Soluble MHC class II-driven therapy for a systemic lupus erythematosus murine experimental in vitro and in vivo model.

Taking into consideration the multiparametric nature of systemic lupus erythematosus (SLE), the severity and variability of symptoms and the lack of effective therapeutic approaches, this study took advantage of the recently described role of soluble major histocompatibility complex class II (sMHCII) molecules in maintaining tolerance to the organism and attempted to apply sMHCII proteins as a treatment to murine SLE experimental models in vitro as well as in vivo. After breaking tolerance to DNA in vitro, which was accompanied by development of specific anti-dsDNA antibodies, syngeneic or allogeneic sMHCII molecules, purified from healthy mouse serum, could significantly reduce the specific antibody levels and drive the system towards immunosuppression, as assessed by specific marker analysis on T cells and cytokine production by flow cytometry and ELISA, respectively. The in vivo experimental model consisted of pristane-induced SLE symptoms to BALB/c mice, which developed maximal levels of anti-dsDNA 2 months after pristane inoculation. Syngeneic or allogeneic sMHCII administration could alleviate pristane-induced symptoms, significantly decrease specific anti-dsDNA antibody production and develop immunosuppression to the host, as manifested by increase of CD4 + CTLA-4 +  and CD4 + CD25 +  cell populations in the spleen. Thus, the results presented in this study introduced the ability of sMHCII proteins to suppress specific autoantigen response, opening new areas of research and offering novel therapeutic approaches to SLE with expanding features to other autoimmune diseases.

Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth.

Patterning of neuronal outgrowth in vitro is important in tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. To date, this has been achieved with the aid of micro- and nanofabrication techniques giving rise to various anisotropic topographies, either in the form of continuous or discontinuous structures. In this study we propose a currently unexplored geometry of a 3D culture substrate for neuronal cell growth comprising discontinuous subcellular microstructures with anisotropic geometrical cross-section. Specifically, using laser precision 3D micro/nano fabrication techniques, silicon substrates comprising arrays of parallel oriented elliptical microcones (MCs) were fabricated to investigate whether a discontinuous geometry comprising anisotropic features at the subcellular level could influence the alignment of peripheral nervous system cell populations. It was shown that both Schwann cells and axons of sympathetic neurons were parallel oriented onto the MCs of elliptical shape, while they exhibited a random orientation onto the MCs of arbitrary shape. Notably, this topography-induced guidance effect was also observed in more complex cell culture systems, such as the organotypic culture whole dorsal root ganglia (DRG) explants. Our results suggest that a discontinuous topographical pattern could promote Schwann cell and axonal alignment, provided that it hosts anisotropic geometrical features, even though the sizes of those range at the subcellular lengthscale. The laser-patterned arrays of MCs presented here could potentially be a useful platform for patterning neurons into artificial networks, allowing the study of neuronal cells interactions under 3D ex-vivo conditions.

Third Harmonic Generation microscopy as a reliable diagnostic tool for evaluating lipid body modification during cell activation: the example of BV-2 microglia cells.

Nonlinear optical processes have found widespread applications in fields ranging from fundamental physics to biomedicine. In this study, we attempted to evaluate cell activation by using the Third Harmonic Generation (THG) imaging microscopy as a new diagnostic tool. The BV-2 microglia cell line with or without activation by lipopolysaccharide was chosen as a representative biological model. The results showed that THG imaging could discriminate between the control versus activated state of BV-2 cells not only as to THG signal intensity but also as to THG signal area, while verifying once more that the majority of the intracellular detected signal corresponds to lipid bodies. Since THG imaging is a real time, non-destructive modality and does not require any prior cell processing and staining, the results presented here provide an important tool for normal versus activated cell discrimination, which could be proved very useful in the study of inflammation.

Microconical silicon structures influence NGF-induced PC12 cell morphology.

Micro-and nanofabrication techniques provide the opportunity to develop new types of cell culture platform, where the effect of various topographical cues on cellular functions such as proliferation and differentiation can be studied. In this study, PC12 cells were cultured on patterned silicon (Si) surfaces comprising arrays of microcones (MCs) exhibiting different geometrical characteristics and surface chemistries. It was illustrated that, in the absence of nerve growth factor (NGF), PC12 cells increased proliferation on all types of patterned surface, as compared to flat Si surfaces. However, in the presence of NGF, PC12 cells showed different responses, depending on the plating surface. Unlike low and intermediate rough MC surfaces, highly rough ones exhibiting large distances between MCs did not support PC12 cell differentiation, independently of the MCs' chemical coatings. These results suggest that the geometrical characteristics of MCs alone can influence specific cellular functions. Tailoring of the physical properties of arrays of Si MCs in order to identify which combinations of MC topologies and spatially defined chemistries are capable of driving specific cellular responses is envisaged.

Do maternity hospital practices support Greek mothers' decision to breastfeed?

AIM: Previous studies have shown that the conditions in Greek maternity hospitals do not support the right of mothers and their children to breastfeed. The aim of the present report was to investigate the degree that Greek maternity hospitals have adopted the 'Ten Steps to Successful Breastfeeding'. METHODS: The study sample comprised 140 mothers living in Athens who had recently given birth and volunteered to fill in specific questionnaires. RESULTS: 40.5% of the mothers did not know what the first meal of their baby was. Regarding hospitals' practices, 68.3% of the mothers mentioned that artificial milk was brought in every meal of the neonate, while 63.6% believed that artificial milk was given to their child without their knowledge, despite the fact that they had already decided to breastfeed. Ninety percent of the mothers giving birth in public maternity hospitals and 60% delivering in private clinics mentioned that health professionals supported breastfeeding (p < 0.05). CONCLUSIONS: It seems that in daily practice, Greece has not yet created an appropriate well informed and supportive environment in regard to breastfeeding.

Characterization of CD25-positive T cells during syngeneic pregnancy: production of stimulatory class II MHC molecules.

Various studies demonstrate that immunosuppression vis-à-vis paternal alloantigens may play a role for successful pregnancy. However, if this theory is true, the question that remains unanswered is how do syngeneic pregnancies manage to produce viable embryos. In allogeneic murine pregnancies immunosuppression is mediated by regulatory CD25(+)/Foxp3/CTLA-4 T cells. In order to evaluate whether these cells also play a role in syngeneic pregnancies, CD25(+)CD4(+) and CD25(+)CD8(+) were isolated from spleens of pregnant mice and examined as to the expression of specific suppressive and stimulatory markers, cytokine and soluble MHC class II antigen production. Interestingly, the CD25(+) cells and their products displayed an MHC-restricted stimulatory activity on total spleen cell proliferation assays. Although the CD25(+)CD4(+) and CD25(+)CD8(+) cells expressed Foxp3, they lacked CTLA-4, while expressing CD28. Non-specific proliferative effect was shown to be mediated by IL-3 and IL-4. The MHC-restricted proliferative effect; however, could be attributed to IA(d) molecules, which were detected in all culture supernatants of the T cell subpopulations tested and their elimination ablated the observed stimulatory activity. The detection of Ea, Eb1, Eb2 in addition to the Aa and Ab transcripts in these cells indicated the possible involvement of other class II gene combinations in this type of regulation. The results indicate that in the absence of paternal MHC alloantigens, maternal CD25(+) cells are involved in the development of immunostimulation to ensure foetal survival.

Endometriosis and infertility: a multi-cytokine imbalance versus ovulation, fertilization and early embryo development.

Endometriosis is tightly linked to infertility which is manifested at very early or more advanced stages of the gestational cycle. Alteration on the production of a great number of cytokines/growth factors can be accused for problems on ovum maturation, fertilization or implantation. Yet, macroscopically these stages are characterized by the inability of conception. A closer look of the cytokinic profile during the conceptional and early gestational cycle could, however, localize the problem and allow a therapeutic approach. In this commentary, going through the cytokine requirement during ovulation, fertilization and the early stages of pregnancy, it became possible to specifically define the harmful endometriosis-induced cytokines for each of the conceptional and early gestational stages. Thus, regulating the levels of interferon-gamma and tumor necrosis-alpha will facilitate ovulation and fertilization, whereas adjusting the levels of interleukin-1beta and colony stimulating gactor-1 will facilitate implantation.

Constitutive or induced elevated levels of L-carnitine correlate with the cytokine and cellular profile of endometriosis.

During the past decade, accumulated evidence indicates an association between endometriosis and an alteration of humoral and cell-mediated immunity. While the role of L-carnitine in the regulation of energy metabolism is well established, it is only recently that L-carnitine has been recognized to modify the immune response in mice after in vitro or in vivo treatment. The present study has examined whether administration of L-carnitine to young female mice alters the percentage of immune cells in peritoneal exudates and the uterus as well as the levels of IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, VEGF, GM-CSF and IGF-I in blood serum, peritoneal fluid and supernatants of uterine cultured cells as tested by immunofluorescence or ELISA techniques, respectively, leading to a pathological disorder resembling human endometriosis. The results showed that, except from infertility, L-carnitine treatment resulted in a significant increase of macrophages and to a lesser degree an increase of T-cells, while elevated levels of IFN-gamma and TNF-alpha were detected in both serum and peritoneal fluid compared to controls. Although levels of L-carnitine measured in mouse serum samples using a radioisotopic method showed an increase as compared to controls, levels of acyl-L-carnitine measured in the murine peritoneal fluid samples showed a decrease similar to that measured in peritoneal fluid samples from patients with endometriosis in stage IV of the disease. These results indicate that L-carnitine administration to female mice alters the cellular and growth factor profile in the uterus and peritoneum towards a phenotypical pathology similar to that of clinical endometriosis.

Serum concentrations of growth factors in women with and without endometriosis: the action of anti-endometriosis medicines.

Endometriosis is a common gynecologic syndrome of unknown etiology and pathogenesis. Growth factors and inflammatory mediators produced by peritoneal leukocytes have recently been postulated to participate in the pathogenesis of endometriosis. Angiogenic factors released from peritoneal macrophages may also play a role in the development of this disease. In the present study, we investigate the soluble levels of vascular endothelial growth factor (VEGF), epidermal growth factor-receptor (EGF-R), granulocyte/macrophage-colony stimulating factor (GM-CSF), Insulin-like growth factor-1 (IGF-1) and interferon-gamma (IFN-gamma) in the serum of 28 women with and 20 without endometriosis. We also compared these levels before, during and after treatment with danazol and leuprorelin acetate depot, the two therapeutic regiments of choice concerning this disease. We found that only sVEGF levels were higher in women with endometriosis in comparison to controls (P < 0.001) while sEGF-R is not present. GM-CSF, IGF-1 and IFN-gamma soluble levels are not affected in either healthy or endometriotic subjects. The 6-month treatment with danazol decreased sVEGF levels (P < 0.02) and increased sEGF-R levels (P < 0.001). These observations support the view that VEGF may be associated with the disease process and that danazol may bring sVEGF levels to a normal threshold. However, future studies will be focused on the anti-angiogenic control of the action of VEGF in patients with endometriosis.

T-regulatory cells: are we re-discovering T suppressors?

Regulatory T cells are shown to originate form the thymus and their role is to maintain self-tolerance to intra-thymic as well as extra-thymic self-antigens. Their mode of action, using in vivo and in vitro systems, has led to different conclusions as to the need of cell-cell interactions or regulation upon suppressive cytokines. The more we study regulatory T cells the more we find similarities to the old notion of the suppressor T cell network. The limited knowledge in molecular technology in the early 70s and 80s discouraged investigators to further scrutinize the issue and the terms T suppressors and contra-suppressors that were coined back then have been forgotten over the years. It is now time to remember the work of these investigators and attempt to explain their findings using the current knowledge and technology.

L-carnitine accelerates the in vitro regeneration of neural network from adult murine brain cells.

The development, growth and regeneration of nerve cells remain an unresolved issue. The up-to-date reported brain repair mechanisms are numerous and evidence suggests that, apart from the required trophism, tropism, microenvironment and specificity of the brain, a plethora of chemical, physiological and immunological compounds can contribute to such events. Among these compounds, we concentrated our interest on L-carnitine (L-Cn), which regulates the beta-oxidation of long chain fatty acids necessary for brain development, myelinization and growth. In contrast to fetal brain cells that grow easily in culture, adult brain cells show limited neurogenesis. Here, using adult brain cells from experimental mice, we show that although L-Cn does not improve their proliferative activity in short-term cultures, it accelerates the growth and differentiation of neurons, astrocytes, oligodendrocytes and ependymal cells from neurospheres in long-term cultures. Thus, the formation of a confluent neural network requires a 2-month period in culture. These observations provide new insights for in vivo use of L-Cn to support brain cell development in cases of injury or brain degenerative diseases.

The role of L-carnitine on a restricted number of myeloid leukemia progenitor cells: generation of atypical cell types.

AIM: Since cellular maturation largely depends on lipid metabolism, we examined whether L-carnitine (L-C), a substance involved in these biochemical pathways, is able to promote differentiation of the promyelocytic cell line HL-60. METHODS: Differentiation was assessed by marker analysis, morphology, immunohistochemistry, proliferation and cellular activity assays. RESULTS: L-C increases HLA-DR and CD14 surface antigens, while morphologic and marker analysis of the treated cells reveals the presence of monocytes, neutrophiles and few dendritic cells. What is important, however, is the induction of cells that have an atypical to this pathway allure staining positive for the neurofilament 3A10 monoclonal antibody, specific for nerve cells and the anti-p75 (Nerve Growth Factor Receptor) monoclonal antibody. The events described concern active and, at the same time, not proliferative senescent cells. CONCLUSIONS: L-C exerts its differentiation action on a certain fraction of the leukemic population yielding a non-negligible number of atypical for the myeloid lineage cells. These findings complement earlier and recent reports that describe the generation of cells of a different lineage irrelevant to their parent line of differentiation indicating that the hemopoietic pool appears to be the source of any kind of cell types according to the stimulus provided. Thus, in the context of the plasticity theory it appears that the HL-60 cell line also possess the potential to differentiate towards unexpected pathways.

L-carnitine modifies the humoral immune response in mice after in vitro or in vivo treatment.

Although the role of L-carnitine (L-Cn) as a cofactor in the oxidation of long-chain fatty acids has been well established, this agent has also been recognized to have an important role in the regulation of carbohydrate metabolism, and consequently, the maintenance of cell membrane structure and cell viability. L-Cn has been reported to reduce the apoptotic levels of CD4+ and CD8+ cells. It has also been demonstrated to interfere with cells of the monocytic lineage by regulating their ability to produce growth factors that ultimately affect both T and B lymphocytic subsets. Therefore, in this study, we examined whether this agent affects the antigenic response of immune cells and determined the relative numbers of immune cells in the murine spleen after in vitro and in vivo treatment. The results showed that L-Cn reduces the relative numbers of CD8+, CD4+ and Ly5+ cells. This observation was consistent in all systems studied including (a) in vitro inoculation of antigen (DNP-HSA) and L-Cn, (b) in vitro priming of spleen cells treated with L-Cn in vivo, and (c) in vivo immunization and L-Cn administration. In all cases, the reduction of T lymphocytes correlated with the decreased production of interleukin-2. L-Cn, however, did not affect the production of specific antibody, which indicates that the observed reduction of Ly5-positive cells is due to cell differentiation of B cells to plasma cells.

Soluble ICAM-1 levels in the serum of endometriotic patients appear to be independent of medical treatment.

Adhesion molecules regulate the interaction of cells with the extracellular matrix and/or other cells. The intercellular adhesion molecule-1 (ICAM-1; CD54) is a member of the immunoglobulin superfamily and expressed by several cell types, including leukocytes and endothelial cells. A circulating form of the usually membrane-bound molecule was identified and characterized in normal human serum and in sera from patients with endometriosis. In the present study, we established the serum-soluble ICAM-1 (sICAM-1) levels in patients with endometriosis. We also studied the effect of danazol and leuprorelin acetate depot on the levels of sICAM-1. Thirty-eight women, 18-45 years of age, with regular menses and documented pelvic endometriosis were recruited from a University Hospital setting. Twenty-two women with endometriosis were randomly divided into two groups. Danazol (600 mg) were given every day for 6 months, and 3.75 mg of leuprorelin acetate depot every 28 days for 6 months. Serum sICAM-1 concentrations were measured before, during and after treatment, and its quantitative determination was performed by an ELISA technique using a specific immunoassay. We found that (1) sICAM-1 levels were higher in women with endometriosis in comparison to healthy subjects; (2) the 6 month treatment with danazol or leuprorelin acetate depot increased sICAM-1 levels (P<0.001); (3) 3 months after termination of both treatments, sICAM-1 levels were unchanged. Although the mechanism leading to the increase of sICAM-1 needs to be further clarified, any benefits of medical treatment of endometriosis such as danazol or leuprorelin appear to be independent of changes in ICAM-1 serum levels.

The possible anti-inflammatory role of circulating human leukocyte antigen levels in women with endometriosis after treatment with danazol and leuprorelin acetate depot.

BACKGROUND: Endometriosis is defined as an inflammatory condition of the female reproductive tract, a state often associated with infertility and miscarriage. Many exogenously administered factors (treatments) control the disease via as yet unknown pathways. Possible candidate molecules involved in these mechanisms could be the serum-soluble human leukocyte antigens (sHIA) that have been detected in a variety of human body fluids and that are associated with several diseases. AIMS: We here examine how danazol and leuprorelin acetate depot treatments exert their anti-inflammatory action. It is plausible that subtle alterations mediated by these treatments and in relation to sHLA may explain the pathophysiology of endometriosis and provide insights towards new therapeutic protocols. METHODS: Indirect enzyme-linked immunosorbent assay (ELISA), using specific monoclonal antibodies, determined serum-soluble class-I and class-II HLA levels. ELISA readings from treated women were compared with normal healthy subjects. RESULTS: Serum-soluble class-I and class-II HLA levels are statistically significantly lower (P < 0.001) in women with endometriosis than in the control groups. However, danazol but not leuprorelin acetate depot administration augments soluble HLA class I and class II (P < 0.01 and P < 0.001, respectively) to normal levels during the treatment period, an increase that may account for the anti-inflammatory effect and the remission observed. CONCLUSIONS: It is shown that one of the underlying causes of endometriosis may be the lack of both circulating class-I and class-II antigen levels. Danazol administration acts via an induced release of these antigens, whose presence correlates with the degree of the inflammatory alleviation obtained. We thus provide evidence that the inflammatory state of the disease appears to be associated with soluble HLA levels because, 3 months after ceasing therapy, the circulating antigens in the serum return to the same levels that correspond to the pathological condition.

Murine ectoplacental cone-derived trophoblast cells express chemokine receptors.

Chemokine receptors (CCRs) have been shown to regulate T cell migration and differentiation as well as the establishment of Th1/Th2 bias. Furthermore, T cells and T cell products are essential to trophoblast development. Thus, postulating that chemokines as well as their receptors may be expressed by trophoblast to move T cells into an interaction with the feto-placental unit, we examined whether CCRs are expressed during the early stages of ectoplacental cone (EPC) formation. For this, murine EPC-derived trophoblast were examined for their ability to express CCRs constitutively or inducible by interferon-gamma (IFN-gamma). Immunofluorescence experiments on EPC-derived trophoblast cells showed that CCR3, CXCR4 and CCR5 are significantly expressed. IFN-gamma accelerated the mobilization of intracellular pools of CCR molecules during early cell culture periods (2-6 h) and, in most cases, increased their expression on EPC-derived trophoblast cells. CCR activity could be detected in the culture supernatants of these cells, inversely proportional to cell surface expression, suggesting the existence of rapid endocytosis and recycling mechanisms. This finding indicates that the level of intracellular CCRs may partly be determined in the extracellular matrix, an event that could play an important role towards neutralization of specific T cell/trophoblast interactions during early stages of pregnancy and protect the fetus against harmful maternal immune responses.

Embryonic immune development and risks of HIV infection.

The importance of the maternal immune system, during both gestation and breastfeeding, for the health of future generations is discussed.

Serum-soluble human leucocyte antigen class I and class II concentrations as an alternative diagnostic test for determining immune indices required for normal pregnancies.

The levels of maternal immunostimulation (required throughout the gestation period) and immunosuppression (needed from the 8th week to labour), as assessed by the mixed lymphocyte reaction (MLR), have been successfully correlated with the outcome of pregnancy. Our laboratory has recently reported that serum-soluble human leucocyte antigen (HLA) class I and II concentrations can be predictive for successful pregnancy outcome. In fact, there is a direct correlation between soluble class II concentrations and maternal immunostimulation because, as expected, these serum HLA concentrations are augmented in the first and second trimester of pregnancy and remain stable thereafter. By the same token, serum HLA class I concentrations are low during the first trimester, correlating with the required absence of immunosuppression, whereas they increase in subsequent trimesters as suppression becomes desirable for counteracting the maternal stimulation, which may otherwise become dangerous to the fetus. In this study, we present biological and statistical evidence that both states of maternal immunostimulation and immunosuppression, reflected by serum soluble HLA class II and class I antigens, do correlate with results obtained by standard MLR and can be predictive of pregnancy failure. The establishment of statistically significant correlations renders the measurement of soluble HLA a reliable test for determining the immunological status of the gestating woman. The unambiguous advantage of such an approach is that soluble HLA testing will no longer require the 1 week delay necessary to obtain MLR results, a period occasionally crucial for applying treatment to women whose immunological indices call for immediate therapeutic intervention.

IFN-gamma facilitates release of class II-loaded intracellular pools in trophoblast cells: a novel property independent of protein synthesis.

Interferon-gamma (IFN-gamma) is an abortion-inducing factor, yet its effects in such a reaction are subject to various levels of regulation. The trophoblast cell line TROPHO-1 can be induced by IFN-gamma to express mRNA and surface class II major histocompatibility complex (MHC) proteins after 8 and 48 h of stimulation, respectively. Untreated cells, however, show an intracellular accumulation of class II antigens earlier (6 h), indicating the existence of MHC pools in the cystosol independent of any induction. On addition of IFN-y, immunofluorescence, subcellular fractionation, and ELISA experiments showed that class II antigen activity detected in the endosomal compartments of the cells could be measured in the culture supernatants. These soluble class II proteins, when isolated and purified using magnetic bead isolation techniques and tested in SDS-PAGE gel and Western blot experiments, had a molecular weight of 70 kDa. Administration of these molecules to pregnant mice as culture supernatants increased the abortion rate and decreased maternal hematocrit levels, effects that could be immunoabsorbed by anti-I-A(d) monoclonal antibodies (mAb). These results indicate that although surface class II molecules are not expressed on trophoblast cells, they accumulate in endosomal compartments and can be released from the cells on addition of IFN-gamma. This new IFN-gamma property, to mobilize intracellular pools of class II MHC antigens in trophoblast cells independent of de novo protein synthesis and induce their release to the extracellular matrix, is a mechanism that appears to be involved in the fetal rejection process, facilitating priming of the maternal organism against the fetal allograft.