Thiosemicarbazone and thiazolylhydrazones of 1-indanone: as a new class of nonacidic anti-inflammatory and antiplatelet aggregation agents

This research based on the anti-inflammatory and antiplatelet aggregation properties of some new thiazolyl hydrazone derivatives of 1-indanone. In this regard a thiosemicabazone and twelve thiazolyl derivatives of 1-indanone have been synthesized. Out of these synthetic compounds seven derivatives 1-3, 6, 11-13 exhibited varying degree of anti-inflammatory action with IC50 esteems going from 5.1+/-1.3 - 78.8+/-4.6µM/mL. Compound 1 [IC50 =5.1+/-1.9µM] displayed potent result than standard ibuprofen [IC50 = 11.2+/-1.9 µM]. In antiplatelet aggregation assay, five compounds 1, 5, 6, 8 and 11 were observed to be dynamic with IC50 esteems observed in the range of 38.34-255.7+/-4.1µM, whereas, aspirin [IC50 = 30.3+/-2.6 µM] was used as standard. However, compound 11 was found to be good active for both anti-inflammatory and antiplatelet aggregation activities [IC50 = 13.9+/-4.9µg/mL] [IC50 = 38.60+/-3.1µM], respectively

Promotion of excisional wound repair by a menstrual blood-derived stem cell-seeded decellularized human amniotic membrane

This is the first study demonstrating the efficacy of menstrual blood-derived stem cell (MenSC) transplantation via decellularized human amniotic membrane (DAM), for the promotion of skin excisional wound repair. The DAM was seeded with MenSCs at the density of 3 × 10⁴ cells/cm² and implanted onto a rat's 1.50 × 1.50 cm² full-thickness excisional wound defect. The results of wound closure and histopathological examinations demonstrated that the MenSC-seeded DAM could significantly improve the wound healing compared with DAM-treatment. All in all, our data indicated that the MenSCs can be a potential source for cell-based therapies to regenerate skin injuries.

Menstrual blood-derived stromal stem cells augment cd4+ t cells proliferation

Background: It is more than sixty years that the concept of the fetal allograft and immunological paradox of pregnancy was proposed and in this context, several regulatory networks and mechanisms have been introduced so far. It is now generally recognized that mesenchymal stem cells exert potent immunoregulatory activity. In this study, for the first time, the potential impact of Menstrual blood Stem Cells [MenSCs], as surrogate for endometrial stem cells, on proliferative capacity of CD4+ T cells was tested

Methods: MenSCs and Bone marrow Mesenchymal Stem Cells [BMSCs] were isolated and assessed for their immunophenotypic features and multi-lineage differentiation capability. BMSCs and MenSCs with or without IFNGamma pre-stimulation were co-cultured with purified anti-CD3/CD28-activated CD4+ T cells and the extent of T cell proliferation at different MenSCs: T cell ratios were investigated by CSFE flow cytometry. IDO activity of both cell types was measured after stimulation with IFNGamma by a colorimetric assay

Results: MenSCs exhibited dual mesenchymal and embryonic markers and multilineage differentiation capacity. MenSCs significantly increased proliferation of CD4+ cells at ratios 1:2, 1:4 and 1:8. IFNGamma pre-treated BMSCs but not MenSCs significantly suppressed CD4+ T cells proliferation. Such proliferation promoting capacity of MenSCs was not correlated with IDO activity as these cells showed the high IDO activity following IFNGamma treatment

Conclusion: Although augmentation of T cell proliferation by MenSCs can be a basis for maintenance of endometrial homeostasis to cope with ascending infections, this may not fulfill the requirement for immunological tolerance to a semi-allogeneic fetus. However, more investigation is needed to examine whether or not the immunomodulatory properties of these cells are affected by endometrial microenvironment during pregnancy

Development and validation of HPLC method for the determination of Candesartan in human plasma

Candesartan [CAN], an ARB-blocker, antihypertensive, was analyzed in human plasma by a simple, accurate and precise RP-HPLC [reverse phase-High performance liquid chromatography assay method which was then validated for its accuracy, specificity and precision. The mobile phase has a constitution of acetone, diethylamine and distilled water, while Phosphoric acid was used to adjust the pH to 2.5 +/- 0.1. This mobile phase was run at 1.1ml/min and the fluorescence wavelength was set to 392 nm. A C-18 HPLC, column particle size [5 [micro]m] Mediterranean Sea ® L x 1.D. 25cm x 4.6 mm [Supelcosil] , with auto sampler injection volume of 30[micro]l ,an internal standard Valsartan was utilized for chromatographic detection. Candesartan took a retention time of 6 +/- 0.5 minutes. This method was validated by the parameters of selectivity, accuracy, precision, repeatability, reproducibility, recovery, linearity and stability. Candesartan's calibration curves were found to be linear in the range of 200ng/ml to 3.125ng/ml and the coefficient of determination [r2] was found to be 0.99. Analytical recovery obtained was above 88%. Hence, this method has been found to be useful for determining Candesartan in plasma

Evaluation of IgY antibody as a polyspecific coombs-reagent

Background: during the last twenty years, the extraction of specific egg yolk [IgY] antibodies from the immunized chickens has been accepted as a useful alternative to the immunization of mammals. The aim of the present study was immunizing the chickens with Human Umbilical Cord Serum [HUCS] and the extraction of specific anti-human globulins [IgG, C3b, and C3d] antibodies from egg yolk in order to obtain polyspecific Coombs reagent

Methods: the novelty of this work was the achievement of a polyclonal reagent through a very cheap alternative method in accordance with all ethical regulations required for obtaining it. Three Leghorn hens [21 weeks old] were immunized four times for a period of 66 days with 20uL of HUCS mixed with PBS/FCA or FIA each time. The extraction of IgY antibodies was performed according to the method of lipid precipitation of yolk and using water soluble fraction as the reagent material. The resulting IgY antibody was characterized by SDS-PAGE and immunoelectrophoresis and tested for the presence of hetero-agglutinins by means of direct agglutination using human erythrocytes of all blood groups treated with 0.1% papain and for indirect Coombs-test to evaluate its specificity to fractions [C3b, C3d, C4d] of human complement and human IgG, respectively

Results: our findings show, that, the reagent obtained contains IgY and other 3 proteins [SDS-PAGE], and reacts specifically with plasma proteins, that migrate in beta and gamma regions. In immunoelectrophoresis, in addition, there is the presence of low hetero-agglutinins levels in IgY-preparation [3 lots], and the possibility to produce high amount [more than 500 ml/egg] of polyspecific Coombs-reagent in chickens is also discussed

Conclusion: IgY-preparation [3 lots], and the possibility to produce high amount [more than 500 ml/egg] of polyspecific Coombs-reagent in chickens with the originality to achieve a polyclonal reagent through a very cheap alternative method in accordance with all ethical regulations required for obtaining it, was also discussed

effect of media supplementation with angiotensin on developmental competence of ovine embryos derived from vitrified-warmed oocytes

Background: This study was aimed to assess the effects of angiotensin II [Ang II] supplementationto the In Vitro Maturation [IVM] and In Vitro Culture [IVC] media ofvitrified-warmed ovine oocytes on their developmental competence and expression ofNa +/K +/ATPase in resulting embryos

Methods: The slaughterhouse-derived immature oocytes [n=1069] were randomly distributedinto four experimental groups: groups I and II] IVM/IVF and IVC of fresh andvitrified oocytes without angiotensin supplementation [Control-Fresh and Control-Vitgroups, respectively]; group III] IVM of vitrified oocytes in the presence of Ang II followedby IVF/IVC [Vit-IVM group]; and group IV] IVM/IVF of vitrified oocytes followedby IVC wherein the embryos were exposed to Ang II on day 4 of IVC [Vit-D4 group].The embryos were immunostained with primary antibodies against Na +/K +/ATPasealpha 1andbeta 1 subunits

Results: In Vit-IVM and Vit-D4 groups, the rates of expanded and total blastocysts onday 7 as well as the proportion of blastocysts on day 8 were increased. The expressionof Na +/K +/ATPasealpha 1 andbeta 1 subunits were positively influenced by the addition of AngII on day 4 [Vit-D4 group].Conclusion: The addition of Ang II to the IVM and IVC media could improve blastocystsformation in vitrified sheep oocytes. This improvement might be related to thegreater expression of Na +/K +/ATPasealpha 1 andbeta 1 subunits when Ang II was added duringIVC

Medium optimization for the production of amylase by Bacillus subtilis RM16 in Shake-flask fermentation

This study was carried out in shaking incubator and covers the optimization of culture conditions of Bacillus subtilis for the maximum production of amylase. Optimal activity was found to be 350 Uml[-1] when soluble starch was used as a substrate. Parameters taken into consideration to observe their effect on the optimum production of amylase include incubation time, incubation temperature, pH, inoculum size, carbon source, nitrogen source and metallic ions. All parameters were monitored in order to obtain high level of the enzyme units in cell-free broth. The established optimized conditions for Bacillus subtiliss train RM16 were found to be: incubation time 24 hours, temperature 40[degree]C and pH 8.0. Inoculum size was 5%, starch [1%] as a carbon source while yeast extract [1.5%] as a nitrogen source. Magnesium ions [0.1%] exerted maximum stimulating effect for the production of amylase which can be further used at large scale applications

Effect of Angiotensin on the quality of In Vitro Produced [IVP] sheep embryos and expression of Na[+] /K[+] /ATPase

Background: The presence of rennin-angiotensin components in mammalian ovaries and their involvement in ovarian physiology have been established. In the present study, effects of angiotensin II [Ang II] on sodium-potassium adenosine triphosphatase [Na[+] /K[+] /ATPase] expression and development of sheep embryos was evaluated

Methods: The abattoir-derived Cumulus Oocyte Complexes [COC] were randomly allocated into three experimental groups; group I] in vitro Maturation [IVM] of oocytes in the presence of Ang II followed by in vitro fertilization [IVF]/in vitro Culture [IVC] [IVM group], group II] IVM/IVF of oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC [D4 group], and group III] IVM/IVF and IVC of oocytes without any angiotensin [Control]. The blastocyst and hatching rates were recorded on days 6 to 8. Day 8 embryos were immunostained with primary and secondary antibodies against Na[+] /K[+] /ATPase alpha 1 and beta 1 subunits

Results: Addition of Ang II during IVM and IVC significantly increased the hatching rate of blastocysts on day 8 compared to the control. The trophectoderm and total blastocyst cells' numbers were significantly increased by addition of Ang II to the IVM and IVC media, though the expression of Na[+] /K[+] /ATPase alpha 1 and beta 1 subunits were positively influenced by the addition of Ang II on day 4 [D4 group]

Conclusion: In conclusion, it seems Ang II through positive effects on embryos, expressed as the greater hatching rate and blastocyst cell number, could increase the sheep embryo developmental rate. These improvements might be partly related to the greater expression of Na[+] /K[+] /ATPase alpha 1 and beta 1 subunits when Ang II was added during IVC

Improved Efficacy of a Dendritic Cell-Based Vaccine against a Murine Model of Colon Cancer: The Helper Protein Effect / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Targeted immunotherapy using dendritic cells (DCs) has been employed in numerous investigations aiming at combating neoplasms. We previously showed that copulsing of an antigen with a helper protein could considerably enhance antigen presenting capacity of ex vivo-generated DCs. In this study, we attempted to administer an effective treatment in a murine model of colon cancer with DCs pulsed with the mixture of a tumor-specific gp70-derived peptide (AH1) and a helper protein, ovalbumin (OVA). MATERIALS AND METHODS: First, the presence of gp70 in CT26 tumor cells and tumor tissues was verified using immunofluorescence and Western blot analyses. Next, DCs were purified from normal mice, loaded ex vivowith AH1 and OVA (DC-Pep-OVA), and injected into tumor-bearing mice. Tumor volume, in vitro antigen (Ag)-specific proliferation of splenic cells, and survival rate were measured to determine the efficacy of DC-Pep-OVA. As the control groups, tumor-bearing mice were vaccinated with DC-Pep, unpulsed DC, and DCs loaded with a mixture of OVA and an irrelevant peptide (P15), or were not vaccinated at all. RESULTS: DC-Pep-OVA showed superior efficacy over other groups, as indicated by smaller tumor volume, higher Ag-specific proliferation rate of splenic cells, and prolonged survival. CONCLUSION: Overall, in the present study we showed for the first time that DCs copulsed with AH1 (tumor Ag) and OVA (helper molecule) could be considered as potentially robust weapons for use in future antitumor immunotherapies.

The use of SPECT-CT improves accuracy of post radioiodine therapy imaging and changes the management strategy in a case of advanced follicular thyroid carcinoma

This is a case of follicular thyroid carcinoma with extensive lung, bone and brain metastases. Multi-modality treatments including total thyroidectomy, modified radical neck dissection, cranial radiotherapy and Iodine-131 (RAI) therapy were instituted. Post RAI therapy planar whole body scan showed RAI avid metastases in the skull, cervical spine, bilateral lungs and abdomen. With the use of SPECTCT imaging, rare adrenal metastasis and additional rib metastasis were identified. Besides, management strategy was altered due to detection of non-RAI avid brain and lung metastatic lesions.

[Survey on intra-peritoneal injection of mercuric chloride on blood albumin and liver enzymes]

The rapid development of the mining and industry activities increased and many toxic metals in the environment of the earth's crust has been dissipated and has taken risks to human exposure, inhalation. Today evidence of many diseases associated with environmental factors harmful to repellent Bio systems is increasing gradually, The majority of these factors were man-made and the activities associated with heavy metals was a major threat for human health. Mercury has the most toxic non-radioactive element that was already known. The purpose of this study was to investigate the effect of mercuric chloride intra peritoneally on blood albumin and some liver enzymes. In this study, 30 male Wistar rats randomized selected into 6 groups [1 control group, and experimental groups of 1, 2, 3, 4, 5]. In control group adequate serum physiology, and in experimental groups a dose of mercuric chloride infused into peritoneal cavity for 7 days. The amount of mercuric chloride infused were 1 mg/kg in 1[st] group, 2 mg/kg in group 2, 5 mg/kg in group 3, 7 mg/kg in group 4, and 10 mg/kg in the fifth group every other day for 7 days. After the 7 days blood samples, were tested and analyzed. In this study, there was a significant relation between decrease in albumin levels in experimental groups compared to the control group and a there was significant relation increase in the amount of transaminases; SGOT and SGPT in the experimental group. This study showed that intra peritoneal injection of mercuric chloride causes the balance were increased, of liver enzymes and serum albumin levels

Case report: combination therapy with mesenchymal stem cells and granulocyte-colony stimulating factor in a case of spinal cord injury

Introduction: Various neuroregenerative procedures have been recently employed along with neurorehabilitation programs to promote neurological function after Spinal Cord Injury [SCI], and recently most of them have focused on the acute stage of spinal cord injury. In this report, we present a case of acute SCI treated with neuroprotective treatments in conjunction with conventional rehabilitation program

Methods: A case of acute penetrative SCI [gunshot wound], 40 years old, was treated with intrathecal bone marrow derived stem cells and parenteral Granulocyte-Colony Stimulating Factor [G-CSF] along with rehabilitation program. The neurological outcomes as well as safety issues have been reported

Results: Assessment with American Spinal Injury Association [ASIA], showed neurological improvement, meanwhile he reported neuropathic pain, which was amenable to oral medication

Discussion: In the acute setting, combination therapy of G-CSF and intrathecal Mesenchymal Stem Cells [MSCs] was safe in our case as an adjunct to conventional rehabilitation programs. Further controlled studies are needed to find possible side effects, and establish net efficacy

Lipopolysaccharide- and lipoteichoic acid-mediated pro-inflammatory cytokine production and modulation of tlr2, tlr4 and myd88 expression in human endometrial cells

Toll-like receptor [TLR]-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells [ESCs] and whole endometrial cells [WECs] to lipopolysaccharide [LPS] and lipoteichoic acid [LTA] Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and production of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene expression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr [p<0.05]. At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs [p<0.05]. LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dose-dependent manner [p<0.05]. Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-alpha in response to LPS activation [p<0.05]. Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus

Analyses of bile from gallbladders of fish [Arius platystomus, Arius tenuispinis, Pomadasys commersonni and Kishinoella tonggol]

Bile from gallbladders of Arius platystomus [Singhara], Arius tenuispinis [Khagga], Pomadasys commersonni [Holoola] and Kishinoella tonggol [Dawan] were derivatised and analysed by GC-MS for identification of bile acids and bile alcohols. Cholic acid and Chenodeoxycholic acid were found as major bile acids in Arius platystomus, Arius tenuispinis and Pomadasys commersonni. Other bile acids identified in Arius platystomus were allochenodeoxycholic acid, allodeoxycholic acid, 3 alpha,7 alpha,12 alpha -trihydroxy-24-methyl-5beta-cholestane-26-oic acid, and 3alpha,7alpha,12alpha, 24-tetrahydroxy-5alpha-cholestane-26-oic acid. Cholesterol was found as major bile alcohol in Arius platystomus, Arius tenuispinis and Pomadasys commersonni. Cholic acid was the major bile acid identified in the bile of Kishinoella tonggol while other bile acids included 3 alpha,7 alpha,12 alpha -tridydroxy-5alpha-cholestanoic acid and 3 alpha,7 alpha,12 alpha -tridydroxy-5beta-cholestanoic acid. Bile alcohol 5beta-cyprinol was present in significant amounts with 5beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol being the other contributors in the bile of Kishinoella tonggol

Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating

The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells [SSCs]. They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture [3, 4, 5, and 6 hr] and discontinuous percoll density with different gradients [20, 28, 30, and 32%]. The difference of percentage of undifferentiated SSCs [PGP9.5 positive] in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat [ver. 3.5]. The highest PGP9.5 [94.6 +/- 0.4] and the lowest c-Kit positive [25.1 +/- 0.7] in Percoll method was significantly [p

effect of pomegranate extract on survival and peritoneal bacterial load in cecal ligation and perforation model of sepsis in rats

Sepsis is one of the major causes of death in intensive care units. Oxidative stress and hyper-inflammation has been shown to be major cause of mortality and morbidity in septic cases. Pomegranate is a fruit considered for its antioxidant and anti-inflammatory properties. The aim of this study is to evaluate the effect of a standard pomegranate fruit liquid extract[POMx], on mortality and peritoneal bacterial load in cecal ligation and perforation [CLP] sepsis model. Male wistar rats were divided into four groups of 24 each: sham; CLP; prevention [consumed POMx [250 mg of polyphenols/kg/day] for 4 weeks before CLP]; treatment [received a single drink of POMx [250 mg of polyphenols/kg] after CLP]. Each group was divided into three subgroups, each containing eight animals, for bacterial load and survival [with and without antibiotics] studies. Sepsis was induced by CLP surgery. Ten day survival rate was recorded. Peritoneal bacterial load was also assessed. Data were analyzed using Log-rank and Kruskal-Wallis tests. There was no significant difference in survival rate of CLP, prevention and treatment groups, in subgroups without antibiotics. However, in subgroups with antibiotics, the prevention group had significantly lower survival rate than sham group [P 0.05]. Conversely, the bacterial load of prevention and treatment groups were significantly higher than sham group [P< 0.01]. Our study demonstrates for the first time that pomegranate extract could increase mortality rate via increasing peritoneal cavity bacterial load, in CLP sepsis model. More studies to assess mechanisms of this effect are warranted

Annona squamosa Linn: Cytotoxic activity found in leaf extract against human tumor cell lines

Cancer is a common cause of death in human populations. Surgery, chemotherapy and radiotherapy still remain the corner stone of treatment. However, herbal medicines are gaining popularity on account of their lesser harmful side effects on non-targeted human cells and biological environment. Annona squamosa Linn is a common delicious edible fruit and its leaf have been used for the treatment in various types of diseases

The objective of present study is to determine the anticancer potential of the organic and aqueous extracts of leaf of Annona squamosa L. MTT [3-[4, 5-dimethylthiazole-2yl]-2, 5-biphenyl tetrazolium bromide] assay against hepatocellular carcinoma cell line BEL-7404, lung cancer line H460, human epidermoid carcinoma cell line KB-3-1, prostatic cancer cell line DU145, breast carcinoma cell line MDA-MB-435, and colon cancer cell line HCT-116 Human primary embryonic kidney cell line HEK293 as control were used for the study

The crude extract [Zed] and Ethyl acetate extract [ZE] were found significant anticancer activity only on human epidermoid carcinoma cell line KB-3-1 and colon cancer cell line HCT-116

effect of sortilin silencing on ovarian carcinoma cells

Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin [NTR3], the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis [27.5 +/- 0.48%] in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation [40.1%] in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart [p<0.05]. As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma

McL[-1] is ip regulated by prenylated coumarin, umbelliprenin in jurkat cells

Chronic lymphocytic leukaemia [CLL] is the most common B-cell malignancy in the western world and exists as subtypes with very different clinical courses. Myeloid cell leukemia 1 [McL[-1]] is one member of Bcl-2 family proteins that has been shown to be expressed in various tissues and malignant cells, including CLL, where its expression is significantly associated with a failure to achieve complete remission following cytotoxic therapy. Induction of apoptosis by prenylated coumarin, umbelliprenin, in Jurkat cells was previously shown. We examined whether umbelliprenin can down-regulate McL[-1] gene and protein in Jurkat cells. In this regard cells were incubated by umbelliprenin, and then down- regulation of McL[-1] gene was studied by Real Time PCR method. Moreover, down-regulation of McL[-1] protein was studied by western blot analysis. We showed that, expression of McL[-1] mRNA was increased from 1 hour to 3 hours incubation, but this increase has a scale down pattern. Moreover umbelliprenin could inhibit McL[-1] protein. In conclusion umbelliprenin treatment modulates McL[-1] expression at both the transcriptional and posttranslational levels

Isolation and partial characterization of human amniotic epithelial cells: the effect of trypsin

Despite the extensive information available in the literature, cell surface marker signature of human Amniotic Epithelial Cells [hAECs] remains controversial. The aim of the present study was to characterize immunophenotypic features, proliferative capacity and immunogenicity of hAECs. We also tested whether expression of some cell surface markers is influenced by the type of trypsin used for tissue digestion. Single cell suspensions of amniotic membranes from four human placentas were isolated by enzymatic digestion and expression of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, CD133, HLA-I, HLA-DR, HLA-G, SSEA-4, STRO-1 and OCT-4 was then evaluated by flow cytometry. The differential impact of four trypsin types on the yield and expression of CD105 and HLA-I was also determined. The proliferative capacity of cultured hAECs was assessed and compared in the presence and absence of Epidermal Growth Factor [EGF]. To test their immunogenicity, hAECs were injected into Balb/c mice and the reactivity of hyperimmunized sera was examined by immunofluorescence staining. Nearly all purified cells expressed mesenchymal markers, CD9, CD10, CD29, and CD73 and the embryonic marker, SSEA-4. A large proportion of the cells also expressed STRO-1 and OCT-4. The purified cells also expressed HLA-G and HLA-I. A very small proportion of hAECs expressed CD34, CD38, CD44, CD133 and HLA-DR. The type of trypsin used for enzymatic digestion affected both the percentage and expression of HLA-I and CD105. hAECs revealed substantial proliferative capacity only when cultured in the medium supplemented with EGF. These cells were shown to be capable of inducing high amounts of anti-donor antibodies. Here we provided evidence that hAECs are immunogenic cells with high level of HLA-I expression. Furthermore, this work highlighted the impact of isolation procedure on the immunophenotype of hAEC