Development of Indirect Immunofluorescence Technique for the Identification of MRC5 Working Seed Cell.

Diploid and continuous cell lines are used to propagate viral vaccines. At Human Viral Vaccine Department of Razi Vaccine and Serum Research Institute, MRC5 diploid cell is used for the development of live attenuated measles, mumps, rubella, and three types of poliovirus vaccines. Additionally, three continuous cell lines (i.e., RK13, HeLa, and Vero) are applied in quality control tests. Accordingly, cell cross-contamination can occur at cell culture labs, hence controlling the identity and specificity of cells is essential. Indirect immunofluorescence is a sensitive, specific, and simple test for cell identification. The present study was designed to develop the in-house indirect immunofluorescence test (IIF) as follows: homemade polyclonal anti-MRC5 serum was prepared in rabbits, and cross-reactive antibodies to RK13, HeLa, and Vero cells were eliminated. The diploid and continuous cell lines were fixed on Teflon slide using cold methanol and acetone. The reproducibility of the in-house IIF test was evaluated using the agreement Kappa test. The purity of the three batches of MRC5 working seed cell at Human Viral Vaccine Department of Razi institute was verified using IIF and no contamination with continuous cell lines was detected.

Association of serum gamma-glutamyltransferase and premature coronary artery disease.

BACKGROUND: Serum gamma-glutamyltransferase (GGT) has been introduced as a predictive factor for cardiovascular disease. In this study, we investigated the association of serum GGT and premature coronary artery disease (CAD) in candidates for coronary angiography. METHODS: In this cross-sectional study, we enrolled male subjects aged ≤45 years and female subjects ≤55 years who were candidates for elective coronary angiography due to typical chest pain or a positive non-invasive test. Baseline characteristics were recorded for all the participants and serum levels of blood glucose, lipid profile and GGT were measured. Patients were divided into CAD and non-CAD groups based on angiography for further comparisons. RESULTS: From a total of 367 patients (age 45.1 ± 6.1 years, 161 males [43.9%]), 176 (47.9%) patients had premature CAD. A high level of GGT was significantly associated with the presence of CAD (p < 0.001). A 10-unit increase in GGT could strongly predict the presence of premature coronary artery disease (OR: 13.34, 95% CI: 7.19-24.78; p < 0.001) after adjustment for confounders. The area under the receiver operating characteristic curve for GGT was 80.9% (range 76.5-85.3) and the sensitivity and specificity of GGT at a cut-point of 22.5 IU/l was 80.1% and 70.2%, respectively. Diagnostic accuracy of GGT was 74.9%. The positive predictive value and negative predictive value for GGT was 71.3 and 79.3, respectively. CONCLUSION: We observed that GGT levels in patients with typical chest pain or positive non-invasive tests could predict the presence of premature CAD in young patients.

IFPA meeting 2015 workshop report IV: placenta and obesity; stem cells of the feto-maternal interface; placental immunobiology and infection.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At the 2015 IFPA annual meeting there were 12 themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology and collectively covered areas of obesity and the placenta, stem cells of the feto-maternal interface, and placental immunobiology and infection.

Turn-on fluorescent chemosensor for determination of lutetium ion.

A turn-on fluorescent chemosensor is introduced for the detection of Lu(3+) ion using N-[3-methyl]-2-[pyridine-2-amido] phenyl] pyridine-2-carboxamide (L) molecule. Fluorescent emission intensity of L enhances after binding to Lu(3+) ions in ethanol-water solution (1:9, v/v). The observed enhancement is the result of a strong covalent binding between Lu(3+) ion and L (the binding constant value is 2.0×10(6) mol(-1) L). The proposed optical chemosensor can be applied for the analysis of Lu(3+) ion in a linear range of 3.3×10(-7) to 1.0×10(-5) mol L(-1). The limit of detection was obtained 8.6×10(-7) mol L(-1). The probe exhibits high selectivity toward Lu(3+) ion in comparison with common metal ions. The proposed fluorescent chemosensor was successfully used in the determination of Lu(3+) ion in some water samples.

Predictors of delayed and no-reflow as recognized with Thrombolysis in Myocardial Infarction [TIMI] flow grade following Primary Percutaneous Coronary Angioplasty.

Background: Initial percutaneous coronary interference (PCI) is still connected by a noticeable incidence of suboptimal coronary flow thrombolysis in infarction of myocardial (TIMI). The predictors of slow and no-reflow in cases that supported initial PCI in our institute was searched for and the relationship of these parameters with major adverse cardiovascular effects (MACE) was assessed. Material and Method: 397 patients with AMI displaying in 24 hours of the sign opening were retrospectively enrolled and underwent primary PCI between March 2006 and March 2012. Demographic, clinical, and procedural data were retrieved from our institutional databank. The baseline and post-PCI flow of blood in the revascularized artery was ranked based on the TIMI grading method. The follow-up visits were performed after one, six and twelve month from hospitalization. All the mortalities and complications were recorded within this period for evaluate the MACE. Results: The frequency of diabetes mellitus and renal failure were importantly larger in cases with a TIMI flow of 0-1 (p=0.03 & p=.01, respectively). Similarly, level of serum creatine were importantly larger in cases with a TIMI flow of 0-1. The predictors for TIMI flow included that utilize of Adenosin or Integrilin, diabetes mellitus, POIT, long tubular lesion, and injury at LAD territory. The incidence of MACE was significantly higher in patients with a TIMI flow of 0-1 (P=0.001) and the survival in this subgroup was significantly poorer (Hazard ratio=4.96; P<0.001). Conclusion: A low TIMI flow is accompanied by a poorer survival and a higher MACE and is influenced by some clinical and vascular characteristics.

Novel isolation strategy to deliver pure fetal-origin and maternal-origin mesenchymal stem cell (MSC) populations from human term placenta.

The placenta is an abundant source of mesenchymal stem/stromal cells (MSC). Although presumed of translationally-advantageous fetal origin, the literature instead suggests a high incidence of either contaminating or pure maternal MSC. Despite definitional criteria that MSC are CD34-, increasing evidence suggests that fetal MSC may be CD34 positive in vivo. We flow sorted term placental digests based on CD34+ expression and exploited differential culture media to isolate separately pure fetal and maternal MSC populations. This method has considerable translational implications, in particular to clinical trials underway with "placental" MSC of uncertain or decidual origin.

Selective recognition of Pr3+ based on fluorescence enhancement sensor.

(E)-2-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)hydrazinecarbothioamide (L) has been used to detect trace amounts of praseodymium ion in acetonitrile-water solution (MeCN/H2O) by fluorescence spectroscopy. The fluorescent probe undergoes fluorescent emission intensity enhancement upon binding to Pr(3+) ions in MeCN/H2O (9/1:v/v) solution. The fluorescence enhancement of L is attributed to a 1:1 complex formation between L and Pr(3+), which has been utilized as the basis for selective detection of Pr(3+). The sensor can be applied to the quantification of praseodymium ion with a linear range of 1.6×10(-7) to 1.0×10(-5) M. The limit of detection was 8.3×10(-8) M. The sensor exhibits high selectivity toward praseodymium ions in comparison with common metal ions. The proposed fluorescent sensor was successfully used for determination of Pr(3+) in water samples.

Decoherence effects on superpositions of chiral states in a chiral molecule.

The superposition of chiral states of chiral molecules, as delocalized quantum states of a many-particle system, can be used for the experimental investigations of decoherence theory. In this regard, a great challenge is the precise quantification of the robustness of these superpositions against environmental effects. The methods so far proposed need the detailed specification of the internal states of the molecule, usually requiring heavy numerical calculations. Here, by using the linearized quantum Boltzmann equation and by borrowing ideas employed for analyzing other quantum systems, we present a general and simple approach, of wide applicability, which can be used to compute the dominant contribution to the decoherence rate for the superpositions of chiral states of chiral molecules, due to environmental scattering.

In vitro antibacterial activity of some Iranian medicinal plant extracts against Helicobacter pylori.

Helicobacter pylori infection causes lifelong chronic gastritis, which can lead to peptic ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. The growing problem of antibiotic resistance by the organism demands the search for novel candidates from plant-based sources. In the present study, we evaluated the in vitro anti-H. pylori activity of some selected medicinal plants on clinical isolates of H. pylori. Gastric biopsy samples were obtained from patients presenting with gastroduodenal complications. Helicobacter pylori was isolated from the specimens following standard microbiology procedures. The disc-diffusion method was used to determine the susceptibility of three H. pylori isolates to methanol extracts of 23 Iranian plants. All tests were performed in triplicate. Among them, the extracts of Punica granatum and Juglans regia had remarkable anti-H. pylori activity with mean of inhibition zone diameter of 39 and 16 mm at 100 µg disc⁻¹, respectively. In view of the results obtained with P. granatum (pomegranate), the peel extracts of nine cultivars of pomegranate (Shirin-e-Pust Sefid, Agha Mohammad Ali-e-Shirin, Sefid-e-Shomal, Sefid-e-Torsh, Shirin-e-Malase, Tabestani-e-Torsh, Shirin-e-Saveh Malase, Alak-e-Shirin, Pust Siyah) were further assayed against the clinical isolates of H. pylori. The results revealed that all Iranian pomegranate cultivars, except for Alak-e-Shirin, showed significant in vitro anti-H. pylori activity against the clinical isolates of H. pylori (mean of inhibition zone diameter ranging from 16 to 40 mm at 50 µg disc⁻¹).

Early spontaneous immortalization and loss of plasticity of rabbit bone marrow mesenchymal stem cells.

OBJECTIVES: Bone marrow-derived mesenchymal stem cells (BM-MSC) have been widely used for cell therapy and tissue engineering purposes. However, there are still controversies concerning safety of application of these cells after in vitro expansion. Therefore, we aimed to investigate the characteristics of rabbit BM-MSC during long-term culture. MATERIALS AND METHODS: In this study, we have examined growth kinetics, morphological changes, differentiation potential and chromosomal abnormalities, as well as tumour formation potential of rabbit BM-MSC in long-term culture. RESULTS AND CONCLUSION: We found that shortly after isolation, proliferation rate of rabbit BM-MSC decreases until they enter a dormant phase. During this period of quiescence, the cells are large and multinucleate. After some weeks of dormancy we found that several small mononuclear cells originated from each large multinucleate cell. These newly formed cells proliferated rapidly but had inferior differentiation potential. Although they were immortal, they did not have the capability for tumour formation in soft agar assay or in nude mice. This is the first report of spontaneous, non-tumorigenic immortalization of BM-MSC in rabbits. The phenomenon raises more concern for meticulous monitoring and quality control for using rabbit BM-MSC in cell-based therapies and tissue engineering experiments.

Synthesis and cloxacillin antimicrobial enhancement of 2-methylsulfonylimidazolyl-1,4-dihydropyridine derivatives.

BACKGROUND AND THE PURPOSE OF THE STUDY: Hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) has been a major problem worldwide in chemotherapy of infection disease. This study was designed to assess the enhancing effects of a new group of dihydropyridine-3,5dicarboxamides, in combination with cloxacillin with distinctly different mechanisms of action against MRSAs. MATERIAL AND METHODS: Dihydropyridine-3,5-dicarboxamides with 2-methylsulfonylimidazole at 4 position 6a-k were synthesized by the reaction of corresponding aldehyde 5 with different N-aryl acetoacetamides 3 in the presence of ammonium hydroxide. Agar disc diffusion method was used to determine the antibacterial and potentiating activity of different synthetic compounds in the presence and absence of cloxacillin to evaluate their activity as modulators of multidrugresistant (MDR). RESULTS AND MAJOR CONCLUSION: The antibacterial effect of cloxacillin was enhanced by compounds 6g and 6h against cloxacillin-resistant strains (MRSA(1) and MRSA(2)). The potentiation was found 1 2 to be statistically significant (p<0.01). Compound 6g at concentration of 1000 µg/disc, caused a 329 percent potentiation of the activity of cloxacillin against MRSA(1).

Synthesis and cytotoxic activity of some 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes.

A series of 2-amino-4-aryl-3-cyano-7- (dimethylamino)-4H-chromenes was synthesized by condensation of 3- (dimethylamino) phenol, an aromatic aldehyde and malonitrile in ethanol containing piperidine. The assignments of the structure of all synthesized compounds were based on spectral data (IR, Mass and(1)H NMR). The cytotoxic activities of the synthesized compounds against six human tumor cell lines were determined by MTT assay. Several compounds showed significant cytotoxic activity.

The effects of deferiprone and deferasirox on the structure and function of beta-thalassemia hemoglobin.

Transfusional iron overload is a major cause of morbidity and mortality in thalassemia, sickle-cell disease and other chronic anemias. To overcome these problems, orally bio available iron chelators, deferiprone and deferasirox, were used for the treatment of patients suffering from thalassemia. The interactions between deferiprone and deferasirox with the carrier protein, beta-thalassemia hemoglobin (Hb), were investigated using fluorescence, circular dichroism (CD) and UV-visible measurements at physiological condition. Strong fluorescence quenching on interactions of the above drugs with beta-thalassemia Hb were observed. Fluorescence quenching data of thalassemia Hb in the presence of deferasirox have shown greater affinity of binding. The number of binding sites to Hb for deferasirox was found to be more relative to those of the deferiprone. The effects of these drugs on the oxygen affinity of the thalassemia Hb were studied by spectroscopic methods using sodium dithionite. Results indicated that deferiprone reduces oxygen affinity (increases oxygen releasing ability) of Hb, while in the presence of deferasirox, oxygen affinity of Hb has significantly increased by dose-dependent manner. As such, deferasirox exhibited opposite effect relative to deferiprone on the function of thalassemia Hb. In clinical dose of deferiprone, CD results showed that, the alpha-helical content of thalassemia Hb significantly increased. By use of the clinical dose of deferasirox, however, a decrease in alpha-helical content of protein was observed, which resulted in decreasing stability of thalassemia Hb. Our study showed that reduction in stability of thalassemia Hb in the presence of deferasirox induced higher conformational changes in protein.

Assessment of endogenous androgen levels in meat, liver and testis of Iranian native cross-breed male sheep and bull by gas chromatography-mass spectrometry.

Androgenic steroids always exist in different animal tissues at trace level, with significant numbers of interfering compounds, which makes their determination difficult. To solve some of the problems in quantification of the natural steroids in those tissues, a new GC-MS method was developed in this study. By using a surrogate analyte approach, which was developed in the authors' previous studies, and extensive sample preparation procedure, which successfully eliminates many of the interfering compounds and resulting in a cleaner extract, accuracy, precision, sensitivity and selectivity of the method for the determination of steroids in complex matrices such as meat, liver and testis were improved. By aid of this method, the levels of androgens in different tissues of Iranian native cross-breed bulls and male sheep were determined. According to the results obtained in the present study, although the androgenic profile (contents and ratios of precursors and metabolites to the main hormones) is similar between the same tissues of both animals, the total androgenic content of each tissue is higher in the bull than the same tissue in male sheep. In addition, in both animals higher amount of androgens were found in liver in comparison with meat and testis.

Effects of novel antituberculosis agents on OmpF channel activity.

Nanopore forming proteins spanning the outer membrane mediate in the diffusion of hydrophilic chemicals through the hydrophobic bacterial cell wall. In this study, the effects of two novel anti-TB derivatives, ethyl alpha-[5-(5-nitro-2-thienyl)-1,3,4-thiadiazole-2-ylthio] acetates and propyl alpha-[5-(5-nitro-2-thienyl)-1,3,4-thiadiazole-2-ylthio] acetates, on OmpF channel reconstituted in artificial bilayers were evaluated by voltage clamp technique. Surprisingly, ethyl derivative (MIC > or = 6.75 microg/ml) showed no effects on OmpF channel activity but the propyl derivative (MIC=0.39 microg/ml) reduced the channel conductance considerably and changed the gating pattern of the channel. The findings obtained here at molecular level, might shed light on better understanding of the actual mechanism(s) by which the novel anti-TB agents permeate through the cell wall of the Mycobacterium tuberculosis.

Structure-activity relationship study of a series of N-substituted piperazinyl-fluoroquinolones as anti-Helicobacter pylori agents.

Helicobacter pylori is now recognized as the primary etiological factor associated with gastritis, peptic ulcer disease and gastric cancers. Fluoroquinolones have been shown to be active against H. pylori. For develop new anti-H. pylori agents, we have investigated the SAR of a series of N-(phenethyl)piperazinyl quinolones for their antimicrobial activity against H. pylori. The anti-H. pylori activity of synthesized compounds along with commercially available anti-H. pylori agents such as metronidazole, and parent quinolones was evaluated by the disc diffusion bioassay. The results indicated that the potency and anti-H. pylori activity profile of the quinolones is highly dependent on the type of substituent at N-1 and the structure of phenethyl unit on piperazine ring. Most compounds containing a cyclopropyl at N-1 exhibited good activity against H. pylori strains. Among them, ciprofloxacin derivative 13 containing 2-methoxyimino-2-(2-chlorophenyl)ethyl moiety was the most active compound.

Electrocatalytic oxidation and determination of deferasirox and deferiprone on a nickel oxyhydroxide-modified electrode.

The electrocatalytic oxidation of two orally administered iron chelator drugs (deferiprone, CP20, and deferasirox, ICL670) was investigated on a nickel oxyhydroxide-modified nickel electrode in alkaline solution. The oxidation process involved and its kinetics were investigated using cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy techniques, as well as steady-state polarization measurements. Voltammetric studies indicated that in the presence of the drugs under study, the anodic peak current of low-valence nickel species increased, followed by a decrease in the corresponding cathodic current. This result indicates that the drugs were oxidized via oxyhydroxide species immobilized on the electrode surface via an EC' mechanism. A mechanism based on the electrochemical generation of Ni(III) active sites and their subsequent consumption by the drugs in question was also investigated. The corresponding rate law under the control of charge transfer was developed, and kinetic parameters were derived. In this context, the charge-transfer resistance accessible both theoretically and through impedancemetry was used as a criterion. The rate constants of the catalytic oxidation of the drugs and the electron-transfer coefficients are reported. A sensitive, simple, and time-saving amperometric procedure was developed for the analysis of deferasirox and deferiprone, with detection limits of 28 and 19 microM, respectively. The electrode was used for the direct assay of deferasirox and deferiprone tablets.

Evaluation of anticancer effects of newly synthesized dihydropyridine derivatives in comparison to verapamil and doxorubicin on T47D parental and resistant cell lines in vitro.

Failure of current anticancer drugs mandates screening for new compounds of synthetic or biological origin to be used in cancer therapy. Multidrug resistance (MDR) is one of the main obstacles in the chemotherapy of cancer. Efflux of cytotoxic agents mediated by P-glycoprotein (P-gp or MDR1) is believed to be an important mechanism of multidrug resistance. Therefore, we decided to investigate the antiproliferative effects of seven newly synthesized 1,4-dihydropyridine (DHP) derivatives in comparison to verapamil (VP) and doxorubicin (DOX) on human breast cancer T47D cells and its MDR1 overexpressed and moderately resistant cells (RS cells) using MTT cytotoxicity assay. We also examined the effects of these compounds on cytotoxicity of DOX in these two cell types. The cytotoxicity assays using MTT showed that most of the tested new DHP derivatives and VP at 10 microM concentration had varying levels of toxicity on both T47D and RS cells. The toxicity was mostly in the range of 10-25%. However, the cytotoxicity of these DHP derivatives, similar to VP, was significantly less than DOX when comparing IC(50) values. Furthermore, these compounds in general had relatively more cytotoxicity on T47D vs RS cells at 10-microM concentration. Among new DHPs, compounds 7a (3,5-dibenzoyl-4-(2-methylthiazol-4-yl)-1,4-dihydro-2,6-dimethylpyridine) and 7d (3,5-diacetyl-4-[2-(2-chlorophenyl)thiazol-4-yl)]-1,4-dihydro-2,6-dimethylpyridine) showed noticeable potentiation of DOX cytotoxicity (reduction of DOX IC(50)) compared to DOX alone in both cells, particularly in RS cells. This effect was similar to that of VP, a known prototype of MDR1 reversal agent. In other words, compounds 7a and 7d resensitized RS cells to DOX or reversed their resistance. Results indicate that compound 7d exerts highest effect on RS cells. Therefore, these two newly synthesized DHP derivatives, compounds 7a and 7d, are promising as potential new MDR1 reversal agents and should be further studied on other highly resistant cells due to MDR1 overexpression and with further molecular investigation.

Antigen-specific lymphocyte proliferation assay and virus neutralization test for measurement of measles-specific immunity in 15-19 years old high school students in Tehran, Iran.

Limited information is available concerning the role of measles-specific cell mediated immunity as a correlate of long-term protection from measles infection. Although serological responses are determined in epidemiological studies and high antibody titer is a good indicator of protection, the role of Cell-Mediated Immunity (CMI) has to be defined more clearly. In this study, Lymphocyte Proliferation (LP) and Viral Neutralization Test (VNT) were used in order to measure measles-specific cellular and humoral immune responses of 100 high school students in Tehran. From total number of subjects studied, 33 were girls and 67 were boys and all were in good health. Of these, 77 had protective neutralizing measles antibody titers and 23 did not have such titer. The results of LP showed that 89 subjects had protective cellular immune responses and 11 did not. A quantitative relationship between humoral and cellular immune responses was not observed. These findings suggest that measles-specific protective CMI is measurable for longer time in comparison to humoral immunity. These data suggest that LP responses may be better sustained than antibody titers in some children.

The application of Shannon's measure of information for a complex chemical system.

Information theory and, specifically, Shannon's measure of information is used to compare the interaction parameters (beta) in regular solution theory (RST) and Ingram's model for the mixture of cetyltrimethylammonium bromide (CTAB) and Triton X-100 (TX100). Results show that beta values from regular solution theory are more accurate than those of Ingram's model. Additionally, the procedure applied in this paper for the calculation of uncertainties in the continuous case, prevents difficulties concerning differential entropy.