Potential of Lactobacillus acidophilus to modulate cytokine production by peripheral blood monocytes in patients with endometriosis.

Background and Objectives: Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. Peripheral blood monocytes cells (PBMCs) may have altered function to some extent in women with endometriosis. Lactobacillus acidophilus is a probiotic bacterium within the human body with the ability of alleviating many inflammatory diseases. Here, we examined the effect of L. acidophilus on PBMCs of endometriosis patients. Materials and Methods: In this study, peripheral blood samples were obtained from endometriosis patients (n=11) and non-endometriosis individuals (n=11). After isolation of peripheral blood mononuclear cells with Ficoll, cells were cultured in the presence and absence of phytohemagglutinin. Also, these cells were co-cultured with 1×106 CFU/ml of L. acidophilus. IL-6 and IL-1 cytokines were measured by ELISA method and the two groups were evaluated and compared. Results: The results showed that in endometriosis patients, the production of pro-inflammatory cytokines, including IL-1 and IL-6, by PBMC was increased compared to non-endometriosis subjects, and stimuli such as PHA intensified this elevation. Also, L. acidophilus increased the levels of pro-inflammatory cytokines including IL-1 and IL-6. However, the production of these cytokines decreased due to the modulatory properties of bacterial cells after 48 h. Conclusion: According to the results of the current study, IL-1 and IL-6 production was significantly increased in PMBCs of endometriosis patients compared to that of the healthy controls. Also, Lactobacillus acidophilus was considered as an antigenic compound and induced IL-1 and IL-6 production. According to these results, probiotics can be further used for the treatment of endometriosis patients and more investigations are needed to confirm these results.

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin potential impacts on peripheral blood mononuclear cells of endometriosis women.

Endometriosis happens following the implantation of endometrial-derived tissues outside the uterine cavity. It has been suggested that 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) is involved in endometriosis development. Furthermore, aryl hydrocarbon receptor (AHR), as a TCDD receptor, has been demonstrated to regulate immune responses. Nonetheless, data regarding the mechanisms, through which TCDD influences the immune system in endometriosis, are still inconclusive. Therefore, frequency of regulatory T cells (Tregs) and the expression of FOXP3, AHR and indoleamine 2, 3-dioxygenase 1 (IDO1) from endometriosis and non-endometriosis individuals were investigated in the absence and presence of TCDD; also, the concentration of IL-6 and kynurenine in the supernatant of cultures was assessed. The impact of TCDD-treated PBMCs on the migration capacity of menstrual blood-derived stromal stem cells (MenSCs) and monocyte chemoattractant protein-1 (MCP-1) and IL-6 production was determined. Here, we found that AHR and IDO1 expression levels were lower in endometriosis PBMCs; however, TCDD treatment increased AHR, FOXP3, IDO1, IL-6, and Treg levels in the endometriosis group (P ≤ 0.05-0.0001). TCDD-treated PBMCs increased the migration capacity of MenSCs and up-regulated MCP-1 and IL-6 levels in the PBMCs/MenSCs co-culture (P ≤ 0.01-0.0001). In conclusion, these results shed light on the probable mechanisms, through which AHR activation by chemical toxicants can impact inflammatory immune mediators involved in the development of endometriosis; also, these data support the idea that TCDD could promote endometriosis progression.

Endometrial mesenchymal stem/stromal cells: The Enigma to code messages for generation of functionally active regulatory T cells.

BACKGROUND: Regulatory T cells (Tregs) play an important role in fine-tuning of immune responses and are pivotal for a successful pregnancy. Recently, the importance of mesenchymal stem cells in regulation of immune responses in general and Tregs in particular has been highlighted. Here, we hypothesized that menstrual stromal/stem cells (MenSCs) contribute to uterine immune system regulation through induction of functionally active Tregs. METHODS: MenSCs were collected from 18 apparently healthy women and characterized. Bone marrow mesenchymal stem cells (BMSCs) served as a control. The effect of MenSCs on proliferation of anti-CD3/CD28-stimulated T CD4 + cells and generation of Tregs with or without pre-treatment with mitomycin C, IFN-γ and IL-1ß was evaluated by flow cytometry. The potential role of IDO, PGE2, IL-6, IL-10, and TGF-ß on proliferation of T CD4 + cells and generation of Tregs was assessed using blocking antibodies or agents. IDO activity was evaluated in MenSCs and BMSCs culture supernatants by a colorimetric assay. IL-10 and IFN-γ production in MenSCs-primed T CD4 + was measured using intracellular staining. To investigate the functional properties of Tregs induced by MenSCs, Treg cells were isolated and their functional property to inhibit proliferation of anti-CD3/CD28-stimulated PBMCs was assessed by flow cytometry. RESULTS: According to the results, proliferation of T CD4 + lymphocytes was enhanced in the presence of MenSCs, while pre-treatment of MenSCs with pro-inflammatory cytokines reversed this effect. PGE2 and IDO were the major players in MenSCs-induced T cell proliferation. Non-treated MenSCs decreased the frequency of Tregs, whereas after pre-treatment with IFN-γ and IL-1ß, they induced functional Tregs with ability to inhibit the proliferation of anti-CD3/CD28-stimulated PBMCs. This effect was mediated through IL-6, IL-10, TGF-ß and IDO. IFN-γ/IL-1ß-treated MenSCs induced IL-10 and IFN-γ production in CD4 + T cells. CONCLUSION: Collectively, these findings indicate that immunomodulatory impact of menstrual blood stem cells (MenSCs) on generation of Tregs and inhibition of T cells proliferation is largely dependent on pre-treatment with IFN-γ and IL-1ß. This is the first report on immunomodulatory impact of MenSCs on Tregs and highlights the pivotal role of endometrial stem cells in regulation of local endometrial immune responses.

Culture density of menstrual blood-derived stromal/stem cells determines the quality of T cell responses: An experimental study.

BACKGROUND: Menstrual blood-derived stromal/stem cells (MenSCs) are a new population of refreshing and highly proliferative stem cells. Immunomodulatory effects of MenSCs profoundly depend on their relative density. OBJECTIVE: To find whether MenSCs cultured at varying numbers would differentially affect the allogenic peripheral blood mononuclear cells (PBMCs) key features. MATERIALS AND METHODS: PBMCs were co-cultured with various MenSCs numbers. PBMCs proliferation was investigated via 3 H-thymidine incorporation. Flow cytometry was used to assess human leukocyte antigen (HLA)-DR, HLA-ABC, HLA-G, and co-stimulatory markers on MenSCs and the percentage of regulatory T cells (Tregs) among PBMCs. The concentration of cytokines was determined in supernatant of co-cultures. RESULTS: The support of PBMCs proliferation at low MenSCs densities correlated with higher levels of pro-inflammatory interferon gamma (IFN-γ) in MenSCs/PBMCs co-culture and increased expression of HLA-DR by MenSCs. On the other hand, the suppressive property of MenSCs at higher densities was independent of Treg frequency, but correlated with a high concentration of Interleukin (IL)-6 and IL-10 in the co-cultures. CONCLUSION: Totally, at different seeding densities, MenSCs could differentially interact with PBMCs leading to significant changes in the level of anti- and/or pro-inflammatory factors. These preliminary in vitro results are suggested to be taken into consideration in experimental models of MenSC-based immunomodulation. Nonetheless, for efficient utilization of MenSCs anti-inflammatory features in pre-clinical disease models, we still need to broaden our knowledge on MenSC-immune system cross-talk; this could play a part in designing more optimized MenSCs injection modalities in the case of future pre-clinical and subsequently clinical settings.

Endometrial and Menstrual Blood Mesenchymal Stem/Stromal Cells: Biological Properties and Clinical Application.

A highly proliferative mesenchymal stem/stromal cell (MSC) population was recently discovered in the dynamic, cyclically regenerating human endometrium as clonogenic stromal cells that fulfilled the International Society for Cellular Therapy (ISCT) criteria. Specific surface markers enriching for clonogenic endometrial MSC (eMSC), CD140b and CD146 co-expression, and the single marker SUSD2, showed their perivascular identity in the endometrium, including the layer which sheds during menstruation. Indeed, cells with MSC properties have been identified in menstrual fluid and commonly termed menstrual blood stem/stromal cells (MenSC). MenSC are generally retrieved from menstrual fluid as plastic adherent cells, similar to bone marrow MSC (bmMSC). While eMSC and MenSC share several biological features with bmMSC, they also show some differences in immunophenotype, proliferation and differentiation capacities. Here we review the phenotype and functions of eMSC and MenSC, with a focus on recent studies. Similar to other MSC, eMSC and MenSC exert immunomodulatory and anti-inflammatory impacts on key cells of the innate and adaptive immune system. These include macrophages, T cells and NK cells, both in vitro and in small and large animal models. These properties suggest eMSC and MenSC as additional sources of MSC for cell therapies in regenerative medicine as well as immune-mediated disorders and inflammatory diseases. Their easy acquisition via an office-based biopsy or collected from menstrual effluent makes eMSC and MenSC attractive sources of MSC for clinical applications. In preparation for clinical translation, a serum-free culture protocol was established for eMSC which includes a small molecule TGFß receptor inhibitor that prevents spontaneous differentiation, apoptosis, senescence, maintains the clonogenic SUSD2+ population and enhances their potency, suggesting potential for cell-therapies and regenerative medicine. However, standardization of MenSC isolation protocols and culture conditions are major issues requiring further research to maximize their potential for clinical application. Future research will also address crucial safety aspects of eMSC and MenSC to ensure these protocols produce cell products free from tumorigenicity and toxicity. Although a wealth of data on the biological properties of eMSC and MenSC has recently been published, it will be important to address their mechanism of action in preclinical models of human disease.

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 IFNγ 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 IFNγ by a colorimetric assay. RESULTS: MenSCs exhibited dual mesenchymal and embryonic markers and multi-lineage differentiation capacity. MenSCs significantly increased proliferation of CD4+ cells at ratios 1:2, 1:4 and 1:8. IFNγ 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 IFNγ 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.

A shift in the balance of T17 and Treg cells in menstrual blood of women with unexplained recurrent spontaneous abortion.

To determine usefulness of menstrual blood for assessment of differential frequency of T cell subsets, peripheral blood (PB) and menstrual blood (MB) from healthy fertile (n=15), unexplained recurrent spontaneous abortion (URSA) women (n=15) and unexplained infertile women (n=8) were collected in the second day of menstrual bleeding. Frequency of T cell subsets was measured by flow cytometry. URSA and unexplained infertile patients had higher frequency of TCRαß(+)CD3(+)CD56(-) and CD45RO(+) T cells as well as CD45RO(+)/CD45RO(-) ratio in PB as compared to MB. Frequency of CD3(+)TCRγδ(+) cells in PB of unexplained infertile and MB of URSA patients were significantly lower than that in fertile group. In all groups MB contained higher percentage of CD4(+)CD25(+)Foxp3(+) regulatory and lower percentages of CD16(+) T cells compared to PB. T17 cells (CD3(+)CD56(-)IL-17(+)) was found to be significantly higher in MB of only fertile and unexplained infertile subjects and not URSA patients in comparison with PB. Compared to PB, only fertile women had higher T17:Treg ratio in their MB. URSA women had significantly lower MB T17 cells and T17:Treg ratio compared to those in fertile and unexplained infertile women. Based on the findings presented here, we speculate that MB has its own unique immune milieu which is not solely continuation of the immune environment of secretory endometrium. Indeed, immunophenotyping of MB immune cells could potentially be a useful tool for investigation of immunological disturbances in pregnancy-related disorders.

Menstrual blood-derived stromal stem cells inhibit optimal generation and maturation of human monocyte-derived dendritic cells.

INTRODUCTION: Menstrual blood stromal stem Cells (MenSCs) have shown promising potential for future clinical settings. Nonetheless, data regarding their interaction with immune cells is still scarce. Here, we investigated whether MenSCs could affect the generation and/or maturation of human blood monocyte-derived dendritic cells (DCs). MATERIALS AND METHODS: MenSCs were isolated from menstrual blood of normal women through culture of adherent mononuclear cells. Magnetically-isolated peripheral blood monocytes were differentiated toward immature DCs (iDC) and mature DCs (mDCs) in the presence or absence of MenSCs. Monocyte-derived cells were assessed for the percentage of monocyte-, iDC-, and mDC-specific markers as well as the expression of costimulatory molecules. IL-6 and IL-10 levels were also determined in supernatants of MenSC-monocytes cocultures. RESULTS: Optimal phenotypic differentiation of monocytes into iDCs was inhibited upon coculture with MenSCs. Moreover, higher levels of IL-6 and IL-10 were detected in these settings. Even though addition of MenSCs to iDC cultures could not prevent iDC maturation, coculture of MenSCs with monocytes from the beginning of differentiation process could effectively hinder generation of fully mature DCs. CONCLUSION: This is the first study to address the inhibitory impact of MenSCs on generation and maturation of DCs. IL-6 and IL-10 could be partly held responsible for this effect. Given the central roles of DCs in regulation of immune responses, these results highlight the importance of further research on the potential modulatory impacts of MenSCs, as rather easily accessible and expandable stem cells, on the immune system-related cells.

Menstrual blood-derived stromal stem cells from women with and without endometriosis reveal different phenotypic and functional characteristics.

Retrograde flow of menstrual blood cells during menstruation is considered as the dominant theory for the development of endometriosis. Moreover, current evidence suggests that endometrial-derived stem cells are key players in the pathogenesis of endometriosis. In particular, endometrial stromal stem cells have been suggested to be involved in the pathogenesis of this disease. Here, we aimed to use menstrual blood, as a novel source of endometrial stem cells, to investigate whether stromal stem cells from endometriosis (E-MenSCs) and non-endometriosis (NE-MenSCs) women differed regarding their morphology, CD marker expression pattern, proliferation, invasion and adhesion capacities and their ability to express certain immunomodulatory molecules. E-MenSCs were morphologically different from NE-MenSCs and showed higher expression of CD9, CD10 and CD29. Furthermore, E-MenSCs had higher proliferation and invasion potentials compared with NE-MenSCs. The amount of indoleamine 2,3-dioxygenase-1 (IDO1) and cyclooxygenase-2 (COX-2) in E-MenSCs co-cultured with allogenic peripheral blood mononuclear cells (PBMCs) was shown to be higher both at the gene and protein levels, and higher IDO1 activity was detected in the endometriosis group. However, NE-MenSCs revealed increased concentrations of forkhead transcription factor-3 (FOXP3) when compared with E-MenSCs. Nonetheless, interferon (IFN)-γ, Interleukin (IL)-10 and monocyte chemoattractant protein-1 (MCP-1) levels were higher in the supernatant of E-MenSCs-PBMC co-cultures. Here, we showed that there are inherent differences between E-MenSCs and NE-MenSCs. These findings propose the key role MenSCs could play in the pathogenesis of endometriosis and further support the retrograde and stem cell theories of endometriosis. Hence, considering its renewable and easily available nature, menstrual blood could be viewed as a reliable and inexpensive material for studies addressing the cellular and molecular aspects of endometriosis.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Evaluation of menstrual blood stem cells seeded in biocompatible Bombyx mori silk fibroin scaffold for cardiac tissue engineering.

Recently, silk fibroin scaffolds have been introduced as novel and promising biomaterials in the field of cardiac tissue engineering. This study was designed to compare infiltration, proliferation, and cardiac differentiation potential of menstrual blood-derived stem cells (MenSCs) versus bone marrow-derived mesenchymal stem cells (BMSCs) in Bombyx mori-derived silk scaffold. Our primary data revealed that the fabricated scaffold has mechanical and physical qualities suitable for cardiac tissue engineering. The MenSCs tracking in scaffolds using immunofluorescent staining and scanning electron microscopy confirmed MenSCs attachment, penetration, and distribution within the porous scaffold matrix. Based on proliferation assay using propidium iodide DNA quantification, the significantly higher level of growth rates of both MenSCs and BMSCs was documented in scaffolds than that in two-dimensional culture (p < 0.01). The expression level of TNNT2, a bona fide cardiac differentiation marker, in BMSCs differentiated on silk scaffolds was markedly higher than those cultured in two-dimensional culture indicating the improvement of cardiac differentiation in the silk scaffolds. Furthermore, differentiated MenSCs exhibited higher expression of TNNT2 compared with induced BMSCs. It seems that silk scaffold-seeded MenSCs could be viewed as a novel, safe, natural, and accessible construct for cardiac tissue engineering.

Suppressive effect of pregnant serum on murine dendritic cell function.

AIM: Tolerance to the semi-allogenic fetal graft by the maternal immune system is a medical enigma. Many aspects of immunoregulation at the feto-maternal interface have been clarified, but systemic effects of pregnancy on the immune system are still elusive. The present study was undertaken to determine whether mid-pregnancy mouse serum has an inhibitory effect on dendritic cells (DC) function. MATERIAL AND METHODS: Mid-gestational sera were obtained from allogenic pregnant Balb/c mice (Balb/c × C57BL/6) on days 9-11 of gestation. Splenic DC were purified from Balb/c mice, and treated with mid-pregnancy mouse serum. Antigen pulsed DC were injected into mice palms. After 5 days, draining lymph nodes were removed, cultured in the presence of cognate antigen, and proliferation of responding cells was measured by (3)H-thymidin incorporation. Interleukin (IL)-10 and interferon-gamma (IFN-γ) production by stimulated lymph node antigen-specific cells was also measured in culture supernatants using sandwich ELISA. RESULTS: Treatment of DC with pregnant mouse serum markedly blocked their ability to induce antigen-specific lymphocyte proliferation and IFN-γ and IL-10 production by primed lymph node cells in comparison with non-pregnant serum-treated DC. CONCLUSION: Pregnant mouse serum has an inhibitory effect on DC capacity to induce antigen-specific proliferation and cytokine secretion by lymph node cells. The suppressive effects of pregnant serum on DC could be considered as one of the mechanisms responsible for the systemic immunomodulation observed during pregnancy.

Effect of menstrual blood-derived stromal stem cells on proliferative capacity of peripheral blood mononuclear cells in allogeneic mixed lymphocyte reaction.

AIM: Menstrual blood stromal stem cells (MBSCs) have been demonstrated to exhibit stem cell properties such as the capability for self-renewal and multipotency, allowing for multilineage differentiation. In addition, this cell type has various immunomodulatory effects. In this study, we examined the potential effect of MBSCs on proliferation of peripheral blood mononuclear cells (PBMCs) in allogeneic mixed lymphocyte reaction (MLR). MATERIALS AND METHODS: Menstrual blood was collected from healthy donors after menstrual blood flow initiated and its mononuclear cell fraction was separated. Cells were subsequently cultured and adherent cells were allowed to propagate and used as stem cells. Flowcytometric immunophenotyping was performed using a panel of monoclonal antibodies including CD44, CD45, CD34, CD9, CD29, CD10, CD38, CD105, CD73, CD133, STRO-1 and Oct-4A. For functional analysis, PBMCs were co-cultured with MBSCs, collected after 4 days and added to allogeneic PBMCs. 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was carried out to evaluate cell proliferation. RESULTS: MBSCs showed surface and intracellular markers of mesenchymal stem cells with the exception of the high expression of Oct-4A. MBSCs affected the proliferative response of PBMC in a dose-dependent manner. At ratio of 1:1 to 1:2, MBSCs inhibited, while at lower ratios (1:32 to 1:64) stimulated the proliferative capacity of allogeneic PBMCs. CONCLUSION: According to the present study, MBSCs exert their immunoregulatory effects on allogeneic PBMCs in a dose-dependent manner. This finding can be considered as a valuable point in future cell therapy strategies, when this cell population is used.

Mutual helper effect in copulsing of dendritic cells with 2 antigens: a novel approach for improvement of dendritic-based vaccine efficacy against tumors and infectious diseases simultaneously.

To develop an efficient dendritic cell (DC)-based immunotherapy protocol, we examined whether simultaneous pulsing of DCs with a given antigen and a third-party antigen could enhance their antigen presentation capacity. Purified splenic DCs of Balb/c mice were pulsed separately with immunoglobulin G, ovalbumin, conalbumin, P15 peptide of Mycobacterium tuberculosis, and prostate-specific antigen or double combinations of the aforementioned antigens. In some settings, DCs pulsed with 1 antigen were mixed equally with those pulsed with another antigen. Antigen-pulsed DCs were injected into the footpad of syngeneic mice and proliferation of whole, CD4 and CD8 depleted lymph node cells was measured after restimulation with cognate antigen. Antigen-specific production of interferon-gamma (IFNgamma) was tested in culture supernatants. Frequency of responding lymph node cells was determined by IFNgamma enzyme-linked immunosorbent spot assay. Our results showed that copulsing of DCs with 2 unrelated antigens increased the capacity of DCs to induce antigen-specific T-cell proliferation against both antigens up to 16-fold. Injection of 2 populations of DCs each pulsed with a different antigen, increased proliferation of primed T cells significantly as well. Both CD4 and CD8 depleted populations showed vigorous proliferative response in copulsing system. In addition, copulsing of DCs with 2 antigens resulted in higher frequency of antigen-specific responding cells and significantly more IFNgamma production. Our results clearly showed that unrelated peptides and proteins could be used to enhance efficacy of DC-based vaccines and in this system, each antigen served to help the other one, a condition that we termed as "mutual helper effect."

The 14kDa protein molecule isolated from garlic suppresses indoleamine 2, 3-dioxygenase metabolites in mononuclear cells in vitro.

A wide range of biological activities of garlic in vitro and in vivo have been verified including its antioxidant, antitumor and anti-inflammatory effects. Indoleamine 2,3-dioxygenase (IDO) is an enzyme widely distributed in mammals and is inducible preferentially by IFN-gamma. IDO degrades the essential amino acid tryptophan to form N-formyl kynurenine. In the present in vitro study, the modulatory effect of 14kDa molecule isolated from garlic on IDO induction was tested. Cultures of mononuclear cells were exposed to 14kDa garlic fraction. Then, their proliferation responses and IDO metabolites were measured. A significant down-regulatory effect of garlic on IDO activity was found and also the proliferation responses of mononuclear cells increased. If these results are verified in vivo, an explanation will be provided on how garlic may interfere in IDO induction, which paves the way for elucidating its specific therapeutic effect in preventing tumor progress.

Immunosuppressive effect of pregnant mouse serum on allostimulatory activity of dendritic cells.

In normal pregnancy, the maternal immune system is directed towards tolerance or suppression in order to prevent rejection of the semi-allogenic fetus. Antigen-presenting cells, especially dendritic cells (DCs), are key cells in initiation and regulation of immune responses. The presence of potent immunostimulatory DCs in the decidual tissue of pregnancy has been demonstrated. The aim of this study was to determine how allostimulatory activity of DCs could be affected during pregnancy. DCs were isolated from spleen of pregnant or non-pregnant Balb/c mice and co-cultured with allogenic T lymphocytes prepared from brachial lymph nodes of C57BL/6 mice. Some cultures of non-pregnant female DCs were treated by 2.5% serum obtained from pregnant mice at early, middle or late gestational periods, and were used in the same mixed lymphocyte reaction (MLR) settings. Cell proliferation was measured by 3H-thymidine incorporation, and cytokine production measured in supernatants of MLR cultures using ELISA. The effect of pregnant mouse serum on expression of DC surface markers was evaluated by flow cytometry. No significant difference was found between stimulatory potential of splenic DCs from pregnant and non-pregnant mice in induction of allogenic T cell proliferative response. Moreover, serum of early or late pregnancy did not have any effect on DC function in comparison with non-pregnant mouse serum, while mid-pregnancy serum significantly inhibited allostimulatory activity of DCs. IFNgamma production in co-culture of DCs treated with pregnant mouse serum was significantly lower than that of the control group; however, no significant difference in IL-10 production was observed. Treatment of DCs with pregnant mouse serum did not influence the percentage of cells expressing MHC-II, CD86, CD8alpha or CD11b. However, a marked reduction of the mean fluorescence intensity of MHC-II was observed. Collectively, our results concerning the diminished capacity of DCs to induce production of Th1 cytokines and allogenic T cell proliferation after treatment with pregnant mouse serum reveal a new way of immunologic tolerance against the semi-allogenic fetus.