Prophylactic vaccines are potent activators of monocyte-derived dendritic cells and drive effective anti-tumor responses in melanoma patients at the cost of toxicity.

Dendritic cell (DC)-based immunotherapy is explored worldwide in cancer patients, predominantly with DC matured with pro-inflammatory cytokines and prostaglandin E2. We studied the safety and efficacy of vaccination with monocyte-derived DC matured with a cocktail of prophylactic vaccines that contain clinical-grade Toll-like receptor ligands (BCG, Typhim, Act-HIB) and prostaglandin E2 (VAC-DC). Stage III and IV melanoma patients were vaccinated via intranodal injection (12 patients) or combined intradermal/intravenous injection (16 patients) with VAC-DC loaded with keyhole limpet hemocyanin (KLH) and mRNA encoding tumor antigens gp100 and tyrosinase. Tumor antigen-specific T cell responses were monitored in blood and skin-test infiltrating-lymphocyte cultures. Almost all patients mounted prophylactic vaccine- or KLH-specific immune responses. Both after intranodal injection and after intradermal/intravenous injection, tumor antigen-specific immune responses were detected, which coincide with longer overall survival in stage IV melanoma patients. VAC-DC induce local and systemic CTC grade 2 and 3 toxicity, which is most likely caused by BCG in the maturation cocktail. The side effects were self-limiting or resolved upon a short period of systemic steroid therapy. We conclude that VAC-DC can induce functional tumor-specific responses. Unfortunately, toxicity observed after vaccination precludes the general application of VAC-DC, since in DC maturated with prophylactic vaccines BCG appears to be essential in the maturation cocktail.

Timeliness of lung cancer diagnosis and treatment in a rapid outpatient diagnostic program with combined 18FDG-PET and contrast enhanced CT scanning.

INTRODUCTION: Delays in the diagnosis of lung cancer are under debate and may affect outcome. The objectives of this study were to compare various delays in a rapid outpatient diagnostic program (RODP) for suspected lung cancer patients with those described in literature and with guideline recommendations, to investigate the effects of referral route and symptoms on delays, and to establish whether delays were related to disease stage and outcome. METHODS: A retrospective chart study was conducted of all patients with suspected lung cancer, referred to the RODP of our tertiary care university clinic between 1999 and 2009. Patient characteristics, tumor stage and different delays were analyzed. RESULTS: Medical charts of 565 patients were retrieved. 290 patients (51.3%) were diagnosed with lung cancer, 48 (8.5%) with another type of malignancy, and in 111 patients (19.6%) the radiological anomaly was diagnosed as non-malignant. In 112 (19.8%) no immediate definite diagnosis was obtained, however in 82 of these cases (73.2%) the proposed follow-up strategy confirmed a benign outcome. The median first line delay was 54 days, IQR (interquartile range) 20-104 days, median patient delay 19 days (IQR 4-52 days), median referral delay was 7 days (IQR 5-9 days), median diagnostic delay 2 days (IQR 1-19 days). In 87% a diagnosis was obtained within 3 weeks after visiting a chest physician and 52.5% started curative therapy within 2 weeks after diagnosis. Patients presenting with hemoptysis had shorter first line delays. The RODP care was generally far more timely compared to literature and published guidelines, except for both referral and palliative therapeutic delay. No specific delay was significantly related to disease stage or survival. CONCLUSIONS: An RODP results in a timely diagnosis well within guideline recommendations. Patient and first line delay account for most of total patient delay. Within the limitations of this retrospective study, we found no association with disease stage or survival.

STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.

BACKGROUND: Chronic mucocutaneous candidiasis (CMC) is characterized by susceptibility to candida infection of skin, nails, and mucous membranes. Patients with recessive CMC and autoimmunity have mutations in the autoimmune regulator AIRE. The cause of autosomal dominant CMC is unknown. METHODS: We evaluated 14 patients from five families with autosomal dominant CMC. We incubated their peripheral-blood mononuclear cells with different combinations of stimuli to test the integrity of pathways that mediate immunity, which led to the selection of 100 genes that were most likely to contain the genetic defect. We used an array-based sequence-capture assay, followed by next-generation sequencing, to identify mutations. RESULTS: The mononuclear cells from the affected patients were characterized by poor production of interferon-γ, interleukin-17, and interleukin-22, suggesting that the defect lay within the interleukin-12 receptor and interleukin-23 receptor signaling pathways. We identified heterozygous missense mutations in the DNA sequence encoding the coiled-coil (CC) domain of signal transducer and activator of transcription 1 (STAT1) in the patients. These mutations lead to defective responses in type 1 and type 17 helper T cells (Th1 and Th17). The interferon-γ receptor pathway was intact in these patients. CONCLUSIONS: Mutations in the CC domain of STAT1 underlie autosomal dominant CMC and lead to defective Th1 and Th17 responses, which may explain the increased susceptibility to fungal infection. (Funded by the Netherlands Organization for Scientific Research and others.).

Determining specificity of motor imagery training for upper limb improvement in chronic stroke patients: a training protocol and pilot results.

Motor imagery (MI) refers to the mental rehearsal of a movement without actual motor output. MI training has positive effects on upper limb recovery after stroke.However, until now it is unclear whether this effect is specific to the trained task or a more general motors kill improvement. This study was set up to advance our insights into the efficacy of MI training and the specificity of its effects. We investigated whether MI training affected the trained hand exclusively, or both hands. Four stroke participants received a 15-min MI training four times a week for 3 weeks. Hand function was measured before and after the training using three measurement of increasing complexity. Hand function improved after MI training, thus confirming the earlier studies. Second, we found specific effects of the MI training for two of the three measurements. These results suggest that MI specificity is dependent on the complexity of the hand function task.

Recognition of fungal pathogens by toll-like receptors.

Toll-like receptors (TLRs) have been identified as a major class of pattern-recognition receptors. Recognition of pathogen-associated molecular patterns (PAMPs) by TLRs, either alone or in heterodimerization with other TLR or non-TLR receptors, induces signals responsible for the activation of innate immune response. Recent studies have demonstrated a crucial involvement of TLRs in the recognition of fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. By studying fungal infection in knock-out mice deficient in either TLRs or TLR-associated adaptor molecules, it appeared that specific TLRs such as TLR2 and TLR4 play differential roles in the activation of the various arms of the innate immune response. Recent data also suggest that TLRs offer escape mechanisms to certain pathogenic microorganisms, especially through TLR2-driven induction of antiinflamatory cytokines. These recent developments provide crucial information for understanding the mechanisms of fungal recognition by cells of the immune system, and provide hope for designing new therapeutical approaches to fungal infections.

Toll-like receptor 4 Asp299Gly/Thr399Ile polymorphisms are a risk factor for Candida bloodstream infection.

Toll-like receptor (TLR)-4 is an important pattern recognition receptor for Candida albicans, playing a role in innate host defense. We investigated whether there is an association between the TLR4 Asp299Gly or TLR4 Thr399Ile polymorphism, and the occurrence of Candida bloodstream infection. We performed a case-control study, involving 43 patients with a Candida bloodstream infection and 166 healthy individuals. TLR4 Asp299Gly and Thr399Ile polymorphisms were assessed, as well as cytokine production after stimulation of peripheral blood mononuclear cells (PBMC) with Candida albicans. We observed that the prevalence of TLR4 Asp299Gly polymorphism was found to be higher in patients with Candida bloodstream infection than in controls (26% versus 10%; OR 3.0; 95%CI 1.3-6.9). All patients bearing the Asp299Gly polymorphism were also positive for the Thr399Ile allele, a linkage well described in literature. IL-10 production was higher in C. albicans-stimulated PBMC from volunteers bearing the TLR4 Asp299Gly polymorphism, and a similar tendency was observed in TLR4 Asp299Gly heterozygous patients who had recovered from candidemia. These findings show that the TLR4 Asp299Gly/Thr399Ile polymorphisms are associated with an increased susceptibility to Candida bloodstream infections, and an increased production of IL-10 is probably involved in this effect.

Nucleotide oligomerization domain 2 (Nod2) is not involved in the pattern recognition of Candida albicans.

Nucleotide-binding oligomerization domain 2 (Nod2) pathways are known to interact with Toll-like receptor 2 (TLR2) and TLR4, which are pattern recognition receptors for Candida albicans. We observed that the prevalence of Nod2 polymorphisms was not increased in patients with Candida infections. Candida-induced cytokine production in individuals with Nod2 polymorphisms was unaffected. We conclude that Nod2 is unlikely to play an important role in the recognition of Candida albicans.

Differential cytokine production and Toll-like receptor signaling pathways by Candida albicans blastoconidia and hyphae.

Toll-like receptors (TLR) are crucial for an efficient antifungal defense. We investigated the differential recognition of blastoconidia and hyphae of Candida albicans by TLRs. In contrast to Candida blastoconidia, which stimulated large amounts of gamma interferon (IFN-gamma), the tissue-invasive Candida hyphae did not stimulate any IFN-gamma by human peripheral blood mononuclear cells (PBMC) or murine splenic lymphocytes. After stimulation with blastoconidia, the production of IFN-gamma was TLR4 dependent, as shown by the significantly decreased IFN-gamma production in anti-TLR4-treated PBMC and in splenic lymphocytes from TLR4-defective ScCr mice. In addition, peritoneal macrophages from ScCr mice produced less tumor necrosis factor alpha (TNF-alpha) than macrophages of control mice did when stimulated with Candida blastoconidia, but not with hyphae, indicating that TLR4-mediated signals are lost during hyphal germination. In contrast, macrophages from TLR2 knockout mice had a decreased production of TNF-alpha in response to both Candida blastoconidia and hyphae. Candida hyphae stimulated production of interleukin-10 through TLR2-dependent mechanisms. In conclusion, TLR4 mediates proinflammatory cytokine induction after Candida stimulation, whereas Candida recognition by TLR2 leads mainly to anti-inflammatory cytokine release. TLR4-mediated proinflammatory signals are lost during germination of Candida blastoconidia into hyphae. Phenotypic switching during germination may be an important escape mechanism of C. albicans, resulting in counteracting host defense.

Functional consequences of the Asp299Gly Toll-like receptor-4 polymorphism.

Toll-like receptor-4 (TLR4) is a pattern-recognition receptor not only for exogenous ligands such as lipopolysaccharide (LPS) of Gram-negative bacteria, but also for endogenous ligands such as fibronectin, heat shock proteins and hyaluronan oligosaccharides. The Asp299Gly allele of the TLR4 gene has been associated with increased risk for severe infections, but reduced progression of atherosclerosis. We have investigated the consequences of the presence of Asp299Gly polymorphism after stimulation of mononuclear cells with lipopolysaccharide (LPS), the non-LPS TLR4 microbial stimuli Aspergillus fumigatus and Cryptococcus neoformans, and the endogenous TLR4 ligand heat shock protein 60. No differences in either production of the proinflammatory cytokine TNF or the antiinflammatory cytokine interleukin-10 were observed between volunteers with the wild-type allele, volunteers heterozygous for the Asp299Gly allele and one volunteer homozygous for the TLR4 variant. In conclusion, the presence of the Asp299Gly TLR4 polymorphism does not result in defective pro and antiinflammatory cytokine production after stimulation with either exogenous (LPS and non-LPS) or endogenous TLR4 ligands, and alternative explanations are likely to be responsible for the epidemiological data showing associations with inflammatory conditions. In addition, this is the first study to demonstrate that even homozygosity for the Asp299Gly mutation does not confer hyporesponsiveness to stimulation with TLR4 stimuli.

Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system.

Toll-like receptors (TLRs) have been identified as a major class of pattern-recognition receptors. Recognition of pathogen-associated molecular patterns (PAMPs) by TLRs, alone or in heterodimerization with other TLR or non-TLR receptors, induces signals responsible for the activation of genes important for an effective host defense, especially proinflammatory cytokines. Although a certain degree of redundancy exists between signals induced by the various TLRs, recent studies have identified intracellular pathways specific for individual TLRs. This leads to the release of cytokine profiles specific for particular PAMPs, and thus, TLRs confer a certain degree of specificity to the innate-immune response. In addition to the activation of the innate-immune response, TLR-mediated recognition represents a link between the innate- and acquired-immune systems, by inducing the maturation of dendritic cells and directing the T helper responses. Alternatively, recent data have also suggested TLR-mediated escape mechanisms used by certain pathogenic microorganisms, especially through TLR2 induction of anti-inflammatory cytokines. Finally, the crucial role of TLRs for the host defense against infections has been strengthened recently by the description of patients partially defective in the TLR-activation pathways.

Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells.

Toll-like receptor (TLR) 2 and TLR4 play a pivotal role in recognition of Candida albicans. We demonstrate that TLR2(-/-) mice are more resistant to disseminated Candida infection, and this is associated with increased chemotaxis and enhanced candidacidal capacity of TLR2(-/-) macrophages. Although production of the proinflammatory cytokines TNF, IL-1alpha, and IL-1beta is normal, IL-10 release is severely impaired in the TLR2(-/-) mice. This is accompanied by a 50% decrease in the CD4+CD25+ regulatory T (Treg) cell population in TLR2(-/-) mice. In vitro studies confirmed that enhanced survival of Treg cells was induced by TLR2 agonists. The deleterious role of Treg cells on the innate immune response during disseminated candidiasis was underscored by the improved resistance to this infection after depletion of Treg cells. In conclusion, C. albicans induces immunosuppression through TLR2-derived signals that mediate increased IL-10 production and survival of Treg cells. This represents a novel mechanism in the pathogenesis of fungal infections.

The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis.

Toll-like receptors (TLRs) represent the main class of pattern-recognition receptors involved in sensing pathogenic microorganisms. The aim of the present study was to assess the role of TLR4 in the defense against Candida albicans infection. The outgrowth of C. albicans was 10-fold higher in TLR4-defective C3H/HeJ mice, compared with that in control C3H/HeN mice (P<.05). Production of tumor necrosis factor (TNF) and interleukin (IL)-1alpha and IL-1beta by mouse macrophages in response to C. albicans stimulation was not affected by TLR4, and the candidacidal capacities of the neutrophils and macrophages of C3H/HeJ mice were normal. In contrast, production of the CXC chemokines KC and macrophage inhibitory protein-2 was 40%-60% lower by the macrophages of C3H/HeJ mice (P<.05), which resulted in a 40% decrease in neutrophil recruitment to the site of infection. Candida-induced TNF and IL-1beta production by human peripheral blood mononuclear cells was significantly inhibited by blocking anti-TLR2 antibodies in vitro. In conclusion, TLR4-defective C3H/HeJ mice are more susceptible to C. albicans infection, and this is associated with impaired chemokine expression and neutrophil recruitment.