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Abstract: Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-γ responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4+ T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8+ T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8+ T cell responses to peptides and peptide pools were well preserved. Loss of both CD4+ and CD8+ T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4+ T cell responses to peptide stimulation and partially restored T cell IFN-γ responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-γ responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4+ T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4+ and CD8+ T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4+ T cell responses and mild skewing of T cell phenotypic marker expression. [Copyright &y& Elsevier]

Summary: Betel quid (BQ) chewing is popular in Taiwan, India, and many southeast-Asian countries. BQ chewing has strong association with the risk of oral leukoplakia (OL), oral submucous fibrosis (OSF), and oral cancer (OC). BQ components exhibit genotoxicity and may alter the structure of DNA, proteins and lipids, resulting in production of antigenicity. BQ ingredients are also shown to induce keratinocyte inflammation by stimulating the production of prostaglandins, TNF-α, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes. These events may provoke tissue inflammation, early cell-mediated immunity (CMI), and immune surveillance in BQ chewers. However, BQ components also directly affect the functional activities of immunocompotent cells, and moreover tumor cells may hypo-respond to the CMI via diverse mechanisms such as induction of apoptosis of lymphocytes, induction of production of suppressor T cells, downregulation of MHC molecules in tumor cells, etc. Clinically, an alteration in lymphocyte subsets, a decrease in total number of lymphocytes, and a reduction in functional activities of CMI have been observed in isolated peripheral blood mononuclear cells (PBMC) and tumor infiltrated lymphocytes (TIL) in patients with OSF, OL or OC. Adaptation of tumor cells to immune system may promote clonal selection of resistant tumor cells, leading to immune tolerance. Future studies on effects of BQ components on CMI and humoral immunity in vitro and in vivo can be helpful for chemoprevention of BQ-related oral mucosal diseases. To elucidate how virus infection, tobacco, alcohol and BQ consumption, and other environmental exposure affect the immune status of patients with oral premalignant lesions or OC will help us to understand the immunopathogenesis of OC and to develop immunotherapeutic strategies for OC. [Copyright &y& Elsevier]

Abstract: In view of the natural resistance to infection by HIV and occasional delayed clinical manifestation of the disease, as also the fact that the virus is able to enter only cells that express CD4 and a co-receptor, we initiated a search for a soluble co-receptor that might compete with its membrane counterpart. Using a sandwich ELISA system, a soluble human CCR5 receptor (sCCR5) was indeed detected in the circulation. Immunoprecipitation of sCCR5-positive plasma samples from Israelis of Ethiopian and non-Ethiopian origin with mAb 2D7, a conformation-dependent anti-CCR5 antibody, revealed the presence of a ∼22kDa protein. A panel of antibodies directed against the membrane receptor was used to characterize the structure of the soluble CCR5: mAb CTC8, recognizing the N-terminal sequence of the protein,10YDIN13; “multidomain” mAbs FAB181B and FAB183B that are dependent upon the presence of Q93 and D95 in ECL1 and K171 and E172 in ECL2A, and mAb FAB182B, recognizing the stretch 184YSQYQF189, which spans the C-terminal part of the second extracellular loop. The presence of short soluble CCR5 in human plasma has not been previously described. Among HIV-negative non-Ethiopian Israelis, 20.4% were sCCR5-positive, as against only 10.5% in HIV-positives. However, 7.1% of HIV-negative Ethiopian Israelis were sCCR5 positive, as were 5.6% HIV-positives. Plasma concentrations of MIP-1β, the CCR5 agonist, were twice as high in sCCR5-positives (140.8 ± 25.8pg/ml) as in the sCCR5-negatives (77.6 ± 11.0pg/ml, P = 0.0157). A significant positive correlation between plasma levels of sCCR5 and MIP-1β was found (Fig. 4, r = 0.8, P < 0.0001). [Copyright &y& Elsevier]

Abstract: Dendritic cells (DC) are currently employed as cellular vaccines in clinical trials of tumor immunotherapy. In most trials, peptide epitopes derived from tumor antigens are being exogenously loaded onto human DC for binding to MHC class I molecules. While this is a convenient method, it suffers from the drawback that the persistence of class I/peptide complexes on the cell surface is in the order of a few hours. This drawback limits the success of vaccination. We have investigated biodegradable poly(d,l-lactide-co-glycolide) microspheres (PLGA-MS) as delivery tools for antigen loading of human monocyte-derived DC (hMoDC). Immature hMoDC readily take up PLGA-MS and present epitopes from encapsulated proteins or peptides both on MHC class I and class II. Interestingly, antigen presentation by hMoDC was markedly prolonged when hMoDC were charged with PLGA-MS-encapsulated as opposed to soluble antigens. The properties of hMoDC with respect to migration, cytokine secretion, survival and allostimulation were not adversely affected by the uptake of PLGA-MS. In this article, we will review the properties of PLGA-MS as an adjuvant and summarize recent data on their potential for antigen delivery to dendritic cells. [Copyright &y& Elsevier]

Abstract: The well known gender-related differences in drug action may partly be explained by changes in activity and expression of drug metabolising enzymes, but also by modulation of active drug transport systems (e.g. P-glycoprotein, Pgp) by sexual steroids, which is yet not well investigated. Because many women are using hormones (e.g. as oral contraceptives) we investigated the influence of different synthetic progestins on Pgp activity. Pgp inhibition of progesterone, medroxyprogesterone, chlormadinone, cyproterone, levonorgestrel, norethisterone, desogestrel, and norgestimate was measured in vitro in two Pgp over-expressing cell lines (L-MDR1, P388/dx cells) and the corresponding parental cell lines by means of calcein assay, and ex vivo in human peripheral blood mononuclear cells (PBMCs) by rhodamine123 efflux. For most progestins tested, concentrations needed to double baseline fluorescence (f2) in L-MDR1 cells were similar to that of the potent Pgp inhibitor quinidine, whereas levonorgestrel and norethisterone did not reach f2. The results in P388/dx cells essentially confirmed our findings in L-MDR1 cells. Additionally, Pgp inhibitory activity of all progestins tested was also shown ex vivo in PBMCs. The potent Pgp inhibition by several synthetic progestins in vitro and ex vivo suggests that such an interaction might be clinically relevant despite generally low plasma concentrations of progestins. The results may be of particular importance for Pgp substrates, such as protease inhibitors and chemotherapeutic agents, for which intracellular concentrations are critical. [Copyright &y& Elsevier]

Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-γ response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-α was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-γ response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-α expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-α or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-γ response to the PRRSV vaccine could be achieved by substituting polyinosinic–polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines. [Copyright &y& Elsevier]

Abstract: Calcitonin inhibits bone resorption by acting on osteoclasts via a specific receptor. The calcitonin receptor (CTR) is also found in many other normal and malignant tissues and cell lines. It has been cloned and sequenced in several species including humans. It belongs to a subclass of seven-transmembrane G protein-coupled receptors. Four human CTR (H-CTR) isoforms generated by alternatively spliced mRNA have previously been described. Two H-CTR encoding DNAs containing an unidentified 50-bp insert are now reported from T47D cells. The 50-bp insert corresponds to a DNA region located between exon 9 and exon 10, and appears to originate from an alternative splicing process. The two H-CTR cDNAs encode 274 and 290 aa long isoforms. Both are deleted from the putative fourth transmembrane domain to C-tail. They differ by the presence (H-CTR5) or absence (H-CTR6) of a previously known 16-aa insert in the putative first intracellular loop. Cell- and tissue-distribution analysis using RT-PCR demonstrates that the shorter one, HCTR6, is more prevalent. The mRNA of both isoforms was detected in giant cell tumor, whereas only H-CTR6 mRNA was detected in TT cells and kidney tissue. Neither H-CTR5 nor H-CTR6 could be detected in peripheral blood mononuclear cells cultured in the presence of RANKL, in MCF7 cells, and in cortical brain and ovarian tissues. When H-CTR6 was transiently expressed in HEK293 cells, CT failed to induce production of cAMP or to bind to the receptor. These suggest either an intrinsic loss of ligand binding function, or an altered intracellular trafficking. Our findings therefore indicate the existence of two novel splice variants of the H-CTR and confirm that multiple splicing patterns could be involved in the post-transcriptional regulation of the gene. [Copyright &y& Elsevier]

Abstract: The activation of human Vα24+Vβ11+natural killer T cells (NKT) cells (Vα24 NKT cells) induces effective antitumor responses with secondary immune effects through activation of conventional T cells and natural killer cells. In this study, we attempted to analyze the characteristics of human NKT cells in lung cancer patients. Vα24 NKT cells stimulated with α-GalCer from healthy volunteers exhibited direct cytotoxic activity against two (RERF-LC-OK and PC-3) of seven human lung cancer cell lines studied. Cytotoxicity by Vα24 NKT cells against human lung cancer cells was dependent on the perforin pathway and independent of Fas/FasL pathway. Intracellular adhesion molecule (ICAM)-1 expression on tumor cells was clearly associated with the cytotoxicity of Vα24 NKT cells. On the other hand, the proportion of Vα24 NKT cells in the patients with lung cancer was lower than that in the healthy volunteers. Furthermore, the proliferative response of Vα24 NKT cells to α-GalCer was significantly lower in the peripheral blood mononuclear cells in the patients with lung cancer. Addition of granulocyte colony-stimulating factor moderately restored the low proliferative response of Vα24 NKT cells in the patients with lung cancer, however the percentage by which the response was restored in these patients was still lower than the natural response in healthy volunteers. These results suggest that Vα24 NKT cells may play a pivotal role or the antitumor response in lung cancer. [Copyright &y& Elsevier]

Recombinant bovine variant of staphylococcal enterotoxin C (SECbovine), produced as a NH2-terminal histidine hexamer fusion protein (His6-tagged SECbovine), expressed at high levels (25%) in Escherichia coli and affinity purified to homogeneity (99.9%), was tested for its diagnostic and therapeutic potentials. His6-tagged SECbovine is antigenically authentic to native SECbovine across host species, as confirmed by antibody-based capture detection assays using human, mouse, rabbit and chicken hyperimmune sera. His6-tagged SECbovine showed significant T-cell stimulation activity in vitro. His6-tagged SECbovine was immunogenic for IgG in mice (intragastric and intravenous routes) and rabbits (intramuscular and subcutaneous routes), dispensing immunoadjuvant coadministration. The formation of neutralizing antibodies reduced the severity of intoxication symptoms in immunized rabbits. Purified anti-recombinant SECbovine rabbit polyclonal IgG neutralized the pyrexic and diarrhoeagenic effects of native SEC/SED and recombinant SEC, tested by the kitten and rabbit bioassays, respectively. [Copyright &y& Elsevier]

Thalidomide has shown to inhibit, selectively and mainly the cytokine tumor necrosis factor-α (TNF-α), thus, thalidomide has inhibitory consequences on other cytokines; this is ascribed as an immunomodulatory effect. Novel thalidomide analogs are reported with immunomodulatory activity. The aim of this work was to synthesize some of these analogs and to assess them as immunomodulatory agents in an acute model of LPS-induced septic challenge in rat. Animal groups received orally twice a day vehicle carboxymethylcellulose (0.9%), or thalidomide in suspension (100mg/kg), or analogs in an equimolar dose. Two hours after last dose, rats were injected with saline (NaCl, 0.9%, i.p.) or LPS (5mg/kg, i.p.). Groups were sacrificed 2h after injection and samples of blood and liver were obtained. TNF-α, interleukin-6, -1β, and -10 (IL-6, IL-1β, IL-10) were quantified by enzyme linked immunosorbent assay (ELISA) and studied in plasma and liver. After 2h of LPS-induction, different patterns of measured cytokines were observed with thalidomide analogs administration evidencing their immunomodulatory effects. Interestingly, some analogs decreased significantly plasma and hepatic levels of LPS-induced proinflammatory TNF-α and others increased plasma concentration of anti-inflammatory IL-10. Thalidomide analogs also showed slight effects on the remaining proinflammatory cytokines. Differences among immunomodulatory effects of analogs can be related to potency, mechanism of action, and half lives. Thalidomide analogs could be used as a pharmacological tool and in therapeutics in the future. [Copyright &y& Elsevier]

Inflammation and tissue damage in systemic lupus erythematosus (SLE) are mediated by class-switched autoantibodies reactive with nucleic acids, nucleic acid-binding proteins, phospholipids and other self-antigens. While some healthy individuals produce IgM antibodies with specificities similar to those of lupus patients, immunoglobulin class switching to mature downstream isotypes appears to be required for the generation of pathogenic autoantibodies. To characterize the cellular and molecular basis of pathogenic autoantibody production in SLE, we studied the capacity of peripheral blood B cells of naïve phenotype from patients with SLE, rheumatoid arthritis (RA) or healthy control subjects to spontaneously switch to IgG and IgA. In addition, we determined the DNA sequences of the upstream evolutionary conserved sequence (ECS)-Iγ promoter regulatory regions that control germline I H -C H transcription and class switch DNA recombination (CSR) to IgG 1 , IgG 2 and IgG 4 . IgM + IgD + B cells from patients with SLE, but not those from RA or healthy control subjects, underwent spontaneous CSR, as assessed by expression of germline Iγ1-Cγ1, Iγ2-Cγ2, Iγ3-Cγ3, Iγ4-Cγ4 and Iα1-Cα1 transcripts, mature (switched) V H DJ H -Cγ1, V H DJ H -Cγ2, V H DJ H -Cγ3 and V H DJ H -Cα1 transcripts and secreted IgG and IgA. Although polymorphic DNA sequences were identified in the ECS-Iγ1, ECS-Iγ2 and ECS-Iγ4 promoter regions, the transcription factor-binding sites that mediate germline Iγ-Cγ transcription were conserved in patients and controls. However, distinct patterns of nuclear protein binding to an ECS-Iγ promoter sequence that contains both positive and negative regulatory elements were observed in SLE patients and controls. These results support a role for exogenous signals, such as through CD40 ligation, rather than altered genomic sequence, in the increased production of class switched autoantibodies in SLE. [ABSTRACT FROM AUTHOR]

In this study, a comprehensive comparative analysis of different evaluation methods of Elispot plates was performed. Three investigators using three different evaluation approaches read 50 randomly selected wells at three different time points. The methods were: (1) manual evaluation using a stereomicroscope, (2) automated evaluation using an image analysis reader system with reading parameters established by each investigator, and (3) automated evaluation using a reader system with preset reading parameters using assay-specific controls. We demonstrate that manual evaluation had the highest variability both within the same method and when comparing all methods, followed by automated evaluation with investigator-dependent parameters. The variability was low only when all investigators used the same parameters established using assay-specific controls. This variability was independent of operator or spot number per well. Based on this study, recommendations for standardization and validation procedures of Elispot assay performance and evaluation procedures are presented. [Copyright &y& Elsevier]

Although a role for monocytes and monocyte-derived dendritic cells (DC) in the activation of T cells is well established, it is less clear to what extent DC and their precursors, monocytes, regulate B cell immune responses. Here we show that regulatory mechanisms similar to those in humans are in place in the bovine immune system. In vitro culture of bovine monocytes with bovine B cells activated by the anti-CD3 triggered CD4+ T cells or through immunoglobulin (Ig) receptor crosslinking induces B cell Ig secretion. Unlike bovine monocyte-derived DC, monocytes do not promote Ig class switching to IgG and IgA in activated peripheral blood B cells. These results suggest that bovine monocytes are capable of directly inducing Ig secretion in activated bovine peripheral blood B cells, but do not provide the signals for B cell Ig class switching. [Copyright &y& Elsevier]

The ontogeny of the human immune system was studied by analyzing fetal and adult tissues for the presence of various lymphocyte populations and activation/maturation markers. CD95 (fas) was expressed in hematopoietic tissues during the final stages of development of monocytes, granulocytes, NK cells and T cells, but to a much lesser extent on B cells. In the periphery, CD95 expression declined on granulocytes and NK cells. CD95 was expressed at a higher level on CD45RA+ peripheral T-cells in the fetus than in the adult. Contrary to the belief that most fetal T-cells are naı¨ve or resting, a notable number of CD45RO+ T-cells were observed as well as an unique CD95−CD45RO+ population. Activation markers CD25, CD122, CD69 and CD80 were also present on fetal T-cells. These findings indicate that in the initial weeks following thymic maturation, a high frequency of T-cells is activated in the periphery of the fetus. [Copyright &y& Elsevier]

Mammalian pregnancy bears many similarities to transplantation, since the fetus is semi-allogenic to mother. Thus, mammals have developed numerous mechanisms to protect the developing fetus from maternal immunologic recognition and attack. We have previously shown that human choriocarcinoma JAR cells, which resemble first trimester trophoblasts, regulate several important mRNAs in activated peripheral blood mononuclear cells (PBMC). We now provide further evidence that communication between maternal and fetal tissues is bi-directional, and that activation of PBMC leads to activation of specific signaling pathways in JAR cells. Activated PBMC were co-cultured with JAR cells for specific time intervals, after which JAR cells were lysed and subjected to western blotting for activated forms of the JNK, Erk 1-2, and p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Erk 1-2, but not JNK or p38, was induced in co-cultures of PBMC and JAR cells. These results were also obtained when JAR cells were incubated with conditioned medium from activated, but not resting, PBMC. Results were confirmed using specific MAPK reporter constructs, using luciferase activity as a measure of Elk-1 phosphorylation. Erk 1-2 phosphorylation was not required for JAR cells to inhibit IL-2 production in activated PBMC. Addition of the specific MAPK inhibitor UO126 to JAR cells prior to the addition of activated PBMC to the cultures did not abolish the capacity of JAR cells to inhibit IL-2 mRNA expression in PBMC. We conclude that there is likely to be significant bi-directional signaling between leukocytes and trophoblasts at the maternal–fetal interface. We propose the existence of a delicate maternal–fetal immunologic homeostasis based on these experimental results. [Copyright &y& Elsevier]

We searched for genes with expressions specific to human monocyte-derived dendritic cells (DCs) using differential display reverse transcription-polymerase chain reaction, and found that N-myc downstream regulated gene 2 (NDRG2), a member of a new family of differentiation-related genes, was expressed in DCs. While DCs derived from CD34+ progenitor cells also showed strong NDRG2 expression, the corresponding mRNA expression was absent in other cell lines including monocytes, B cells, and NK cells. The inhibition of DC differentiation by dexamethasone or vitamin D3 treatment down-regulated the expression of the NDRG2 gene in DCs. In addition, gene expression was induced in a myelomonocytic leukemia cell line, which is capable of differentiating into DCs in cytokine-conditioned culture. The level of NDRG2 gene expression in DCs was significantly higher than that of other members of the NDRG gene family. Finally, in contrast to the stable NDRG2 expression in CD40-stimulated DCs, the induction of DC maturation by lipopolysaccharide (LPS) resulted in the down-regulation of NDRG2 gene expression. This down-regulation is likely to be due to a modification and subsequent destabilization of NDRG2 mRNA, because co-treating with actinomycin D and LPS significantly blocked this LPS effect. Taken together, our results indicate that NDRG2 is expressed during the differentiation of DCs, and that NDRG2 gene expression is differentially regulated by maturation-inducing stimuli. [Copyright &y& Elsevier]

Background/Aims: Patients with recurrent hepatitis C virus (HCV)-infection after liver transplantation (OLTx) could develop an early, multispecific, preferentially intrahepatic CD4+ T cell response. We asked now whether there is a correlation between the HCV-specific CD4+ T cell response and treatment outcome in patients who receive interferon (IFN)-α/ribavirin.Methods: Liver- and blood-derived T cell lines of 20 patients were studied in parallel before, under, at the end and after antiviral treatment. Virus-specific IFN-γ production at a single cell level to HCV-proteins (core, non-structural protein (NS)3/4, NS5) was determined by enzyme-linked immunospot assay.Results: In 6/7 non-responders a weak HCV-specific CD4+ T cell response was detectable. All six sustained responders developed a strong, at NS3/4 and NS5 directed and long-lasting CD4+ T cell response which was mainly detected in peripheral blood mononuclear cells. This reaction was significantly stronger: (1) in the responders than in the non-responders; and (2) within the responders at the end of treatment than before (P<0.03). Seven transient-responders showed a weak and/or transient HCV-specific CD4+ T cell response.Conclusions: In patients with recurrent HCV-infection after OLTx, who receive antiviral treatment, a strong, at NS3/4 and NS5 directed and long-lasting CD4+ T cell response is associated with HCV-elimination whereas no or a weak/transient response is associated with treatment failure. [Copyright &y& Elsevier]

Women mount more vigorous antibody- and cell-mediated immune responses following either infection or vaccination than men. The incidence of most autoimmune diseases is also higher in women than in men; however, during pregnancy many autoimmune diseases go into remission, only to flare again in the early post-partum period. Successful pregnancy requires that the female immune system tolerate the presence of a semi-allogeneic graft for 9 months. Oral contraceptive use can increase susceptibility to certain genital tract infections and sexually transmitted diseases in women. Moreover, treatment of mice and rats with female sex hormones is required to establish animal models of genital tract Chlamydia, Neisseria and Mycoplasma infection. This review describes what is currently known about the effects of the female sex hormones oestradiol and progesterone on innate and adaptive immune responses in order to provide a framework for understanding these sex differences. Data from both human and animal studies will be reviewed. [Copyright &y& Elsevier]

Exposure to cadmium (Cd) can cause a variety of biological effects including alterations of immune responses in animals and humans. Both immunosuppression and immunoenhancement have been reported. The present study was aimed at investigating the consequences of exposure to Cd on the human immunoglobulin (Ig) E synthesis, using purified peripheral blood B lymphocytes and IL-4 and anti-human CD40 monoclonal antibody (a-CD40 mAb) as stimuli. Low concentrations of Cd (0.1–10 μM) markedly inhibited production of IgE in a concentration-dependent manner. IgG production, in contrast to IgE, showed a tendency towards being enhanced by Cd, although with a certain individual variability; IgM production was not affected. Cd failed to alter immediate surface expression of the activation markers CD69 and CD23 indicating that early activation events were not impaired. However, the portion of activated B cells was diminished by Cd after stimulation for more than 24 h, paralleled by a concomitant decrease in viability and a subsequent reduction in proliferation. These data suggest that the mechanism of Cd action on activated B cells involved pathways that interrupted an effectively initiated cell activation and induced a cytotoxic signal. Results from this study thus provide further evidence for and new information on the immunotoxic and immunomodulatory effects of Cd on human immune responses. [Copyright &y& Elsevier]

Objectives: Ischemia/reperfusion injury is characterized by endothelial cell activation leading to increased expression of adhesion molecules such as inter-cellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, endothelial- and platelet-selectin (E- and P-selectin), and to the subsequent recruitment of leukocytes. The aim of the present study was to investigate the respective effects of a proinflammatory cytokine (tumor necrosis factor alpha , TNF-α), hypoxia and/or reoxygenation on adhesion molecule expression and natural killer (NK) cell adhesion in an in vitro model of I/R. Methods: Human aortic endothelial cells (HAEC) were stimulated in vitro for 8h with TNF-α (1000 U/ml) and exposed to hypoxia (1% O2), reoxygenation (21% O2) or different combinations thereof. Cell surface expression of ICAM-1, VCAM-1 and E-/P-selectin on HAEC was analyzed by flow cytometry, and culture supernatants were tested for soluble adhesion molecules by ELISA. Rolling adhesion of NK cells on HAEC was determined using a rotating assay. Results: Untreated HAEC constitutively expressed ICAM-1 on their surface but neither expressed E-/P-selectin, VCAM-1, nor shedded soluble adhesion molecules. Exposure of HAEC to hypoxia or hypoxia and reoxygenation did not upregulate cell surface expression or shedding of adhesion molecules. In contrast, TNF-α significantly upregulated cell surface expression of ICAM-1, VCAM-1, and E-/P-selectin and led to the shedding of ICAM-1 and E-selectin. Combined treatment of HAEC with TNF-α, hypoxia and reoxygenation reduced E-/P-selectin surface expression and enhanced E-selectin shedding, but did not further influence ICAM-1 and VCAM-1. Soluble VCAM-1 was not detected. NK cell adhesion on HAEC increased 4-fold after TNF-α stimulation, but was not affected by hypoxia or hypoxia and reoxygenation. Conclusions: Both the expression of endothelial adhesion molecules and rolling NK cell adhesion was upregulated by TNF-α but not by hypoxia alone or hypoxia followed by reoxygenation supporting the view that anti-inflammatory treatment may reduce ischemia/reperfusion injury. [Copyright &y& Elsevier]

Background/Aims: Association of hepatitis C virus (HCV) with increased autoantibodies, mixed cryoglobulinaemia, non-Hodgkin''s B-cell lymphoma and increased peripheral innate (CD5pos) B cells suggests a role for B-lymphocytes in the pathogenesis of HCV-infection.Methods: Flow cytometry was used to estimate CD5pos B cell levels and CD81 co-expression in chronic HCV infection. Viral load was assessed using PCR.Results: We demonstrate expansion of innate B cells in HCV-infected liver from patients with fibrosis score less than stage II (39%, % of total B cells, P=0.002) and end stage HCV cirrhosis (20%, P<0.05) compared with normal liver (8%). Expression of CD81, a signal transducing molecule and putative HCV receptor, was significantly increased on peripheral blood CD5pos B cells compared with conventional B cells (P=0.0001). Higher levels of CD81 on CD5pos B cells were more dramatic in the liver of HCV-infected individuals. However, no significant difference was observed in the viral load of CD5posCD81High B cells and CD5negCD81Low B cells.Conclusions: Increased expression of CD81 on innate B cells, a population that is expanded in the livers and peripheral blood of chronic HCV-infected patients, suggests a role in viral specific activation and clonal proliferation in chronic HCV infection. [Copyright &y& Elsevier]

This review critically discusses data on immunology of colorectal cancer, starting from pathology and molecular biology, and then considering the molecular characterisation of colon cancer antigens and the clinical trials of immunotherapy. A careful evaluation of histopathological studies on intra-epithelial infiltration by T cells in primary tumours, together with the analysis of HLA expression by colorectal cancer cells, suggest that anti-tumour T cell immune responses may take place in vivo in those patients, influencing prognosis and shaping the tumour immunological profile. Moreover, the molecular characterisation of tumour antigens expressed by colorectal carcinomas, together with improved understanding of mechanisms of the immune response and more sensitive methods for the in vivo detection of T cell responses, are now allowing researchers to design new and more effective vaccination protocols, with encouraging preliminary results. By drawing together the experimental evidence from different research fields, this review provides support for the concept that colorectal carcinoma is immunogenic and may reasonably be considered as a target for immunotherapy, and attempts to address critical issues and envisage future developments in this challenging research field. [Copyright &y& Elsevier]

Increased levels of serum IgE have been described in gliadin-intolerant patients; however, biological mechanisms implicated in this immunoglobulin production remained unknown. In this study, we demonstrated that in vitro crude gliadins and gliadin lysates (Glilys) promoted the IL-4-induced IgE production by human peripheral blood mononuclear cells (PBMC), indicating that the biological process related to gliadin intolerance and/or allergy may lead to IgE production in vivo. It was found that crude gliadin and Glilys potentiated, after 13 days of culture in a dose-dependent manner, IL-4-induced IgE production and, to a lesser extent, the IgG production, while they did not affect IgA or IgM productions. This promoting effect of gliadin and Glilys on the IL-4-induced activation of normal human PBMC was also observed on the early release (2 days) of the soluble fraction of CD23, suggesting its possible involvement in IgE potentiation. The promoting effect of crude gliadin and Glilys appeared to be indirect because they did not modify purified B-lymphocytes IgE production after IL-4 and anti-CD40 monoclonal antibody stimulation.In addition, as revealed by luminol-dependent chemiluminescence, we demonstrated that crude gliadin and Glilys promoted a substantial production of free radicals by normal human PBMC, treated or not with IL-4. This redox imbalance associated with an increased IgE production led us to evaluate the effect of pharmacological antioxidants (N-acetyl-cysteine (NAC) and Cu/Zn-superoxide dismutase (SOD1)) on IgE production by human PBMC. The NAC and the intracellularly delivered SOD1 were found to suppress the IL-4±crude gliadin or Glilys-induced IgE production by normal human PBMC. Taken together, these data indicated that gliadin specifically enhanced IL-4-induced IgE production by normal human PBMC, probably by the regulation of redox pathways, and that this ‘pro-allergenic’ effect could be counteracted by natural antioxidants: thiols and/or vectorized SOD1. [Copyright &y& Elsevier]

The particular immune system of the camel has been but little investigated. In this work circulating camel peripheral blood mononuclear cells (PBMC) were studied by flow cytometry. Monoclonal antibodies (mAbs) raised against ruminant leukocytes were used for the detection of cell surface antigens.Monoclonals to T-cell markers, CD4 (CACT138A) and CD8 (CACT80C), exhibited no reactivity towards camel PBMC in contrast to their reactivity to PBMC of other ruminant species and those of cattle in particular. A relatively high percentage (29.1±8.9%) of camel PBMC reacted with a non-immunoglobulin cell surface marker, B-B2, comparable to the reactivity of bovine PBMC. The B-B7 cell marker revealed 22.4±10.0% of reactive camel PBMC while the CD45 leukocyte common antigen was identified only on 19.4±3.1% of camel PBMC as compared to 74.7±4.9% for bovine PBMC. IgM (PIg45A) was detected on 9.1±1.4% of camel PBMC and on 46.6±19.5% of the bovine PBMC. Double fluorescent labeling with two B-cell markers and an anti-ruminant λ light-chain mAb revealed 7–9% of cells bearing both B and λ L-chain markers. Light chain reactivity was also assessed using an anti-goat F(ab′)2 antiserum. The values obtained, 14.3±5.8% for the camel and 47.8±2.7% for the cattle, are close to the values observed for surface IgM. These data suggest that camels, like other ruminants, possess L-chain bearing cells of the B-cell lineage. However, in the camel, Igs are different in that in addition to regular four chain Igs, about 65% of them possess two heavy chain Igs devoid of light chains. Because different sets of VH gene segments are used by four and two chain Igs, it is possible that there might be two lineages of B-cells each secreting a different form of antibodies. [Copyright &y& Elsevier]

More than 10 splice variants of the Fc receptor for IgA (FcαR, CD89) have been identified in human myeloid cells. In this study, we quantified FcαR splice transcripts ΔEC2 and Δ66EC2, which lack the entire and a part of the homologous immunoglobulin-like extracellular domain 2 (EC2), respectively. Tumor necrosis factor-α was found to specifically increase the ratio of ΔEC2 to the wild type CD89 in neutrophils and conversely decrease the ΔEC2 ratio in monocytes. We also observed a significant decrease in the neutrophil ΔEC2/CD89 ratio in pneumonia patients. These results suggest that ΔEC2 is differentially regulated and could be involved in immunoregulation of IgA-mediated host defense. [Copyright &y& Elsevier]

Background: While in the past human cytomegalovirus (HCMV) represented the major viral opportunistic pathogen in patients with AIDS, incidence of HCMV disease in HIV-infected patients drastically dropped after introduction of highly active antiretroviral therapy (HAART). However, cases of HCMV disease in HIV-infected patients treated with HAART have been reported. Objective: A 38-year-old HIV-infected patient developed HCMV retinitis in May 1999 after reaching a nadir of 69 CD4+ T cells/μl. HAART and anti-HCMV treatments with parenteral ganciclovir (GCV) were started, resulting in HIV viremia suppression, rise in CD4+ T cell count to >300 cells/μl and recovery from retinitis. Notwithstanding the apparent immune reconstitution, every attempt to discontinue GCV maintenance treatment was followed by a relapse of retinal lesions. Thus, HCMV-specific CD4+ cellular immune response was investigated. Results: Lymphoproliferation assay and cytokine flow cytometry analysis were performed repeatedly from November 1999 showing absolute lack of HCMV specific CD4+ T cell response, in the presence of an efficient lymphoprolipherative response against another pathogen (Candida) or a mitogen (Phytohemoagglutinin). Conclusion: In some patients, immune reconstitution after HAART may be only partial, since lack of pathogen-specific CD4+ T cell response may persist even in the case of a significant rise in the absolute CD4+ T cell count. This case suggests that immunologic assays investigating specific immune response against HCMV in HIV infected patients may be more useful than the CD4+ T cell count alone in assessing immune function reconstitution after HAART and in deciding interruption of anti-HCMV secondary prophylaxis. [Copyright &y& Elsevier]

The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid-like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen-free conditions. The protein was enzymatically active in converting 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 μmol min−1 mg−1 at pH 7.5 and 55°C. The kcat value was 0.4 s−1 and the Km value for HMBPP 0.42 mM. [Copyright &y& Elsevier]

Status of tumor progression (either remaining in situ, or becoming invasive/metastatic) may be defined largely by subtle interactions (‘cross-talk’) in a microenvironment formed by interfacing tumor cell and host cell membrane domains (termed ‘glycosynapses’) involved in glycosylation-dependent cell adhesion and signaling. Functional roles of tumor-associated gangliosides, organized in glycosynapses of three types of tumor cell lines, are discussed. Gangliosides function as adhesion receptors or as ‘sensors’ that can be stimulated by antibodies, with consequent activation of signal transducers leading to enhanced motility and invasiveness. [Copyright &y& Elsevier]

Prenatal and early postnatal immune system development has been studied in minipigs. First leukocytes were observed in the yolk sac and fetal liver (FL) on the 17th day of gestation, the majority of them being SWC3+. The colonization of the thymus (TH) with leukocytes was observed 21 days later. Two waves of fetal TH colonization with pro-T cells were deduced from the frequency of thymocyte subsets. Thymic B cells and immunoglobulin-secreting cells (Ig-SC) were studied by flow cytometry and ELISPOT, respectively. When the total numbers of fetal Ig-SC were compared, the TH was identified as the main source of natural antibodies and the only site of IgA and IgG synthesis. In germ-free animals, the TH also represented the major site of IgG and IgA production and the number of Ig-SC was not influenced by colonization with microflora. FL and bone marrow were identified as primary B lymphopoietic sites. The phenotype of B precursors was characterized and pre-B II cells were shown to be the dominant mononuclear fraction between DG50 and DG105. In the periphery, relative proportions of lymphocyte subsets were determined. Studies in gnotobiotic piglets have revealed that the appearance of CD4+CD8+ T cells and CD2− B cells is absolutely dependent on the contact of immune system with live viruses and bacteria, respectively. [Copyright &y& Elsevier]

Monocytic adaptor (Mona) also known as Gads is a Grb2-related adaptor whose expression is restricted to hematopoietic cells. It plays an important role in intracellular signaling in T cells, monocytic cells, and platelets. Here we investigated the regulatory aspects of Mona expression in human hematopoietic cells. This was carried out by combining nucleotide sequence analyzes and experimental approaches. We confirmed that Mona expression is restricted to T-cell, myeloid and platelet lineages. In the various cells examined, we detected two major Mona transcripts (1.9 and 4 kb), likely resulting from the alternative use of two polyadenylation sites. Consequently, Mona transcripts of the same size have identical 3′ untranslated region (UTR), irrespective of the cell type. In contrast, Mona transcripts contain either 5′ UTR-1A or -1B exons, that were detected in a cell-lineage specific manner. Thus, T cells and several myeloid cell lines express 5′ UTR-1A-containing transcripts, whereas platelets and cell lines exhibiting megakaryocytic potential express 5′ UTR-1B-containing transcripts. Interestingly, 5′ UTR-1A is generated from an exon located approximately 45 kb upstream of exon 1B. This suggested that lineage-restricted transcription of the Mona gene is controlled by specific promoters. Indeed, 2-kb genomic fragments upstream of each 5′-UTR showed lineage-restricted ability to drive expression of luc reporter gene. [Copyright &y& Elsevier]

A recombinant bovine interleukin-12 (boIL-12) that contains a histidine hexamer, rboIL-12His, was produced, purified and administered to calves. We first tried the purification of heterodimer IL-12 from a mixture of p40 homodimer, p40 monomer, and p40–p35 heterodimer with a p35 subunit tagged with a histidine hexamar at its C-terminal (p35His). A recombinant baculovirus expressing p35His was generated and used for superinfection with a recombinant baculovirus expressing p40 subunit. The expressed subunits, p40 and p35His, were assembled into a 70 kDa heterodimer in insect cells, released into culture medium, and then purified using a nickel chelate column. The purified rboIL-12His was bioactive for induction of IFN-γ in bovine peripheral blood mononuclear cells (PBMCs) in vitro.The purified rboIL-12His was then administered to calves with inactivated Salmonella Typhimurium (ST). When sera were assayed by ELISA, specific anti-ST IgG1 antibodies were detected in all ST immunized calves, but, specific anti-ST IgG2 antibodies were detected only in calves administered ST along with rboIL-12His, indicating a possible switch to a Th1 response. Administration of commercially available Salmonella vaccine did not elicit IgG2 antibodies in calves. These results suggest that co-administration of IL-12 with inactivated ST cells could induce a Th1-type response in calves. [Copyright &y& Elsevier]

HLA class II molecules play pivotal roles in antigen presentation to CD4+ T cells. We investigated signaling via HLA-DR molecules expressed on CD4+ T cells. When HLA-DR or CD3 molecules on cloned CD4+ T cells were cross-linked by solid-phase mAbs, T cells proliferated, and this resulted in anergy. Whereas cross-linking of HLA-DR and CD3 resulted in secretion of the same levels of IFN-γ and IL-8, secretion of IL-10 induced by cross-linking of HLA-DR was less than that induced by cross-linking of CD3 on CD4+ T cells. Interestingly, expression of p27Kip1 but not p21Cip1 increased after stimulation by either anti-HLA-DR or anti-CD3 mAb. This was indeed the case, when T cells were rendered anergic using a soluble form of antigenic peptide. In contrast, T cells stimulated by peptide-pulsed PBMC expressed little p27Kip1. We propose that signaling via HLA-DR molecules on CD4+ T cells at least in part contributes to the induction of T cell anergy, through the upregulated expression of the p27Kip1. The implication of our finding is that HLA-DR molecules play a role in human T cell anergy induced by a soluble form of antigenic peptide. [Copyright &y& Elsevier]

Theileria annulata is a tick-transmitted protozoan that causes tropical theileriosis, an often fatal leukoproliferative disorder of cattle. To characterize and identify parasite proteins suitable as diagnostic antigens and/or vaccine candidates, a cDNA clone encoding a macroschizont stage protein was isolated and characterized (here designated TaSP). The gene, present as a single copy within the parasite genome, is transcribed in the sporozoite and schizont stage and codes for a protein of about 315 amino acids, having a predicted molecular weight of 36 kDa. Allelic variants were found within single parasite isolates and between isolates originating from different geographical regions. The N-terminal part contains a predicted signal peptide and the C-terminal section encodes membrane-spanning regions. Comparison of a number of cDNA clones showed that both these sequence regions are conserved while the central region shows both size and amino acid sequence polymorphism. High identity of the N- and C-terminal regions with the polymorphic immunodominant molecule (PIM) of Theileria parva (identity of 93%), the existence of a central polymorphic region and two short introns within genomic clones suggest that the presented gene/protein may be the T. annulata homologue of PIM. However, the central region of TaSP has no significant identity with PIM, contains no repetitive peptide motifs and is shorter, resulting in a lower molecular weight. The existence of the predicted secretion signal peptide and membrane spanning regions suggest that TaSP is located at the parasite membrane. [Copyright &y& Elsevier]

The anti-CD4 MAb 5A8 is a potent inhibitor of CD4-mediated infection of HIV-1. CD4 is obligatory for infection with primary HIV-1 isolates. Humanized 5A8 (hu5A8) was constructed to reduce the potential immunogenicity and enhance the in vivo half-life when used in humans. hu5A8 is a molecularly engineered human IgG4 antibody retaining the binding and functional properties of the murine version of 5A8 (mu5A8). This humanized MAb has been shown to be very effective in inhibiting HIV-1 infection of human CD4+ T cells and macrophages in vitro and to reduce viral load in rhesus monkeys chronically infected with simian immunodeficiency virus (SIV). 5A8 was evaluated in a good laboratory practice (GLP)-compliant tissue cross-reactivity study on human (three donors/37 tissues) and rhesus monkey (two donors/37 tissues) tissues. hu5A8 bound to the surface of human T cells and macrophages, but only to T cells from rhesus monkeys. The antibody did not cross react with other tissues. The highly identical staining patterns of hu5A8 in human and rhesus monkey tissues support the use of rhesus monkeys as a preclinical model for humans. In a GLP-compliant safety study in rhesus monkeys, weekly administration of hu5A8 at 5 mg/kg or 25 mg/kg for 8 weeks was shown to be safe and well tolerated in all monkeys. Although hu5A8 induced anti-hu5A8 antibody response in healthy rhesus monkeys, it was not immunogenic in chimpanzees. Together, the results from these preclinical studies support the studies of the anti-HIV-1 effect of hu5A8 in HIV-1 infected individuals. [Copyright &y& Elsevier]

STAT5A and STAT5B genes belong to the signal transducer and activators of transcription (STAT) family of transcription factors. They show a high degree of sequence homology at levels of mRNA, however, in spite of their supposed redundancy, each STAT5 has distinct biological functions mainly related to the immune system, hematopoiesis, growth and mammary development. We isolated and sequenced both STAT5A and STAT5B encoding human genes finding that they are segmented in 20 and 19 exons, respectively, of comparable size except for the extreme 5′ exons and the 3′ exons. Two CpG islands, 23.2% CpG for STAT5A and 30.2% for STAT5B, are present at the 5′ of both STAT5 genes covering the 5′ untranslated regions. More surprisingly, the two genes share two major regions of almost identical sequence which diverge between the different species indicating an intra-species specific mechanism of preservation. Furthermore, we identified two alternative 5′ exons in STAT5B genes and thus two alternative promoters. The second putative promoter is not embedded in a CpG island and it shows a tissue specific pattern of expression. Finally, the STAT5B gene was assessed as a candidate gene in a human disorder related to growth failure. [Copyright &y& Elsevier]

9 kDa granulysin is a protein present in the granules of human CTL and NK cells, with cytolytic activity against microbes and tumors. Previous work from our group demonstrated that this granulysin isoform induced apoptosisin vitroon hematological tumor cells and on primary tumor cells from B-CLL patients. In the present work, recombinant 9 kDa granulysin was used as an anti-tumoral agent to study itsin vivoeffect on tumor development in athymic “nude” mice models bearing human breast adenocarcinoma MDA-MB-231 or multiple myeloma NCI-H929–derived xenografts. Granulysin prevented thein vivodevelopment of detectable MDA-MB-231-derived tumors. In addition, recombinant granulysin was able to completely eradicate NCI-H929-derived tumors. All granulysin-treated tumors exhibited signs of apoptosis induction and an increased NK cell infiltration inside the tumor tissue comparing to control ones. Moreover, noin vivodeleterious effects of the recombinant 9 kDa granulysin doses used in this study were observed on the skin or on the internal organs of the animals. In conclusion, granulysin was able to inhibit the progression of MDA-MB-231-derived xenografts and also to eradicate multiple myeloma NCI-H929-derived xenografts. This work opens the door to the initiation of preclinical and possibly clinical studies for the use of 9 kDa granulysin as a new anti-tumoral treatment. [ABSTRACT FROM AUTHOR]

Autologous dendritic cell (DC) therapy is an experimental cellular immunotherapy that is safe and immunogenic in patients with advanced melanoma. In an attempt to further improve the therapeutic responses, we treated 15 patients with melanoma, with autologous monocyte-derived immature DC electroporated with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively active TLR4 (caTLR4) together with mRNA encoding a tumor-associated antigen (TAA; respectively gp100 or tyrosinase). In addition, DC were pulsed with keyhole limpet hemocyanin (KLH) that served as a control antigen. Production of this DC vaccine with high cellular viability, high expression of co-stimulatory molecules and MHC class I and II and production of IL-12p70, was feasible in all patients. A vaccination cycle consisting of three vaccinations with up to 15×106DC per vaccination at a biweekly interval, was repeated after 6 and 12 months in the absence of disease progression. mRNA-optimized DC were injected intranodally, because of low CCR7 expression on the DC, and induced de novo immune responses against control antigen. T cell responses against tyrosinase were detected in the skin-test infiltrating lymphocytes (SKIL) of two patients. One mixed tumor response and two durable tumor stabilizations were observed among 8 patients with evaluable disease at baseline. In conclusion, autologous mRNA-optimized DC can be safely administered intranodally to patients with metastatic melanoma but showed limited immunological responses against tyrosinase and gp100. [ABSTRACT FROM PUBLISHER]

Chronic pancreatitis (CP) is a risk factor of pancreatic ductal adenocarcinoma (PDAC) and characterized by a pronounced desmoplastic reaction with CD4+T cells accounting for the majority of the stromal T cell infiltrate. Epithelial-mesenchymal-transition (EMT) is a critical process for metastasis by which epithelial/carcinoma cells become enabled to disseminate probably prior to tumor formation. To investigate whether CD4+T cells induce EMT in human pancreatic ductal epithelial cells, premalignant H6c7 cells were mono- or co-cultured with human CD4+CD25+CD127−CD49d−regulatory T cells (T-regs) or CD4+CD25−T-effector cells (T-effs) being isolated by negative magnetic bead separation from blood of healthy donors. Particularly in the presence of activated T-effs, H6c7 cells acquired a spindle-shaped morphology, reduced E-cadherin expression, and elevated expression of the mesenchymal proteins vimentin, L1CAM, and ZEB-1. This was accompanied by an increased invasive behavior. Moreover, activated T-effs exerted similar effects in the PDAC cell line T3M4. Blocking of TNF-α and IL-6 being released at greater amounts into supernatants during co-cultures with activated T-effs attenuated the EMT-associated alterations in H6c7 cells. Supporting these findings, EMT-associated alterations (exemplified by reduced E-cadherin expression and enhanced expression of vimentin and L1CAM) were predominantly detected in ductal epithelium of CP tissues surrounded by a dense stroma enriched with CD4+T cells. Overall this study points to a novel role of CD4+T cells beyond their immune function in pancreatic tumorigenesis and underscores the view that EMT induction in pancreatic ductal epithelial cells represents an early event in PDAC development being essentially promoted by inflammatory processes. [ABSTRACT FROM PUBLISHER]

Indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO) represent some of the key immune regulators. Their increased activity has been demonstrated in a number of human malignancies but not yet in chronic myeloid leukemia (CML). In the present study, the activity of these enzymes was tested in 29 CML patients and 28 healthy subjects by monitoring the kynurenine (KYN)/tryptophan ratio. Serum samples taken prior to the therapy displayed a highly significant difference in KYN levels between the patient and control groups. However, increased KYN levels were detected in only 13 (44.8%) of these CML patients. The KYN levels in pretreatment sera of the patients correlated with the tumor burden. There was also a strong correlation between KYN levels and uric acid levels (UA). This suggests but does not prove the possible involvement of UA in activating IDO family of enzymes. Whenever tested, the increased KYN levels normalized in the course of the therapy. Patients with normal KYN levels in their pretreatment sera and subsequently treated with interferon-α, showed a transitory increase in their KYN levels. The present data indicate that CML should be added to the malignancies with an increased activity of the IDO family of enzymes and suggest that IDO inhibitors may be used in the treatment of CML patients. [ABSTRACT FROM PUBLISHER]

In melanoma, both the induction of immunosuppression by tumor cells and the inflammatory antitumor response can induce an upregulation of counter-regulatory mechanisms such as indoleamine 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1) and CTLA-4+regulatory T-cells (Tregs) in the tumor microenvironment. Even though these immunosuppressive mediators are targets for immunotherapy, research investigating their expression in the peripheral blood is lacking. We therefore, performed flow cytometry on PBMCs of stage I–IV melanoma patients. IDO expression was detected in plasmacytoid dendritic cells (pDC) and monocytic myeloid-derived suppressor cells (mMDSC), and increased in advanced disease stage (p= 0.027). Tryptophan breakdown confirmed the functional activity of IDO and was linked with increased PD-L1+ cytotoxic T-cells (p= 0.009), relative lymphopenia (p= 0.036), and a higher mDC/pDC ratio (p= 0.002). High levels of circulating PD-L1+ cytotoxic T-cells were associated with increasedCTLA-4expression by Tregs (p= 0.005) and MDSC levels (p= 0.033). This illustrates that counter-regulatory immune mechanisms in melanoma should be considered as one interrelated signaling network. Moreover, both increased PD-L1+ T-cells andCTLA-4expression in Tregs conferred a negative prognosis, indicating theirin vivorelevance. Remarkably, circulatingCTLA-4, IDO, and pDC levels were altered according to prior invasion of the sentinel lymph node and IDO expression in the sentinel was associated with more IDO+ PBMCs. We conclude that the expression of IDO, PD-L1, andCTLA-4in the peripheral blood of melanoma patients is strongly interconnected, associated with advanced disease and negative outcome, independent of disease stage. Combination treatments targeting several of these markers are therefore likely to exert a synergistic response. [ABSTRACT FROM PUBLISHER]

Both IL-17 and Th17 cells have been ascribed tumor promoting as well as tumor suppressing functions. We reviewed the literature on correlations between IL-17 versus Th17 cells and survival in human cancer, following the PRISMA guidelines. Serum, formalin-fixed, paraffin-embedded (FFPE) tissue and peripheral blood samples were most frequently studied. High IL-17 quantities were correlated with poor prognosis, whereas high Th17 cell frequencies were correlated with improved prognosis. Since Th17 cells are a subpopulation of IL-17+cells and had a different correlation with prognosis than total IL-17, we substantiate that a distinction should be made between Th17 and other IL-17+cells. [ABSTRACT FROM PUBLISHER]

Background.Glioblastoma (GBM) is the most common malignant brain tumor in adults and is nearly always fatal. Emerging evidence suggests that humanCytomegalovirus(HCMV) is present in 90–100% of GBMs and that add-on antiviral treatment for HCMV show promise to improve survival. Methods.In a randomized, placebo-controlled trial of valganciclovir in 42 GBM patients, blood samples were collected for analyses of HCMV DNA, RNA, reactivity against HCMV peptides, IgG, and IgM at baseline and at 3, 12, and 24 weeks of treatment. Results.All 42 tumors were positive for HCMV protein. All patients examined had at least one blood sample positive for HCMV DNA, 63% were HCMV RNA positive, and 21% were IgM positive. However, 29% of GBM patients were IgG negative for HCMV. Five of these samples were positive in an enzyme-linked immunosorbent assay (ELISA) that used antigens derived from a clinical isolate. Blood T cells from 11 of 13 (85%) HCMV IgG-negative GBM patients reacted against HCMV peptides. Valganciclovir did not affect IgG titers, DNA, or RNA levels of theHCMV immediate early (HCMV IE)gene in blood. Conclusion.In GBM patients, HCMV activity is higher than in healthy controls and serology is a poor test to define previous or active HCMV infection in these patients. [ABSTRACT FROM PUBLISHER]

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme. Remarkably, we discovered IDO-specific T cells that can influence adaptive immune reactions in patients with cancer. Further, a recent phase I clinical trial demonstrated long-lasting disease stabilization without toxicity in patients with non-small-cell lung cancer (NSCLC) who were vaccinated with an IDO-derived HLA-A2-restricted epitope. [ABSTRACT FROM PUBLISHER]

CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset involved in the induction of antitumor immune responses. Here, we provide a view on the recent observation that Vγ9Vδ2-T cells, through trogocytosis of CD1d-containing membrane fragments, have the capacity to act as antigen presenting cells for iNKT. [ABSTRACT FROM AUTHOR]

Tumor-associated stromal myofibroblasts are essential for the progression and metastatic spread of solid tumors. Corresponding myeloid cell infiltration into primary tumors is a negative prognostic factor in some malignancies. The aim of this study was to define the exact role of stromal myofibroblasts and stromal factors in early prostate carcinoma (PCa) regulating monocyte infiltration and differentiation into dendritic cells (DCs). Epithelial and stromal primary cultures were generated from PCa biopsies and their purity confirmed. Stromal cells produced significantly more of the (C‒C) motif chemokine ligand 2 (CCL2), interleukin 6 (IL-6) and transforming growth factor β (TGFβ) than epithelial cells. Monocyte chemoattraction was predominantly due to stromal-derived factors, mainly CCL2. DCs generated in the presence of stromal (but not epithelial) factors upregulated CD209, but failed to downregulate the monocyte marker CD14 in a signal transducer and activator of transcription 3 (STAT3)-dependent manner. Monocytes exposed to stromal factors did not produce detectable amounts of IL-10, however, upon lipopolysaccharide stimulation, stromal factor generated dendritic cells (sDC) produced significantly more IL-10 and less IL-12 than their conventional DC counterparts. sDC failed to cross-present tumor-antigen to CD8+T cells and suppressed T-cell proliferation. Most importantly, sDC expressed significantly elevated levels of programmed cell death ligand-1 (PD-L1) in a primarily STAT3 and IL-6-dependent manner. In parallel with our findingsin vitro, tumor-infiltrating CD14+cellsin situwere found to express both PD-L1 and CD209, and a higher percentage of tumor-associated CD3+T cells expressed programmed cell death-1 (PD-1) molecules compared to T cells in blood. These results demonstrate a hitherto undescribed, fundamental contribution of tumor-associated stromal myofibroblasts to the development of an immunosuppressive microenvironment in early PCa. [ABSTRACT FROM AUTHOR]