Hematopoietic prostaglandin D synthase defines a proeosinophilic pathogenic effector human T(H)2 cell subpopulation with enhanced function.

BACKGROUND: IL-5(+) pathogenic effector T(H)2 (peT(H)2) cells are a T(H)2 cell subpopulation with enhanced proinflammatory function that has largely been characterized in murine models of allergic inflammation. OBJECTIVE: We sought to identify phenotype markers for human peT(H)2 cells and characterize their function in patients with allergic eosinophilic inflammatory diseases. METHODS: Patients with eosinophilic gastrointestinal disease (EGID), patients with atopic dermatitis (AD), and nonatopic healthy control (NA) subjects were enrolled. peT(H)2 and conventional T(H)2 (cT(H)2) cell phenotype, function, and cytokine production were analyzed by using flow cytometry. Confirmatory gene expression was measured by using quantitative RT-PCR. Prostaglandin D2 levels were measured with ELISA. Gut T(H)2 cells were obtained by means of esophagogastroduodenoscopy. RESULTS: peT(H)2 cells were identified as chemoattractant receptor-homologous molecule expressed on T(H)2 cells-positive (CRTH2(+)), hematopoietic prostaglandin D synthase-positive CD161(hi) CD4 T cells. peT(H)2 cells expressed significantly greater IL-5 and IL-13 than did hematopoietic prostaglandin D synthase-negative and CD161(-) cT(H)2 cells. peT(H)2 cells were highly correlated with blood eosinophilia (r = 0.78-0.98) and were present in 30- to 40-fold greater numbers in subjects with EGID and those with AD versus NA subjects. Relative to cT(H)2 cells, peT(H)2 cells preferentially expressed receptors for thymic stromal lymphopoietin, IL-25, and IL-33 and demonstrated greater responsiveness to these innate pro-TH2 cytokines. peT(H)2 but not cT(H)2 cells produced prostaglandin D2. In patients with EGID and those with AD, peT(H)2 cells expressed gut- and skin-homing receptors, respectively. There were significantly greater numbers of peT(H)2 cells in gut tissue from patients with EGID versus NA subjects. CONCLUSION: peT(H)2 cells are the primary functional proinflammatory human T(H)2 cell subpopulation underlying allergic eosinophilic inflammation. The unambiguous phenotypic identification of human peT(H)2 cells provides a powerful tool to track these cells in future pathogenesis studies and clinical trials.

The retinoic acid receptor-α modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression.

BACKGROUND: Th2 cytokine responses are enhanced by all trans retinoic acid (ATRA), the bioavailable form of vitamin A. Retinoic acid receptor alpha (RARα) is the high affinity receptor for ATRA that mediates these pro-Th2 effects. We have previously characterized two major human Th2 subpopulations: IL-5- Th2 (IL-5-, IL-4+, IL-13+) and IL-5+ Th2 cells (IL-5+, IL-4+, IL-13+), which represent less and more highly differentiated Th2 cells, respectively. We hypothesized that the pro-Th2 effects of ATRA may differentially affect these Th2 subpopulations. METHODS: Specific cytokine producing Th2 subpopulations were identified using intracellular cytokine staining. Proliferation was measured using the Cell Trace Violet proliferation tracking dye. Apoptotic cells were identified using either annexin-V or active caspase 3 staining. Th2 gene expression was measured using quantitative polymerase chain reaction. RESULTS: ATRA increased the output of Th2 cells from house dust mite allergen (HDM) specific short-term cell lines, and this enhancement was limited to the IL-5+ Th2 subpopulation. Conversely, the RARα antagonist Ro415253 decreased Th2 cell output from these cultures, and this effect was again limited to the IL-5+ Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited Th2 cell proliferation, and this affect was more pronounced for the IL-5+ vs. IL-5- Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited the expression of IL5 in a significant manner, which was not found for IL4 or IL13. CONCLUSIONS: We report that the reciprocal regulation of Th2 cytokine expression and proliferation by RARα modulators are largely limited to modulation of IL-5 gene expression and to proliferation of the highly differentiated IL-5+ Th2 subpopulation. These results suggest that RARα antagonism is a potential means to therapeutically target allergic inflammation. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01212016.

Incidental Histopathologic Finding of Sinonasal Inverted Papilloma Among Surgically Excised Polyps Increases the Risk of Tumor Recurrence.

Inverted papilloma (IP) is a benign tumor remarkable for its tendency toward recurrence. Local relapse implicates incomplete resection concerning the bone adjacent to tumor base. The high false negative rates on biopsies, mainly when nasal polyps coexist, may affect the surgical management and outcomes. Our objective was to study the impact of preoperative histologic diagnosis in IP recurrence, particularly in patients with pre-surgical diagnosis of inflammatory polyps. A retrospective analysis of 62 patients treated for IP was conducted. Demographic data and information about smoking status, alcohol intake, tumor location, histology, presence of nasal polyps, staging, malignancy, previous biopsies and surgical approach were evaluated to identify factors associated with recurrence. Prevalence of nasal polyps was higher in patients with recurrence. Smoking history, alcohol abuse, staging, histologic type, malignancy and surgical approach were not associated with recurrence. The presence of nasal polyps at endoscopy was inversely associated with the diagnosis of IP at incisional biopsy. Incidental histologic diagnosis of IP after surgery increased the risk of recurrence more than tenfold. Biopsy reporting the diagnosis of IP previous to surgery was inversely associated to recurrence. In patients with IP, coexistence of nasal polyps at initial endoscopy and lack of pathological IP diagnosis prior to surgery are strongly associated with a higher risk of recurrence. When excisional biopsy reports IP incidentally, an early revision surgery should be considered in order to avoid future aggressive surgeries because of tumor recurrence.

Publisher Correction: PhIP-Seq characterization of serum antibodies using oligonucleotide-encoded peptidomes.

The version of this paper originally published contained typesetter-introduced errors in some of the code commands, consisting of conversion of a closing backslash (\) to a forward slash (/). These errors have been corrected in the HTML and PDF versions of the protocol.

Comprehensive Profiling of HIV Antibody Evolution.

This study evaluates HIV antibody responses and their evolution during the course of HIV infection. A phage display system is used to characterize antibody binding to >3,300 HIV peptides in 57 adults with early- to late-stage infection. We find that the number of unique epitopes targeted ("antibody breadth") increases early in infection and then stabilizes or declines. A decline in antibody breadth 9 months to 2 years after infection is associated with subsequent antiretroviral treatment (ART) initiation, and a faster decline in antibody breadth is associated with a shorter time to ART initiation. We identify 266 peptides with increasing antibody reactivity over time and 43 peptides with decreasing reactivity over time. These data are used to design a prototype four-peptide "serosignature" to predict duration of HIV infection. We also demonstrate that epitope engineering can be used to optimize peptide binding properties for applications such as cross-sectional HIV incidence estimation.

PhIP-Seq characterization of serum antibodies using oligonucleotide-encoded peptidomes.

The binding specificities of an individual's antibody repertoire contain a wealth of biological information. They harbor evidence of environmental exposures, allergies, ongoing or emerging autoimmune disease processes, and responses to immunomodulatory therapies, for example. Highly multiplexed methods to comprehensively interrogate antibody-binding specificities have therefore emerged in recent years as important molecular tools. Here, we provide a detailed protocol for performing 'phage immunoprecipitation sequencing' (PhIP-Seq), which is a powerful method for analyzing antibody-repertoire binding specificities with high throughput and at low cost. The methodology uses oligonucleotide library synthesis (OLS) to encode proteomic-scale peptide libraries for display on bacteriophage. These libraries are then immunoprecipitated, using an individual's antibodies, for subsequent analysis by high-throughput DNA sequencing. We have used PhIP-Seq to identify novel self-antigens associated with autoimmune disease, to characterize the self-reactivity of broadly neutralizing HIV antibodies, and in a large international cross-sectional study of exposure to hundreds of human viruses. Compared with alternative array-based techniques, PhIP-Seq is far more scalable in terms of sample throughput and cost per analysis. Cloning and expression of recombinant proteins are not required (versus protein microarrays), and peptide lengths are limited only by DNA synthesis chemistry (up to 90-aa (amino acid) peptides versus the typical 8- to 12-aa length limit of synthetic peptide arrays). Compared with protein microarrays, however, PhIP-Seq libraries lack discontinuous epitopes and post-translational modifications. To increase the accessibility of PhIP-Seq, we provide detailed instructions for the design of phage-displayed peptidome libraries, their immunoprecipitation using serum antibodies, deep sequencing-based measurement of peptide abundances, and statistical determination of peptide enrichments that reflect antibody-peptide interactions. Once a library has been constructed, PhIP-Seq data can be obtained for analysis within a week.

An extract of Morinda citrifolia interferes with the serum-induced formation of filamentous structures in Candida albicans and inhibits germination of Aspergillus nidulans.

An aqueous extract of Morinda citrifolia was shown to interfere with the serum-induced morphological conversion of Candida albicans from a cellular yeast to a filamentous form in vitro. The conversion of C. albicans from a cellular yeast to a filamentous form in vivo is associated with pathogenicity. No significant effect on growth in serum-free media was seen at the concentrations used to interfere with the morphological change. The same extract also inhibited the germination of Apergillus nidulans spores. These results demonstrate that M. citrifolia contains a water-soluble component or components that interfere with the morphological conversion of C. albicans and the germination of A. nidulans and may have potential therapeutic value with regard to candidiasis and aspergillosis.

A protein-farnesyl transferase inhibitor interferes with the serum-induced conversion of Candida albicans from a cellular yeast form to a filamentous form.

A commercially available, cell permeable, protein-farnesyl transferase inhibitor interfered with the serum-induced morphological change in Candida albicans from a cellular yeast form to a filamentous form. The inhibitor has a negligible effect on the growth of C. albicans cells in the cellular yeast form, at the levels used to interfere with the morphological change. Conversion of C. albicans from the yeast form to filamentous form is associated with pathogenicity and hence protein-farnesyl transferase inhibitors are potentially of therapeutic value against C. albicans infection.

Separation of the filamentous and cellular yeast forms of C. albicans following serum induction.

Following serum induction of filamentous structures in Candida albicans, the budding and filamentous forms of the organism are separated and the total protein in each form is determined. The method is useful for testing potential chemotherapeutics aimed at interference with the formation of the pathogenic, filamentous form of C. albicans.

Toward a comprehensive and systematic methylome signature in colorectal cancers.

CpG Island Methylator Phenotype (CIMP) is one of the underlying mechanisms in colorectal cancer (CRC). This study aimed to define a methylome signature in CRC through a methylation microarray analysis and a compilation of promising CIMP markers from the literature. Illumina HumanMethylation27 (IHM27) array data was generated and analyzed based on statistical differences in methylation data (1st approach) or based on overall differences in methylation percentages using lower 95% CI (2nd approach). Pyrosequencing was performed for the validation of nine genes. A meta-analysis was used to identify CIMP and non-CIMP markers that were hypermethylated in CRC but did not yet make it to the CIMP genes' list. Our 1st approach for array data analysis demonstrated the limitations in selecting genes for further validation, highlighting the need for the 2nd bioinformatics approach to adequately select genes with differential aberrant methylation. A more comprehensive list, which included non-CIMP genes, such as APC, EVL, CD109, PTEN, TWIST1, DCC, PTPRD, SFRP1, ICAM5, RASSF1A, EYA4, 30ST2, LAMA1, KCNQ5, ADHEF1, and TFPI2, was established. Array data are useful to categorize and cluster colonic lesions based on their global methylation profiles; however, its usefulness in identifying robust methylation markers is limited and rely on the data analysis method. We have identified 16 non-CIMP-panel genes for which we provide rationale for inclusion in a more comprehensive characterization of CIMP+ CRCs. The identification of a definitive list for methylome specific genes in CRC will contribute to better clinical management of CRC patients.

Correction of age-associated deficiency in germinal center response by immunization with immune complexes.

In aging, both primary and secondary antibody responses are impaired. One of the most notable changes in age-associated immune deficiency is the diminished germinal center (GC) reaction. This impaired GC response reduces antibody affinity maturation, decreases memory B cell development, and prevents the establishment of long-term antibody-forming cells in the bone marrow. It is of great importance to explore novel strategy in improving GC response in the elderly. In this study, the efficacy of immunization with immune complexes in overcoming age-associated deficiency in GC response was investigated. We show that the depressed GC response in aged mice can be significantly elevated by immunization with immune complexes. Importantly, there is a significant improvement of B cell memory response and long-lived plasma cells. Our results demonstrate that immune complex immunization may represent a novel strategy to elicit functional GC response in aging, and possibly, to overcome age-related immune deficiency in general.

Rectification of age-associated deficiency in cytotoxic T cell response to influenza A virus by immunization with immune complexes.

Decline in cellular immunity in aging compromises protection against infectious diseases and leads to the increased susceptibility of the elderly to infection. In particular, Ag-specific cytotoxic T lymphocyte (CTL) response against virus is markedly reduced in an aged immune system. It is of great importance to explore novel strategy in eliciting effective antiviral CTL activity in the elderly. In this study, the efficacy and mechanisms of immunization with immune complexes in overcoming age-associated deficiency in cellular immunity were investigated. In this study, we show that the severely depressed CTL response to influenza A in aged mice can be significantly restored by immunization with immune complexes consisting of influenza A virus and mAb to influenza A nucleoprotein. The main mechanisms underlying this recovery of CTL response induced by immune complex immunization in aged mice are enhanced dendritic cell function and elevated production of IFN-gamma in both CD4(+) Th1 and CD8(+) CTLs. Thus, these results demonstrate that immune complex immunization may represent a novel strategy to elicit effective virus-specific cytotoxic response in an aged immune system, and possibly, to overcome age-related immune deficiency in general.

Overexpression of Bcl(XL) in B cells promotes Th1 response and exacerbates collagen-induced arthritis.

B cells play a pathogenic or regulatory role in many autoimmune diseases through production of autoantibodies, cytokine production, and Ag presentation. However, the mechanisms that regulate these B cell functions under different autoimmune settings remain unclear. In the current study, we found that when B cells overexpress an antiapoptotic gene, Bcl(XL), they significantly increased production of IFN-gamma and enhanced Th1 response. Consistently, Bcl-x(L) transgenic mice developed more severe and sustained collagen-induced arthritis due to the enhanced Th1 response. The production of autoantibodies in Bcl(XL) transgenic mice was comparable to that in wild-type mice. Thus, our results indicate a novel role of Bcl(XL) in regulating B cell functions and immune responses. In patients with rheumatoid arthritis, arthritogenic B cells often up-regulate Bcl(XL) expression, which may not only render B cells resistant to apoptosis but also alter the ability of the autoreactive B cells to produce cytokines and modulate the inflammatory response. This may have therapeutic implications if Bcl(XL) expression can be down-regulated in autoreactive B cells.

CXCL13 neutralization reduces the severity of collagen-induced arthritis.

OBJECTIVE: To investigate the role of CXCL13 in the development and pathogenesis of collagen-induced arthritis (CIA), and to determine the mechanisms involved in the modulation of arthritogenic response by CXCL13 neutralization. METHODS: Mice were immunized with type II collagen (CII) and treated with anti-CXCL13 or control antibodies during boosting. Mice were monitored for the development and severity of arthritis. The effects of CXCL13 neutralization on immune response to CII were evaluated by cytokine production by CII-specific T cells and CII-specific antibody production. Follicular response in the spleen and in synovial tissue was determined by in situ immunohistology. RESULTS: Mice receiving neutralizing antibodies to CXCL13 developed significantly less severe arthritis compared with mice injected with phosphate buffered saline or control antibodies. Follicular response both in the spleen and in synovial tissue was inhibited by anti-CXCL13 treatment. Injection with anti-CXCL13 antibodies did not significantly affect antigen-specific recall lymphocyte proliferation or type 1 cytokine production in vitro. Antibody response specific to CII was not inhibited by anti-CXCL13 treatment. However, anti-CXCL13 treatment induced significantly higher levels of interleukin-10 production after in vitro CII stimulation. CONCLUSION: Neutralization of CXCL13 inhibits the development of CIA and reduces follicular response in both lymphoid and nonlymphoid tissues. These findings may have important implications regarding the pathogenesis and treatment of autoimmune arthritis.

Blockade of lymphotoxin pathway exacerbates autoimmune arthritis by enhancing the Th1 response.

OBJECTIVE: To study the role of the lymphotoxin (LT) signaling pathway in the development and pathogenesis of collagen-induced arthritis (CIA), and to understand the mechanisms by which blockade of the LT pathway influences the arthritogenic response to type II collagen (CII). METHODS: LTalpha-deficient and wild-type C57BL/6 mice were immunized with CII. Male DBA/1 mice were immunized with CII and treated with LTbeta receptor immunoglobulin fusion protein (LTbetaR-Ig) or control Ig. Mice were monitored for the development and severity of arthritis. The effects of LT blockade on immune responses were evaluated by cytokine production and antigen-specific proliferation in vitro, the delayed-type hypersensitivity (DTH) response, and serum levels of CII-specific antibodies. RESULTS: CIA that developed in LTalpha-deficient mice was more severe and prolonged than that which developed in wild-type mice. Blocking LT signaling with LTbetaR-Ig significantly exacerbated the disease. Exacerbation of CIA was associated with an enhanced Th1-type response, including increased type 1 cytokine production, an enhanced DTH response, and elevated production of CII-specific IgG2a antibodies. CONCLUSION: Blockade of the LT signaling pathway exacerbates the development and progression of CIA, probably by skewing the Th1/Th2 balance that determines the outcome of autoimmune responses.

Carvone and perillaldehyde interfere with the serum-induced formation of filamentous structures in Candida albicans at substantially lower concentrations than those causing significant inhibition of growth.

Carvone and perillaldehyde were shown to inhibit the transformation of Candida albicans to a filamentous form at concentrations far lower and more biologically relevant than the concentrations necessary to inhibit growth. This morphological transformation is associated with C. albicans pathogenicity; hence these naturally occurring monoterpenes are potential lead compounds in the development of therapeutic agents against C. albicans infection.