Dendritic cells loaded with killed breast cancer cells induce differentiation of tumor-specific cytotoxic T lymphocytes

BACKGROUND: Early clinical trials, mostly in the setting of melanoma, have shown that dendritic cells (DCs) expressing tumor antigens induce some immune responses and some clinical responses. A major difficulty is the extension to other tumors, such as breast carcinoma, for which few defined tumor-associated antigens are available. We have demonstrated, using both prostate carcinoma and melanoma as model systems, that DCs loaded with killed allogeneic tumor cell lines can induce CD8(+ )T cells to differentiate into cytotoxic T lymphocytes (CTLs) specific for shared tumor antigens. METHODS: The present study was designed to determine whether DCs would capture killed breast cancer cells and present their antigens to autologous CD4(+ )and CD8(+ )T cells. RESULTS: We show that killed breast cancer cells are captured by immature DCs that, after induced maturation, can efficiently present MHC class I and class II peptides to CD8(+ )and CD4(+ )T lymphocytes. The elicited CTLs are able to kill the target cells without a need for pretreatment with interferon gamma. CTLs can be obtained by culturing the DCs loaded with killed breast cancer cells with unseparated peripheral blood lymphocytes, indicating that the DCs can overcome any potential inhibitory effects of breast cancer cells. CONCLUSION: Loading DCs with killed breast cancer cells may be considered a novel approach to breast cancer immunotherapy and to identification of shared breast cancer antigens

Associations of IL-4, IL-4R, and IL-13 Gene Polymorphisms in Coal Workers' Pneumoconiosis in China: A Case-Control Study

Background: The IL-4, IL-4 receptor (IL4R), and IL-13 genes are crucial immune factors and may influence the course of various diseases. In the present study, we investigated the association between the potential functional polymorphisms in IL-4, IL-4R, and IL-13 and coal workers ’ pneumoconiosis (CWP) risk in a Chinese population. Methods: Six polymorphisms (C-590T in IL-4, Ile50Val, Ser478Pro, and Gln551Arg in IL-4R, C-1055T and Arg130Gln in IL-13) were genotyped and analyzed in a case-control study of 556 CWP and 541 control subjects. Results: Our results revealed that the IL-4 CT/CC genotypes were associated with a significantly decreased risk of CWP (odd

Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages

BACKGROUND: Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. METHODS: Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. RESULTS: IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.CONCLUSION: AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response

Optimizing the diagnostic work-up of acute uncomplicated urinary tract infections

<p>Abstract</p> <p>Background</p> <p>Most diagnostic tests for acute uncomplicated urinary tract infections (UTIs) have been previously studied in so-called single-test evaluations. In practice, however, clinicians use more than one test in the diagnostic work-up. Since test results carry overlapping information, results from single-test studies may be confounded. The primary objective of the Amsterdam Cystitis/Urinary Tract Infection Study (ACUTIS) is to determine the (additional) diagnostic value of relevant tests from patient history and laboratory investigations, taking into account their mutual dependencies. Consequently, after suitable validation, an easy to use, multivariable diagnostic rule (clinical index) will be derived.</p> <p>Methods</p> <p>Women who contact their GP with painful and/or frequent micturition undergo a series of possibly relevant tests, consisting of patient history questions and laboratory investigations. Using urine culture as the reference standard, two multivariable models (diagnostic indices) will be generated: a model which assumes that patients attend the GP surgery and a model based on telephone contact only. Models will be made more robust using the bootstrap. Discrimination will be visualized in high resolution histograms of the posterior UTI probabilities and summarized as 5^th, 10^th, 25^th50^th, 75^th, 90^th, and 95^thcentiles of these, Brier score and the area under the receiver operating characteristics curve (ROC) with 95% confidence intervals. Using the regression coefficients of the independent diagnostic indicators, a diagnostic rule will be derived, consisting of an efficient set of tests and their diagnostic values.</p> <p>The course of the presenting complaints is studied using 7-day patient diaries. To learn more about the natural history of UTIs, patients will be offered the opportunity to postpone the use of antibiotics.</p> <p>Discussion</p> <p>We expect that our diagnostic rule will allow efficient diagnosis of UTIs, necessitating the collection of diagnostic indicators with proven added value. GPs may use the rule (preferably after suitable validation) to estimate UTI probabilities for women with different combinations of test results. Finally, in a subcohort, an attempt is made to identify which indicators (including antibiotic treatment) are useful to prognosticate recovery from painful and/or frequent micturition.</p

Keratinocytes Determine Th1 Immunity during Early Experimental Leishmaniasis

Experimental leishmaniasis is an excellent model system for analyzing Th1/Th2 differentiation. Resistance to Leishmania (L.) major depends on the development of a L. major specific Th1 response, while Th2 differentiation results in susceptibility. There is growing evidence that the microenvironment of the early affected tissue delivers the initial triggers for Th-cell differentiation. To analyze this we studied differential gene expression in infected skin of resistant and susceptible mice 16h after parasite inoculation. Employing microarray technology, bioinformatics, laser-microdissection and in-situ-hybridization we found that the epidermis was the major source of immunomodulatory mediators. This epidermal gene induction was significantly stronger in resistant mice especially for several genes known to promote Th1 differentiation (IL-12, IL-1β, osteopontin, IL-4) and for IL-6. Expression of these cytokines was temporally restricted to the crucial time of Th1/2 differentiation. Moreover, we revealed a stronger epidermal up-regulation of IL-6 in the epidermis of resistant mice. Accordingly, early local neutralization of IL-4 in resistant mice resulted in a Th2 switch and mice with a selective IL-6 deficiency in non-hematopoietic cells showed a Th2 switch and dramatic deterioration of disease. Thus, our data indicate for the first time that epidermal cytokine expression is a decisive factor in the generation of protective Th1 immunity and contributes to the outcome of infection with this important human pathogen

PGE2 Induces IL-6 in Orbital Fibroblasts through EP2 Receptors and Increased Gene Promoter Activity: Implications to Thyroid-Associated Ophthalmopathy

BACKGROUND: IL-6 plays an important role in the pathogenesis of Graves' disease and its orbital component, thyroid-associated ophthalmopathy (TAO). Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE(2), underlying the inflammatory phenotype of these cells. METHODOLOGY/PRINCIPAL FINDINGS: Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE(2) on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP(2) receptors and increased steady-state IL-6 mRNA levels. The pre-translational up-regulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE(2)-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE(2) provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP) preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE(2) promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts. CONCLUSION/SIGNIFICANCE: These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO

Avian Pathogenic Escherichia coli (APEC) Infection Alters Bone Marrow Transcriptome in Chickens

Avian pathogenic Escherichia coli (APEC) is a major cause of disease impacting animal health. The bone marrow is the reservoir of immature immune cells; however, it has not been examined to date for gene expression related to developmental changes (cell differentiation, maturation, programming) after APEC infection. Here, we study gene expression in the bone marrow between infected and non-infected animals, and between infected animals with mild (resistant) versus severe (susceptible) pathology, at two times post-infection. We sequenced 24 bone marrow RNA libraries generated from the six different treatment groups with four replicates each, and obtained an average of 22 million single-end, 100-bp reads per library. Genes were detected as differentially expressed (DE) between APEC treatments (mild pathology, severe pathology, and mock-challenged) at a given time point, or DE between 1 and 5 days post-infection (dpi) within the same treatment group. Results demonstrate that many immune cells, genes and related pathways are key contributors to the different responses to APEC infection between susceptible and resistant birds and between susceptible and non-challenged birds, at both times post-infection. In susceptible birds, lymphocyte differentiation, proliferation, and maturation were greatly impaired, while the innate and adaptive immune responses, including dendritic cells, monocytes and killer cell activity, TLR- and NOD-like receptor signaling, as well as T helper cells and many cytokine activities, were markedly enhanced. The resistant birds’ immune system, however, was similar to that of non-challenged birds. The DE genes in the immune cells and identified signaling models are representative of activation and resolution of infection in susceptible birds at both post-infection days. These novel results characterizing transcriptomic response to APEC infection reveal that there is combinatorial activity of multiple genes controlling myeloid cells, and B and T cell lymphopoiesis, as well as immune responses occurring in the bone marrow in these early stages of response to infection

Regulation of Inflammatory Gene Expression in PBMCs by Immunostimulatory Botanicals

Many hundreds of botanicals are used in complementary and alternative medicine for therapeutic use as antimicrobials and immune stimulators. While there exists many centuries of anecdotal evidence and few clinical studies on the activity and efficacy of these botanicals, limited scientific evidence exists on the ability of these botanicals to modulate the immune and inflammatory responses. Using botanogenomics (or herbogenomics), this study provides novel insight into inflammatory genes which are induced in peripheral blood mononuclear cells following treatment with immunomodulatory botanical extracts. These results may suggest putative genes involved in the physiological responses thought to occur following administration of these botanical extracts. Using extracts from immunostimulatory herbs (Astragalus membranaceus, Sambucus cerulea, Andrographis paniculata) and an immunosuppressive herb (Urtica dioica), the data presented supports previous cytokine studies on these herbs as well as identifying additional genes which may be involved in immune cell activation and migration and various inflammatory responses, including wound healing, angiogenesis, and blood pressure modulation. Additionally, we report the presence of lipopolysaccharide in medicinally prepared extracts of these herbs which is theorized to be a natural and active component of the immunostimulatory herbal extracts. The data presented provides a more extensive picture on how these herbs may be mediating their biological effects on the immune and inflammatory responses

Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R

Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove useful as an adjunctive treatment for tuberculosis