Evaluation of antiviral activity of different origin compounds by flow cytometry

Against many viral diseases caused for example by HSV, EBV, CMV, HIV, RSV, HCV for which vaccines are not available, chemiotherapeutics seem to have the principal significance. High progress in development of new antiviral compounds is observed. In addition to synthetic compounds a large number of naturally occurring substances have been shown to posses antiviral activity. One of such substance is tannic acid. In this study comparison of antiviral activity of tannic acid, acyclovir (ACV) and ganciclovir (GCV) against cytomegalovirus (CMV) is presented. The MRC5 cells infected with CMV and treated with different compounds were analyzed by flow cytometry and cythopatic effect inhibition test for inhibition of virus replication and by MTT assay for cytotoxity. It has been shown that tannic acid has antiviral activity against cytomegalovirus and that expression of virus antigens measured as median fluorescence intensity (MFI) by flow cytometry can be used for evaluation of virus replication.     

Evaluation of platelet function by flow cytometry

Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.     

Evaluation of adenoviral vectors by flow cytometry

Biological assays for adenoviral gene therapy vectors have included conventional procedures initially developed to detect wild-type adenoviruses. Standard virological assays to quantitate adenoviruses rely on the virus to infect and replicate in the host cell until a cytopathic effect is observed. The appearance of plaques, colonies of rounded, enlarged cells containing infectious virions, usually takes 2 to 3 weeks to reach an endpoint. We describe a flow cytometric bioassay for adenovirus which shortens the time from when the infection takes place to the time that biological titer is determined. A fluorescent focus-forming assay was one of the first rapid adenoviral bioassays developed. Virus titer was determined using fluorescence immunocytochemistry to detect adenovirus proteins and microscopy to count fluorescent foci in cultures of adenovirus-infected cells. In this study, we describe a flow cytometric assay performed on cells stained for adenovirus hexon capsid protein, where virus titer is determined based on the dose-dependent appearance of hexon-positive cells. Adenovirus hexon detection in infected cells can provide data to determine virus titer, inducible promoter function in vector-complementing cells, and vector replication in complementation-deficient cells.     

Estimation of thyroid gland activity in the Snell dwarf mouse by ultrastructural observation of the thyroid gland, measurement of plasma thyroxine concentration and thyroid hormone binding capacity

An ultrastructural study of the thyroid gland of the Snell dwarf mouse showed cellular activity to be very low. Follicle cell diameters were significantly lower than in controls whilst the nucleocytoplasmic ratio was significantly higher. The observed cellular activity of the thyroid cells was associated with circulating levels of thyroxine which were found to be significantly lower than in controls. Measurement of the free thyroxine index showed very little free hormone available for tissue uptake. No differences in thyroid function due to age or sex in the dwarf mice were seen. Possible endocrine imbalances contributing to the low thyroid activity in the Snell dwarf mouse are discussed.     

Central nervous system mediated stimulation by thyrotropin-releasing hormone of microcirculation in thyroid gland of rats.

The blood flow of thyroid, adrenal cortex and renal cortex in the pentobarbital anesthetized rat was assessed from hydrogen gas desaturation curve. The microcirculation of thyroid was markedly augmented within 2 min after an intraventricular injection of Thyrotropin-Releasing Hormone (TRH) while Met-Enkephalin (ENK) failed to influence. Both TRH and ENK stimulated the microcirculation of adrenal cortex moderately. ENK diminished the microcirculation of renal cortex whereas TRH did not exert any effect. The response of thyroid to TRH was abolished by vagotomy, thus the existence of a specific TRH-vagus -thyroid connection was indicated.     

Immunohistochemical localization of thyroid hormone in rat thyroid gland.

Direct and indirect immunofluorescence techniques were used to localize the thyroid hormones triidothyronine (T3) and thyroxine (T4) in adult rat thyroid gland. Optimum dilutions of the antisera were established and four tissue fixatives were investigated for usefulness in this technique. Use of antibodies specific for either T3 or T4 resulted in brilliant fluorescence in the colloid pools and apical cytoplasm of follicular cells. In all cases, the adjacent parathyroid gland was devoid of fluorescence. This report demonstrates that these dipeptide hormones can be localized by using immunofluorescence techniques.     

Deiodase activity in rats following an overdose of thyroid hormone]

The L-DIT and L-T4 deiodinating activity in supernatants from liver or kidney homogenates of normal rats or rats loaded with T4, T3 or TSH, was investigated. Deiodination of L-DIT occurs in liver supernatants twice as much as in liver, referring to mg of protein of g of tissue, while the deiodination of L-T4 seems rather equal. The over all rate of deiodination, however, reaches in liver for L-DIT the threefold, for L-T4 the fivefold value, as compared with kidney. Short periods of loading do not alter deiodination of L-DIT in liver or kidney at all; but lead to significant elevation of L-T4-deiodination in liver tissue. Higher doses or longer periods of loading cause a significant rise of L-DIT-deiodination in liver and kidney, while the L-T4-deiodination in liver is significantly decreased and in kidney significantly elevated.     

Evaluation of fresh and frozen-thawed fowl semen by flow cytometry

The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.     

Measuring activity of endocytosis-regulating factors in T-lymphocytes by flow cytometry

Elucidation of the mechanisms regulating membrane traffic of lymphocyte receptors is of great interest to manipulate the immune response, as well as for accurately delivering drugs and nanoprobes to cells. Aiming to detect and characterize regulators of endocytosis and intracellular traffic, we have modified the FACS-based endocytosis assay to measure and quantify the activity of putative endocytic regulators as EGFP chimeras. To study the activity of putative endocytosis regulators, we transfected Jurkat T-lymphocytes with EGFP-tagged constructs of the regulators to be tested. Cells were then incubated with a αCD3^APC antibody, and were allowed to internalize the label. After acid-washing the cells, APC fluorescence was measured by flow cytometry in cells gated for EGFP⁺, as well as in their EGFP⁻ (transfection-resistant) counterparts that were taken as internal controls. This approach facilitated intra- and inter-assay normalization of endocytic rates/loads by comparison with the internal control. We have used this assay to test the regulatory activity of polarity kinase EMK1, and here we substantiate a role for EMK1 in the control of receptor internalization in T-lymphocytes. The method here presented gives quantitative measures of internalization, and will facilitate the development of tools to modulate endocytic rates or the intracellular fate of internalized materials.     

Functional activity of thyroid gland in male rats with acute and mild streptozotocin diabetes

Type 1 diabetes mellitus (DM1) and dysfunction of the thyroid gland (TG) are the most common endocrine diseases, which are interrelated. However, the molecular mechanisms of thyroid dysfunction in DM1 and the role of adenylyl cyclase signaling system (ACSS) in this process remain poorly understood. Typically for studying etiology and pathogenesis of thyroid diseases in DM1 the models of acute DM1 induced by high doses of streptozotocin (STZ) are used. At the same time, a suitable model for this purpose is the model of mild DM1 initiated by moderate doses of STZ, which more closely resembles human DM1. The aim of this study was a comparative study of the functional state of the thyroid gland in rats with 30-day acute DM1 induced by injection of STZ at a dose of 65 mg/kg, and in rats with 30- and 210-day mild DM1 induced by three consecutive injections of STZ at medium doses (30–40 mg/kg). For this purpose in diabetic animals the levels of thyroid hormones and TSH and the functional activity of hormone-sensitive ACSS in membranes isolated from thyroid gland were studied. It was shown that in blood of rats with acute DM1 the levels of fT₄, fT₃, and tT₃ were decreased by 45, 23 and 19%, respectively, while the level of TSH did not change significantly. In rats with the 30-day mild DM1 the concentration of fT₄ was decreased by 32%, while the levels of tT₄, tT₃, and TSH were similar to that in control. In rats with prolonged mild DM1 after 150 and 210 days following the first treatment with STZ the levels of tT₄, fT₄, and tT₃ were significantly reduced, but the concentration of TSH in rats with 210-day mild DM1 was increased by 119%. The results obtained in the study of thyroid status and TSH levels in rats with prolonged mild DM1 are in good agreement with the data obtained in the study of thyroid diseases in patients with DM1. It was found that the AC basal activity in the membranes isolated from the thyroid gland of diabetic rats did not change, except for the rats with the prolonged mild DM1 where this activity was increased by 21%. In all groups of diabetic rats the decrease of AC stimulating effects of GppNHp (10^?5 M) and TSH (10^?8 M) was found, and in the rats with prolonged mild DM1 the AC effect of PACAP-38 (10^?6 M) was also reduced. The decrease of AC effect of TSH varied among different groups of the diabetic animals: in the rats with acute DM1 this effect was reduced by 46% and in the rats with 30- and 210-day mild DM1-by 18 and 34%. Thus, it was concluded that the key cause of the thyroid resistance to TSH under conditions of DM1 is a weakening of the signal transduction generated by TSH via the ACSS.     

In vitro protein kinase activity measurement by flow cytometry

Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-μ, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample.     

Detection of acid-beta-galactosidase activity in viable human fibroblasts by flow cytometry

The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was used to detect acid beta-galactosidase in intact cultured human fibroblasts. The accumulation of intracellular fluorescein, as measured by flow cytophotometry was linear with the incubation time in three control strains. The two fibroblast strains from patients with acid beta-galactosidase deficiency did not show an accumulation of intracellular fluorescence. Within one control cell population there was a positive correlation between the amount of accumulated intracellular fluorescein fluorescence and the specific acid beta-galactosidase activity as measured biochemically on sorted cells from different zones of the fluorescence distribution. No correlation was found between the specific acid beta-galactosidase activity and the fluorescein fluorescence of three different control cell strains.     

Evaluation of androgen action on fibroblast growth by flow cytometry

The action of androgen (dihydrotestosterone-DHT-) on fibroblast growth was evaluated by 3H thymidine incorporation in DNA, by DNA assay using 3-5 diaminobenzoic acid fixation, and by a more sophisticated technique: flow cytometry. Cell DNA and proteins were stained with propidium iodide and fluorescein isothyocyanate, respectively. We did not observe any detectable DNA variation when fibroblats were incubated in the presence of DHT. Moreover, DHT did not modify DNA and protein distribution, either in the total cell cycle or in each phase of the cell cycle. These results suggest that androgens do not induce total protein synthesis nor increase DNA in target cells. It is likely that they induce specific protein synthesis.     

Stimulation by thyrotropin-releasing hormone of vagal outflow to the thyroid gland

An intracerebroventricular (i.c.v.) injection of TRH to the urethane anesthetized rat stimulates the activity of the superior laryngeal nerve (n.sl) which is a vagal ramus terminating at the thyroid gland and adjacent muscles. The response to TRH, a tonic increase in the n.sl outflow, was dose dependent in the 0.005-5.0 micrograms/100 g B.W. range. In contrast to this, methionine-enkephalin (ENK), neurotensin (NT) and somatostatin (SRIF) (5 micrograms/100 g, i.c.v.) all caused a transient decrease in n.sl activity. SRIF showed the highest attenuating effect when injected alone and was capable of diminishing the increased activity produced by a prior injection of TRH. ENK and NT failed to affect the TRH-induced increased activity. When injected concomitantly with TRH, SRIF blocked the response to TRH while ENK and NT both failed to affect the response to TRH. Pretreatment with triiodothyronine for 5 days strongly inhibited the response of the n.sl outflow to TRH. On the other hand, pretreatment with atropine, haloperidol, propranolol, phenoxybenzamine and p-chlorophenylalanine failed to block the stimulating effect of TRH although the response was diminished by some antagonists. It therefore seemed that TRH transmission is involved in central stimulation and SRIF is antagonistic in this regulation of n.sl outflow to the thyroid gland.