Evaluation of membrane-bound and soluble forms of human leucocyte antigen-G in systemic sclerosis.

Systemic sclerosis (SSc) is a complex disease characterized by immune dysregulation, extensive vascular damage and widespread fibrosis. Human leucocyte antigen-G (HLA-G) is a non-classic class I major histocompatibility complex (MHC) molecule characterized by complex immunomodulating properties. HLA-G is expressed on the membrane of different cell lineages in both physiological and pathological conditions. HLA-G is also detectable in soluble form (sHLA-G) deriving from the shedding of surface isoforms (sHLA-G1) or the secretion of soluble isoforms (HLA-G5). Several immunosuppressive functions have been attributed to both membrane-bound and soluble HLA-G molecules. The plasma levels of sHLA-G were higher in SSc patients (444·27 ± 304·84 U/ml) compared to controls (16·74 ± 20·58 U/ml) (P < 0·0001). The plasma levels of transforming growth factor (TGF)-ß were higher in SSc patients (18 937 ± 15 217 pg/ml) compared to controls (11 099 ± 6081 pg/ml; P = 0·003), and a significant correlation was found between TGF-ß and the plasma levels of total sHLA-G (r = 0·65; P < 0·01), sHLA-G1 (r = 0·60; P = 0·003) and HLA-G5 (r = 0·47; P = 0·02). The percentage of HLA-G-positive monocytes (0·98 ± 1·72), CD4+ (0·37 ± 0·68), CD8+ (2·05 ± 3·74) and CD4+ CD8+ double-positive cells (14·53 ± 16·88) was higher in SSc patients than in controls (0·11 ± 0·08, 0·01 ± 0·01, 0·01 ± 0·01 and 0·39 ± 0·40, respectively) (P < 0·0001). These data indicate that in SSc the secretion and/or shedding of soluble HLA-G molecules and the membrane expression of HLA-G by peripheral blood mononuclear cells (PBMC) is clearly elevated, suggesting an involvement of HLA-G molecules in the immune dysregulation of SSc.

Successful antiviral therapy determines a significant decrease in squamous cell carcinoma antigen-associated (SCCA) variants' serum levels in anti-HCV positive cirrhotic patients.

Aberrant squamous cell carcinoma antigen (SCCA) expression is an early event in hepatocarcinogenesis, and increasing serum levels of SCCA variants IgM immune complexes (SCCA-IgM IC) have been found in cirrhotic patients developing hepatocellular carcinoma (HCC). We longitudinally evaluated a cohort of cirrhotic patients with hepatitis C virus infection (HCV) who underwent pegylated interferon (PEG-IFN) and ribavirin treatment. SCCA-IgM IC levels were assessed in the sera of 33 cirrhotic patients with HCV (21 males, median age 57 years) before, at the end and at 6-month and 1-year follow-up after treatment with PEG-IFN and ribavirin. SCCA-IgM IC serum levels (arbitrary units/mL, AU/mL) were evaluated according to treatment outcome: sustained virological response (SVR) vs nonresponse (NR). Overall, 15 patients obtained a SVR to antiviral therapy (45%). There was no significant difference in baseline SCCA-IgM IC serum levels between SVR and NR patients. When compared to baseline (451.2 AU/mL), SVR patients showed a significant decrease in median SCCA-IgM IC serum levels at the end of treatment (186.8 AU/mL, P = 0.013) and at both 6-month (96.8 AU/mL, P < 0.001) and 1-year follow-up (52.4 AU/mL, P < 0.001), while no significant modification was observed in NR patients. In patients with HCV-related liver cirrhosis, successful antiviral therapy is associated with a dramatic and significant decrease in SCCA-IC serum levels. Because of the pathophysiological correlation between SCCA and liver carcinogenesis, it is hypothesized that in patients with liver cirrhosis, SVR may be accompanied by a decreased proliferative stimulation.

HLA-G Expressing Immune Cells in Immune Mediated Diseases.

HLA-G is a HLA class Ib antigen that possesses immunomodulatory properties. HLA-G-expressing CD4+ and CD8+ T lymphocytes, NK cells, monocytes, and dendritic cells with immunoregulatory functions are present in small percentages of patients with physiologic conditions. Quantitative and qualitative derangements of HLA-G+ immune cells have been detected in several conditions in which the immune system plays an important role, such as infectious, neoplastic, and autoimmune diseases as well as in complications from transplants and pregnancy. These observations strongly support the hypothesis that HLA-G+ immune cells may be implicated in the complex mechanisms underlying the pathogenesis of these disorders.

IGF-I induced rapid recruitment of integrin beta1 to lipid rafts is Caveolin-1 dependent.

Caveolin-1 (Cav-1) regulates both insulin like growth factor receptor (IGF-IR) and integrin beta1 function. However, the role of Cav-1 in IGF-IR/integrin beta1 cross talk remains to be established. In this study, we observed that IGF-I did not induce integrin beta1 internalization but its plasma membrane reorganization. In particular, we found a rapid and transient association between integrin beta1 and Cav-1 followed by the enrichment of integrin beta1 in lipid rafts. To determine the role of Cav-1 in this process, we transfected Hacat cells with small interfering RNA specific for Cav-1 (siRNA-Cav-1) and with a scrambled siRNA as control (siRNA-Ctr). Cav-1 down regulated Hacat cells were then stimulated with IGF-I and analyzed by immunofluorescence and flow cytometry. We found that Cav-1 silencing abolished the recruitment of integrin beta1 to lipid rafts in the presence of IGF-I. These data demonstrate that IGF-IR/integrin beta1 cross talk is followed by integrin beta1 lipid raft compartmentalization and that Cav-1 is required for this process.

Soluble HLA-G and HLA-A,-B,-C serum levels in patients with allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is characterized by Th2-polarized immune response. Soluble HLA (sHLA) molecules play an immunomodulatory activity. So far, however, no study investigated them in AR. OBJECTIVE: The aim of this study was to evaluate sHLA-G and sHLA-A,-B,-C serum levels in AR patients with pollen allergy and in a group of healthy controls. METHODS: Forty-nine AR patients were enrolled. A group of healthy nonallergic subjects was considered as control. sHLA-G and sHLA-A,-B,-C serum levels were determined by immunoenzymatic method. The study was conducted during the winter, such as outside the pollen season. RESULTS: Allergic patients had significantly higher levels of both sHLA-G (P < 0.0001) and sHLA-A,-B,-C (P = 0.011) molecules than normal controls. Moreover, there was a significant relationship between these two soluble molecules (r = 0.69) in allergic patients. CONCLUSION: The present study provides the first evidence that both sHLA-G and sHLA-A,-B,-C serum levels are significantly increased in AR patients with pollen allergy.

In vivo apoptosis of CD8(+) lymphocytes in acute myeloid leukemia patients: involvement of soluble HLA-I and Fas ligand.

In this study, we show that high serum levels of soluble human leukocyte antigens (HLA) class I molecules (sHLA-I, range: 0.7-1.7 micro g/ml) and soluble Fas ligand (FasL, range: 0.4-1.9 ng/ml) are detected in patients with acute myeloid leukemia (AML) at diagnosis, compared with healthy donors (HD) (sHLA-I, range: 0.1-0.6 micro g/ml; sFasL, range: 0.1-0.4 ng/ml). Patients' sera were able to induce transcription and secretion of FasL in CD8(+) T cells, followed by apoptosis in vitro; this apoptosis was inhibited by anti-HLA-I-specific monoclonal antibodies, suggesting that sHLA-I is responsible for cell death. These findings closely relate to the in vivo upregulation of FasL transcription observed in peripheral blood (PB) lymphocytes from AML patients; in the same cells, mRNA for the antiapoptotic proteins Bcl-2 and Bcl-x(L) was downregulated. Interestingly, caspase-8 and caspase-3, both downstream mediators of death receptor-induced apoptosis, were activated in CD8(+) cells of AML patients; one-third of these cells were already apoptotic in vivo, at variance with lymphocytes of HD. These data strongly suggest that in AML, increased levels of sHLA-I molecules may contribute to the elimination of potentially anti-tumor effector cells through a FasL/Fas interaction.

Soluble HLA class I/CD8 ligation triggers apoptosis in EBV-specific CD8+ cytotoxic T lymphocytes by Fas/Fas-ligand interaction.

In the present study, we report that allogeneic soluble HLA class I (sHLA-I) molecules isolated from serum induce apoptosis on EBV-specific CD8(+) Fas(+) cytotoxic T lymphocytes (CTL). CTL apoptosis is induced by the binding of sHLA-I molecules to CD8 and its extent depends on the time of incubation with sHLA-I molecules. Apoptosis is triggered by the interaction of Fas(+) CTL with soluble Fas-ligand, which is released following the binding of sHLA-I antigens to CD8 molecules. These results suggest that sHLA-I molecules may regulate immune responses by inducing apoptosis in virus-specific CTL.

Serum HLA class I levels in elderly humans. Utilization in following the response to influenza vaccine.

Some healthy elderly people (30%-40%) do not respond to influenza vaccination. Subjects who respond to influenza vaccination show a significant increase in serum HLA class I levels 2 weeks after immunization. In the present study, serum HLA class I levels were measured prior to vaccination and 4 and 6 weeks after vaccination. The responders (HAI titers > or = 40) had higher prevaccination HLA class I levels (2.03 +/- 0.12 microgram/ml) than nonresponders (HAI titers < 40) (1.57 +/- 0.14 microgram/ml) (P = 0.03). The responders' sHLA levels remained stable at 4 (1.98 +/- 0.12 microgram/ml) and 6 (2.13 +/- 0.12 microgram/ml) weeks postvaccination. In contrast, nonresponders' sHLA levels increased at 4 weeks (1.85 +/- 0.18 microgram/ml) but declined to prevaccination levels at 6 weeks (1.59 +/- 0.16 microgram/ml). Lymphocytes isolated from the responders (n = 4) 4 weeks after vaccination had a higher in vitro proliferative response (mean SI = 20) than lymphocytes from nonresponders (n = 4, mean SI = 6.4) to the influenza vaccine. This proliferative response was significantly inhibited (mean SI = 6.3, P = 0.04) in the responders by the addition of an anti-HLA class I mAb and was associated with increased cell surface expression of HLA class I molecules. Two forms of sHLA class I molecules of relative mass of M(r) 42,000 and 40,000 were immunoprecipitated from the serum of nonresponders whereas only the M(r) 40,000 form was detected in the sera of young controls and of elderly responders.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble HLA class I and class II molecule levels in serum and cerebrospinal fluid of multiple sclerosis patients.

Increased concentrations of soluble HLA class I and class II molecules (sHLA-I and sHLA-II) have been observed in infectious, inflammatory, and autoimmune diseases. Because autoimmune mechanisms are considered to play a role in the pathogenesis of multiple sclerosis (MS), we decided to dose sHLA-I and sHLA-II in serum and cerebrospinal fluid (CSF) of MS patients comparing their concentrations with those observed in serum and CSF of patients with other neurologic diseases (OND) without evidence of neuroradiologic involvement of central nervous system (CNS) and in serum of healthy donors. The serum concentrations of sHLA-I were higher in both MS and OND patients than in healthy donors (P < 0.05) whereas sHLA-II serum concentrations were lower in MS patients than in both OND patients and healthy donors (P < 0.01). Detectable amounts of sHLA-II were observed in the CSF of 45% of MS patients and in CSF of only 6% of OND patients (P < 0.001). In MS patients a significant correlation between sHLA-I serum and CSF concentrations was observed (P < 0.01), whereas sHLA-II serum and CSF levels did not correlate. In conclusion, alterations of sHLA-I and sHLA-II serum and CSF concentrations are present in MS patients and could be involved in the induction of enhanced susceptibility to develop MS or in MS pathogenesis.

Downregulation of HLA class I antigen expression in CD4+ T lymphocytes from HIV type 1-infected individuals.

The expression of HLA class I antigens is downregulated in CD4+ T cells following in vitro HIV-1 infection. We determined whether the expression of HLA class I antigens is downmodulated in peripheral blood lymphocytes (PBLs) of HIV-1-positive subjects and whether this defect correlates with disease progression. A cohort of 62 HIV-1-seropositive individuals in different stages of disease was studied. Among these, four subjects were evaluated at yearly intervals for 6 years. The expression of HLA class I, HLA class II, and CD38 antigens was analyzed in PBLs and in CD4+ and CD8+ T lymphocyte subpopulations. The percentage of HLA class I-positive cells and the membrane density of HLA class I antigens were significantly lower in PBLs from HIV-1-positive individuals than in PBLs from HIV-negative controls, proportionally decreased with disease progression, and significantly correlated with the decrease in CD4+ T lymphocytes. Furthermore, the percentage of HLA class I-positive cells and the membrane density of HLA class I antigens were significantly lower in CD4+ T lymphocytes from AIDS patients with respect to CD4+ T lymphocytes from HIV-negative controls and to CD8+ T lymphocytes from HIV-negative controls and AIDS patients. By contrast, the expression of HLA class II and CD38 antigens was upregulated in CD4+ and CD8+ T lymphocytes from HIV-1-positive subjects. The defective expression of HLA class I antigens could impair the lysis of HIV-infected CD4+ cells by virus-specific HLA class I-restricted cytotoxic T lymphocytes and contribute to the progression of disease.

Serum HLA class I antigen levels in allogeneic bone marrow transplantation: a possible marker of acute GVHD.

The levels of serum HLA class I antigens were determined at weekly intervals up to 5 weeks in 46 patients who had undergone allogeneic BMT. In patients with GVHD grade I or with GVHD grade I and fever of unknown origin (FUO), serum HLA class I antigen levels did not change during the observation period. In patients with GVHD grade II-IV serum HLA class I antigen level significantly increased in the week before the onset of GVHD, was maximal at the onset of GVHD and then persisted unchanged in the following 2 weeks. In patients with GVHD grade I or GVHD grade II-IV and infections whose onset coincided with that of acute GVHD a significant increase of serum HLA class I antigen level was found 2 weeks after the onset of the infectious episode. An increase of serum HLA class I antigen level was also found before the onset of repetitive GVHD grade II-IV episodes as well as during and after infectious episodes whose onset occurred after the onset of acute GVHD. The mean +/- s.d. concentrations of serum HLA class I antigens during GVHD grade II-IV episodes (9.4 +/- 3.4 micrograms/ml) and 2 weeks after the onset of infectious episodes (7.1 +/- 1.6 micrograms/ml) are significantly (P < 0.01 and P < 0.05, respectively) higher than that found 2 weeks before the onset of GVHD (3.0 +/- 0.5 micrograms/ml). The results of the present investigation suggest that measurement of serum HLA class I antigen level may be a possible marker to detect an acute GVHD following BMT.

Modulation of MHC Gene Expression by Glucocorticoid Hormones.

The effects of the glucocorticoid hormones prednisone (PDN) and deflazacort (DFC) on MHC gene products membrane expression and mRNA level were evaluated on peripheral blood lymphocytes and on the melanoma M14 cell line. The modulatory effect of the association of PDN and DFC with interferon-γ was also evaluated. PDN and DFC inhibited MHC gene expression in lymphocytes and M14 cells both on the cell membrane and at the mRNA level. Interferon-γ was able to counteract this inhibitory effect. These results may contribute to clarify the role played by glucocorticoid hormones in the modulation of immune responses.

Soluble HLA class I and Fas ligand molecules in blood components and their role in the immunomodulatory effects of blood transfusions.

It has been known for many years that blood transfusions may have immunomodulatory effects, however an ultimate explanation of this phenomenon is lacking. In the present paper we report that the concentrations of soluble HLA class I (sHLA-I) and soluble Fas ligand (sFasL) molecules in supernatants of blood components which contain elevated numbers of residual donor leukocytes, like red blood cells and random-donor platelets, are significantly higher than in other blood components. Elevated amounts of sFasL molecules are also found in some commercial immunoglobulin preparations. sHLA-I and sFasL molecules in blood components and in immunoglobulin preparations are biologically active in vitro as they inhibit mixed lymphocyte responses and cytotoxic T cell activity in allogeneic and autologous combinations and induce apoptosis in Fas-positive cells. If these results are paralleled in vivo the amount of sHLA-I and sFasL molecules should be taken into account in clinical practice in order to select the blood component and the immunoglobulin preparation which could induce the desired immunomodulatory effect in the recipient.

dsDNA-, nucleohistone- and DNASE I-reactive T lymphocytes in patients affected by systemic lupus erythematosus: correlation with clinical disease activity.

OBJECTIVE: To demonstrate the involvement of T lymphocytes reactive to autoantigens in the pathogenesis of autoimmune diseases and to analyse their clinical relevance. METHODS: The frequency of T cell clones reactive to double strand DNA (dsDNA), Nucleohistone (NH) complex and Dnase I was calculated for the peripheral blood mononuclear cells (PBMC) of 15 SLE patients and 9 healthy subjects by proliferation assay. RESULTS: DsDNA- and NH-specific T cell clones were found in the majority of the patients analysed (frequency ranging from 2 to 50 clones/10(7) PBMC), while their absence or very low frequency (2 clones/10(7) PBMC) was observed in the control PBMC. Their frequency significantly correlated with decreased serum concentrations of C3 and C4 and with the systemic lupus erythematosus disease activity index (P = 0.03). A very low frequency of Dnase I-reactive T cell clones was observed in both SLE and healthy subjects. CONCLUSION: Our results suggest that dsDNA- and NH-reactive T lymphocytes may be involved in the pathogenesis of SLE and that their quantification in the peripheral blood of patients could be a useful tool to follow the clinical course of the disease.

Immunoregulatory role of soluble HLA molecules: a new skin for an old subject?

The available evidence suggests that measurement of the level of total sHLA-1 antigens and of donor-derived and recipient-derived allospecificities as well as the characterization of their variants in recipient's serum may provide useful information to differentiate graft rejections from infections in allograft recipients. Moreover, a significant progress has been made in our understanding of the functional properties of sHLA-I antigens in serum and of their potential role in the modulation of immune responses. If these preliminary results will be confirmed, then sHLA-I antigens are likely to become important reagents to monitor and treat graft recipients.

Involvement of reactive oxygen species in the bactericidal activity of activated carbon fibre supporting silver; Bactericidal activity of ACF(Ag) mediated by ROS.

An activated carbon fibre supporting silver (ACF(Ag)) was tested for its antibacterial capacity against Escherichia coli (E. coli). Water that has passed through ACF(Ag) demonstrated strong bactericidal ability. This activity decreased over the time suggesting that generated bactericidal species were short lifespan. Since formation of reactive oxygen species (ROS) might be catalysed by silver impregnated and/or ACF itself, implication of ROS and silver was evaluated by the use of ROS scavengers and a silver ions neutralizing agent. The role of ROS in the E. coli mortality was confirmed by the use of a molecular approach which revealed a strong expression of oxidative stress genes.

Increased levels of interleukin-10 in saliva of Sjögren's syndrome patients. Correlation with disease activity.

The aim of the study was to determine the levels of interleukin (IL)-10, IL-2, IL-4, and interferon-gamma in the saliva of patients with Sjögren's syndrome and to correlate them with laboratory and clinical parameters of disease activity. The levels of IL-2, IL-4, IL-10, and interferon-gamma were measured in salivary samples, obtained directly from the Stenone duct of 14 Sjögren's syndrome patients and 26 healthy controls by ELISA. A significant elevation of IL-10 was found in salivary fluids of Sjögren's syndrome patients compared with healthy controls (P=0.007). Elevated interferon-gamma levels were found in some patients. IL-2 and IL-4 were undetectable in all saliva samples. In patients, IL-10 levels significantly correlated with the degree of xerophthalmia and xerostomia (P=0.02 and P=0.01, respectively) and with the erythrocyte sedimentation rate (P=0.006). Our data suggest that elevated IL-10 levels are detectable in the saliva of Sjögren's syndrome patients and correlate with the severity of the disease.

[The immunomodulatory effect of blood transfusions and intravenous immunoglobulins: the role of the soluble molecules of the Class-I major histocompatibility complex and of the Fas ligand]. / Effetto immunomodulatorio delle emotrasfusioni e delle immunoglobuline per via endovenosa: ruolo delle molecole del complesso maggiore di istocompatibilità di classe I e Fas-ligando solubili.

Allogeneic blood transfusions may have immunomodulatory effects including improved allograft acceptance and increased risk for cancer recurrence or post-operative bacterial infections. These effects are associated with the presence of leukocytes in transfused blood and are reduced by pre-storage leuko-reduction. However, the precise mechanism of this effect has not yet been elucidated. We report that the concentrations of soluble major histocompatibility complex class I and soluble Fas-ligand molecules are significantly higher in supernatants of blood components containing elevated numbers of residual donor leukocytes, such as red blood cells and random-donor platelets, than in other blood components. Elevated amounts of soluble Fas-ligand molecules are also found in some intravenous immunoglobulin preparations. Soluble molecules detected in blood components and in immunoglobulin preparations are biologically active in vitro. In fact, they inhibit mixed lymphocyte responses and cytotoxic T cell activity in allogeneic and autologous combinations and induce apoptosis in Fas-positive cells. These results should be taken into account in clinical practice to select the blood component or the immunoglobulin preparation in order to induce or prevent an immunosuppressive effect in the recipient.

OxLDL- and HSP-60 antigen-specific CD8(+) T lymphocytes are detectable in the peripheral blood of patients suffering from coronary artery disease.

Inflammatory and immunologic mechanisms are important for the initiation and the progression of atherosclerotic lesions. OxLDL and HSP-60 antigens are involved in the pathogenesis of atherosclerotic disease by triggering immune cells within the plaques. Through the MHC pentamer assays, we investigated the presence of OxLDL- and HSP-60-specific CD8(+) T lymphocytes in twenty HLA-A2-positive patients suffering from coronary artery disease (10 NSTEMI and 10 stable angina). Similarly, 10 age- and sex-matched healthy subjects were enrolled as controls. Biological samples were collected within 6 h of admission to hospital, at 30 days and at 180 days. OxLDL- and HSP-60-specific CD8(+) T lymphocytes were never detectable in the peripheral blood from all the healthy controls. On the contrary, at each scheduled time point, both of these specific cells could be detected in peripheral blood from all enrolled patients. More in detail, the flow cytometric analysis of MHC-1 pentamer OxLDL-specific CD8(+) T lymphocytes revealed a sharp and significant increase at the hospital admission, within 6 h from the chest pain onset, followed by an evident decline to lower levels at 30 days and at 180 days from the enrollment in the study. On the contrary, although MHC-1 pentamer HSP-60 CD8(+) T lymphocytes were detectable in enrolled patients, almost no variance could be detectable during the follow-up scheduled evaluations. On the whole, this finding indicates that HSP-60- and OxLDL-specific CD8(+) T lymphocytes could play a role in the maintenance or worsening of the atherosclerotic coronary disease.