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Kaposi's sarcoma (KS) is characterized by hyperproliferation of spindle cells that have an endothelial origin and assume their characteristic features upon infection with human herpesvirus-8, the causative agent for KS. The multifocal nature of KS suggests that spindle cells derive from circulating HHV8-infected precursors that yet lack identification. We investigated whether endothelial colony-forming cells (ECFCs) obtained from KS patients may be putative precursors of spindle cells by assessing whether their in vitro behavior may evoke the in vivo behavior of KS spindle cells. We isolated and cultured ECFCs from the blood of 83 patients with classic KS and compared them with ECFCs obtained from 86 healthy donors. ECFCs were highly increased in the blood of classic KS patients; they showed higher proliferative and vasculogenic potential and higher production of IL-6 than control ECFCs. Similarly to spindle cells in KS lesions, a variable proportion of cells within each ECFC colony expressed the human herpesvirus-8 latency-associated nuclear antigen. ECFCs obtained from classic KS patients evoked KS spindle cell behavior, thus supporting the hypothesis that ECFCs may be putative precursors of spindle cells. ECFCs can therefore represent a noninvasive tool for studying KS and screening drug activity, thus possibly guiding personalized care for KS patients., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Dietary guidelines aim to inform people of the types of foods and quantities they should consume each day or week to promote and maintain health. The aim of this study was to describe children's dietary behaviors in terms of adherence to the Swiss Society for Nutrition (SSN) dietary guidelines and possible determinants., Methods: A cross-sectional study was conducted in September 2010 with 568 children aged 6-12 years old living in Ticino Switzerland. Food intake was collected using 7-day food logs. Adherence with the dietary guidelines from the SSN was assessed according to age group., Results: With the exception of fish and cereal/potato intake (adherence rates of 68.5% and 47.9%, respectively), adherence to SSN guidelines was low: 26.9% for meat; 22.7% for eggs; 10.4% for fruit; 9.5% for sweets, snacks & soft drinks; 3.5% for milk & dairy, and 0% for vegetables. Multivariate analysis showed no consistent association between the child or their parent's socio-demographic characteristics and adherence to SSN guidelines. Girls had a higher likelihood of adhering with fruit and meat guidelines: multivariate adjusted odds ratio (95% confidence interval) 1.98 (1.10-3.56) and 1.80 (1.08-2.99), respectively. Children aged 10 to 12 had a lower likelihood of adhering with cereals and potatoes 0.48 (0.29-0.78), and a higher likelihood of adhering with the guideline for eggs 1.78 (1.00-3.15)., Conclusion: Dietary intake of Ticinese children shows poor adherence with SSN guidelines. Given the lack of specific socio-demographic factors associated with adherence, population-wide interventions to improve dietary intake are necessary.

Hepatitis C virus (HCV) has a high propensity to establish chronic infections. Failure of HCV-infected individuals to activate effective antiviral immune responses is at least in part related to HCV-induced impairment of dendritic cells (DCs) that play a central role in activating T cell responses. Although the impact of HCV on DC phenotype and function is likely to be more prominent in the liver, major HCV-induced alterations are detectable in peripheral blood DCs (pbDCs) that represent the most accessible source of DCs. These alterations include numerical reduction, impaired production of inflammatory cytokines and increased production of immunosuppressive IL10. These changes in DCs are relevant to our understanding the immune mechanisms underlying the propensity of HCV to establish persistent infection. Importantly, the non-invasive accessibility of pbDCs renders the analysis of these cells a convenient procedure that can be serially repeated in patient follow-up. Accordingly, the study of pbDCs in HCV-infected patients during conventional treatment with pegylated interferon and ribavirin indicated that restoration of normal plasmacytoid DC count may represent an additional mechanism contributing to the efficacy of the dual therapy. It also identified the pre-treatment levels of plasmacytoid DCs and IL10 as putative predictors of response to therapy. Treatment of chronic HCV infection is changing, as new generation direct-acting antiviral agents will soon be available for use in interferon-free therapeutic strategies. The phenotypic and functional analysis of pbDCs in this novel therapeutic setting will provide a valuable tool for investigating mechanisms underlying treatment efficacy and for identifying predictors of treatment response.

Preterm birth is the leading cause of neonatal morbidity and mortality. Although the underlying causes of pregnancy-associated complication are numerous, it is well established that infection and inflammation represent a highly significant risk factor in preterm birth. However, despite the clinical and public health significance, infectious agents, molecular trigger(s), and immune pathways underlying the pathogenesis of preterm birth remain underdefined and represent a major gap in knowledge. Here, we provide an overview of recent clinical and animal model data focused on the interplay between infection-driven inflammation and induction of preterm birth. Furthermore, here, we highlight the critical gaps in knowledge that warrant future investigations into the interplay between immune responses and induction of preterm birth., (© Society for Leukocyte Biology.)

Objective: The aim of this study was to investigate the biological effects of belimumab on B cells in the first phase II open-label trial with belimumab in patients with primary SS (pSS) (BELISS)., Methods: Peripheral blood B cell subsets and their B cell activating factor-receptor (BAFF-R) expression were analysed by multicolour flow cytometry in 10 pSS patients either before or after 24 and 52 weeks of therapy with belimumab. Serum BAFF levels were analysed by ELISA., Results: At baseline, pSS patients showed a significant increase in circulating B cells compared with healthy donors matched for age and sex, with a predominant expansion of transitional and naive B cell subsets. pSS patients also showed higher serum BAFF levels and lower B cell BAFF-R expression. Therapy with belimumab in pSS patients induced a significant reduction in transitional and naive B cell subsets to levels similar to those observed in healthy donors. Furthermore, belimumab normalized BAFF-R expression in all B subsets comprised within the memory compartment. The restoration of B cell frequency and subset composition in response to belimumab was also associated with a decrease in serum levels of Ig, RF, ANAs, and with an increase in the C4 complement fraction. All of these belimumab-mediated effects were observed after 24 weeks of therapy and maintained until the end of the therapeutic protocol., Conclusion: Taken together, our findings show that targeting BAFF with belimumab is successful in normalizing B cell frequency, phenotype and functions in pSS., Trial Registration: clinicaltrials.gov; https://clinicaltrials.gov/; NCT01008982., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

CD91 is a scavenger receptor expressed by different immune cells and its ligands defensins have been demonstrated to contribute to immune responses against infections and tumours. We previously demonstrated that CD91 is expressed on human monocyte-derived dendritic cells (moDCs) and that human defensins stimulate in vitro the activation of these cells. In this study, we observed that CD91 is expressed at different levels on two distinct moDC subsets: CD91(dim) and CD91(bright) moDCs. Although CD91(bright) moDCs represented a small proportion of total moDCs, this subset showed higher levels of activation and maturation markers compared with CD91(dim) moDCs. The frequency of CD91(bright) moDCs increased by ~ 50% after in vitro stimulation with recombinant human neutrophil peptide-1 (rHNP-1) and recombinant human β defensin-1 (rHBD-1), while lipopolysaccharide (LPS) stimulation decreased it by ~ 35%. Both defensins up-regulated moDC expression of CD80, CD40, CD83 and HLA-DR, although to a lower extent compared with LPS. Notably, upon culture with rHNP-1 and rHBD-1, CD91(bright) moDCs maintained their higher activation/maturation status, whereas this was lost upon culture with LPS. Our findings suggest that defensins promote the differentiation into activated CD91(bright) DCs and may encourage the exploitation of the CD91/defensins axis as a novel therapeutic strategy to potentiate antimicrobial and anti-tumour immune response., (© 2014 John Wiley & Sons Ltd.)

Spatiotemporal interactions play important roles in tissue development and function, especially in stem cell-seeded bioscaffolds. Cells interact with the surface of bioscaffold polymers and influence material-driven control of cell differentiation. In vitro cultures of different human progenitor cells, that is, endothelial colony-forming cells (ECFCs) from a healthy control and a patient with Kaposi sarcoma (an angioproliferative disease) and human CD133+ muscle-derived stem cells (MSH 133+ cells), were seeded onto polyglycolic acid-polylactic acid scaffolds. Three-dimensional (3D) images were obtained by X-ray phase-contrast microtomography (micro-CT) and processed with the Modified Bronnikov Algorithm. The method enabled high spatial resolution detection of the 3D structural organization of cells on the bioscaffold and evaluation of the way and rate at which cells modified the construct at different time points from seeding. The different cell types displayed significant differences in the proliferation rate. In conclusion, X-ray synchrotron radiation phase-contrast micro-CT analysis proved to be a useful and sensitive tool to investigate the spatiotemporal pattern of progenitor cell organization on a bioscaffold.

Natural Killer (NK) cells are important players of the innate arm of the immune system and provide an early defense against pathogens and tumor-transformed cells. Peripheral blood NK (PB-NK) cells were first identified because of their ability to spontaneously kill tumor-cell targets in vitro without the need for specific antigen priming, which is the reason that they were named 'natural killer' cells. The characterization of NK cells in human tissues and body organs represented another important step forward to better understand their physiology and physiopathology. In this regard, many reports revealed over the past decade a differential anatomic distribution of NK cell subsets in several sites such as the intestine, lung, cervix, placenta and liver as well as in secondary lymphoid organs such as spleen, lymph nodes and tonsils. Among all these tissues, the liver is certainly unique as its parenchyma contains an unusually high number of infiltrating immune cells with 30-50% of total lymphocytes being NK cells. Given the constant liver intake of non-self antigens from the gastrointestinal tract via the portal vein, hepatic NK (H-NK) cells must retain a certain degree of tolerance in the context of their immune-surveillance against dangers to the host. Indeed, the breakdown of the tolerogenic state of the liver-associated immune system has been shown to induce autoimmunity. However, the role of NK cells during the course of autoimmune liver diseases is still being debated mainly because a complete characterization of H-NK cells normally resident in healthy human liver has not yet been fully disclosed. Furthermore, the differences in phenotype and functions between human and mouse H-NK cells often preclude translation of results obtained from murine models into experimental approaches to be performed in humans. Here, we provide an extensive characterization of the phenotype of H-NK cells physiologically resident in the human liver by both mentioning data available in literature and including a set of original results recently developed in our laboratory. We then review our current knowledge in regard to the contribution of H-NK cells in regulating local immune homeostasis and tolerance as well as in inducing the development of liver autoimmunity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Pulmonary VO2 on-kinetics during light-to-moderate-intensity constant-work-rate exercise, an experimental model mirroring energetic transitions during daily activities, has been shown to speed up with aerobic exercise training (AET) in normal subjects, but scant data are available in chronic heart failure (CHF)., Methods and Results: Thirty CHF patients were randomized to 3 months of light-to-moderate-intensity AET (CHF-AET) or control (CHF-C). Baseline and end-protocol evaluations included i) one incremental cardiopulmonary exercise test with near infrared spectroscopy analysis of peak deoxygenated hemoglobin+myoglobin concentration changes (Δ[deoxy(Hb+Mb)]) in vastus lateralis muscle, ii) 8 light-to-moderate-intensity constant-work-rate exercise tests for VO2 on-kinetics phase I duration, phase II τ, and mean response time (MRT) assessment, and iii) circulating endothelial progenitor cell (EPC) measurement. Reference values were obtained in 7 age-matched normals (N). At end-protocol, phase I duration, phase II τ, and MRT were significantly reduced (-12%, -22%, and -19%, respectively) and peak VO2, peak Δ[deoxy(Hb+Mb)], and EPCs increased (9%, 20%, and 98%, respectively) in CHF-AET, but not in CHF-C. Peak Δ[deoxy(Hb+Mb)] and EPCs relative increase correlated significantly to that of peak VO2 (r=0.61 and 0.64, respectively, p<0.05)., Conclusions: Light-to-moderate-intensity AET determined a near-normalization of pulmonary VO2 on-kinetics in CHF patients. Such a marked plasticity has important implications for AET intensity prescription, especially in patients more functionally limited and with high exercise-related risk. The AET-induced simultaneous improvement of phase I and phase II, associated with an increase of peak peripheral oxygen extraction and EPCs, supports microcirculatory O2 delivery impairment as a key factor determining exercise intolerance in CHF., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The pathophysiology of chronic heart failure (CHF) involves multiple hystologic and molecular alterations. To determine the effects of physical training on circulating endothelial progenitor cells (EPCs), angiogenesis (angiogenin, angiopoietin-1 and -2, VEGF, Tie-2, SDF-1α) and inflammation (IL-6, CRP), we compared data obtained from 11 CHF pts before and after 3 months aerobic exercise training, to those from 10 non trained CHF pts (CHF-C group, age 64 + 2 years, NYHA 2). At the end of the study, EPCs count and AP-2 serum levels significantly increased in the CHF-TR group. These preliminary data suggest a significant effect of even a short program of physical training on angiogenic activation and endothelial dysfunction.

Background: Endothelial colony-forming cells (ECFCs), are circulating endothelial progenitor cells increasingly studied in various diseases because of their potential for clinical translation. Experimental procedures for their ex vivo culture still lack standardization. In particular two different extracellular matrix proteins, either fibronectin or collagen, are commonly used by different Authors for coating plastic plates, both allowing to obtain cells that have all the features of ECFCs. However, possible differences in the impact of each substrate on ECFCs have not been analysed, so far. Therefore, in this study we investigated whether fibronectin and collagen may differentially affect ECFC cultures., Methodology/principal Findings: ECFCs were isolated and cultured from peripheral blood mononuclear cells of healthy donors. The impact of fibronectin compared with collagen as the only variable of the experimental procedure was analysed separately in the phase of isolation of ECFC colonies and in the following phase of cell expansion. In the isolation phase, although similar frequencies of colonies were obtained on the two substrates, ECFC colonies appeared some days earlier when mononuclear cells were seeded on fibronectin rather than collagen. In the expansion phase, ECFCs cultured on collagen showed a longer lifespan and higher cell yields compared with ECFCs cultured on fibronectin, possibly related to the higher levels of IL-6 and IL-8 measured in their supernatants. ECFCs cultured on both substrates showed similar immunophenotype and ability for in vitro tube formation., Conclusions/significance: Overall, the results of this study indicate that, although both fibronectin and collagen efficiently sustain ECFC cultures, each of them brings some advantages within individual steps of the entire process. We suggest that colony isolation performed on fibronectin followed by cell expansion performed on collagen may represent a novel and the most efficient strategy to obtain ECFCs from adult peripheral blood samples.

Classic Kaposi's sarcoma (cKS) is a human herpesvirus-8 (HHV-8)-associated lympho-angioproliferative tumor typically occurring in the elderly. It is associated with HHV-8-driven perturbed balance of peripheral B-cell subsets, which may have an impact on immune responses to antigenic stimulation. We took advantage of the common practice of cKS patients to undergo seasonal influenza vaccination because of advanced age and analyzed the immunogenicity and safety of licensed trivalent influenza vaccine in 46 cKS patients and 44 matched controls. Licensure criteria for immunogenicity were fulfilled in both groups. Four weeks after vaccination, hemagglutination-inhibition antibody titers against each viral strain contained in the vaccine increased in patients and controls (all P<0.001). Protection against at least one strain was achieved by 96% of cKS and 91% of control subjects. Protection against all strains persisted after 12 weeks, demonstrating a long-lasting response to vaccination. The vaccine was equally well tolerated by patients and controls, as assessed by evaluating solicited local and systemic reactions to the vaccine, and appearance or increase of antinuclear autoantibodies. HHV-8 virological rebound was observed in four cKS patients, but was not accompanied by progression of KS lesions. We conclude that seasonal influenza vaccine given to cKS patients is immunogenic and safe.

Natural cytotoxicity receptors (NCRs) were originally identified as specific natural killer cell activating receptors that, on binding to their endogenous ligands, trigger the killing of tumor cell targets. We recently described the differentiation of a novel subset of NCR(+) Vδ1 T cells characterized by a remarkably high cytolytic potential against cancer cells. Here we demonstrate that the engagement of NKp30, one of the NCRs expressed de novo on Vδ1 T cells after stimulation, triggers the production of high levels of CCL3/MIP-1α, CCL4/ MIP-1β, and CCL5/RANTES but not of CXCL12/SDF-1. In turn, this NKp30-induced secretion of cc-chemokines is able to significantly suppress the replication of a CCR5 tropic strain of HIV-1 in CD4(+)/CCR5(+) infected PM1 cell lines. This experimental evidence disclosing an unanticipated antiviral function of NCR(+) Vδ1 T cells opens new avenues for understanding the pathogenic role and for manipulating the function of γδ T cells in HIV-1 infection.

There are hints that hypoxia exposure may affect the number of circulating endothelial progenitor cells (EPCs) in humans. To test this hypothesis, the concentration of EPCs was determined by flow cytometry in the peripheral blood of 10 young healthy adults before (0 h), at different times (0.5 h, 1 h, 2 h and 4 h) during a 4 h normobaric hypoxic breathing simulating 4100 m altitude, and in the following recovery breathing room air. Results were interpreted mainly on the basis of the changes in surface expression of CXC chemokine receptor-4 (CXCR-4, a chemokine receptor essential for EPC migration and homing) and the percentage of apoptotic cells, the plasmatic levels of markers of oxidative stress induced by hypoxic breathing. Compared to 0 h, the concentration of EPCs, identified as either CD45(dim)/CD34(+)/KDR(+) or CD45(dim)/CD34(+)/KDR(+)/CD133(+) cells, decreased from 337 ± 83 ml(-1) (mean ± SEM) to 223 ± 52 ml(-1) (0.5 h; P < 0.005) and 100 ± 37 ml(-1) (4 h; P < 0.005), and from 216 ± 91 to 161 ± 50 ml(-1) (0.5 h; P < 0.05) and 45 ± 23 ml(-1) (4 h; P < 0.005), respectively. Upon return to normoxia, their concentration increased slowly, and after 4 h was still lower than at 0 h (P < 0.05). During hypoxia, CXCR-4 expression and plasmatic stromal derived cell factor-1 (SDF-1) increased abruptly (0.5 h: +126% and +13%, respectively; P < 0.05), suggesting cell marginalization as a possible cause of the rapid hypoxia-induced EPC reduction. Moreover, hypoxia exposure induced an increase in EPC apoptosis and markers of oxidative stress, which was significantly evident only starting from 2 h and 4 h after hypoxia offset, respectively, suggesting that EPC apoptosis may contribute to the later phase of hypoxia-induced EPC reduction. Overall, these observations may provide new insights into the understanding of the mechanisms operated by EPCs to maintain endothelial homeostasis.

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

Background: Human herpesvirus-8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS) and of some lymphoproliferative disorders of B cells. Most malignancies develop after long-lasting viral dormancy, and a preventing role for both humoral and cellular immune control is suggested by the high frequency of these pathologies in immunosuppressed patients. B cells, macrophages and dendritic cells of peripheral lymphoid organs and blood represent the major reservoir of HHV-8. Due to the dual role of B cells in HHV-8 infection, both as virus reservoir and as agents of humoral immune control, we analyzed the subset distribution and the functional state of peripheral blood B cells in HHV-8-infected individuals with and without cKS., Methodology/principal Findings: Circulating B cells and their subsets were analyzed by 6-color flow cytometry in the following groups: 1- patients HHV-8 positive with classic KS (cKS) (n = 47); 2- subjects HHV-8 positive and cKS negative (HSP) (n = 10); 3- healthy controls, HHV-8 negative and cKS negative (HC) (n = 43). The number of B cells belonging to the preimmune/natural effector compartment, including transitional, pre-naïve, naïve and MZ-like subsets, was significantly higher among HHV-8 positive subjects, with or without cKS, while was comparable to healthy controls in the antigen-experienced T-cell dependent compartment. The increased number of preimmune/natural effector B cells was associated with increased resistance to spontaneous apoptosis, while it did not correlate with HHV-8 viral load., Conclusions/significance: Our results indicate that long-lasting HHV-8 infection promotes an imbalance in peripheral B cell subsets, perturbing the equilibrium between earlier and later steps of maturation and activation processes. This observation may broaden our understanding of the complex interplay between viral and immune factors leading HHV-8-infected individuals to develop HHV-8-associated malignancies.

Defensins are endogenous defense peptides with well defined antimicrobial activity against a broad spectrum of pathogens including bacteria, fungi, viruses, and parasites.Several lines of evidence suggest that defensins might also contribute to the regulation of host innate and adaptive immunity, but their immunomodulatory functions are still poorly understood. Herein, we studied the impact of human defensins on multiple functions of DCs, which are a central player in all immune responses, bridging innate and adaptive immunity. We challenged DCs differentiated in vitro from human moDCs with HNP-1 alpha-defensin or HBD-1. HNP-1 and HBD-1 were chemotactic for moDCs. Both defensins promoted the activation and maturation of moDCs, as assessed by up-regulation of surface expression of the costimulatory molecules CD80, CD86, and CD40, the maturation marker CD83, and HLA-DR. HNP-1 and HBD-1 also enhanced the production of the proinflammatory cytokines TNF-alpha, IL-6, and IL-12p70 but did not affect the production of the regulatory cytokine IL-10. According to these stimulatory effects, HNP-1 and HBD-1 increased the allostimulatory activity of moDCs significantly. Finally, HNP-1 and HBD-1 promoted the up-regulation of CD91 on the DC surface. CD91 is a scavenger receptor involved in the recognition of multiple ligands including defensins, thus suggesting that defensins may amplify their own effects through the activation of an autocrine loop. Taken together, our observations may provide new insight into the immunomodulatory properties of human defensins and may aid the exploration of new therapeutic strategies to potentiate antimicrobial and antitumor immunity.

Analysis of peripheral blood dendritic cells (PBDCs) is increasingly reaching clinical relevance in a wide range of pathologies, in which investigating the capacity of DC subsets to respond adequately to specific stimuli may aid the comprehension of underlying immunopathologic mechanisms. The evaluation of PBDC responses directly challenged in whole blood (WB) samples offers many advantages over other methods that require DC isolation and culture, but it is limited in multiparametric analysis, currently based on 3- or 4-color assays. Therefore, in this study we developed a 6-color assay dedicated to the analysis of PBDC responses upon WB stimulation. We incubated WB samples with ligands to toll-like receptors (TLRs) with a clear-cut distribution on myeloid DCs (mDCs) or plasmacytoid (pDCs) and analyzed DC responses in terms of upregulation of activation/maturation markers, as well as production of a wide range of regulatory cytokines. Four colors were used to gate on mDCs and pDCs that were identified as lineage-/HLA-DR+/CD11c+ and lineage-/HLA-DR+/CD123+, respectively, and two further colors were used to analyze either the surface expression of CD80, CD86, CD40 or CD83, or the intracellular accumulation of IL-12, tumor-necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IL-6, IL-10 or IL-4. With this method, we could directly compare in the same flow cytometric tube the responses of mDCs and pDCs to TLR stimulation, and investigate the reciprocal coexpression of distinct activation markers or regulatory cytokines. We suggest that the 6-color WB assay presented here may represent a novel tool for investigating the complex biology of DCs.

Background: Flow cytometric analysis of peripheral blood dendritic cells (PBDCs) and their myeloid (mDCs) and plasmacytoid (pDCs) subsets is a less invasive procedure that is acquiring growing clinical relevance. Because dendritic cells (DCs) lack unique lineage markers, current methods that are based on 3- or 4-color assays do not allow multiparametric analysis of DC subsets. In this study a dedicated 6-color assay was developed., Methods: mDCs and pDCs were counted and characterized for the expression of activation/maturation markers by using a single-platform 6-color assay. Whole-blood samples from 20 healthy controls were directly stained with either CD80-FITC/CD40-PE/lineage-PerCP-Cy5.5/CD123-PE-Cy7/CD11c-APC/HLA-DR-APC-Cy7 or CD86-FITC/CD83-PE/lineage-PerCP-Cy5.5/CD123-PE-Cy7/CD11c-APC/HLA-DR-APC-Cy7 combination, in the presence of commercial fluorospheres. A dual-platform 3-color assay currently in use was run in parallel for comparison., Results: The 6-color assay provided mDCs and pDCs counts similar to counts obtained by the 3-color assay. Only the 6-color assay could show differential expression of activation markers by mDCs and pDCs, with pDCs expressing lower levels of costimulatory molecules and HLA-DR, but higher levels of CD83., Conclusions: The 6-color assay described here may be a sensitive tool for assessing possible variations in the number and features of mDCs and pDCs whose reciprocal balance is critical in understanding the more detailed orchestration of immune responses.

Background: Vasculopathy is one of the hallmarks of systemic sclerosis (SSc), characterized by endothelial activation and over expression of adhesion molecules. A preliminary in vitro study has suggested that aminaftone, a naphtohydrochinone used in the treatment of capillary disorders, may downregulate the expression of adhesion molecules in endothelial cells., Objective: This study investigated the ex vivo effects of aminaftone on soluble adhesion molecule concentrations in patients with SSc., Methods: This randomized, open-label pilot study was conducted in patients with SSc. Patients received baseline treatment for Raynaud's phenomenon (eg, calcium channel blockers and IV cyclic iloprost) with (test) or without (control) aminaftone 75 mg or placebo TID for 12 weeks. Standard treatment for Raynaud's phenomenon was allowed as long as the dose was stable for >or=3 months prior to randomization. Concentrations of soluble E-selectin adhesion molecule 1 (sELAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), and soluble intracellular adhesion molecule 1 (sICAM-1) were measured at baseline and 12 weeks, and their variation was tested using the analysis of variance for repeated measures with statistical correction. Laboratory analyses were performed by experienced personnel blinded to treatment assignment., Results: A total of 24 patients were enrolled (21 women, 3 men; mean age, 53.4 years; aminaftone, 12 patients; control, 12 patients). Decreases in mean (SD) sELAM-1 and sVCAM-1 concentrations were significantly greater in treated patients (sELAM-1, from 17.0 [7.8] to 11.9 [9.0] pg/mL; sVCAM-1, from 51.2 [12.9] to 40.8 [13.8] ng/mL) compared with controls (sELAM-1, from 20.3 [9.9] to 20.4 [10.5] pg/mL; sVCAM-1, from 56.8 [49.6] to 62.7 [40.6] ng/mL) (both, P < 0.05 [analysis of variance or repeated measures after Bonferroni correction]). No significant changes in sICAM-1 concentrations versus controls were observed., Conclusions: In this small pilot study in this select group of patients with SSc, aminaftone was associated with downregulation of sELAM-1 and sVCAM-1 concentrations. Studies evaluating the potential role of aminaftone in the treatment of vascular sclerodermal disease and SSc are warranted.

Keyhole limpet hemocyanin (KLH) is a xenoantigen largely used in vitro as an immunogen to study primary antigen-specific T cell responses and in vivo as a vaccine component with optimal carrier qualities. So far, the mechanisms by which KLH exerts its immunostimulatory properties are still largely unknown. In particular, although dendritic cells (DCs) play a central role in the initiation and activation of immune responses, the effects of KLH on these cells have been poorly explored. In the present study we investigated the effects of KLH on DCs differentiated in vitro from human monocytes. We observed that KLH promotes the activation and maturation of DCs, as assessed by up-regulation of the surface expression of CD80, CD86, CD40, HLA-DR and CD83. Moreover, even if KLH stimulated the production of IL-12 and IL-10 by DCs, the final balance was clearly in favour of IL-12. According to these stimulatory effects, KLH significantly increased the allostimulatory activity of DCs. To verify whether these effects of KLH may be related to the binding of this highly glycosilated molecule to mannose receptor (MR), we performed inhibition experiments with anti-MR antibody. Results showed that the stimulatory activity of KLH is indeed partially mediated by its interaction with MR. Taken together, our results seem to indicate that KLH does promote the maturation of DCs endowed with the ability to stimulate cell-mediated immune responses. We suggest that this property of KLH may represent a novel further mechanism by which this molecule may exert its efficacy when co-administered with others antigens in immunotherapeutic protocols.

Background: The cellular reservoirs of Kaposi's sarcoma-associated herpesvirus (KSHV) and the exact nature of the putative KSHV-infected circulating precursor of spindle cells of Kaposi's sarcoma (KS) still remain poorly defined. Because KS spindle cells are thought to be of endothelial origin, and because mature endothelial cells do not sustain persistent KSHV-infection, our attention was focalized on circulating hematopoietic precursors able to differentiate into endothelial lineage., Methods and Findings: Late endothelial progenitor cells (late-EPCs) were cultured from the peripheral blood mononuclear cells of 16 patients with classic KS. The presence and load of KSHV genomes were analyzed by real-time polymerase chain reaction in DNA extracted from cells and supernatants of late-EPC cultures obtained from 7 patients. Endothelial colonies cultured from the peripheral blood of KS patients were found to satisfy all requisites to be defined late-EPCs: they appeared from the CD14-negative fraction of adherent cells after 11-26 days of culture, could be serially expanded in vitro, expressed high levels of endothelial antigens but lacked leukocyte markers. Late-EPC cultures were found to harbor KSHV-DNA at variable levels and to retain the virus after multiple passages in cells as well as in supernatants, suggesting that a quote of KSHV lytic infection may spontaneously occur. Lytic phase induction or hypoxia could amplify virus release in supernatants., Conclusion: Our results suggest that circulating endothelial progenitors from KS patients are KSHV-infected and support viral productive replication and may therefore represent potential virus reservoirs and putative precursors of KS spindle cells.

Through the production of stimulatory and suppressive cytokines, dendritic cells (DCs) regulate virus-specific immune responses that are crucial to virus eradication. To explore a possible role of DCs in the persistence of hepatitis C virus (HCV) infection, in this study we analysed peripheral blood DCs (PBDCs) in patients with chronic hepatitis C (CHC) compared with those in both healthy seronegative (HSN) controls and a group of subjects who had spontaneously resolved infection, defined as healthy HCV-seropositive (HSP), and we evaluated the relationships between PBDCs and HCV-specific CD4(+) T-cell reactivity. The number of PBDCs, their immunophenotype and expression of regulatory cytokines were evaluated by flow cytometry on whole-blood samples. HCV-specific CD4(+) T-cell activation, proliferation and cytokine production were evaluated in cultures of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with HCV peptides. We found that PBDCs from CHC subjects were numerically reduced and showed lower interleukin-12 (IL-12) and higher IL-10 expression than those from HSN controls. PBDCs from HSP subjects were similar to those from HSN controls. HCV-specific CD4(+) T-cell proliferation was less frequent and vigorous in CHC than in HSP patients and was directly related to the number of PBDCs and their IL-12 production but inversely related to their IL-10 production. Taken together, these results seem to suggest that cytokines of DC origin contribute to the regulation of HCV-specific immunity in CHC patients and indicate that PBDCs may represent a novel non-invasive tool for immune monitoring of these patients.

Although the immune system is clearly capable of recognizing and eliminating tumor cells, it usually fails to provide adequate protective antitumor responses. It is becoming increasingly clear that modifications of dendritic cells (DCs), which are central regulators of immune responses, could be a strategy exploited by tumor cells to escape from immune control. This review focuses on the current understanding of DC defects occurring in cancer patients, mechanisms responsible for the induction of such defects, consequences of DC defects on antitumor immune response and current concepts of DC-based immunotherapy. An improved understanding of how tumors interact with DCs to escape from immune control may enable the design of improved DC-based immunotherapeutic strategies for patients with cancer.

Dendritic cells (DCs) are the single most central player in all immune responses. To assess whether DC alterations may contribute to the immune dysregulation that affects the elderly, we investigated the effects of ageing on DCs. We analyzed the number, phenotype and function of peripheral blood DCs from 70 healthy subjects aged 20-92 years by using flow cytometric methods that allow cell characterization directly in whole blood samples. We demonstrated that the number of myeloid DCs progressively declines with age. This finding was accompanied by a decrease of CD34+ precursors and increase of circulating monocytes, suggesting that the entire differentiation process of antigen presenting cells is partially dysregulated in the elderly. DCs from aged individuals also appeared to have a more mature phenotype and impaired ability to produce IL-12 upon stimulation. These results may help to clarify the contribution of innate immunity to the development of immunosenescence.

Activation-induced cytidine deaminase (AID), an enzyme with homology to members of the APOBEC family, is involved in somatic hypermutation (SHM) of immunoglobulin (Ig) genes, either by direct deamination of DNA or by an indirect action through its putative RNA editing activity. AID is able to mutate both Ig-like reporter constructs and selected non-Ig genes in normal B cells and in other cells when ectopically overexpressed in mammalian cells and transgenic mice. However, in spite of the fact that in these transgenic animals AID activity was driven by an ubiquitous promoter, only T lymphomas and lung adenomas occurred. In the present work, we constructed three sets of transgenic mice in which AID was under the control of lck, HTLV-I and MMTV promoters, respectively. The lck/AID mice developed thymic lymphomas with variable but high efficiency, while no tumor was detected in HTLV-I/AID mice after two years of monitoring. Four MMTV/AID founder mice died with an atypical clinical picture, although no mammary tumor was found. These findings suggest that additional factors, present in thymocytes but not in other tissues or in lymphoid cells at different stages of differentiation, are needed for AID to fully manifest its tumorigenic potential in mouse. Alternatively, the display of full AID mutagenic and transforming activity could be related to the existence of physiologic DSBs which occur in both thymocytes and switching B cells.

In this study, we investigated whether dendritic cells (DCs) are altered in classic Kaposi's sarcoma (cKS), a lympho-angioproliferative disorder associated with human herpesvirus-8 (HHV-8) infection. By direct analysis of peripheral blood DCs (PBDCs), we demonstrated that cKS patients have lower frequency of myeloid and plasmacytoid DCs than controls. This reduction was greater in patients with advanced stages of disease. PBDCs from cKS patients also showed up-regulated expression of the scavenger receptor CD91 and impaired IL-12 expression. PB monocytes that represent DC precursors in vivo and in vitro showed the same alterations; accordingly, DCs differentiated in vitro from cKS monocytes were similarly affected. The same alterations were induced by addition of cKS plasma during DC differentiation from control monocytes. These results indicate that PBDCs and their precursors are altered in cKS and suggest that soluble circulating factors participate in this process. The study may provide new insights into the pathogenesis of cKS.

We investigated the apoptosis of myelin basic protein (MBP)-specific T lymphocytes in multiple sclerosis (MS) patients with acute (AMS) or stable (SMS) MS by evaluating the expression of apoptosis markers on peripheral cells. Cells of healthy controls (HC) were evaluated as well. Results showed that mitogen-stimulated apoptosis was comparable among patients and controls, whereas MBP-stimulated CD4+ and CD8+ 7-AAD+ and 7-AAD+ Fas+ cell (apoptotic cells) were significantly reduced in AMS patients. A reduction of the apoptotic rate of myelin-specific CD4+ and CD8+ T lymphocytes could be involved in the immune-mediated destruction of the myelin sheath seen in AMS patients.

Background/aims: T-lymphocyte reactivity against viral antigens may represent the only immunological marker of host contact with a virus. Aim of the present study was to investigate whether vertical exposure to hepatitis C virus (HCV) could activate HCV-specific T-cell responses that may represent a biomarker of previous contact with the virus, and possibly contribute to the low rate of vertical HCV transmission., Methods: We studied 28 children born from chronically HCV-infected mothers. HCV-specific activation and proliferation of CD4+-lymphocytes and cytokine production were evaluated in cultures of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with HCV-peptides., Results: HCV-specific CD4+-cell reactivity was observed in 20 out of the 28 children (71%). The proliferation of HCV-specific CD4+-cells was more frequent and vigorous in children than in their mothers. In children, but not in the mothers, activation of CD4+-cells upon stimulation with HCV-peptides was directly correlated with proliferation. Early upon stimulation with HCV-peptides, lymphocytes from children produced lower levels of IL-10 than lymphocytes from the mothers., Conclusions: Vertical exposure to HCV induces the development of viral-specific CD4+-cell-mediated immune responses, possibly endowed with protective function against infection, which may contribute to the low rate of vertical HCV transmission.

Effective therapy for prion diseases is currently unavailable. Recently, vaccination was shown to be effective in mouse models of a particular neurodegenerative conditions: Alzheimer's disease (AD). Here, we report that vaccination with synthetic oligopeptides homologous to the hamster (Mesocricetus auratus) prion protein augments survival time in animals infected intraperitoneally with 263K scrapie agent. For each hamster included in the study, prion-specific serum antibodies as well as deposition of pathological prion protein (PrP(res)), glial fibrillary acidic protein (GFAP), and mRNA expression for cytokines (TNF alpha, IL-1beta, IL-10) in brain tissues were evaluated. In immunized animals, increased survival after challenge was associated with a reduction of cerebral lesion, PrP deposition and GFAP expression; in these animals, anti-prion protein peptide antibody levels were increased, and the expression of pro-inflammatory cytokines (TNF alpha and IL-1beta) was reduced. Vaccination could be an effective therapeutic approach to postpone disease onset.

The effects of interleukin 16 (IL-16) on dendritic cell (DC) generation from human CD34(+) progenitor cells are not known. Here, we show that IL-16 added to a basal cocktail comprised of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, Flt-3 ligand (Flt3L), and tumor necrosis factor alpha (TNF-alpha) does induce the CD34(+) hematopoietic cells to proliferate in vitro and to differentiate into phenotypically and functionally mature DCs. IL-16 exerts this function more efficiently than stem cell factor (SCF) as a control, thrombopoietin (TPO), or IL-16 plus TPO. Moreover, we show that the combination of IL-16 plus TPO induces the generation of tolerogenic DCs, able to induce an anergic state in T cells that persists when T cells are rechallenged with immunogenic DCs. An altered pattern of cytokine production, a reduced expression of the C-type lectin DC-SIGN, and an increased surface expression of the inhibitory molecules immunoglobulin-like transcript 2 (ILT-2), ILT-3, and ILT-4 may all contribute to confer the tolerogenic properties of these DCs. Generation of tolerogenic DCs may aid the exploration of new therapeutic strategies to promote tolerance to autoantigens and prevent disease development.

Monocyte-derived dendritic cells (DCs) generated in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4-DCs) are used to enhance antitumor immunity in cancer patients, although recent evidence suggests that their functional repertoire may be incomplete; in particular, IL-4-DCs appear unable to induce type 2 cytokine-producing T helper (Th) cells. To assess whether type 1 interferon (IFN) could replace IL-4 and generate DCs with a more complete repertoire, we characterized in detail DCs generated from human monocytes cultured with GM-CSF and IFN-alpha (IFN-DCs). We found that IFN-alpha induces DC differentiation more efficiently than IL-4, yielding similar numbers of DCs in a shorter time and that this differentiation persists upon removal of cytokines. Although IFN-DCs had a more mature immunophenotype than IL-4-DCs, showing higher expression of CD80, CD86, and CD83, they still preserved comparable endocytic and phagocytic capacities and responsiveness to maturation stimuli. IFN-DCs had strong antigen-presenting capacity, inducing intense proliferation of T cells to alloantigens or influenza virus. Moreover, IFN-DCs produced lower levels of IL-12p70 and higher levels of IFN-alpha, IL-4, and IL-10 than IL-4-DCs. As a consequence of this different pattern of cytokine secretion, IFN-DCs induced T cells to produce type 1 (IFN-gamma) and type 2 (IL-4 and IL-10) cytokines, and as expected, IL-4-DCs induced only Th1 differentiation. As immune responses with extreme Th1 bias are considered inadequate for the induction of optimal, systemic antitumor immunity, the ability of IFN-DCs to promote more balanced cytokine responses may suggest the advisability to consider these cells in the development of future, DC-based immunotherapy trials.

Aim: Kaposi's sarcoma (KS) is a lympho-angioproliferative disorder characterized by angiomatous nodules and plaques that mainly affect the skin. The disease is consistently associated with human herpesvirus-8 (HHV8) and with a state of preexistent immunosuppression. Dendritic cells (DCs) have an instrumental role in the activation and function of both innate and adaptative immune responses. At least 2 distinct subsets have been characterized in peripheral blood based on phenotypic markers: myeloid DCs (CD11c+), associated with Ag uptake, T cell activation and ability to secrete IL-12, and plasmacytoid DCs, high virus-induced IFN-alpha producing cells. Because of the role of both DC subtypes in antiviral and antitumor induced responses, we hypothesized that DCs could be involved in the onset and evolution of KS., Methods: Thirty-five patients with mediterranean KS assigned to different clinical stages were compared with 51 healthy control subjects. Peripheral blood DCs were quantified and functionally characterised by flow cytometry directly on whole blood samples. The production of the regulatory cytokines, IL-12 and IL-10, was assessed as intracellular accumulation after incubation with or without lipopolysaccharide (LPS)., Results: Myeloid DCs identified as lineage-/HLA-DR+/CD11c+ cells were significantly lower in KS patients than in controls (0.54+/-0.25 vs 0.69 +/-0.26% of the peripheral blood mononuclear cells; p<0.017). Furthermore, CD11c+ DCs were lower in patients with more diffuse disease. Plasmacytoid DCs, identified as lineage-/HLA-DR+/CD123+ cells, were lower in KS patients (0.23+/-0.19 vs 0.36+/-0.17; p<0.001). DCs from KS patients were more mature, as assessed by expression of the maturation marker CD83, and showed an impaired ability to produce IL-12 upon LPS stimulation, as compared with controls., Conclusion: The numerical and functional alterations of peripheral blood DCs observed in KS patients suggest an involvement of these cells in the onset and evolution of the disease.

Objective: To investigate interactions of immune cells with vascular endothelium in patients with systemic sclerosis (SSc) and in patients with idiopathic or autoimmune Raynaud's phenomenon (RP)., Methods: Lymphocytes obtained from 11 patients with SSc, 9 with RP and 14 control subjects were pre-stimulated in vitro with alloantigens and cultured together with human umbilical vein endothelial cells (HUVECs). Lymphocyte adhesion and induction of endothelial HLA-class 11 molecules were measured by flow cytometry. Lymphocyte cytotoxicity against HUVECs was also evaluated. In some cases cells were cultured under experimental conditions of hypoxia and reoxygenation., Results: Lymphocyte adhesion and induction of endothelial cell expression of HLA-DR molecules were similar in controls and SSc patients, but significantly lower in RP (p < 0.05 and p < 0.03, respectively). Cytotoxic activity of lymphoblasts against endothelial cells was negligible in all patient groups. Under experimental conditions of hypoxia and reoxygenation lymphocyte adhesion was significantly greater than in normoxic conditions in SSc patients, while it was similar to normoxia in control subjects and RP patients., Conclusion: These results suggest that in RP patients there may be regulatory mechanisms of lymphocyte response able to control the processes that lead to lymphocyte adhesion and endothelial HLA-DR molecule induction. These mechanisms could play an important role in RP, and might possibly be lost in clinically evident SSc.

The aim of the present study was to analyse the in vitro proliferation and cytokine production by alloantigen-stimulated peripheral blood mononuclear cells (PBMC) obtained from patients affected by systemic sclerosis (SSc) and patients with Raynaud's phenomenon (RP). In SSc patients the proliferation of PBMC stimulated in vitro with alloantigens was significantly increased compared with healthy subjects, while no differences were observed for RP patients. Lymphocytes from SSc patients also produced larger amounts of IFN-gamma compared with healthy controls. However, patients with clinically active disease had lower IFN-gamma levels than those found in clinically stable patients. Patients affected by RP showed significantly higher levels of IFN-gamma than healthy subjects. Analysis at the clonal level of the lymphocyte subsets involved in alloantigen stimulation in one patient affected by active SSc, and one subject with RP confirmed the results obtained using PBMC. In particular, in the RP patient but not in the SSc patient, we observed a population of CD4+ T cells which proliferated to alloantigens in vitro and produced high levels of IFN-gamma. We suggest that T lymphocytes producing high levels of IFN-gamma might play a protective role in RP patients and in established scleroderma.

In this study we analyzed the proliferative response to the extracellular domain of thyrotropin receptor (TSHR-ECD) of T-cell lines raised from healthy subjects. We found high frequencies of cell lines reactive to TSHR-ECD, ranging from 12% to 37%. The response of the cell lines to a set of overlapping peptides of TSHR-ECD showed that the most recognized epitopes by T lymphocytes are on the C-terminal portion. In particular, the regions of residues 360-396 and 258-277 are immunodominant in T-lymphocyte reactivity. A group of cell lines specific for the peptides of TSHR-ECD lost the response to the peptides during time in culture. However, these lines were still responsive to TSHR extracellular domain. The cloning of one of these lines showed three types of T-cell clones: (1) CD4+ clones (n = 4) highly responsive to the TSHR-ECD; (2) CD4+ clones (n = 4) low responsive to TSHR-ECD; (3) CD8+ clones (n = 9) not responsive to TSHR-ECD. The first group of clones was stable during time in culture, while the second group was characterized by the loss of the specific response to TSHR-ECD after some weeks from the first analysis. The observation of a spontaneous anergy in the second group of CD4+ clones suggests that mechanisms of control of the lymphocyte response to TSHR-ECD could be activated in vitro.

Cyclo-oxygenase pathway metabolites released in the microenvironment by activated platelets and endothelial cells are potential local modulators of the immune response. In the present study, we have investigated the modulatory role of PGE2, iloprost (prostacyclin analogue), U-46619 (thromboxane analogue) on the release of IL-2, IFN-gamma, TNF-alpha and IL-6 by T lymphocytes. Our results show that PGE2 and prostacyclin differ in the regulation of cytokine production. PGE2 inhibited the release of IL-2 and IFN-gamma, while iloprost did not affect their production. The addition of PGE2 or iloprost greatly decreased the amount of TNF-alpha measured in the supernatants, although the rates of inhibition differed according to the kind of stimulation. Unlike that of PGE2, inhibition by iloprost was stronger in alloactivated cultures than in PHA-stimulated ones. In vitro IL-6 production was stimulated by PGE2 in alloactivated cultures and by iloprost, whatever the stimulus. These results are probably due to other cellular subsets contaminating the T-lymphocyte preparations. After complete removal of monocytes from cell cultures, there were inhibitory effects of lloprost and PGE2 on IL-6 released in the supernatants. We did not observe any significant effect of thromboxane analogue on the production of either cytokine.

Objective: To compare the long-term effects of intermittent infusion of iloprost with those of oral nifedipine on the in vitro production of cytokines in patients with systemic sclerosis (SSc), and to evaluate their relationship with the effects of the two treatments on clinical parameters., Methods: The production of cytokines by alloactivated circulating mononucleated cells was assessed before and after one year of treatment in a subset of 31 patients enrolled in a 12-month randomized clinical trial. Nineteen patients were treated with a 5-day (8 hr per day), 2.0 ng/kg per minute infusion followed by a 1-day infusion every 6 weeks; 12 patients were treated with an oral slow-release formulation of nifedipine, 20 mg twice daily. Quantitative determinations of interleukin-1 beta (IL1-beta) and interleukin-6 (IL6) in the culture supernatants were performed with a commercial ELISA; the levels of tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) were measured by specific radioimmunometric assays., Results: The production of IL1-beta was significantly lower in the iloprost group than in the nifedipine group. Both the cutaneous fibrosis and the capillaroscopic patterns were better in patients treated with iloprost than in patients treated with nifedipine. There was a significant positive covariance between IL1-beta changes and the changes in both the skin score and the capillaroscopic score., Conclusion: There are several mechanisms by which iloprost could exert its clinical efficacy. Vasodilatation and inhibition of platelet aggregation are certainly important, but they are transient. We suggest that the long-lasting modulation of the cytokine network observed in the present study could be another potential mechanism responsible for the persistent efficacy of iloprost despite its intermittent administration.

The authors present a case of a patient who developed recurrent bacterial upper respiratory and pulmonary infections and marked hypogammaglobulinemia with a gradual decrease of serum IgG, IgA and IgM some months after acute Epstein-Barr virus infection. Test for identification of lymphocyte subpopulation showed increased CD8+ T-cells with a surface phenotype (CD8+, CD57+, HLA-DR+) characteristic of virus-induced, activated cytotoxic cells. Viral investigations showed a positive anti-EBNA titer, an IgG titer anti-VCA of 1:40, a negative IgG titer anti-EA and human immunodeficiency virus negativity. The authors conclude that these clinical features are indicative of possible common variable immunodeficiency following Epstein-Barr virus infection.

The aim of the present study was to investigate the in vitro mitotic response and cytokine production after allogeneic stimulation of peripheral blood mononuclear cells (PBMC) from healthy elderly subjects. Interleukin-1 (IL-1), interleukin-2 (IL-2), gamma-Interferon (gamma-IFN), and Tumor Necrosis Factor alpha (TNF-alpha) were detected in the supernatants of mixed lymphocyte cultures (MLC). The in vitro proliferative response was significantly reduced in the elderly subjects. The amount of IL-2 detected in the supernatant from cultures of cells from elderly donors was higher than for young controls, as were the production of cytokines predominantly secreted by the macrophage population (IL-1 and TNF-alpha). There was no difference in the production of gamma-IFN by cells of elderly and young subjects.

Hypercoagulability can be defined as a condition of procoagulant imbalance due to heightened enzymatic activation of coagulation zymogens, but with no laboratory evidence of fibrin deposition nor clinical signs of thrombosis. The imbalance can be detected by measuring the plasma levels of prothrombin fragment 1 + 2 (F1 + 2), fibrinopeptide A (FPA) and thrombin-antithrombin III (TAT) complexes. The aims of this study were to establish the frequency of existence and biochemical pattern of hypercoagulability in patients with cancer and autoimmune disorders, clinical conditions associated with an increased risk of thrombosis, and to ascertain the most sensitive method for its diagnosis. In approximately one-fourth of the patients hypercoagulability was identified by finding high levels of FPA F1 + 2 or TAT unaccompanied by signs of fibrin deposition (expressed by normal levels of D-dimer). In a smaller proportion of patients (approximately 10%), the concomitant presence of high levels of D-dimer indicated that the activation of the coagulation cascade had gone beyond the stage of heightened enzymatic activity to the point of cross-linked fibrin deposition. Of the markers used to detect hypercoagulability. FPA seems to be the most sensitive, being significantly increased in all clinical conditions studied.

In our Dialytic Unit, the prevalence of antibodies to HCV (anti-HCV) was 16.4% in 1990 (73 hemodialyzed) and 21% in 1991 (105 hemodialyzed). The incidence of seroconversion was 13.1%. The transaminase behavior was similar in a group of anti-HCV-positive and a group of anti-HCV-negative patients and with previous HBV infection. Blood transfusions and dialytic age did not affect anti-HCV positivity.