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

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.

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

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

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

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.

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

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

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

Immune reactivity of Brucella melitensis-vaccinated rabbit serum with recombinant Omp31 and DnaK proteins

Brucella melitensis infection is still a major health problem for human and cattle in developing countries and the Middle East. In this study, in order to screen immunogenic candidate antigens for the development of a Brucella subunit vaccine, a cytoplasmic protein [DnaK] and an outer membrane protein [Omp31] of B. melitensis were cloned, expressed in E.coli BL21 and then purified using Ni-NTA agarose. Immunized serum was prepared from a rabbit inoculated with attenuated B. melitensis. It was proved that immunized serum contains antibodies against recombinant Omp31 [rOmp31] and DnaK [rDnaK] by Western blot and ELISA assays. The results may suggest the importance of these proteins as subunit vaccines against B. melitensis as well as targets for immunotherapy

Production and characterization of a marine monoclonal antibody against human ferritin

Ferritin is an iron storage protein, which plays a hey role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody [mAb] against human ferritin was reported. Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma [clone: 2F9-C9] was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity [2.34 10[9] A/[1]] and the isotype was determined to be lgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic bit if other requirements of the hit are met

Isolation and culture of human spermatogonial stem cells derived from testis biopsy

In cancer patients, chemo and radiotherapy can cause infertility by damaging spermatogenesis process. This process is based on self-renewal and differentiation of a rare population of the testicular cells called Spermatogonial Stem Cells [SSCs]. Scientists have tried to isolate, enrich and culture Human spermatogonial stem cells, hoping to resolve infertility problems in cancer recovered patients in the future. Spermatogonial stem cells were isolated and purified from human testicular biopsies sample consisting of at least 500,000 and at most 2,000,000 cells. Two enzymatic digestion steps were performed. Enriching methods, differential plating, and specific culture in serum-free medium with added growth factors: human GDNF, bFGF, EGF and LIF was performed on coated dishes. Human spermatogonial stem cell clusters were observed after 7 to 10 days in specific culture, then after several passages and successful expanding duration of 52 days, the cells were evaluated by three layer immunocytochemistry test [LSAB] to stain GPR125 protein as a surface marker in human spermatogonial stem cells. In current study human spermatogonial stem cell were isolated and expanded with the least manipulations in comparison with the other usual isolation methods like florescent or magnetic activated cell sorting. In contrast to the other SSCs isolation and culture methods, this system is based on the testicular biopsies against large samples, thus suggested method in this study is closer to clinical usage in the future

Umbelliprenin from Ferula szowitsiana activates both intrinsic and extrinsic pathways of apoptosis in jurkat T-CLL cell line

Umbelliprenin is a prenylated compound, which belongs to the class of sesquiterpene coumarins. It is extracted from dried roots of Ferula szwitsiana collected from the mountains of Golestan forest [Golestan Province, north of Iran]. Induction of apoptosis in Jurkat T-CLL cells has been previously shown. In this study, effect of umbelliprenin on proapoptotic caspases [caspase-8 and -9] and antiapoptotic Bcl-2 family protein was studied. Jurkat cells were incubated with umbelliprenin. Cells were then lysed and activation of proteins was studied by Western blot analysis. In this study, we showed that umbelliprenin activates intrinsic and extrinsic pathways of apoptosis by the activation of caspase-8 and -9 respectively. Inhibition of Bcl-2 was also shown. In conclusion, umbelliprenin induced apoptosis in Jurkat cells through caspase-dependent apoptosis pathway

Combination of thrombophilic gene polymorphisms as a cause of increased the risk of recurrent pregnancy loss

Recurrent pregnancy loss is [RPL] a heterogeneous condition. While the role of acquired thrombophilia has been accepted as an etiology for RPL, the contribution of specific inherited thrombophilic gene polymorphisms to the disorder has been remained controversial. One hundred women with a history of two or more consecutive abortions and 100 women with at least two live births and no miscarriages were included in the study and evaluated for the presence of 11 thrombophilic gene polymorphisms [Factor V LEIDEN, Factor V 4070 A/G, Factor V 5279 A/G, Factor XIII 103 G/T, Factor XIII 614 A/T, Factor XIII 1694 C/T, PAI-1-675 4G/5G, ITGB3 1565 T/C, ?-Fibrinogen-455G/A, MTHFR 677 C/T, MTHFR 1298 A/C] using PCR-RFLP technique. The data were statistically analyzed using Mann-Whitney test and logistic regression model. There was no relation between factor XIII 103G/T gene polymorphism with increased risk of RPL. However, the other 10 gene polymorphisms were found to be associated with increased/decreased risk of RPL. Multiple logistic regression model for analyzing the simultaneous effects of these polymorphisms on the risk of RPL showed that six of these 11 polymorphisms [Factor V 1691G/A, Factor V 5279A/G, Factor XIII 614A/T, ?-Fibrinogen-455G/A, ITGB3 1565T/C, and MTHFR 1298A/C] were associated with RPL. It is possible to calculate the risk of abortion in a patient with RPL by determining only six of the 10 polymorphisms that are individually associated with RPL

Lymphocyte cytotoxicity of oxLDL in patients with atherosclerosis

Atherosclerosis, a chronic inflammatory disease of the vessel wall is characterized by local and systemic immune responses to a variety of antigens. Oxidized low-density lipoprotein [oxLDL] is considered as an important determining factor in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the degree of peripheral blood mononuclear cells [PBMC] vulnerability to in vitro oxLDL-induced cytotoxicity from atherosclerotic patients in comparison to healthy individuals. Thirty patients with atherosclerotic lesions, confirmed by angiography, and 30 matched healthy individuals were investigated. PBMC was prepared from individuals' blood samples which were further stimulated with low dose [1 micro g/mL] and high dose [50 micro g/mL] of extensively oxidized LDL. MTT assay was utilized to measure cell viability and proliferation. Stimulation index [SI] was calculated as mean ratio of optical density [OD] of the stimulated cells divided by OD of untreated cells. Low dose oxLDL treatment caused no significant proliferative or cytotoxic effect in the control group; however, similar treatment caused significant cytotoxic effect in the patient group compared to the controls [p=0.026]. High dose oxLDL treatment induced more significant cytotoxicity in the patient compared to the control group [p=0.006]. Comparison of the SI between the two groups of patients and controls showed significantly lower index by either the low [p=0.03] or the high dose [p<0.001] oxLDL in the patients compared to the controls. PBMC from patients with atherosclerosis showed increased susceptibility to oxLDL-induced cytotoxicity. Our results imply that prolonged exposure to elevated levels of circulating oxLDL could weaken the cellular defense mechanisms by progressive depletion of the pool of antiapoptotic proteins, rendering the cells more vulnerable to oxLDL-induced cell death

Cytokine profile in the endometrium of normal fertile and women with repeated implantation failure

Repeated Implantation Failure [RIF] is one of the most intricate obstacles in assisted reproduction. The cytokine and chemokine composition of uterine cavity seem to play important roles in the implantation process. To compare the cytokine profile in the endometrium of normal fertile women and those with repeated implantation failure. After enzymatic digestion of endometrial tissues, whole endometrial cells and endometrial stromal cells from RIF and normal fertile women were cultivated and stimulated for cytokine secretion. The levels of IL-10, TGF-beta, IFN-gamma, IL-6, IL-8 and IL-17 in culture supernatants of the two groups were assayed by ELISA and compared together. Endometrial stromal cells and whole endometrial cells of normal fertile women produced higher levels of IL-6, IL-8 and TGF-beta compared to RIF group, although this difference was statistically significant only in endometrial stromal cells [p=0.005, 0.002 and 0.001, respectively]. In addition, endometrial stromal cells of normal fertile women produced lower levels of IL-10 in comparison with RIF group [p<0.005]. Disturbances in cytokine production at the feto-maternal interface could be a cause of implantation failure. A pro-inflammatory cytokine milieu seems to be pivotal for successful implantation

Extraction, purification and characterization of lipopolysaccharide from Escherichia coli and Salmonella typhi

Lipopolysaccharide [LPS] is an important structural component of the outer cell membrane complex of gram negative microorganisms. Its causative role in gram negative bacteria-induced diseases and broad applications in different kinds of cell stimulation experiments provided a conceptual basis for studies directed at the isolation, purification, and detailed chemical characterization of LPS. The main problem with LPS purification protocols is the contamination of the end product with nucleic acids and proteins in variable proportions which could potentially interfere with downstream applications. In this study, a simple procedure for purification of LPS from Escherichia coli [E.coli] and Salmonella typhi [S.typhi] with high purity and very low contaminating nucleic acids and proteins based on the hot phenol-water extraction protocol has been introduced. The purity of extracted LPS was evaluated by silver and coomassie blue staining of SDS-PAGE gels and HPLC analysis. Limulus Amebocyte Lysate [LAL] coagulation activity and rabbit pyrogen assay were exploited to monitor the functionality of purified LPS. The results showed that DNase and RNase treatment of the sample is essential after the sonication step to eliminate nucleic acid contamination in the LPS fraction. Silver staining demonstrated ladder pattern which is characteristic of LPS. No contaminating protein was found as assessed by coomassie blue staining. HPLC fractionation revealed high degree of purity comparable with commercial LPS. Parenteral administration of purified LPS resulted in substantial increase of rabbits' body temperature [mean: 1.45°C]. LAL coagulation assay confirmed the functional activity of the purified LPS. In conclusion, the protocol presented here could be employed for isolation of LPS with high purity and functional activity

Peptide-based Polyclonal Antibody Production against P110 Protein of Mycoplasma genitalium

Mycoplasma genitalium [M.genitalium] is a sexually transmitted pathogen. Detection of this microorganism in clinical specimens by culture is rather difficult and time consuming. The aim of this study was to produce polyclonal antibody against a synthetic peptide from P110 protein of M.genitalium in order to develop a diagnostic tool for detection of this microorganism in clinical specimens. A synthetic peptide from P110 protein was conjugated to Keyhole Limpet Hemocyanin]KLH[and used for immunization of a white New Zealand rabbit. The produced antibody was purified by affinity chromatography and its specific interaction with immunizing peptide was determined by ELISA. Immunoreactivity of the antibody was also tested by Western blotting in bacterial cell lysate prepared from M.genitalium G-37. To confirm its application as a diagnostic tool, indirect immunofluorescent staining method was performed on M.genitalium-infected PBMC using anti-P110 as the primary antibody. The results showed that produced antibody has excellent reactivity with immunizing peptide and also detected a single band of 110 kDa corresponding to P110 protein. M.genitalium-infected PBMC showed a bright fluorescent signal in IF staining. This antibody might be used as a tool in diagnostic applications