Duodenal lymphocytosis in functional dyspepsia.

BACKGROUND AND STUDY AIMS: Functional dyspepsia is an exclusion diagnosis requiring different tests, including endoscopy, often repeated over time. Duodenal biopsies are frequently resorted to, not rarely revealing duodenal microscopic inflammation. Aim of the study is to confirm a previously supposed role of antro-duodenal low-grade inflammation in functional dyspepsia, evaluating the frequency of duodenal lymphocytosis, H. pylori infection and their association in a group of patients with functional dyspepsia compared to asymptomatic control subjects. PATIENTS AND METHODS: A cross-sectional, observational study has been conducted screening all the patients who underwent duodenal biopsies during upper endoscopy, in a 30 months period. All the patients without endoscopic lesions were analysed. The study group consisted of patients compatible with the diagnosis of functional dyspepsia (Rome III criteria). The control group consisted of healthy asymptomatic subjects in the population subjected to endoscopy. The presence of duodenal lymphocytosis and of H. pylori infection in the two groups was evaluated. RESULTS: 216 patients were enrolled: 161 in the functional dyspepsia group and 55 as asymptomatic control group. The frequency of duodenal lymphocytosis was similar between cases and control groups (25.47% vs 25.45%; p = 0.99), as well as H. pylori infection (26.71% vs 23.64%; p = 0.78). Duodenal lymphocytosis was significantly associated with functional dyspepsia only in H. pylori positive dyspeptic patients (p = 0.047). 94% of the subjects with both lymphocytosis and H. pylori infection suffer from dyspepsia. Duodenal intraepithelial lymphocytosis is significantly associated with bloating (p = 0.0082). CONCLUSIONS: In our cohort of dyspeptic patients, duodenal lymphocytosis is significantly associated with bloating and the simultaneous presence of duodenal lymphocytosis and H. pylori infection is significantly more prevalent than in control subjects.

HLA-G expression and regulation during Pseudomonas aeruginosa infection in cystic fibrosis patients.

BACKGROUND: Deregulated immune response fails to control biofilm-forming bacteria, as Pseudomonas aeruginosa, in the lungs of cystic fibrosis (CF) patients. HLA-G is an immune-modulatory molecule involved in respiratory diseases and infections. MATERIALS & METHODS: HLA-G mRNA and protein were analyzed in plasma and exhaled breath condensate from CF patients undergoing intravenous antibiotic treatment, CF cell line and murine model. RESULTS: Therapy normalizes HLA-G plasmatic in CF patients suggesting a systemic anti-inflammatory role while in CF airway system, higher expression of HLA-G is associated with P. aeruginosa infection. CF cell line and murine model expressed higher HLA-G molecules in the presence of P. aeruginosa. CONCLUSION: Plasmatic and lung HLA-G expression suggest a role in reducing systemic inflammation and supporting P. aeruginosa infection.

Matrix metalloproteinase-2 (MMP-2) generates soluble HLA-G1 by cell surface proteolytic shedding.

Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.

HLA-G may predict the disease course in patients with early rheumatoid arthritis.

The current management of early rheumatoid arthritis (ERA) is to start an intensive treatment as soon as possible. To avoid under/overtreatment, it is important to identify reliable ERA evolution biomarkers. HLA-G molecules has been associated with rheumatoid arthritis, suggesting a role in disease regulation. HLA-G antigens are expressed as membrane bound and soluble isoforms (mHLA-G, sHLA-G) that act as ligand for immune-inhibitory receptors (ILT2, ILT4, KIR2DL4). Expression of HLA-G is influenced by a 14 bp insertion/deletion polymorphism in exon 8 of the gene, where the deletion is associated with mRNA stability. We analyzed 23 ERA patients during a 12 months follow-up disease treatment for sHLA-G, IL-1beta, IL-6, IL-10 and TNF-alpha levels in plasma samples by ELISA, mHLA-G and ILT2 expression on peripheral blood CD14 positive cells by flow cytometry and typed HLA-G 14 bp deletion/insertion polymorphism by Real-Time PCR. Disease status (DAS28), ultrasonography with power Doppler and laboratory data were checked. Cytokine levels confirmed the anti-inflammatory effect of the treatment. sHLA-G, mHLA-G and ILT2 expression inversely correlated with DAS28 disease scores. The frequency of 14 bp deletion allele increased in patients with disease remission. Based on these results, HLA-G may be a candidate biomarker to evaluate early prognosis and disease activity in ERA patients.

Role of HLA-G 14bp deletion/insertion and +3142C>G polymorphisms in the production of sHLA-G molecules in relapsing-remitting multiple sclerosis.

HLA-G is believed to act as an anti-inflammatory molecule in Multiple Sclerosis (MS). The 3' untranslated region of the HLA-G gene is characterized by two polymorphisms, DEL/INS14bp and +3142C>G, which control soluble HLA-G (sHLA-G) production. The influence of these two HLA-G variants on sHLA-G serum and cerebrospinal fluid (CSF) levels was investigated in 69 Relapsing-Remitting MS patients grouped in magnetic resonance imaging (MRI) inactive and active disease. Serum and CSF sHLA-G levels were more elevated in high than in low DEL/INS 14bp and +3142C>G sHLA-G producers and were different among the various combined HLA-G genotypes in both MRI inactive and active diseases. The highest and the lowest sHLA-G values were identified in MS patients with C/C,DEL/DEL and G/G,INS/INS genotypes, respectively. Our preliminary findings suggest that serum and CSF sHLA-G levels in MS could be influenced by HLA-G polymorphisms irrespective of the inflammatory microenvironment.

Umbilical cord blood CD34(+)cell-derived progeny produces human leukocyte antigen-G molecules with immuno-modulatory functions.

Human umbilical cord blood units (UCBs) are an alternative source in allogeneic-stem-cell transplantation. Human leukocyte antigen (HLA)-G is a tolerogenic molecule with a possible implication in UCB immunoregulatory effect. HLA-G expression was observed in UCB myeloid and plasmacytoid dendritic cells; in contrast, CD34(+) cells did not produce this molecule. CD34(+) cells are primitive hematopoietic progenitor cells that are present in UCB and are necessary for long-term engraftment via production of immunoregulatory molecules and a hematopoietic progeny that supports cellular recovery. The role of these cells in UCB transplantation needs further evaluation of HLA-G expression in CD34(+) cells and their hematopoietic progeny. We confirmed the absence of HLA-G expression in CD34(+) cells, whereas CD34(+)-derived progeny secreted HLA-G molecules and expressed HLA-G mRNA in in vitro cultures. Furthermore, soluble HLA (sHLA)-G molecules purified from the culture supernatants of CD34(+)-derived progeny were able to suppress lymphoproliferative response in an HLA-G dose-dependent manner. Overall these results identify CD34(+)-derived hematopoietic progeny as producers of HLA-G molecules and support a role of this antigen as an immuno-modulatory factor in UCB.

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential.

BACKGROUND AIMS: The beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC). METHODS: Thirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-(3)H)thymidine. RESULTS: The results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502). CONCLUSIONS: We propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.

Soluble human leukocyte antigen-g expression and glucose tolerance in subjects with different degrees of adiposity.

CONTEXT: Type 2 diabetes mellitus (T2DM) and obesity are characterized by a low-grade inflammation, which might be related to the development of insulin resistance. Human leukocyte antigen-G (HLA-G) shows antiinflammatory and tolerogenic properties, including the modulation of CD8+ T-cell cytotoxicity and regulation of CD4+ T-lymphocyte function. These functions are partially shared with IL-10, whose levels are reduced in insulin-resistant states. OBJECTIVE: The aim was to explore the relationship between HLA-G and the metabolic and inflammatory pattern of obesity or T2DM. PATIENTS AND MAIN OUTCOME MEASURES: Soluble HLA-G, IL-6, and IL-10 were measured and related with metabolic and biochemical parameters in 230 volunteers with normal glucose tolerance, impaired glucose tolerance, or T2DM by oral glucose tolerance test. RESULTS: sHLA-G, detected in 144 subjects (sHLA-G positive), was more frequent in T2DM or impaired glucose tolerance subjects than in normal glucose tolerance (chi(2) =18.6; P < 0.0001), and its plasma levels increased progressively across the classes of glucose tolerance. sHLA-G-positive individuals had higher body mass index, systolic blood pressure, and cholesterol levels; a reduced degree of insulin sensitivity; and almost 2-fold higher levels of IL-6, a cytokine related to insulin sensitivity, whereas IL-10 was similar. In the sHLA-G-positive subgroup, by a multivariate regression model, sHLA-G was significantly related to 2-h glucose, the area under insulin curve, and IL-6 levels (multiple r(2) = 0.14; P < 0.001), independently of age, gender, and body mass index. CONCLUSIONS: A frequent expression of sHLA-G, linked to a typical biomarker of insulin resistance like IL-6, seems to characterize subjects with an impaired glucose metabolism.

Human leukocyte antigen-G molecules are constitutively expressed by synovial fibroblasts and upmodulated in osteoarthritis.

Human leukocyte antigen (HLA)-G molecules are nonclassical HLA class I antigens expressed as membrane bound and soluble isoforms (sHLA-G) with a restricted tissue distribution and anti-inflammatory functions. Because inflammation is involved in the pathogenesis of osteoarthritis (OA), we have analyzed the expression and production of HLA-G molecules in in vitro cultured synovial fibroblasts (SFs) from OA patients and control subjects. We have analyzed the levels of sHLA-G1 and HLA-G5 isoforms by immunoenzymatic assay (enzyme-linked immunosorbent assay) in the SF culture supernatants from six OA patients and six control subjects in 70-day in vitro cultures and after the addition of lipopolysaccharide or recombinant interleukin (IL)-10 (rIL-10). We have confirmed HLA-G modulation by cytofluorimetry and immunofluorescence. The results have demonstrated the spontaneous production of sHLA-G1 molecules by both OA and control SFs. The expression was confirmed by cytofluorimetry and immunofluorescence. OA SFs produce both sHLA-G1 and HLA-G5 molecules during the first 23 days of culture and higher levels of sHLA-G1 during the first 40 days of in vitro culture and after lipopolysaccharide or rIL-10 activation compared with control SFs. The production of HLA-G1 molecules, constitutively expressed by control and OA SFs, is significantly increased in OA, suggesting a possible mechanism to counteract the inflammation of the synovial joints.

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. METHOD OF STUDY: Soluble HLA-G (sHLA-G) can be detected in maternal blood, and in this study, two different isoforms of sHLA-G, namely sHLA-G1 generated by shedding of membrane-bound HLA-G1 and HLA-G generated by specific HLA-G transcripts, have been investigated early [median of 16.4 weeks of gestation (GW)] and late (median: 38.9 GW) in pregnancy in an original cohort of 580 pregnant Caucasian women. RESULTS: Lower concentrations of sHLA-G1 were found late in pregnancy (>32 GW) in a group of women with severe pre-eclampsia compared with controls with uncomplicated pregnancies (P = 0.029, P(C) = 0.09; Mann-Whitney; Logistic regression analysis: P = 0.024, OR = 0.920, 95% CI: 0.855-0.989). However, this was not the case with HLA-G5, and significantly more of the cases with severe pre-eclampsia had detectable plasma HLA-G5 compared with that of the control group (P = 0.013, P(C) = 0.04; Mann-Whitney). Similar findings were not observed in women with gestational hypertension or existing hypertension continuing into pregnancy. Furthermore, there was a trend toward lower maternal plasma sHLA-G1 in a group of women with premature birth (<37 GW) compared with that of the control group (P = 0.028, P(C) = 0.17; Mann-Whitney). On the contrary, HLA-G5 was lower in the control group compared with that in the premature group (P = 0.004, P(C) = 0.02; Mann-Whitney). CONCLUSION: This study shows in line with other published studies that a high, detectable soluble HLA-G concentration in maternal plasma or serum is not mandatory for a successful pregnancy. However, complications during pregnancy, such as (severe) pre-eclampsia, spontaneous abortion, IUGR, and premature birth, are associated with a low or undetectable level of soluble HLA-G in the maternal blood circulation. Also, this study indicates that sHLA-G1 is the interesting soluble HLA-G isoform in pre-eclampsia, and that low or undetectable levels of HLA-G5 at the end of pregnancy seem to be associated with an uncomplicated normal pregnancy, whereas in severe pre-eclampsia and possibly other pregnancy complications, such as preterm birth and IUGR, the level of HLA-G5 is higher.

Extracellular ATP acting at the P2X7 receptor inhibits secretion of soluble HLA-G from human monocytes.

Bacterial LPS induces the release of ATP from immune cells. Accruing evidence suggests that extracellular ATP participates in the inflammatory response as a proinflammatory mediator by activating the inflammasome complex, inducing secretion of cytokines (IL-1, IL-18) and cell damaging agents such as oxygen radicals, cationic proteins, and metalloproteases. It is not known whether ATP can also act as a proinflammatory mediator by inhibiting production of molecules down-modulating the immune response. Here, we show that extracellular ATP impairs in an IL-10-dependent fashion the expression of the tolerogenic soluble and membrane-bound HLA-G Ag in human monocytes. The effect of ATP was mimicked by BzATP (3'-O-(4-benzoyl)benzoyl-ATP) and greatly reduced by pretreatment with oATP (periodate-oxidized ATP), KN-62 (1-[N,O-bis(5-isoquinoline-sulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine), and an anti-P2X(7) mAb, thus pointing to a specific role of the P2X(7) receptor. The effect of ATP was time- and dose-dependent and was not due to a decrease in expression of IL-10 receptor. Inhibition by ATP was reverted by supplementation of culture medium with exogenous IL-10. Due to the well-known immunosuppressive activity of IL-10 and soluble HLA-G, this novel effect of ATP might be relevant for the pathophysiology and therapy of inflammatory disorders.

Possible role of human leukocyte antigen-G molecules in human oocyte/embryo secretome.

Human leucocyte antigen-G (HLA-G) is a nonclassical HLA class I molecule observed for the first time in human cytotrophoblast. Several investigations have demonstrated the presence of soluble HLA-G in oocyte/embryo secretome. A focus on the possible role of HLA-G in oocyte/embryo context will be developed in this review. In addition, the possible use of HLA-G in assisted reproductive technology will be treated.

Allergic women have reduced sHLA-G plasma levels at delivery.

PROBLEM: HLA-G antigen maintains a tolerogenic condition at the foeto-maternal interface, counteracts inflammation in autoimmune diseases and soluble HLA-G (sHLA-G) levels decrease in allergic-asthmatics. Taking into consideration these findings, we analyzed if sHLA-G and interleukin-10 (IL-10) could be influenced by pregnancy and labour in allergic and non-allergic women. METHOD OF STUDY: sHLA-G isoforms and IL-10 levels were determined in the plasma samples of 43 women (15 non-allergic, 28 allergic) during third trimester, at delivery and 2 years after pregnancy by immunoenzymatic assays. RESULTS: A significant increase in sHLA-G and IL-10 levels was documented at delivery in both allergic and non-allergic women. Allergic women showed lower sHLA-G concentrations. sHLA-G1 was evidenced as the predominant plasma isoform. CONCLUSION: The data showed increased sHLA-G and IL-10 concentrations at delivery, regardless of the allergic status. The sHLA-G1 isoform is mainly responsible for the increased sHLA-G levels at delivery.

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.

Reduced production of anti-inflammatory soluble HLA-G molecules in styrene exposed workers.

HLA-G antigens are non-classical HLA-class I anti-inflammatory molecules. Since styrene exposure has been suggested to induce immune alteration, we analyzed plasma levels and "in vitro" peripheral blood mononuclear cell (PBMC) production of soluble HLA-G (sHLA-G) and interleukin-10 (IL-10) molecules after lipopolysaccharide (LPS) stimulation, in styrene exposed workers and healthy subjects. Exposed workers showed reduced plasma levels of sHLA-G and IL-10 in comparison to healthy controls. Similarly, lower levels of sHLA-G and IL-10 molecules were observed in PBMC culture supernatants after LPS activation. These data propose styrene exposure as a mediator of impaired sHLA-G production.

Decreased production of human leukocyte antigen G molecules in sinonasal polyposis.

BACKGROUND: Sinonasal polyposis (SNP) is a chronic inflammatory pathology of nasal and paranasal cavities. Human leukocyte antigen (HLA) G molecules are nonclassic class I antigens with anti-inflammatory and tolerogenic properties. As most theories consider polyps to be the manifestation of chronic inflammation, there could be a possible implication of HLA-G molecules in SNP. The purpose of this study was to investigate the possible correlation between SNP and the production of soluble HLA-G (sHLA-G) by peripheral blood mononuclear cells (PBMCs). METHODS: The study involved 22 SNP patients (11 with no evidence of disease [NED] after surgery and 11 with relapse [RE]) and 20 healthy subjects. The presence of sHLA-G in PBMC lipopolysaccharide (LPS)-stimulated culture supernatants was analyzed. The levels of interleukin (IL) 10, one of the main up-regulators of sHLA-G production, were determined. Exogenous IL-10 was added to the SNP PBMC cultures to reconstitute the impairment in sHLA-G production. RESULTS: Increased IL-10 levels in LPS-activated PBMC culture supernatants were found in NED patients in comparison with healthy subjects (p = 0.0184). No sHLA-G production was observed in either of the patient subgroup supernatants (p < 0.0001). The addition of exogenous IL-10 showed the reconstitution of sHLA-G production in NED and in a lower amount in RE patients. CONCLUSION: The results show a defect in sHLA-G production in SNP patients mainly related to the IL-10/HLA-G pathway. Given the anti-inflammatory functions of HLA-G molecules, this impairment could increase the susceptibility to the disease. The different sHLA-G production after exogenous IL-10 addition between NED and RE SNP could represent a marker of disease severity.