Statistics: All Together Now, One Step at a Time. Microcirculation 18(4), 312. “
“Please cite this paper as:

Drummond and Tom (2011). How Can We Tell If Frogs Jump Further? Microcirculation 18(6), 512–515. “
“Please cite this paper as: Cracowski (2011). Female Hormones and Skin this website Microvascular Function. Microcirculation 18(5), 356–357. “
“Extensive vascular adaptations occur during pregnancy, and these result in the formation of a low-resistance placental circulation that maintains high blood flow to the developing fetus. These adaptations encompass both functional and structural alterations, including altered vasoreactivity of resistance vessels, arterial remodeling and angiogenesis. This Special Topics issue presents a collection of expert reviews that summarize the current state of knowledge on the regulation of the structural and functional changes that occur within the fetoplacental circulation, as well as introduce emerging

research questions and tools. Emphasis is placed on defining the mechanisms that underlie these physiological adaptations, as a foundation for applying this knowledge to the development of improved early detection markers and treatments for pathological conditions such as preeclampsia, gestational diabetes mellitus, and fetal growth restriction. Pregnancy evokes a complex temporal series of vascular adaptations that includes an extensive expansion of the vasculature that supplies the uterus and fetus, and the de novo formation of vascular networks within the placenta. SSR128129E These adaptations promote the ultimate establishment of a low-resistance placental circulation, which is critical to enable the substantive increase in fetoplacental Raf inhibitor review blood flow [9, 10] that is necessary for sustaining the developing fetus with an effective supply of oxygen and nutrients, and adequate removal of metabolic waste products. Remodeling of the vasculature occurs at multiple levels of the vascular tree (macro- and micro-vessels) and encompasses both functional and structural adaptations. Vasodilation and the circumferential enlargement of (hypertrophy) of the uterine vessels greatly facilitate the increased blood supply to the developing placenta and fetus [11].

Neovascularization of the placenta supports the development of this new organ, and also contributes to the establishment of high placental blood flow [14]. The fetoplacental vasculature represents a unique system to study physiological mechanisms underlying vascular remodeling within the adult. Beyond its value in the investigation of physiological adaptive processes, the application of this knowledge to studying disease states may help to identify early markers, and/or to develop effective treatments, for pathological conditions that endanger the health of both fetus and mother. Despite these potential benefits, the regulation of these adaptive events within the fetoplacental circulation has been understudied in comparison to other vascular beds.

To test this possibility, we immunized a cohort of WT and dnRAG1 mice with either NP-AECM-FICOLL or NP-CGG, which serve as models for thymus-independent and thymus-dependent antigens, respectively,35,36 and analysed NP-specific IgM or IgG antibody responses either 7 days after primary immunization or 14 days after a subsequent booster immunization (day 21). LEE011 mw We find that both IgM and IgG anti-NP responses to NP-AECM-FICOLL, but not NP-CGG, are significantly reduced in dnRAG1 mice compared with their WT counterparts

(Fig. 6c). These data suggest that dnRAG1 mice have a selective defect in responding to thymus-independent antigens, but are capable of mounting robust immune responses to thymus-dependent antigens. The impaired progression of B-cell development at the Talazoparib purchase immature-to-mature transition observed in dnRAG1 mice suggests that dnRAG1 expression interferes with the receptor editing process that occurs during this important stage of B-cell development.37 To test this possibility more directly, we bred dnRAG1 mice to mice bearing an anti-dsDNA specific immunoglobulin heavy chain transgene, called 3H9H56R, knocked into the endogenous heavy chain locus (56Rki mice) to determine whether dnRAG1 expression impedes the extensive light chain receptor editing that occurs in 56Rki mice

to obtain an ‘editor’ light chain capable of neutralizing the anti-dsDNA reactivity of the heavy chain.12 The 56ki model has the added feature of allowing us to determine whether editing of the 3H9H56R transgene through heavy chain gene replacement,38 which is thought to occur earlier in B-cell development,39 is also impaired by dnRAG1 triclocarban expression, and whether CD19+ B220lo B-cell accumulation in dnRAG1 mice depends on BCR specificity. A comparison of the various B-cell subsets

in WT, dnRAG1, 56Rki and double-transgenic (DTG) mice revealed several interesting results (see Supplementary material, Table S2). First, in contrast to dnRAG1 mice, DTG mice failed to accumulate splenic B220lo CD19+ B cells (Fig. 7a), clearly indicating that this population arises in dnRAG1 mice through selection based on BCR specificity. Interestingly, however, B1a B cells are still evident in the peritoneal cavity of DTG mice (Fig. 7a). Second, compared with both dnRAG1 and 56Rki mice, DTG mice show a significantly lower percentage and absolute number of IgM+ IgD+ mature B cells in the bone marrow (Fig. 7b; see Supplementary material, Fig. S4a). Third, DTG mice resemble 56Rki mice more closely than dnRAG1 mice in terms of the absolute number of cells in each of the transitional and mature B-cell subsets in the spleen, except for MZ B cells, which are significantly more abundant in DTG mice than in 56Rki mice (Fig. 7b).

A similar pattern is seen in other recently published data of B-lymphocyte subpopulations in healthy children [18]. Two papers have been published examining the EUROclass classification in children with CVID. Van de Ven et al. showed that two of nine children with CVID and heterozygous TACI

mutations belonged to the EUROclass high-risk group based on immunophenotyping results (smB-Trhigh) [36]. Yong et al. showed the correlation in a small group of children with CVID: children with few or absent switched memory B-lymphocytes (<5/ml; n = 24) exhibited a more severe clinical phenotype and more autoimmune cytopenia (21% vs. 0%) than those with higher FK228 order numbers of switched memory B-lymphocytes (n = 21) [37]; but this cohort is too small to extrapolate the data to the entire paediatric population. However, the great changes of these populations during development emphasize that a classification developed in adults cannot simply be extrapolated to classify the prognosis of children. A large, multicenter study is needed to evaluate the immunophenotyping characteristics of children with CVID and to correlate these with their clinical phenotype to create a reliable paediatric CVID classification.

Nearly 10% of CVID patients show a disease-modifying mutation in the gene encoding for TACI (TNFRSF13B), a tumour necrosis factor receptor expressed SCH727965 manufacturer mainly by activated B-lymphocytes (like marginal zone and memory B-lymphocytes), activated T-lymphocytes, monocytes, and dendritic cells. It mediates isotype switching, promotes plasma cell differentiation, and is essential for thymus-independent antibody responses, but also has

an inhibitory role in B-cell homeostasis [14]. Lack of TACI-expression can be used as a screening method before performing genetic analysis for the gene. There is little information about normal TACI-expression in healthy adults [38], and none in children, however. Plasma levels of BAFF and APRIL (both ligands of TACI) are significantly higher in patients with CVID, and correlate inversely with age in healthy subjects [39], suggesting Vildagliptin a positive age effect for TACI. Preterm neonatal naive B-lymphocytes show lower BAFF-R fluorescence intensity compared to adult naive B-lymphocytes, but in the same study no significant difference between TACI-expression on naive B-lymphocytes was found between cord blood and adults [38]. However, a lower gene expression of TACI determined by RT-PCR was seen in preterm cord blood compared to adult blood [38]. We found lower percentages of TACI+ B-lymphocytes in younger children compared to older children and adults. We did not find any effect of age on the BAFF-R expression on B-lymphocytes. This means that a low number of TACI-positive B-lymphocytes in young children is not indicative of a potential TACI-mutation.

The proliferation of the DO11·10 hybridoma cell line transfectants expressing SOCS-3 mRNA is also inhibited by stimulation of specific antigens, which confirms EPZ015666 order that IL-2 can inhibit T lymphocyte immunity through up-regulating the expression of SOCS-3 mRNA. However, SOCS-3 proteins, not mRNA, have the same effect in lymphocytes, and it would be interesting to perform this at proteic level on primary lymphocyte cells. SOCS-3

is a critical negative feedback regulatory factor of the JAK/STAT signalling transduction pathway, which plays a critical negative regulatory role in maintaining the balance of immunity. It has been shown that SOCS-3 can inhibit the proliferation of lymphocyte lines to the stimulation of specific antigens [16,19,22,24]. However, inhibition of the proliferation

of allogeneic lymphocytes with allogeneic antigen stimulation has not been reported. In this study, our results showed that the proliferation of B6 naive CD4+ T cells inducibly expressing SOCS-3 mRNA by IL-2 to the stimulation of allogeneic antigen was inhibited, suggesting the possibility of the initial inhibition of aGVHD. Further studies also demonstrated that the Th1-type polarization of B6 naive CD4+ T cells inducibly expressing SOCS-3 mRNA by IL-2 to the stimulation of allogeneic antigen was inhibited. These results support further that B6 naive CD4+ T cell inducibly expressing SOCS-3 mRNA by IL-2 could inhibit aGVHD, but selleck chemicals we do not know whether B6 naive CD4+ T cell transfectants expressing SOCS-3 can inhibit aGVHD. This will need further study. These results will help us to understand the mechanisms of the inhibitory effect on aGVHD. We hypothesized that whether Dichloromethane dehalogenase IL-2 signalling promotes or inhibits immunity might be related to the state of the CD4+ T cell. If the target cells of IL-2 signalling are activated CD4+ T cells, which express the high-affinity IL-2 receptor (IL-2R) with IL-2Rα (CD25), the IL-2 signal activates the JAK/STAT signalling

transduction pathway after IL-2 binds with high-affinity IL-2R. At the same time, down-regulation of SOCS-3 expression induced by antigen-TCR-mediated signals attenuates inhibition to the JAK/STAT signalling transduction pathway [16]. Activation of the JAK/STAT signalling transduction pathway leads to STAT phosphorylation and activation of genetic transcription, which can drive T cell proliferation and promote immunity. If the target cells of IL-2 signalling are naive CD4+ T cells which express low-affinity IL-2R without IL-2Rα (CD25), but with IL-2Rβ and IL-2Rγ, the IL-2 signal up-regulates expression of the negative feedback regulatory factor SOCS-3 when IL-2 binds with low-affinity IL-2R. Up-regulation of SOCS-3 expression can enhance inhibition to the JAK/STAT signalling transduction pathway and inhibit STAT phosphorylation and genetic transcription. This leads to the inhibition of T cell proliferation and immunity.

Visceral leishmaniasis (VL), caused by Leishmania donovani (L. donovani) and L. infantum, is the most severe systemic disease among the three main categories of leishmaniasis [1]. Moreover,

co-infection of Leishmania–HIV constitutes an emerging and serious public health problem [2, 3]. Despite considerable advances, there are still no efficient vaccines available against human leishmaniasis [4, 5]. DNA-based vaccines offer practical advantages, mostly because of the capacity of developing countries to cheaply and rapidly produce pDNA from bacteria. Furthermore, it is possible to formulate several antigens from different stages of the parasite life cycle or different subspecies as one shot vaccine [6]. A2 was first identified in L. donovani as a gene family that is expressed specifically in PLX4032 manufacturer the amastigote stage [7] and is associated with the visceralization process [8]. The protective response generated by recombinant A2 protein immunization was associated with a mixed Th1/Th2 response, production of IFN-γ in response to A2 antigen and an anti-A2 humoral response [9]. Also A2-expressing recombinant L. tarentolae shows promise as an effective live vaccine against L. infantum infection [10]. Among other L. infantum vaccine candidates, Daporinad mw cysteine proteinases type I (CPB) and II (CPA)

have been administrated in experimental vaccinations in both mouse and dog models and showed acceptable level of protection [11, 12]. Furthermore, it has been proved that the CPA/CPB cocktail is more protective against cutaneous and visceral Leishmania infections than CPA or CPB alone [11-13]. In general, DNA delivery methods can be subdivided into two categories: first, the use of biological vectors and second, systems employing Parvulin either chemical or physical approaches. Among the most investigated physical methods, electroporation for gene delivery has attracted

considerable attention recently, because of both the site-specific nature of the delivery and the high efficiency of the method. Electroporation, traditionally used for gene delivery, is believed to be a gold standard and is defined as the application of controlled electric fields to facilitate cell permeabilization, leading to the enhancement of gene uptake into cells after injection of naked DNA [14]. However, different factors such as dose of DNA, electrode shape and number, electrical field strength and duration must be optimized for antigen expression [14, 15]. Therefore, despite versatility [16], efficiency [16-18] and lower amount of required DNA [19], this technique presents several disadvantages like cell damage or rupture [18, 20], nonspecific transport leading to improper cell function and cell death [20] and even degradation of the plasmid DNA [21]. For these reasons chemical DNA delivery systems have been used to demonstrate increased plasmid uptake and reduced tissue damage.

There are currently insufficient

data to support guideline recommendations on the use of DES specific to patients with CKD or those on dialysis. Similarly there has been limited assessment of outcomes following the use of stents in transplant recipients. a. We recommend that all CKD patients, including haemodialysis, peritoneal dialysis and transplant patients, should be treated as per the general population when presenting with an acute coronary syndrome (ACS) ST-elevation myocardial infarction (STEMI) or non-ST-elevation acute coronary syndrome (NSTE-ACS) with regards to reperfusion therapy, antiplatelet Panobinostat chemical structure therapy (aspirin and clopidogrel), anticoagulant therapies (heparin, thrombin and glycoprotein IIb/IIIa inhibitors), beta-blockers and angiotensin-converting enzyme inhibitors (ACEi) (1C). c. We recommend that all CKD patients, including haemodialysis, peritoneal dialysis and transplant patients, should be treated for chronic stable CAD as the general population with regards to antiplatelet therapies, beta-blockers, ACEi and angiotensin receptor blockers (ARB)* (1D). *For angiotensin-converting ICG-001 enzyme inhibitors

and angiotensin receptor blockers refer to The KHA-CARI Guidelines: ‘Cardiovascular effects of blood pressure lowering in patients with chronic kidney disease.’ (summarized in Section 3 below). d. We recommend that all patients with CKD with an estimated glomerular filtration rate (eGFR) <60 mL/min, and specifically

those with an eGFR <30 mL/min undergoing antiplatelet or anticoagulant therapy, are considered as being at increased risk of bleeding. Dose adjustment of specific antiplatelet and anticoagulant drugs, specifically enoxaparin, bivalirudin, and glycoprotein IIb/IIIa inhibitors eptifibatide and tirofiban, is recommended (1A). Because of the ease of reversibility, unfractionated heparin (UFH) may be used in place of low molecular weight heparin Docetaxel order (LMWH) particularly in patients with a eGFR ≤30 mL/min, with standardized monitoring of clotting times (activated partial thromboplastin time, APPT) (ungraded). (Note: Data support an increased risk for bleeding with the use of LMWH or UFH in patients with increasing degrees of renal dysfunction, and in particular those with a CrCl ≤30 mL/min; however, they do not support an increased risk of bleeding with the use of LMWH compared with UFH within subgroups of CKD. The increased risk of bleeding in patients with eGFR ≤30 mL/min on LMWH is possibly abrogated by the use of anti-Xa adjusted dosing schedules, but these strategies have not been well tested in patients with renal insufficiency.) There is a two- to six-fold increased risk of cardiovascular events in patients with CKD,[6] with approximately 40–50% of the mortality of patients with stage 5 CKD on renal replacement treatment being attributed to CVD.

This study was approved by Research Ethics Committee of the Institute of Rheumatology in Warsaw. Genetic analysis of polymorphisms.  Genomic DNA was extracted from whole blood collected in tubes containing EDTA from patients with RA and the

control group using standard phenol/chloroform extraction method and the QIAapm DNA Blood Mini Kit (Qiagen, Hilden, Germany). The single nucleotide polymorphisms (SNPs) in the IL-17F gene were detected by the PCR–RFLP method. Amplification reaction was performed click here with 200 ng of genomic DNA in a 50-μl PCR mixture using 10 pmol of each primer: forward: 5′-GTG TAG GAA CTT GGG CTG CAT CAA T-3′ and reverse: 3′-AGC TGG GAA TGC AAA CAA AC-3′. Other conditions were as follows: 0.25 mm each dNTP (Qiagen) and 1.5 U HiFi Taq Polymerase (Novazym, Poznań, Poland) and 1× PCR buffer (containing 1.5 μm magnesium chloride, Sigma, MO, USA). The DNA was denatured at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s; 55.2 °C for 1 min and 72 °C for 1 min with a final extension at 72 °C for 10 min. Five microlitres of the amplification products, 470 basepairs (bp), were digested with 1 μl of AvaII (Fermentas, Burlington, Canada) for FK228 solubility dmso the Glu126Gly polymorphism, and with NlaIII (Fermentas) for the His161Arg polymorphism at 37 °C and separated by size on agarose gel. AvaII digestion of PCR product yielded 470 bp

for allele A and 75 and 395 bp for allele G (Fig. 1A). However, NlaII digestion of PCR product yielded 52, 130 and 288 bp for allele A, whereas for allele G 52 and 418 bp fragments were observed (Fig. 1B). Statistics.  Comparison of genotypes distribution and allele frequencies between patients with RA and healthy subjects was evaluated by the Chi-square (χ2) test with Yate’s correction. For genetic association analyses, both polymorphisms were tested

for deviations from Hardy–Weinberg equilibrium using the HWE program (http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Molecular motor The compared continued variables (clinical and laboratory parameters), the Wilcoxon test and χ2 test with Yate’s correction were used. Results were presented as the mean, standard deviation and the median. Linkage disequilibrium (LD) between His161Arg and Glu126Gly alleles was evaluated using the CubeX – Cubic Exact Solution program [27], and the frequency differences of haplotypes in patients with RA and controls were compared using the χ2 test with Yate’s correction. Statistical significance was considered to be indicated by a P-value lower than 0.05. The distribution genotypes of the both IL-17F polymorphisms were all in Hardy–Weinberg low in both the RA and control groups (P > 0.05). The genotype and allele frequencies in RA groups and in controls are shown in Table 2. In patients with RA, the homozygous wild genotype for His161Arg (AA genotype) was found in 88.

Here, we studied the role of the TNF family member 4-1BB ligand (4-1BBL) during the interaction of NK cells with chronic lymphocytic leukemia (CLL) cells. 4-1BBL

was highly expressed on patient B-CLL cells in all 56 investigated cases. Signaling via 4-1BBL following interaction with 4-1BB, which was detected on NK cells of CLL patients but not healthy individuals, led to the release of immunoregulatory cytokines including TNF by CLL cells. CLL patient sera contained elevated levels of TNF and induced 4-1BB upregulation on NK cells, which in turn impaired direct and Rituximab-induced NK-cell reactivity against 4-1BBL-expressing targets. NK-cell reactivity was not only enhanced by blocking the interaction of NK cell-expressed 4-1BB Selleckchem RAD001 with 4-1BBL expressed by CLL cells, but also by preventing 4-1BB upregulation on NK cells via neutralization of TNF in patient

serum with Infliximab. Our data indicate that 4-1BBL mediates NK-cell immunosubversion in CLL, and thus might contribute to the reportedly compromised efficacy of Rituximab to induce NK-cell reactivity in the disease, and that TNF neutralization may serve to enhance the efficacy of Rituximab treatment in CLL. “
“Drug-induced liver injury [DILI] is often caused by innate and adaptive host immune responses. Characterization of inflammatory infiltrates in the liver may improve understanding of the underlying pathogenesis of DILI. To characterize leukocytes infiltrating Selleck Napabucasin liver tissue from subjects with acute DILI [n = 32] vs. non-DILI causes of acute liver injury [n = 25]. Immunostains for CD11b/CD4 (Kupffer and T helper cells),

CD3/CD20 (T and B cells), and CD8/CD56 (T cytotoxic and NK cells) were evaluated in biopsies from subjects with acute DILI, either immuno-allergic [IAD] or auto-immune [AID] and idiopathic autoimmune (AIH) and viral hepatitis (VH) and correlated with clinical and pathologic features. All biopsies showed numerous CD8+ T cells and macrophages. DILI cases had significantly fewer B-lymphocytes than AIH and VH and significantly fewer NK cells than VH. Prominent plasma cells were unusual in IAD (3/10 cases), but were strongly associated with AIH (8/9) and also observed in most with AID (6/9). They were also found in Dynein 5/10 cases with VH. Liver biopsies from subjects with DILI were characterized by low counts of mature B cells and NK cells in portal triads in contrast to VH. NK cells were only found in cases of VH, whereas AIH and VH both showed higher counts of B cells than DILI. Plasma cells were most strongly associated with AIH and less so with AID, but were uncommon in IAD. “
“T. gondii is a highly successful global pathogen that is remarkable in its ability to infect nearly any nucleated cell in any warm-blooded animal. Infection with T.

The IL-17E-mediated Th2 responses are inhibited in the absence of either chain.71,74,98 In agreement with

the pre-clinical data signifying a role in Th2 biology, elevated expression of IL-17RB is detected in human asthmatic lung tissue, and the 5661G-A polymorphism within the IL-17RB gene, has been identified to be protective against asthma.64,107 Database mining for proteins homologous to IL-17RA led to the identification of IL-17RC.97 Biochemical analyses demonstrate high-affinity LY2109761 research buy interactions between IL-17RC–IL-17A and IL-17RC–IL-17F.66 There have been no reports of IL-17RC binding to other IL-17 family members. Similar to IL-17RA, IL-17RC expression is elevated in patients with RA, emphasizing the PD0325901 role of this pathway in autoimmune disease pathology.94,95 Intriguingly, alternative splice variants of IL-17RC have been detected in prostrate cancer tumours, but the function of these proteins is unclear.108,109 The function of IL-17RC has only been reported in the context of IL-17A and IL-17F biology.

In agreement with the essential role for IL-17RC in IL-17A and IL-17F responses, genetic deletion or antibody-mediated blockade of this chain abrogates IL-17A and IL-17F responses such as pro-inflammatory cytokine induction.11,110 Similar to the il17ra−/− mice, il17rc−/−mice display delayed onset and milder disease in the MOG-EAE model, and increased susceptibility to fungal infections.111,112 The biology of these IL-17R family members, which were also identified through database searches, is unknown. Interleukin-17RD was detected in endothelial cells and epithelial

cells (Table 2).113 Similar to IL-17RB and IL-17RC, IL-17RD has also been demonstrated to co-localize and complex with IL-17RA.114 Biochemical data suggest that this interaction mediates IL-17A function, as mutations within the cytoplasmic domain almost of IL-17RD prevent IL-17A induction of the 24p3 luciferase reporter.114 Other studies suggest that this receptor may have inhibitory effects, as over-expression suppresses fibroblast growth factor-mediated Ras and phosphatidyl inositol 3-kinase signalling. The significance of IL-17RD in vivo remains to be determined. Likewise, the biology of IL-17RE is undetermined. It has been reported that IL-17C binds to IL-17RE, although the import of this interaction is not understood.7 The specific cellular populations that are IL-17RE+ have not been defined.115 Although multiple splice variants of the IL-17RE gene have been identified, the biological significance of these isoforms is unknown.115 A role in MAPK activation has been detected, but further studies are required to understand the significance of this observation. Although substantial efforts have elucidated the biological functions of this unique family, there is still much to be discovered. In particular, the significance of the newer family members in host defence and inflammation needs to be addressed.

Transfected cells were added to antibiotic-free EGM-2 in 12-well costar multiwell cell culture plates and incubated overnight at 37°C. The medium was then replaced with complete EGM-2 medium containing 2% fetal bovine

serum. Two hours later, cells were either infected with AdVIFI16, AdVLacZ (MOI of 300) or mock-infected. After 36 h, protein extracts were prepared and chemiluminescence was measured using the Dual Luciferase Reporter Assay System kit (Promega) at the Lumino luminometer (Stratec Biomedical Systems, Birkenfeld, Germany). Preconfluent HUVEC were washed once with PBS and incubated with either AdVIFI16 or AdVLacZ (used as a control) at an MOI of 300 in EGM. After 2 h at 37°C, find more the virus was washed off and fresh medium was added.

After 60 h of incubation, supernatants were collected, centrifuged and transferred to new tubes for the chemokine/cytokine analysis according to the manufacturer’s instructions. The RayBio human cytokine array (G Series 2000 Ab arrays; RayBiotech, Norcross, GA, USA) is a glass slide format. The signals from G series arrays are detected using a laser scanner for the detection of 174 human cytokines in single experiment. In brief, after blocking, the arrays were incubated with the indicated samples. Unspecific bound proteins were removed Selumetinib in vivo and the arrays were incubated with a cocktail of biotin-Ab and then fluorescent dye-conjugated streptavidin. Spots were visualized using detection buffer loaded Sodium butyrate to cover the entire surface and incubated for 5 min. Image fluorescence signals were scanned and a software used that allows the fluorescence from all samples to be detected simultaneously or each sample to be detected on an individual basis as required. Spots were digitized into pixel densities. The densities were exported into spreadsheet software (Excel; Microsoft, Redmond, WA, USA) and the background intensity subtracted. The data were normalized to the positive control values provided by the manufacturer as 100% 26. LacZ- and IFI16-infected samples were compared for significance

using Student’s t-tests. p-Values of <0.05 were considered statistically significant. CCL4, CCL5 and CCL20 chemokines were quantified in LacZ- and IFI16-infected HUVEC supernatants by ELISA (R&D Systems, by SPACE, Milan, Italy) in accordance with the manufacturer's instructions. Human PBMC were isolated from venous blood of voluntary healthy donors using HistoPaque (Sigma) density gradient centrifugation. L-DC were generated as described previously 27 starting from monocytes purified with a monocyte isolation kit II (Miltenyi Biotech, Bologna, Italy) by negative selection. After 6 days of culture, cells were >95% CD1a+ and almost CCR6+ (from 65 to 85%) and langerin+ (from 50 to 70%) as determined by FACSCalibur (BD Bioscences, Milano, Italy).