Olavio R. Baricordi

HLA-G expression in early embryos is a fundamental prerequisite for the obtainment of pregnancy.

Different mechanisms mediated by the expression of the HLA‐class Ib HLA‐G products are suggested to account for the induction of immune tolerance against the paternal antigens of the fetus during pregnancy. Soluble HLA‐G antigens, mainly produced by cytotrophoblast cells at the materno‐fetal interface and circulating in the body fluids, show a capacity analogous to that of membrane‐boundstructures to inhibit NK cells. In the present report we have investigated, using specific ELISA, the presence of sHLA‐G molecules in culture supernatants of early embryos obtained by in vitro fertilization (IVF) before transfer. The data obtained from the analysis of 285 supernatants corresponding to 101 IVF procedures (43 IVF, 58 intracytoplasmic sperm injection) identify two groups of patients on the basis of sHLA‐G antigen presence. No differences in clinical parameters were observed between the groups, but positive embryo implantations occurred only in women showing sHLA‐G molecules in culture supernatants (Fishers exact p value 2.56×10–3). The results obtained indicate that expression of HLA‐G products in embryo cells is a mandatory, but not sufficient, prerequisite for the development of pregnancy.

Cytolytic P2X purinoceptors

Anedoctal evidence accumulated over almost 20 years has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. The plasma membrane receptors involved have been pharmacologically characterized and cloned during the last 3 years, and named purinergic P2X. P2X receptors share an intriguing structural relatedness with Caenorhabditis elegans degenerins and mammalian amiloride-sensitive Na channels (ENaCs). Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. The intracellular pathways activated are poorly known, but the perturbation in intracellular ion homeostasis clearly plays a major role. ICE proteases (caspases) are also triggered, nonetheless their activation is not requested for ATP-dependent cell death. The physiological meaning of P2X receptor-dependent cytotoxicity is not understood, but an involvement in immune-mediated reactions is postulated.

Presence of detectable levels of soluble HLA-G molecules in CSF of relapsing–remitting multiple sclerosis: relationship with CSF soluble HLA-I and IL-10 concentrations and MRI findings

We have investigated the presence of non-classical soluble HLA-G molecules (sHLA-G) in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and the possible relationships between CSF levels of sHLA-G, classical soluble HLA-I (sHLA-I) molecules, IL-10 amounts and Magnetic Resonance Imaging (MRI) findings were evaluated. We studied by ELISA technique the sHLA-I, sHLA-G and IL-10 levels in CSF of 50 relapsing-remitting (RR) MS patients stratified according to clinical and MRI evidence of disease activity. Thirty-six patients with other inflammatory neurological disorders (OIND) and 41 with non-inflammatory neurological disorders (NIND) were used as controls. CSF mean levels were significantly higher in MS and OIND than in NIND for sHLA-I (p<0.001) and in MS than in controls for sHLA-G (p<0.001), with no differences among the various groups for IL-10 mean concentrations. An increase in CSF sHLA-I was found in MS patients with Gd-enhancing lesions (p<0.01), while sHLA-G and IL-10 were more represented in MS patients without lesional activity on MRI scans (p<0.02). In MRI-inactive MS, CSF IL-10 mean concentrations were significantly greater in patients with CSF-detectable levels of sHLA-G than in those without any evidence of CSF sHLA-G expression (p<0.05). Our findings suggest that CSF classical sHLA-I and non-classical sHLA-G levels may modulate MS activity as assessed by MRI acting in opposite directions. The association observed between sHLA-G and IL-10 when Gd-enhancing lesion resolved indicates a potential immunoregulatory role for IL-10 in the control of MS disease activity by shifting the sHLA-I/sHLA-G balance towards sHLA-G response.

Soluble human leukocyte antigen-G isoforms in maternal plasma in early and late pregnancy.

Problem Human Leukocyte Antigen (HLA)‐G is a class Ib gene located in the human major histocompatibility complex (MHC). Several lines of investigation indicate that the HLA‐G molecule is involved in the maternal acceptance of the semi‐allogenic fetus during pregnancy and in the development of tolerance. Expression of soluble HLA‐G (sHLA‐G) is positively correlated with successful in vitro fertilization (IVF) treatments, and aberrant expression of HLA‐G in certain complications of pregnancy, such as pre‐eclampsia and spontaneous abortion, has been reported. The main purpose of this study was to investigate the levels of different soluble HLA‐G isoforms in maternal plasma in early and late pregnancy.

A decreased positivity for CD90 on human mesenchymal stromal cells (MSCs) is associated with a loss of immunosuppressive activity by MSCs

Biologic and clinical interest in human mesenchymal stromal cells (hMSC) has risen over the last years, mainly due to their immunosuppressive properties. In this study, we investigated the basis of immunomodulant possible variability using hMSC from different sources (amniotic membrane, chorion, and bone marrow from either healthy subjects or patients with hematological malignancies, HM) and having discordant positivity for several immunological markers. The CD90+ hMSC reduced lymphoproliferative response in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) via sHLA‐G and IL‐10 up‐modulation. On the contrary, hMSC showing a significantly lower expression for CD90 antigen, elicited a lymphoproliferative allogeneic response in PHA/PBMCs without any increase in soluble HLA‐G and IL‐10 levels. These data seems to suggest that CD90 molecule may be considered a novel predictive marker for hMSC inhibitory ability, and might cooperate with HLA‐G molecule in regulating suppressive versus stimulatory properties of hMSC. These results may have clinical implication in either transplantation or in regenerative medicine fields.

Emerging topics and new perspectives on HLA-G

Following the Fifth International Conference on non-classical HLA-G antigens (HLA-G), held in Paris in July 2009, we selected some topics which focus on emerging aspects in the setting of HLA-G functions. In particular, HLA-G molecules could play a role in: (1) various inflammatory disorders, such as multiple sclerosis, intracerebral hemorrhage, gastrointestinal, skin and rheumatic diseases, and asthma, where they may act as immunoregulatory factors; (2) the mechanisms to escape immune surveillance utilized by several viruses, such as human cytomegalovirus, herpes simplex virus type 1, rabies virus, hepatitis C virus, influenza virus type A and human immunodeficiency virus 1 (HIV-1); and (3) cytokine/chemokine network and stem cell transplantation, since they seem to modulate cell migration by the downregulation of chemokine receptor expression and mesenchymal stem cell activity blocking of effector cell functions and the generation of regulatory T cells. However, the immunomodulatory circuits mediated by HLA-G proteins still remain to be clarified.

ORIGINAL ARTICLE: Soluble Human Leukocyte Antigen-G Isoforms in Maternal Plasma in Early and Late Pregnancy

Problem Human Leukocyte Antigen (HLA)‐G is a class Ib gene located in the human major histocompatibility complex (MHC). Several lines of investigation indicate that the HLA‐G molecule is involved in the maternal acceptance of the semi‐allogenic fetus during pregnancy and in the development of tolerance. Expression of soluble HLA‐G (sHLA‐G) is positively correlated with successful in vitro fertilization (IVF) treatments, and aberrant expression of HLA‐G in certain complications of pregnancy, such as pre‐eclampsia and spontaneous abortion, has been reported. The main purpose of this study was to investigate the levels of different soluble HLA‐G isoforms in maternal plasma in early and late pregnancy.

Non-classic sHLA class I in human oocyte culture medium.

Soluble human leukocyte antigen (sHLA) class I molecules have been described in all human fluids. These molecules play a significant role in immune function. sHLA has been shown to produce tolerance and to induce apoptosis in cytotoxic alloreactive T cells. They are also present in the supernatant of many cultured cells. Similarly, non-classic HLA class I antigens in soluble form are present in human fluids. Among these, HLA-G is the most important because of its location in fetal tissue that suggests maternal immunological tolerance of the fetal semiallograft. In our present study we show that using two monoclonal antibodies, w6/32 and TP25.99, in the enzyme-linked immunosorbent assay allows the detection of non-classic sHLA class I molecules in the medium from human embryo cultures. The sample were collected from oocytes cultures. Oocyte donors were 11 women attending the in vitro fertilization program. The results showed a significant association (chi2 = 9.66, p = 0.002) between sHLA antigens and the oocyte cleavage rate measured 48 h after fertilization.

A functional role for soluble HLA-G antigens in immune modulation mediated by mesenchymal stromal cells.

BACKGROUND It has been suggested that soluble factors produced by bone marrow (BM) mesenchymal stromal cells (MSC) play a fundamental role in mediating immune modulation. HLA-G antigens (Ag) are major histocompatibility complex (MHC) class Ib molecules characterized by a limited polymorphism and a splicing mechanism that regulates the production of membrane-bound and soluble isoforms. Interleukin-10 (IL-10) cytokine is one of the main up-modulators of soluble HLA-G Ag (sHLA-G) production by CD14+ peripheral blood monocyte cells and increased IL-10 levels are reported to be associated with MSC immune modulation. METHODS We investigated, by specific enzyme-linked immunosorbent assay (ELISA), the possible role of sHLA-G molecules in the inhibition of the peripheral blood mononuclear cell (PBMC) response to phytohemagglutinin (PHA) mediated by MSC from different sources. RESULTS There was a significant correlation between the presence of increased levels of sHLA-G and IL-10 in the MSC/PBMC/PHA culture supernatants and lymphoproliferative inhibition. Neutralizing experiments performed with monoclonal Ab directed against HLA-G and IL-10 molecules confirmed the inhibitory ability of sHLA-G Ag. Furthermore, exogenous IL-10 induced sHLA-G molecule secretion by MSC alone in a polymorphic way, while a longitudinal analysis confirmed the loss of MSC inhibitory functions in relation to in vitro MSC aging. DISCUSSION Overall the results obtained suggest a functional role for sHLA-G molecules in inhibiting the PBMC response mediated by MSC. Moreover, the ability of IL-10 to induce sHLA-G Ag production by MSC alone could be proposed as a marker of MSC functional ability.

Purinoceptor function in the immune system

Immune cells express plasma membrane receptors for extracellular adenosine and purine and pyrimidine nucleotides. Overwhelming evidence suggests that these receptors have a pivotal role in the modulation of several immune cell responses, and may therefore be very important in the overall economy of the immune network. Adenosine can be either an activatory or an inhibitory mediator. Nucleotides have a more complex effect, depending not only on the P2 receptor subtype activated but also on doses applied and the length of stimulation. Both metabotropic (G‐protein‐linked) and ionotropic (intrinsic ion channels) P2 receptors are present in immune cells and their expression is modulated by inflammatory cytokines and differentiation agents. The physiological meaning of the P2 receptors is still obscure; however, converging evidence from different laboratories suggest that they may be involved in a wide variety of responses such proliferation, differentiation, cell death, and cytokine release. Drug Dev. Res. 39:319–329, 1996.