Adoptive cell therapy with induced regulatory T cells normalises the abortion rate in abortion-prone mice.

Ovarian hormones drive invivo generation of regulatory T cells (Tregs) during pregnancy. Little is known about the therapeutic potential of invitro hormone-derived Tregs in pregnancy loss. We investigated the effects of hormone-induced Tregs in a murine model of abortion. CD4+CD25- T cells were isolated from the spleens of CBA/J mice and stimulated with either 17ß-oestradiol (E2), progesterone (P4) or transforming growth factor-ß1 (TGFB1) plus retinoic acid (RA) for 4 days to generate induced Tregs (iTregs). On Days 1-4 of gestation, DBA/2-mated pregnant CBA/J female mice (abortion prone) were injected intravenously with iTregs or Tregs isolated from normal BALB/c-mated pregnant CBA/J mice (np-Tregs). On Day 14, the number of resorbed fetuses was assessed. Serum interferon (IFN)-γ and uterine forkhead box p3 (Foxp3) expression was analysed by ELISA and immunohistochemistry respectively. Using a 3H-thymidine incorporation assay, isolated CD4+CD25+ Tregs induced by the different treatments suppressed the proliferation of CD4+CD25- T cells. Adoptive transfer of iTregs (from all induction groups) significantly decreased fetal resorption in abortion-prone mice. There were no significant changes in serum IFN-γ concentrations after the adoptive transfer of iTregs or np-Tregs. Immunohistochemistry revealed significantly higher Foxp3 expression in gravid uteri from mice injected with np-Tregs and P4-induced iTregs than in the phosphate-buffered saline-treated group. The findings of this study indicate a potential therapeutic benefit of invitro-induced Tregs in patients with recurrent abortion.

Vaccination with human amniotic epithelial cells confer effective protection in a murine model of Colon adenocarcinoma.

As a prophylactic cancer vaccine, human amniotic membrane epithelial cells (hAECs) conferred effective protection in a murine model of colon cancer. The immunized mice mounted strong cross-protective CTL and antibody responses. Tumor burden was significantly reduced in tumor-bearing mice after immunization with hAECs. Placental cancer immunotherapy could be a promising approach for primary prevention of cancer. In spite of being the star of therapeutic strategies for cancer treatment, the results of immunotherapeutic approaches are still far from expectations. In this regard, primary prevention of cancer using prophylactic cancer vaccines has gained considerable attention. The immunologic similarities between cancer development and placentation have helped researchers to unravel molecular mechanisms responsible for carcinogenesis and to take advantage of stem cells from reproductive organs to elicit robust anti-cancer immune responses. Here, we showed that vaccination of mice with human amniotic membrane epithelial cells (hAECs) conferred effective protection against colon cancer and led to expansion of systemic and splenic cytotoxic T cell population and induction of cross-protective cytotoxic responses against tumor cells. Vaccinated mice mounted tumor-specific Th1 responses and produced cross-reactive antibodies against cell surface markers of cancer cells. Tumor burden was also significantly reduced in tumor-bearing mice immunized with hAECs. Our findings pave the way for potential future application of hAECs as an effective prophylactic cancer vaccine.

Overexpression of KCNJ3 gene splice variants affects vital parameters of the malignant breast cancer cell line MCF-7 in an opposing manner.

BACKGROUND: Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K(+) channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. METHODS: In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced. Subsequently, selected cardinal neoplasia associated cellular parameters were assessed and compared. RESULTS: Adhesion to fibronectin coated surface as well as cell proliferation remained unaffected. Other vital parameters intimately linked to malignancy, i.e. wound healing, chemoinvasion, cellular velocities / motilities and angiogenesis were massively affected by GIRK1 overexpression. Overexpression of different GIRK1 splice variants exerted differential actions. While GIRK1a and GIRK1c overexpression reinforced the affected parameters towards malignancy, overexpression of GIRK1d resulted in the opposite. Single channel recording using the patch clamp technique revealed functional GIRK channels in the plasma membrane of MCF-7 cells albeit at very low frequency. DISCUSSION: We conclude that GIRK1d acts as a dominant negative constituent of functional GIRK complexes present in the plasma membrane of MCF-7 cells, while overexpression of GIRK1a and GIRK1c augmented their activity. The core component responsible for the cancerogenic action of GIRK1 is apparently presented by a segment comprising aminoacids 235-402, that is present exclusively in GIRK1a and GIRK1c, but not GIRK1d (positions according to GIRK1a primary structure). CONCLUSIONS: The current study provides insight into the cellular and molecular consequences of KCNJ3 overexpression in breast cancer cells and the mechanism upon clinical outcome in patients suffering from breast cancer.

Regulation of luteinizing hormone receptor in hippocampal neurons following different long-lasting treatments of castrated adult rats.

The aim of this study was to investigate the effects of different Luteinizing hormone (LH) and steroid hormones levels on LH receptor (LHR) expression in the hippocampal cells. Rats (24 males and 24 females) were assigned to four groups: one control and three experimental [gonadectomy (GDX), gonadectomy + gonadotropin releasing hormone analogue (GDX+GnRHa) and GDX+GnRHa+estradiol (E2) or testosterone (T)] independently for each gender. All experimental rats were gonadectomized; then GnRHa was administrated to GDX+GnRHa group, and GnRHa plus steroid hormone to GDX+GnRHa+E2 or T group in both genders for four-month. LHR mRNA expression and its protein level in hippocampal cells were measured using QRT-PCR and Western blotting. Quantification of mRNA revealed a decrease in LHR transcripts level in GDX+GnRHa group of females. A significant change was observed between GDX groups and GDX+GnRHa+E2 or T versus GDX+GnRHa group in females. High levels of LH decreased significantly the immature isoform of LHR in GDX group compared to control group in both genders, but low LH concentrations in GDX+GnRHa group induced immature LHR isoform production only in females. Therefore increased LH concentration induces production of incomplete LHR transcripts in hippocampal cells and decreases immature LHR at the protein level. This implies that LH decreases the efficiency of translation through either producing non-functional LHR molecules or preventing their translation.

Pyrvinium pamoate inhibits proliferation and invasion of human endometriotic stromal cells.

Endometriosis is characterized by the presence of functional endometrial tissue in other pelvic organs. This gynecologic problem occurs in 35-50% of women with pain and infertility. Endometriotic cells share some characteristics such as proliferation, migration, and invasion with tumor cells. Pyrvinium pamoate, an anthelmintic drug approved by the Food and Drug Administration, could inhibit the Wnt/ß-catenin signaling pathway and its anticancer effects were examined by several researchers. In this study, 12 ectopic and eutopic endometrial biopsies from females with ovarian endometrioma and 12 endometrial biopsies from nonendometriotic females were obtained. Ectopic (EESCs), eutopic (EuESCs), and control (CESCs) endometrial stromal cells were isolated. Then, the effect of pyrvinium pamoate on the proliferation and invasiveness of in vitro cultured cells was evaluated. The proliferation of CESCs, EuESCs, and EESCs was significantly decreased after treatment with pyrvinium pamoate. In addition, treatment with pyrvinium pamoate significantly inhibited the invasiveness of CESCs, EuESCs, and EESCs compared to nontreated groups. The results of the present research showed that pyrvinium pamoate inhibits the proliferation and invasion of human endometriotic stromal cells in vitro, further investigations on the therapeutic potential of this compound in endometriosis are required.

Expression profiling of plac1 in murine cancer cell lines

AIM: Placenta-specific 1 (PLAC1) is among recently-discovered placental antigens which exerts fundamental role in placental function and development. Increasing body of literature shows that PLAC1 is frequently activated and expressed in a wide variety of human cancers and promote cancer progression. However, no data is available regarding the expression of mouse orthologue, plac1, in murine cancer cell lines. Materials and Methods: We investigated the expression of plac1 in a series of murine cell lines from different histological origins, mammary carcinoma (4T1), melanoma (B16F10), colorectal carcinoma (CT26), renal carcinoma (Renca), glioma (GL26), B-cell lymphoma (A20 and BCL1) and also two fibroblast cell lines (NIH3T3 and L929), using RT-PCR, Western blotting and flow cytometry. Results: Our data demonstrated that plac1 transcript and plac1 protein were expressed in all examined cell lines, as judged by RT-PCR and Western blot, respectively. The molecular weight of mouse plac1 was experimentally observed to be approximately 24 kD. Flow cytometric analysis showed surface expression of plac1 in aforesaid cell lines ranging from 2% to 42.5%. Conclusion: Based on the ubiquitous expression of plac1, the investigated cancer cell lines or immortalized cell lines can be used to examine the role of plac1 in the process of immortalization.

Role of rotaviruses in children with acute diarrhea in Tehran, Iran.

BACKGROUND: Rotavirus illness is associated with significant cause of morbidity and is a common cause of hospitalization worldwide. OBJECTIVE: This study was performed to assess the role of rotaviruses in children presenting with acute diarrhea in two main Children's Medical Centers and one general hospital in Tehran. STUDY DESIGN: Stool specimens from 704 children less than 5 years of age suffering from diarrhea were tested for the presence of rotaviruses by a monoclonal antibody-based enzyme immunoassay. A total of 176 fecal specimens collected from healthy children in similar age group were studied as controls. RESULTS: Rotavirus antigen was detected in 15.3% of patients. Infants between 6 and 12 months of age were most frequently affected. Rotavirus infection was significantly less frequent in breast-fed than among bottle-fed babies. Watery diarrhea was present in 68.5% of children. Detection rate was highest in the spring and lowest in summer. Rotavirus can be regarded as a major etiologic agent of acute diarrhea in infants and children up to 5-years-old in Iran, immunization at birth may protect the children before their first symptomatic infection.

Modified protocol for improvement of differentiation potential of menstrual blood-derived stem cells into adipogenic lineage.

OBJECTIVES: To characterize potency of menstrual blood-derived stem cells (MenSCs) for future cell therapies, we examined differentiation potential of MenSCs into adipocytes. MATERIALS AND METHODS: Differentiation potential of MenSCs in comparison to bone marrow stem cells (BMSCs) was assessed in conventional culture medium. Differentiation potential of MenSCs into adipocytes was improved using different combinations of growth factors and hormones. RESULTS: First, we demonstrated that MenSCs preserve their appearance and karyotypic stability during passages. Although these cells express mesenchymal stem cells markers, they cannot simply be classified as mesenchymal stem cells due to expression of embryonic stem cells marker, OCT-4. Oil red O staining showed that differentiated MenSCs in conventional medium with/without retinoic acid (protocols 1 and 2) did not attain adipocyte characteristics, whereas differentiated BMSCs in conventional medium accumulated oil vacuoles typically. Nevertheless, real-time RT-PCR results showed that LPL gene expression was up-regulated in both protocols 1 and 2, whereas LEPR was up-regulated only in protocol 2 (fortified with retinoic acid). Surprisingly, protocol 3 (including rosiglitazone) had odd influence on mRNA expression of all genes (LEPR, LPL and PPAR-γ). Oil red O staining confirmed fat-producing ability of MenSCs under protocol 3. CONCLUSIONS: Presented data suggest an efficient differentiation protocol for in vitro production of MenSC-derived adipocytes. These cells are suggested to be an apt alternative to BMSCs for future stem cell therapy of soft tissue injuries.

Expression profiling of vitamin D receptor in placenta, decidua and ovary of pregnant mice.

OBJECTIVES: The presence of vitamin D receptor (VDR) and the identification of localized vitamin D3 synthesis in placenta and decidua implicate the importance of vitamin D3 in reproductive function. There is, however, no data on the expression profile of VDR in the mouse placenta and endometrium throughout the pregnancy period. STUDY DESIGN: In the present work expression of VDR in reproductive tissues of pregnant mice at different gestational phases has been addressed. Expression of VDR was determined by semi-quantitative RT-PCR, Western blotting and immunohistochemistry. RESULTS: The results showed that VDR mRNA and protein were expressed in decidua, placenta and ovary throughout the pregnancy. VDR gene expression in placenta was significantly elevated in late pregnancy when compared to that of mid pregnancy. Additionally, VDR expression level in decidua rose significantly as pregnancy progressed from early to mid stages. VDR expression in decidua of pregnant mice was higher in comparison to endometrium of non-pregnant mice. Immunohistochemical analysis revealed that VDR protein is consistently expressed by luminal and glandular epithelial cells of decidua, giant cells, glycogen rich cells and labyrinth cells of placenta and by almost all follicular cell types of ovary. Surveying the expression of VDR at the protein level by Western blotting confirmed PCR results. CONCLUSION: It seems that expression of VDR in reproductive organs is finely tuned during pregnancy indicating its eminent role in reproductive biology.

Indoleamine 2,3-dioxygenase is expressed in the endometrium of cycling mice throughout the oestrous cycle.

Indoleamine 2,3-dioxygenase (IDO), an enzyme responsible for tryptophan catabolism, is thought to be required to prevent the rejection of the allogenic fetus by maternal T cells and to protect against intra- and extra-cellular pathogens. Consequently, we studied the expression of IDO in the endometrium of female Balb/c mice during the oestrous cycle. At each phase, the endometrium was peeled away and the relative expression of IDO mRNA was detected by semi-quantitative RT-PCR. The presence of IDO protein was confirmed in each phase by Western blotting and immunohistochemistry. Our results showed that IDO is expressed in the endometrium of cycling mice during all the phases of oestrous cycle. The expression of IDO was highest at the oestrus and lowest at the dioestrus. By means of Western blotting and immunohistochemistry, we obtained evidence that IDO protein is synthesised in the endometrium of cycling mice throughout the oestrous cycle. In accordance with RT-PCR results, IDO protein was predominant at the oestrus phase. IDO protein was mainly localised in the glandular and luminal epithelial cells. Our results support the concept of IDO providing a mechanism of innate immunity to protect from ascending infections of the female reproductive tract. In addition, considering the fact that mating only occurs during the oestrus phase, the high expression of IDO in this phase is likely to be a mechanism that induces immunological tolerance of the fetus.

Epitope mapping of recombinant hepatitis B surface antigen by murine monoclonal antibodies.

Hepatitis B surface antigen (HBsAg) induces a potent protective antibody response in immunized healthy individuals. The antibody response in humans is largely directed to a restricted conformational immunodominant region of HBsAg, identified as "a" determinant. Our aim was generation and characterization of murine monoclonal antibodies (MAbs) against recombinant HBsAg and their use for epitope mapping of the antigen. Hybridoma cells were established from Balb/c mice immunized with recombinant HBsAg of the "adw" subtype and cloned by limiting dilution. Specificity of MAbs was studied by indirect ELISA and immunoblotting. Topology of the epitopes was analyzed by competitive and inhibition ELISA. Eight hybridoma clones producing MAbs specific for the immunogen were established. Five of the MAbs recognized overlapping conformational epitopes, whereas the remaining three MAbs were found to identify linear epitopes. Cross-inhibition studies suggest recognition of mutually exclusive epitopes by these MAbs. Our data suggest that, similar to the human system, the mouse antibody response is largely directed to restricted conformational overlapping epitopes of HBsAg.

Morphological and cytochemical characteristics of purified murine splenic dendritic cells.

Dendritic cells function as the main cellular population responsible for professional antigen presentation and hence for induction of primary immune responses. Although they are present in virtually every tissue, nevertheless their number is usually so low that it makes their isolation for studies very difficult. In this study, we purified dendritic cells from mouse spleen by a three-step enrichment method and evaluated morphological and cytochemical characteristics of isolated cells. We showed that isolated dendritic cells from mouse spleen had all lobulated nuclei with multiple cytoplasmic projections and their morphological features changed after an overnight incubation. It was also shown that typical dendritic cells lacked both Myeloperoxidase (MPO) and Non Specific Esterase (NSE) activity. In conclusion, for reaching a reasonable purity in isolation of dendritic cells from lymphoid tissues, many enrichment steps should be taken, and for determining the purity of isolated cells, we recommend that a combination of morphological and cytochemical studies be used.