Single-nucleotide polymorphisms in 3'-untranslated region inducible costimulator gene and the important roles of miRNA in alopecia areata.

Background: Alopecia areata (AA) spares the stem cell compartment and attacks only the base of the hair follicle, which is surrounded by infiltrating lymphocytes. AA is associated with polymorphisms in immune-related genes and with decreased function of CD4+CD25+ T regulatory (Treg) cells. Treg function is modulated by the costimulatory molecules, like inducible costimulator (ICOS) that are crucial in orienting T cell differentiation and function so that they strongly impact on the immunologic decision between tolerance or autoimmunity development. Objective: The aim of our study was to investigate the possible association of AA with single-nucleotide polymorphisms (SNP) present in the ICOS 3'-untranslated region (3'UTR) region and to elucidate how SNPs modulate ICOS gene expression by affecting miRNA binding sites. Methods: This is a case-control study performed in 184 patients with AA and 200 controls. ICOS gene and miRNA expression were analyzed by real-time polymerase chain reaction. Results: The genotype carrying the rs4404254(C) [p = 0.012, OR (95% CI): 0.5 (0.3-0.8)] and rs4675379(C) [p = 0.015, OR (95% CI): 0.3 (0.1-0.8)] 3' UTR alleles was more frequently observed in AA patients than in controls and correlated with a reduced ICOS expression. miR-1276 significantly suppressed ICOS expression by binding to the 3'UTR of ICOS mRNA. Also, we observed that, miR-101 and miR-27b are upregulated, while miR-103 and miR-2355-3p are downregulated in peripheral blood mononuclear cells of AA patients compared to controls. Conclusion: Our data show that rs4404254 and rs4675379 SNPs of ICOS gene are associated with AA and also reveal that the presence of rs4404254 polymorphism correlates with ICOS post-transcriptional repression by microRNA binding.

Single nucleotide polymorphisms in the promoter regions of Foxp3 and ICOSLG genes are associated with Alopecia areata.

Alopecia areata (AA), an autoimmune disease affecting anagen stage hair follicles, is associated with polymorphisms in immune-related genes and with decreased number of CD4+ CD25+ T regulatory cells (Treg). Treg function is modulated by the forkhead box protein 3 (FOXP3) transcription factor and by inducible costimulator (ICOS), through interaction with the relative ligand, ICOSLG, whose genes are polymorphic. The aim of the study was to investigate whether specific single nucleotide polymorphisms (SNPs) of the rs2294020 FOXP3 and/or rs378299 ICOSLG genes may be associated with AA. A case-control study was performed in 120 AA patients and 84 controls. SNPs were analyzed by gene sequencing. FOXP3 and ICOSLG gene expressions were analyzed by real-time PCR. Increased frequencies of the genotype carrying the FOXP3 rs2294020-3675(A) [P = 0.002, OR (95 % CI): 2.55 (1.2-2.7)] or the ICOSLG rs378299-509(C) [P = 0.01, OR (95 % CI): 2.21 (1.1-2.6)] allelic variants were observed in AA patients than in controls. The genotype carrying the combination of the FOXP3 rs2294020-3675(A) and ICOSLG rs378299-509(C) allelic variants with the HLA DQB1*03 allele was more frequently present in AA patients than in controls (P = 0.04). The presence of the FOXP3 rs2294020-3675(A) or the ICOSLG rs378299-509(C) allelic variant was associated with reduced relative gene expression in AA patients. These data suggest that rs2294020 SNP of FOXP3 gene and rs378299 SNP of ICOSLG gene are associated with AA and with a reduced expression of the FOXP3 and ICOSLG genes in alopecia patients.

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.

The role of AIRE polymorphisms in melanoma.

Polymorphisms of AIRE, a transcription factor that up-regulates intrathymic expression of tissue-specific antigens including melanoma-associated antigens (MAAs), may variably affect the selection of MAAs-specific thymocytes, generating T-cell repertoires protecting or predisposing individuals to melanoma. We found that AIRE single nucleotide polymorphisms (SNPs) rs1055311, rs1800520 and rs1800522 were significantly more frequent in healthy subjects than in melanoma patients, independently from sex, age and stages of melanoma. The presence of these SNPs was associated with increased frequency of two T-cell clonotypes specific for MAGE-1 linking their protective effect to selection/expansion of MAA-specific T cells. Interestingly, mRNA transcribed on the rs1800520 SNP showed increased free energy than the wild type suggesting that its reduced stability may be responsible for the different activity of the polymorphic AIRE molecule. This finding may contribute at identifying subjects with increased risk of developing melanoma or patients with melanoma that may take benefit from immunotherapy.

Safety and immunogenicity of two influenza virus subunit vaccines, with or without MF59 adjuvant, administered to human immunodeficiency virus type 1-seropositive and -seronegative adults.

The objective of this study was to evaluate and compare both the safety and tolerability and the humoral and cell-mediated immune responses for two influenza virus subunit vaccines, one with MF59 adjuvant (Fluad) and one without an adjuvant (Agrippal), in healthy and in human immunodeficiency virus type 1 (HIV-1)-infected adult individuals. To achieve this aim, an open, randomized, comparative clinical trial was performed during the 2005-2006 season. A total of 256 subjects were enrolled to receive one dose of vaccine intramuscularly. Blood samples were taken at the time of vaccination and at 1 and 3 months postvaccination. A good humoral antibody response was detected for both vaccines, meeting all the criteria of the Committee for Medical Products for Human Use. After Beyer's correction for prevaccination status, Fluad exhibited better immunogenicity than Agrippal, as shown from the analysis of the geometric mean titers, with significant differences for some virus strains; however, no definitive conclusions on the clinical significance of such results can be drawn, because the method used to estimate antibody response is currently nonstandard for influenza virus vaccines. Significant induction of an antigen-specific CD4+ T-lymphocyte proliferative response was detected at all time points after immunization, for both the vaccines, among HIV-1-seronegative subjects. This was different from what was observed for HIV-1-infected individuals. In this group, significance was not reached at 30 days postvaccination (T30) for those immunized with Agrippal. Also when data were compared between treatment groups, a clear difference in the response at T30 was observed in favor of Fluad (P = 0.0002). The safety profiles of both vaccines were excellent. For HIV-1-infected individuals, no significant changes either in viremia or in the CD4+ cell count were observed at any time point. The results showed good safety and immunogenicity for both vaccines under study for both uninfected and HIV-1-infected adults, confirming current recommendations for immunization of this high-risk category.

Association of -318 C/T and +49 A/G cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms with a clinical subset of Italian patients with systemic sclerosis.

Systemic sclerosis (SSc) is a complex and heterogeneous autoimmune disorder with a multi-factorial pathogenesis. Like other autoimmune disorders, the possible role of specific cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms in predisposing to SSc has been hypothesized, but it remains controversial. CTLA-4 promoter (-318C/T) and exon 1 (+49 A/G) polymorphisms have been analysed in 43 Italian females with SSc and in 93 unrelated matched healthy controls by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method. No significant association has been found with either polymorphisms.Nevertheless, SSc patients without concomitant Hashimoto's thyroiditis (HT) were carrying both the -318T allele (P = 0.031) and the +49 G allele (P = 0.076) more frequently than SSc patients with HT [defined by positivity for anti-thyroperoxidase (TPO) and anti-thyroglobulin (TGA) autoantibodies] than controls. Haplotype analysis confirms this association (P = 0.028), and suggests the predominant role of the -318T, whereas that of the +49 G, if any, seems weak. Thus, in Italian SSc patients the CTLA-4 -318C/T promoter polymorphism appears to be associated with the susceptibility to develop SSc without thyroid involvement. Larger studies are needed to confirm these findings and to clarify whether the -318C/T polymorphism is the functional responsible or whether it reflects the presence of another linked genetic element in the same chromosomal region.

AIRE gene polymorphisms in systemic sclerosis associated with autoimmune thyroiditis.

Mutations in the autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Systemic sclerosis (SSc) is a non-organ-specific autoimmune disease mainly characterized by cutaneous involvement, that is frequently associated with other autoimmune manifestations common to APECED. Nineteen SSc patients, 22 patients affected by SSc associated with autoimmune thyroiditis, and 100 healthy controls were analyzed. We identified 11 AIRE gene variants, one of which has never previously been described. Intronic polymorphism G11107A was significantly correlated to SSc/thyroiditis. Data show that variants of the AIRE gene might be correlated to different clinical manifestations in SSc patients.

Disruption of immunological tolerance: role of AIRE gene in autoimmunity.

The mechanism underlying the generation of T and B autoreactive clones in autoimmune diseases is still unknown. Among genetic factors implicated in autoimmunity, Autoimmune Regulator gene (AIRE) is one of the candidates to better understand the complex scenario of autoimmune manifestations. AIRE mutations are responsible for the development of autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) with monogenic autosomal recessive inheritance; it has been shown that AIRE regulates the negative selection of autoreactive T cells clones, driving the transcription of tissue-specific antigens in thymic epithelial cells. In various autoimmune manifestations correlated or not to APECED, AIRE variants act in a semidominant manner, leading to a reduction in AIRE protein amount per cell, and consequently to a marked decrease in ectopic proteins expression in the thymus. The co-occurrence of autoimmune diseases in the same individual has prompted several studies aimed to recognize shared patho-physiological mechanisms; in this scenario small reductions in function could explain the predisposition to autoimmunity in AIRE-heterozygous carriers of missense mutations; further studies to investigate whether the AIRE gene is involved in determining these autoimmune manifestations should be carried out.

Non-antigen-specific CD8(+) T suppressor lymphocytes in diseases characterized by chronic immune responses and inflammation.

Recent studies on regulatory lymphocytes demonstrate that CD8(+) T suppressor (Ts) cells may have great relevance in controlling immune system homeostasis and avoiding development of chronic inflammatory diseases. Among the three subpopulations of CD8(+) Ts cells so far recognized in humans, the type 2 (non-antigen-specific) cell is characterized by the capacity to inhibit both T cell proliferation and cytotoxic T lymphocyte activity through secretion of soluble factors. Previous work has shown the impairment of in vitro generation of type 2 CD8(+) Ts cells from the peripheral blood of relapsed patients with multiple sclerosis, systemic lupus erythematosus, or systemic sclerosis. Here, similar findings are demonstrated for patients with human immunodeficiency virus or chronic hepatitis C virus infection. Furthermore, the presence of type 2 CD8(+) Ts cells infiltrating diseased tissues in patients with autoimmune thyroiditis or cancer is shown. Collectively, these findings suggest that type 2 CD8(+) Ts cells may be involved in the control of pathologic chronic immune responses, contributing in some cases to the pathogenesis of the disease.

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.

Blunted coronary flow reserve in systemic sclerosis.

OBJECTIVES: We investigated whether the non-invasive determination of coronary flow reserve (CFR), as evaluated by transthoracic Doppler echocardiography, might be a potential method to detect early dysfunction of cardiovascular system in patients affected by systemic sclerosis (SSc) without clinical signs or symptoms of cardiac impairment. The possible correlations between the CFR values and the duration of the disease, specific autoantibodies and cutaneous involvement subsets were investigated. METHODS: Forty-four consecutive patients affected by SSc were analysed. The CFR was detected in the distal left anterior descending coronary artery by contrast-enhanced transthoracic second harmonic Doppler in all SSc patients and in 16 healthy controls. CFR was assessed at rest and during hyperaemia induced by administration of adenosine at 0.14 mg/kg/min over 5 min. The CFR was calculated as the ratio between hyperaemic (peak adenosine infusion) and resting peak diastolic velocity (PdvCFR) and resting velocity time integral (VtiCFR). Past medical history was carefully investigated. RESULTS: Both PdvCFR and VtiCFR were significantly reduced in SSc patients when compared with controls (P<0.0001). In particular, both PdvCFR and VtiCFR were significantly lower in patients with dSSc when compared with patients affected by lSSc (P<0.02 and P<0.04 respectively). No statistically significant correlation was found between CFR values and history of smoking, serum levels of cholesterol or triglycerides, blood pressure, age of patients, duration of SSc and serum autoantibody positivity for ANA, ACA and Scl70. CONCLUSIONS: CFR is often reduced in SSc patients. CFR was lower in patients with dSSc than in those affected by lSSc. A reduced CFR value should be considered an indirect sign of heart involvement in scleroderma, but its clinical and prognostic implications need to be clarified.

Non-antigen specific CD8+ T suppressor lymphocytes.

The homeostasis of peripheral immune system function is maintained by the activity of regulatory lymphocytes. Among these cells, a subset of CD8+CD28- T suppressor lymphocytes has recently been characterized for the capacity to mediate their effects without antigen restriction. These non-antigen-specific CD8+ T suppressor lymphocytes originate from circulating CD8+CD28- T lymphocytes after stimulation with interleukin-2 and interleukin-10. CD8+ suppressor cells inhibit both antigen-specific CD4+ T cell proliferation and cellular cytoxicity through secretion of cytokines such as interferon-gamma, interleukin-6, and interleukin-10. The function of CD8+ suppressor cells is impaired in patients with systemic lupus erythematosus in relapse as well as in patients with systemic sclerosis with disease progression, suggesting the involvement of CD8+ suppressor cells in the pathogenesis of autoimmune diseases. Interestingly, CD8+ suppressor cells have been found among tumor-infiltrating lymphocytes, which could be related to tumor-induced-immunosuppression. Failure to generate CD8+ suppressor cells from the peripheral blood is frequently observed in HIV-infected patients. It remains to be clarified whether this phenomenon is due to depletion and/or functional impairment of this cell subset or to their compartmentalization in peripheral tissues and immunocompetent organs where they could contribute to the induction of immunodeficiency.

Modifications of immunological and neuro-endocrine parameters induced by antiorthostatic bed-rest in human healthy volunteers.

AIM: Space flight has profound effects on immunological and neuroendocrine parameters. Microgravity plays a major role in the induction of these changes. The aim of the present study was the evaluation on ground of the effects induced by antigravitary posture on immune and neuroendocrine functions. METHODS: Eight healthy male volunteers (mean age 24+/-1 years) were maintained in antigravitary posture (-10 degrees) for 72 hours. Four of them were also maintained in supine posture for 72 hours as controls. The following immunological and neuroendocrine parameters have been analysed: peripheral white blood cells count, CD11b integrin expression and H(2)O(2) production by neutrophils, lymphocyte and monocyte phenotype, intracytoplasmic cytokine (IFN-gamma, TNF-alpha and IL-4) pattern, lymphocyte proliferation to mitogens and antigens, cortisol, ACTH, catecholamines, GH, LH, prolactin and testosterone plasma levels. RESULTS: In subjects maintained in antigravitary posture, norepinephrine, dopamine, cortisol, ACTH, GH and prolactin plasma levels increased whereas H(2)O(2) production by neutrophils, lymphocyte proliferation, NK cells number and intracytoplasmic IFN-g expression decreased. No significant modifications were observed in subjects maintained in supine posture. CONCLUSION: The results of this study indicate that several neuroendocrine and immunological parameters are modulated by a prolonged antigravitary posture on ground and may negatively affect astronauts defenses against pathogens during space flights.

Human CD4 internal antigen anti-idiotypic monoclonal antibody. immunochemical and sequence analysis.

The mouse mAb2 16D7 recognizes the paratope of the syngeneic anti-human CD4 mAb HP2/6 (mAb1 of our idiotypic cascade) and mimics CD4 in xenogeneic settings in humans. Immunochemical and sequence analyses were performed to define the minimum structural requirement for this mimicry. Binding assay of mAb1 with isolated naive 16D7 H and L chains showed that only the second reacted with mAb1. Specificity was indicated by the lack of reactivity of mAb1 with the L chain of mAb2 14D6, which also recognizes mAb1-paratope. It is likely that the 16D7-L mAb1-specific epitope is "sequence-dependent", since fully denatured 16D7-L still reacted with mAb1. Sequence analysis of 16D7 and mAb1 showed a high degree of homology of their VH. as both were coded by the same gene family (V/II), whereas CDR3 showed the greatest diversity. Alignment of 16D7-H CDR3 with CD4, however, produced no similarity. In contrast, analyses of the 16D7 VL sequence (XX/V) defined a CDR3 6-mer peptide with a 50% identity (83% of similarity) to the CD4 stretch 218-223. This peptide seems a suitable replacement for 16D7 in active immunotherapy as it did not match any protein fragment retrieved from the n-r database (NCBI) and both the peptide and the corresponding CD4 amino acid stretch are surface accessible. Based on their immunochemical profiles and similarity to CD4, four additional 16D7-derived peptides were designed for synthesis. The data indicate that CD4 mimicry by mAb2 can be obtained at the level of primary structure and provide useful information for the synthesis of peptide(s) with bioactive potential.

In vitro immunosuppressive activity of soluble HLA class I and Fas ligand molecules: do they play a role in autologous blood transfusion?

BACKGROUND: The immunomodulatory effects of allogeneic blood transfusion may contribute to a poor prognosis in patients with cancer who are undergoing surgery, and clinical trials have been carried out to investigate whether these patients would benefit from autologous blood donation. As the immunomodulatory effects of allogeneic blood transfusion have been related to soluble molecules released from residual WBCs during storage, the in vitro immunomodulatory activity of soluble molecules detected in supernatants from stored autologous blood was evaluated. STUDY DESIGN AND METHODS: Blood was donated by four healthy volunteers. Packed WBC-reduced RBCs were obtained and stored for 30 days, and supernatants were collected. FFP and serum were also obtained. The concentration of soluble molecules was determined by immunoenzymatic assays. The in vitro immunomodulatory activity of undiluted blood component supernatant was assessed by antigen-specific cytotoxic T-cell activity and mixed lymphocyte reactions in autologous combinations and by apoptosis induction in Fas+ cells. RESULTS: The concentrations of soluble Fas-ligand and HLA class I molecules were higher in packed RBCs than in WBC-reduced RBCs, FFP, and serum. Undiluted supernatants of packed RBCs strongly inhibited functional assays and induced apoptosis in Fas+ cells. The immunomodulatory effects were correlated with the amount of soluble Fas ligand and HLA class I molecules. CONCLUSION: The results of the present study are comparable with those already reported in allogeneic blood components, and they indicate that undiluted supernatants of autologous blood components may exert immunosuppressive effects in vitro.

Identification of key mutations in HIV reverse transcriptase gene can influence the clinical outcome of HAART.

Therapeutic failures due to in vivo loss of drug sensitivity are still a major problem in AIDS care. Currently, the role of and methods for detecting resistant mutant strains in patients before therapeutic choices are still under debate. To investigate the relevance of screening for key mutations alone the commercial INNO-LiPA HIV-1 RT method was applied retrospectively to analyzing several HIV codons correlated with resistance to RTI (reverse-transcriptase inhibitors) in sera from 62 patients before starting HAART protocols, selected on the basis of clinical parameters. INNO-LiPA detected several resistant mutant strains, which were strictly consistent with previous selective pressure in the patients. A significant correlation between genotype pattern and response to HAART was found. The presence of key mutations associated with resistance to one or two RTI included in the protocol correlated with a decrease in treatment benefits, whereas patients with wild-type or non-resistant viral strains exhibited better response to HAART. Even if this information had been available when treatment was started, 45 of the patients would not have received different treatment. When compared with the total number of patients, the subgroup receiving a treatment that was considered retrospectively as consistent with the key mutation pattern exhibited a significantly better outcome. Although the interpretation of resistance-related key mutations needs improvement, this surrogate LiPA method seems to maintain a predictive role in the management of HIV infection, and is less expensive.

Impairment of CD8+ T suppressor cell function in patients with active systemic lupus erythematosus.

Alteration of T cell suppression function has been recognized in patients with systemic lupus erythematosus (SLE). Recently, CD8(+) T suppressor lymphocytes (CD8(+) Ts) have been generated in vitro by incubating purified CD8(+) T cells with IL-2 and GM-CSF. Using this method, we generated CD8(+) Ts from patients affected by SLE. No major differences were found in the CD8(+) Ts phenotype between SLE patients and healthy subjects. CD8(+) Ts from SLE patients with active disease did not inhibit the anti-CD3 mAb-induced proliferation of autologous PBMC, whereas CD8(+) Ts from SLE patients in remission exerted an inhibitory activity comparable to normal subjects. The inhibitory effect of CD8(+) Ts cells was neither mediated by cytotoxic activity nor by apoptosis induction. Two cytokines, IFN-gamma and IL-6, were found to be responsible for the function of CD8(+) TS: In fact, counteraction of CD8(+) Ts suppression activity was obtained by blocking IFN-gamma with a specific Ab or by inhibiting CD8(+) Ts-mediated IL-6 secretion by an antisense oligonucleotide. Interestingly, CD8(+) Ts from SLE patients showed a peculiar cytokine pattern characterized by an impaired secretion of IL-6 and an increased secretion of IL-12. Thus, it appears that an altered balance between inhibitory (IL-6) and stimulatory (IL-12) cytokines might be responsible for the functional impairment of CD8(+) Ts in SLE patients.