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1.  A laboratory test based on determination of cytokine profiles: a promising assay to identify exposition to contact allergens and predict the clinical outcome in occupational allergic contact dermatitis 
BMC Immunology  2015;16:4.
Background
Para-phenylenediamine (PPD) is the main allergen causing adverse reactions to hair dyes and a frequent cause of occupational-related skin sensitization among hairdressers and beauticians. The immunologic mechanism of the disease relies on the production of inflammatory cytokines by allergen-specific T cells, while regulatory T cells are thought to down-modulate the allergic response. This study was aimed at investigating the expression of effector or regulatory cytokines in exposed subjects in order to verify whether different cytokine profiles might predict distinct clinical outcomes. Peripheral blood mononuclear cells (PBMC) from 21 subjects occupationally exposed or not (10) to PPD were kept in short term cultures in the presence of optimized concentrations of NiSO4 × 6H2O or PPD. The production of IFN-γ and IL-10 elicited by antigens were analyzed by the ELISpot assay.
Results
The presence of IFN-γ responses toward PPD was significantly correlated with a positive patch test (P = 0.002) and allergic symptoms, while IL10 responses were invariably found in PPD-exposed but clinically asymptomatic subjects with negative patch testing. We found concordance between the different cytokine profiles and patch test results. No false-positive results were found for the different cytokine profiles induced by PPD, resulting in 100% specificity. The sensitivity of the test was 87.5% (95% CI 65.9-100.0) with an overall test accuracy of 93.3%. Although larger prospective-retrospective studies are necessary to validate the predictive potential of the test, the negative and positive predicted values for PPD in this study were NPV = 87.5% and PPV = 100%, respectively.
Conclusions
These data indicate that distinct cytokine profiles are associated with different clinical manifestations. The test, which is based on a simple and rapid profiling of cytokine responses by T lymphocytes against allergens, has proven to be a promising laboratory tool, useful for both the identification of previous contact with allergens and the etiologic diagnosis of contact allergies as well as capable of predicting the clinical outcome (development of an allergic or tolerant response).
doi:10.1186/s12865-015-0066-3
PMCID: PMC4335538  PMID: 25651756
Allergic contact dermatitis; Cytokines; ELISpot; Nickel; Occupation; Para-phenylenediamine; Patch test
2.  Analysis of the ORFK1 hypervariable regions reveal distinct HHV-8 clustering in Kaposi’s sarcoma and non-Kaposi’s cases 
Background
Classical Kaposi’s Sarcoma (cKS) is a rare vascular tumor, which develops in subjects infected with Human Herpesvirus-8 (HHV-8). Beside the host predisposing factors, viral genetic variants might possibly be related to disease development. The aim of this study was to identify HHV-8 variants in patients with cKS or in HHV-8 infected subjects either asymptomatic or with cKS-unrelated cutaneous lymphoproliferative disorders.
Methods
The VR1 and VR2 regions of the ORF K1 sequence were analyzed in samples (peripheral blood and/or lesional tissue) collected between 2000 and 2010 from 27 subjects with HHV-8 infection, established by the presence of anti-HHV-8 antibodies. On the basis of viral genotyping, a phylogenetic analysis and a time-scaled evaluation were performed.
Results
Two main clades of HHV-8, corresponding to A and C subtypes, were identified. Moreover, for each subtype, two main clusters were found distinctively associated to cKS or non-cKS subjects. Selective pressure analysis showed twelve sites of the K1 coding gene (VR1 and VR2 regions) under positive selective pressure and one site under negative pressure.
Conclusion
Thus, present data suggest that HHV-8 genetic variants may influence the susceptibility to cKS in individuals with HHV-8 infection.
doi:10.1186/s13046-014-0119-0
PMCID: PMC4311464  PMID: 25592960
HHV-8; Kaposi’s sarcoma; Phylogenesis; HHV-8 variants; Selective pressure
4.  The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study 
Retrovirology  2014;11:49.
Background
Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination.
Findings
Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization.
Conclusions
Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy.
doi:10.1186/1742-4690-11-49
PMCID: PMC4087126  PMID: 24961156
HIV progression; Tat; Antibodies; CD4+ T cells; Viral load
5.  Challenges in HIV Vaccine Research for Treatment and Prevention 
Many attempts have been made or are ongoing for HIV prevention and HIV cure. Many successes are in the list, particularly for HIV drugs, recently proposed also for prevention. However, no eradication of infection has been achieved so far with any drug. Further, a residual immune dysregulation associated to chronic immune activation and incomplete restoration of B and T cell subsets, together with HIV DNA persistence in reservoirs, are still unmet needs of the highly active antiretroviral therapy, causing novel “non-AIDS related” diseases that account for a higher risk of death even in virologically suppressed patients. These “ART unmet needs” represent a problem, which is expected to increase by ART roll out. Further, in countries such as South Africa, where six millions of individuals are infected, ART appears unable to contain the epidemics. Regretfully, all the attempts at developing a preventative vaccine have been largely disappointing. However, recent therapeutic immunization strategies have opened new avenues for HIV treatment, which might be exploitable also for preventative vaccine approaches. For example, immunization strategies aimed at targeting key viral products responsible of virus transmission, activation, and maintenance of virus reservoirs may intensify drug efficacy and lead to a functional cure providing new perspectives also for prevention and future virus eradication strategies. However, this approach imposes new challenges to the scientific community, vaccine developers, and regulatory bodies, such as the identification of novel immunological and virological biomarkers to assess efficacy end-points, taking advantage from the natural history of infection and exploiting lessons from former trials. This review will focus first on recent advancement of therapeutic strategies, then on the progresses made in preventative approaches, discussing concepts, and problems for the way ahead for the development of vaccines for HIV treatment and prevention.
doi:10.3389/fimmu.2014.00417
PMCID: PMC4157563  PMID: 25250026
HIV-1 vaccine; therapeutics; HAART; functional cure; clinical studies; preclinical and clinical proof-of-concept studies
6.  Assessment of T Regulatory Cells and Expanded Profiling of Autoantibodies May Offer Novel Biomarkers for the Clinical Management of Systemic Sclerosis and Undifferentiated Connective Tissue Disease 
In order to identify disease biomarkers for the clinical and therapeutic management of autoimmune diseases such as systemic sclerosis (SSc) and undifferentiated connective tissue disease (UCTD), we have explored the setting of peripheral T regulatory (T reg) cells and assessed an expanded profile of autoantibodies in patients with SSc, including either limited (lcSSc) or diffuse (dcSSc) disease, and in patients presenting with clinical signs and symptoms of UCTD. A large panel of serum antibodies directed towards nuclear, nucleolar, and cytoplasmic antigens, including well-recognized molecules as well as less frequently tested antigens, was assessed in order to determine whether different antibody profiles might be associated with distinct clinical settings. Beside the well-recognized association between lcSSc and anti-centromeric or dcSSC and anti-topoisomerase-I antibodies, we found a significative association between dcSSc and anti-SRP or anti-PL-7/12 antibodies. In addition, two distinct groups emerged on the basis of anti-RNP or anti-PM-Scl 75/100 antibody production among UCTD patients. The levels of T reg cells were significantly lower in patients with SSc as compared to patients with UCTD or to healthy controls; in patients with lcSSc, T reg cells were inversely correlated to disease duration, suggesting that their levels may represent a marker of disease progression.
doi:10.1155/2013/390563
PMCID: PMC3681301  PMID: 23818915
7.  Immunomodulatory effects of total intravenous and balanced inhalation anesthesia in patients with bladder cancer undergoing elective radical Cystectomy: preliminary results 
Background
Although surgery and anesthesia induce immunesuppression, remains largely unknown whether various anesthetic techniques have different immunosuppressive effects on cancer patients. Therefore, the aim of this study was to investigate the influence of total intravenous anesthesia with target-controlled infusion (TIVA-TCI) and balanced inhalation anesthesia (BAL) on the peri-operative levels of inflammatory cytokines and regulatory T cells (Tregs) in patients with bladder cancer undergoing surgery.
Methods
Twenty eight consecutive patients with bladder cancer who underwent radical cystectomy were prospectively randomized into two groups to receive TIVA-TCI (n = 14) or BAL (n = 14). Before the induction of anesthesia (T0), 6–8 hours (T1) post-surgery, and 5 days post-surgery (T2), Tregs and serum levels of interleukin -1beta (IL-1β), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin −2 (IL-2), interleukin −6 (IL-6), and interleukin −10 (IL-10) were measured.
Results
In the peri-operative period all cancer patients showed a marked and significant increase in IL-6. Moreover, TIVA-TCI patients also showed a higher increase in IFN-γ, whereas in BAL patients Tregs were reduced by approximately 30% during surgery. The incidence of infections, metastases, and death was similar in both groups.
Conclusions
The increase in the Th1 response in the TIVA-TCI group and the reduction in Tregs in the BAL group seem to balance the immunosuppressive effect induced by IL-6. Therefore TIVA-TCI and BAL can be both used in major surgery in patients with bladder cancer without worsening the outcome.
doi:10.1186/1756-9966-32-6
PMCID: PMC3577511  PMID: 23374147
Total intravenous anesthesia with target-controlled infusion (TIVA-TCI); Balanced inhalation anesthesia (BAL); Urinary bladder carcinoma; Cytokines; Tregs
8.  HIV-1 Tat Promotes Integrin-Mediated HIV Transmission to Dendritic Cells by Binding Env Spikes and Competes Neutralization by Anti-HIV Antibodies 
PLoS ONE  2012;7(11):e48781.
Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.
doi:10.1371/journal.pone.0048781
PMCID: PMC3496724  PMID: 23152803
9.  Anal cytological abnormalities and epidemiological correlates among men who have sex with men at risk for HIV-1 infection 
BMC Cancer  2012;12:476.
Background
The incidence of anal cancer, a Human Papillomavirus (HPV)-related neoplasia, has been increasing in recent decades, mainly in men who have sex with men (MSM). Cytological changes of the anal epithelium induced by HPV can be detected through an anal pap smear. This study aimed to evaluate the prevalence and epidemiological correlates of anal cytological abnormalities among relatively young MSM at risk for HIV-1 infection, to help clarify whether or not this population deserves further investigation to assess the presence of anal cancer precursor lesions.
Methods
MSM were recruited among attendees of a large STI clinic for a HIV-1 screening program. Anal samples, collected with a Dracon swab in PreservCyt, were used both for liquid-based cytology and HPV testing by the Linear Array HPV Genotyping Test. Data regarding socio-demographic characteristics and sexual behavior were collected in face-to-face interviews.
Results
A total of 346 MSM were recruited (median age 32 years). Overall, 72.5% of the individuals had an anal HPV infection, with 56.1% of them being infected by oncogenic HPV genotypes. Anal cytological abnormalities were found in 29.8% of the cases (16.7% ASC-US and 13.1% L-SIL). Presence of ASC-US+ was strongly associated with infection by any HPV type (OR=4.21, 95% CI: 1.97-9.23), and particularly by HPV 16 and/or 18 (OR=5.62, 95% CI: 2.33-13.81). A higher proportion of ASC-US+ was found in older MSM, in those with a higher number of lifetime partners and in those with a history of ano-genital warts. However, none of these variables or the others analyzed showed any significant association with abnormal cytological findings.
Conclusions
The presence of anal cytological abnormalities in about one third of the recruited MSM and their strong association with HPV infection, in particular that caused by HPV 16 and/or 18, might provide a further complement to the data that now support the introduction of HPV vaccination among MSM to protect them from the development of HPV-associated diseases. Additional studies are needed to determine whether and how screening for anal cancer precursor lesions should be performed in younger MSM.
doi:10.1186/1471-2407-12-476
PMCID: PMC3517502  PMID: 23072547
Anal cytology; HPV infection; Men who have sex with men
10.  Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model 
Background
Antigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours. Therefore assays capable at exploring the generation and function of cytotoxic T lymphocytes represent an important objective for both clinical and experimental settings.
Methods
Here we show a simple and reproducible assay for the evaluation of antigen-specific CD8+ cytotoxic T lymphocytes based on a LysiSpot technique for the simultaneous determination of antigen-specific IFN-γ production and assessment of tumor cytolysis. The assay was developed within an experimental model of colorectal carcinoma, induced by the colorectal tumor cell line DHD-K12 that induces tumors in BDIX rats and, in turn, elicits a tumor- specific immune response.
Results
Using DHD-K12 cells transfected to express Escherichia coli β-galactosidase as target cells, and by the fine setting of spot colours detection, we have developed an in vitro assay that allows the recognition of cytotoxic T lymphocytes induced in BDIX rats as well as the assessment of anti-tumour cytotoxicity. The method highlighted that in the present experimental model the tumour antigen-specific immune response was bound to killing target cells in the proportion of 55%, while 45% of activated cells were not cytotoxic but released IFN-γ. Moreover in this model by an ELISPOT assay we demonstrated the specific recognition of a nonapeptide epitope called CSH-275 constitutionally express in DHD-K12 cells.
Conclusions
The assay proved to be highly sensitive and specific, detecting even low frequencies of cytotoxic/activated cells and providing the evaluation of cytokine-expressing T cells as well as the extent of cytotoxicity against the target cells as independent functions. This assay may represent an important tool to be adopted in experimental settings including the development of vaccines or immune therapeutic strategies
doi:10.1186/1756-9966-31-9
PMCID: PMC3395825  PMID: 22296726
LysiSpot; ELISpot; Tumor antigens; CTLs; BDIX rats; Colon cancer
11.  Molecular and Immunological Characterization of Staphylococcus aureus in Pediatric Atopic Dermatitis: Implications for Prophylaxis and Clinical Management 
S. aureus represents a critical cofactor in atopic dermatitis (AD). In this paper, the prevalence of S. aureus infection/colonization was evaluated in 117 children as well as in their cohabitants, in order to assess the value of S. aureus characterization in predicting disease onset and severity and in providing indications for prophylaxis. Results showed that children with AD as well as their cohabitants had a significantly greater incidence of S. aureus infection/colonization as compared to controls. The genetic characterization showed a virtual identity of the bacteria strains collected at different sites of the patients with those found in the cohabitants, suggesting both a direct transmission between the nasal reservoir and the lesions in the same atopic subject and a risk for reinfection within family cohabitants. These data stress the need of preliminary laboratory assessment and posttherapy control in both AD patients and their close contacts for effective S. aureus eradication.
doi:10.1155/2011/718708
PMCID: PMC3205653  PMID: 22110527
13.  Therapeutic Immunization with HIV-1 Tat Reduces Immune Activation and Loss of Regulatory T-Cells and Improves Immune Function in Subjects on HAART 
PLoS ONE  2010;5(11):e13540.
Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4+ T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4+ and CD8+ cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4+ T cells and B cells with reduction of CD8+ T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4+ and CD8+ T cells were accompanied by increases of CD4+ and CD8+ T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the OBS population. These findings support the use of Tat immunization to intensify HAART efficacy and to restore immune homeostasis.
Trial registration
ClinicalTrials.gov NCT00751595
doi:10.1371/journal.pone.0013540
PMCID: PMC2978690  PMID: 21085635
14.  Nickel, palladium and rhodium induced IFN-gamma and IL-10 production as assessed by in vitro ELISpot-analysis in contact dermatitis patients 
BMC Immunology  2008;9:19.
Background
Recent attempts to diminish nickel use in most industrial products have led to an increasing utilization of alternative metal compounds for destinations such as the alloys used in orthopaedics, jewellery and dentistry. The present study was undertaken with the aim to evaluate the potential for an allergic response to nickel, palladium and rhodium on the basis of antigen-specific induction of inflammatory/regulatory cytokines, and to characterize, according to the cytokine profiles, the nature of simultaneous positive patch tests elicited in vivo.
Peripheral blood mononuclear cells (PBMC) from 40 patients with different patch test results were kept in short term cultures in the presence of optimized concentrations of NiSO4 × 6H2O, PdCl2 and Rh(CH3COO)2. The production of IFN-γ and IL-10 elicited by metal compounds were analyzed by the ELISpot assay.
Results
We found a specific IFN-γ response by PBMC upon in vitro stimulation with nickel or palladium in well recognized allergic individuals. All controls with a negative patch test to a metal salt showed an in vitro IL-10 response and not IFN-γ production when challenged with the same compound. Interestingly, all subjects with positive patch test to both nickel and palladium (group 3) showed an in vitro response characterized by the release of IFN-γ after nickel stimulation and production of IL-10 in response to palladium.
Conclusion
These results strongly suggest that the different cytokine profiles elicited in vitro reflect different immune responses which may lead to the control of the allergic responses or to symptomatic allergic contact dermatitis. The development of sensitive and specific in vitro assays based on the determination of the cytokine profiles in response to contact allergens may have important diagnostic and prognostic implications and may prove extremely useful in complementing the diagnostic limits of traditional patch testing.
doi:10.1186/1471-2172-9-19
PMCID: PMC2409297  PMID: 18482439
15.  Decreased levels of metalloproteinase-9 and angiogenic factors in skin lesions of patients with psoriatic arthritis after therapy with anti-TNF-α 
Background
Inflammation represents an early and key event in the development of both the cutaneous psoriasis and psoriatic arthritis. Compelling evidences indicate that the production of TNF-α plays a central role in psoriasis by sustaining the inflammatory process in the skin as well as in the joints. Among the multiple effects produced by TNF-α on keratinocytes, the induction of matrix metalloproteinase-9 (MMP-9), a collagenase implicated in joint inflammatory arthritis which acts as an angiogenesis promoting factor, might represent a key mechanism in the pathogenesis of the disease. Aims of the present study were to investigate a) the role of MMP-9 in the development of psoriasis by assessing the presence of MMP-9 in lesional skin and in sera of psoriatic patients; b) the association of MMP-9 with the activity of the disease; c) the relationship between MMP-9 and TNF-α production.
Methods
Eleven psoriatic patients, clinically presenting joint symptoms associated to the cutaneous disease, were included in a therapeutic protocol based on the administration of anti-TNF-α monoclonal antibody (Infliximab). Sera and skin biopsies were collected before treatment and after 6 weeks of therapy. Tissues were kept in short term cultures and production soluble mediators such as TNF-α, MMP-9, MMP-2, VEGF and E-Selectin, which include angiogenic molecules associated to the development of plaque psoriasis, were measured in the culture supernatants by immunoenzymatic assays (ng/ml or pg/ml per mg of tissue). MMP-9 concentrations were also measured in the sera. The cutaneous activity of disease was evaluated by the Psoriasis Area and Severity Index (PASI).
Results
Clinical and laboratory assessment indicated that all but one patients had a significant improvement of the PASI score after three months of therapy. The clinical amelioration was associated to a significant decrease of MMP-9 (P = 0.017), TNF-α (P = 0.005) and E-selectin (P = 0.018) levels, spontaneously released by lesional biopsies before and after therapy. In addition, significant correlations were found between the PASI measurements and TNF-α (r2 = 0.33, P = 0.005), MMP-9 (r2 = 0.25, P = 0.017), E-selectin (r2 = 0.24, P = 0.018) production. MMP-9 levels were significantly correlated with those of TNF-α (r2 = 0.30, P = 0.008). A significant decrease of MMP-9 in the sera, associated to the clinical improvement was also found.
Conclusion
Our findings show the existence of a direct relationship between MMP-9 and TNF-α production strongly suggesting that MMP-9 may play a key role in the skin inflammatory process in psoriasis.
doi:10.1186/1740-2557-3-5
PMCID: PMC1601955  PMID: 17022813

Results 1-15 (15)