addition, using the fluorescence indicator 2',7'-bis(carboxyethyl)carboxyfluorescein to monitor intracellular pH, it was found that oleic acid itself acidifies the cytosol by about 0.3 to 0.4 pH units. Acidification is probably necessary, but is not sufficient to inhibit the calcium rise. Stearic acid (18:0), even at concentrations that correspond to a factor of two to three more bound to the cell than for oleic acid, had no effect on either the [Ca2+]i or intracellular pH responses. Oleic acid was found to bind to cells with single site kinetics and with a number of sites and affinity corresponding to membrane lipid binding sites. Esterification of added oleic acid was negligible in the time (seconds to minutes) required to induce inhibition of the [Ca2+]i response. Inasmuch as added FFA primarily binds to membrane lipid, is not appreciably esterified, and the inhibition is reversed by treatment with fatty acid free BSA, it is likely that the oleic acid effects are due to a physical perturbation of membrane lipid. Furthermore, oleic acid does not affect Con A binding or the production of inositol phosphate metabolites, suggesting that the inhibition of the response is distal to surface recognition events or receptor-phospholipase C coupling. Given the relatively low levels of FFA at which these effects occur it is possible, under conditions in which FFA levels are elevated, that FFA perturbation may modulate CTL activity.     

Free fatty acids are produced in and secreted from target cells very early in cytotoxic T lymphocyte-mediated killing

Conjugation of CTL with their cognate targets elicits a number of early changes within the target cell that are thought to play an important role in the lytic mechanism. We now report that at times earlier than 5 min after conjugation with allospecific CTL, free fatty acids (FFA) are produced in and then secreted from alloantigen-bearing target cells. Using murine CTL clones with different alloantigen specificities, stimulation of FFA production from target cells was found to be Ag specific. FFA production does not appear to be specific for any particular FFA species. Indeed, a wide spectrum of cis unsaturated as well as saturated FFA are produced. FFA production is well correlated with, and specific for, CTL-mediated target cell lysis. Other means of perturbing or lysing target cells, including freeze/thaw disruption, detergent solubilization, or increasing membrane permeabilization with ionomycin, do not stimulate FFA production. In particular, FFA production is not stimulated by treatment with pore-forming granules under conditions that cause more than 90% target cell lysis. These results suggest that FFA production plays an important role in CTL-mediated lysis because stimulation of FFA release specifically requires an event that is CTL induced, occurs very early after conjugation, and is strongly correlated with CTL-mediated lysis.     

Neurocognition,Insight and Medication Nonadherence in Schizophrenia: A Structural Equation Modeling Approach

Objective

The aim of this study was to examine the complex relationships among neurocognition, insight and nonadherence in patients with schizophrenia.

Methods

Design: Cross-sectional study. Inclusion criteria: Diagnosis of schizophrenia according to the DSM-IV-TR criteria. Data collection: Neurocognition was assessed using a global approach that addressed memory, attention, and executive functions; insight was analyzed using the multidimensional ‘Scale to assess Unawareness of Mental Disorder;’ and nonadherence was measured using the multidimensional ‘Medication Adherence Rating Scale.’ Analysis: Structural equation modeling (SEM) was applied to examine the non-straightforward relationships among the following latent variables: neurocognition, ‘awareness of positive symptoms’ and ‘negative symptoms’, ‘awareness of mental disorder’ and nonadherence.

Results

One hundred and sixty-nine patients were enrolled. The final testing model showed good fit, with normed χ² = 1.67, RMSEA = 0.063, CFI = 0.94, and SRMR = 0.092. The SEM revealed significant associations between (1) neurocognition and ‘awareness of symptoms,’ (2) ‘awareness of symptoms’ and ‘awareness of mental disorder’ and (3) ‘awareness of mental disorder’ and nonadherence, mainly in the ‘attitude toward taking medication’ dimension. In contrast, there were no significant links between neurocognition and nonadherence, neurocognition and ‘awareness of mental disorder,’ and ‘awareness of symptoms’ and nonadherence.

Conclusions

Our findings support the hypothesis that neurocognition influences ‘awareness of symptoms,’ which must be integrated into a higher level of insight (i.e., the ‘awareness of mental disorder’) to have an impact on nonadherence. These findings have important implications for the development of effective strategies to enhance medication adherence.     

Cell cycle analysis with bromodeoxyuridine pulselabeling in asynchronous hybridoma cultures

Summary The cell cycle kinetics of a mouse hybridoma was examined by immunocytochemical staining of incorporated bromodeoxyuridine in an asynchronous culture. The cell cycle phase traverse times were extracted from a time series of bivariate distributions of incorporated bromodeoxyuridine and total DNA content; the G₁, S and G₂/M phase traverse times were 7, 9 and 4 h, respectively in the exponential growth phase.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

Weekly vs. tri-weekly cisplatin based chemoradiation in carcinoma cervix: a prospective randomized study of toxicity and compliance

BackgroundAddition of chemotherapy to radiation has improved 5-year survival by 6%. However, the optimal dose and schedule of concurrent cisplatin is not well defined, though widely accepted practice is the weekly schedule of 40 mg/m² for 5 weeks. Repeated admissions for weekly cisplatin drain the limited resources in high volume centres. We intended to study the compliance and toxicity of two cisplatin schedules in our patients diagnosed with carcinoma cervix.Materials and methodsBetween 2007–2011, 212 patients, histologically proven squamous cell carcinoma with stages IIB to IIIB were randomized into two arms. All patients were planned for external beam radiotherapy 45 Gy/25 frs over 5 weeks followed by Intracavitary or Interstitial brachytherapy to a total BED dose of 75–85 Gy. Single agent cisplatin given concomitantly, was scheduled weekly (40 mg/m²/cycle, 5 cycles) in an arm A and three weekly (100 mg/m²/cycle, 2 cycles) in an arm B. Toxicity and compliance were evaluated weekly according to the RTOG guidelines. Analysis of the compiled data was done using SSPS version 20.ResultsOf the evaluable 212, 109 patients received weekly cisplatin chemotherapy and 103 patients received three weekly cisplatin. The most common acute toxicity observed was grade I–II leucopoenia. The upper and lower gastrointestinal reactions were high in three weekly arms, which was statistically significant (57% and 42.7%, p < 0.05). Proctitis was observed in 10% of patients in both of the arms and only two patients had Gr1 Cystitis after 6 months of treatment.ConclusionsTri-weekly cisplatin based concurrent chemoradiation can be adopted in high volume centres with manageable haematological and gastrointestinal acute toxicities.     

Short term exposure to cis unsaturated free fatty acids inhibits degranulation of cytotoxic T lymphocytes

Degranulation of CTL stimulated by alloantigen-bearing target cells is shown to be inhibited by short term exposure to low concentrations of long chain cis unsaturated free fatty acids (FFA), whereas saturated FFA have no effect. The Ag-specific (TCR mediated) stimulation of cloned murine CTL was monitored by changes in intracellular calcium concentrations [( Ca2+]i) using the fluorescence indicator acetoxymethylester of fura-2 and by degranulation as measured by the release of BLT-esterase. Treatment of the CTL cells with any of the physiologically important FFA; oleic (18:1), linoleic (18:2), linolenic (18:3), or arachidonic (20:4) acid, at concentrations between 1 and 10 microM inhibits the target cell-mediated rise in [Ca2+]i which occurs within seconds of stimulation and the release of BLT-esterase, which occurs over a period of 1 to 3 h. These inhibitory effects are observed within seconds to minutes of FFA addition. Inhibition can be reversed by treating cells with fatty acid free BSA and, in agreement with our previous studies, indicates that the effects of FFA are due to physical perturbations of cellular components. To determine the locus of this perturbation, the effect of FFA on the lipid order of CTL plasma membrane was determined using fluorescence polarization of the membrane impermeable probe trimethylammoniumdiphenylhexatriene. Cis unsaturated FFA were found to disorder the lipid acyl chains and the degree of disorder was found to increase with the degree of cis unsaturation. These results, together with the previous studies, suggest that inhibition results from a physical perturbation of plasma membrane lipid order. Moreover, because degranulation requires elevated levels of [Ca2+]i, it is likely that inhibition of degranulation results from a FFA-induced decrease in Ca2+ permeability through the membrane.     

Genetic diversity and relationships in mulberry (genusMorus) as revealed by RAPD and ISSR marker assays

Background  

The genus Morus, known as mulberry, is a dioecious and cross-pollinating plant that is the sole food for the domesticated silkworm, Bombyx mori. Traditional methods using morphological traits for classification are largely unsuccessful in establishing the diversity and relationships among different mulberry species because of environmental influence on traits of interest. As a more robust alternative, PCR based marker assays including RAPD and ISSR were employed to study the genetic diversity and interrelationships among twelve domesticated and three wild mulberry species.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.